Cytochrome b5 and its physiological significance

Pharmac. Tl~er.A, Yol. 2, pp. 477-515, 1978. Pergamon Press. Printed in Great Britain

Specialist Subject Editors: JOHN B. SCHENKMAN a n d DAVID KUPFER

CYTOCHROME PHYSIOLOGICAL NOZOMU

b5 A N D I T S SIGNIFICANCE OSHINO

Research and Development Department, Nihon Schering K. K., 2-6-64 Nishimiyahara, Yodogawa-ku, Osaka 532, Japan

1. INTRODUCTION Despite its early discovery and a large amount of work on its biochemistry, cytochrome b5 did not draw much attention from pharmacologists until recently. With recent progress in the investigation of microsomal drug metabolizing systems, it has become obvious that microsomal cytochrome b5 is able to mediate electron transfer from reduced pyridine nucleotides to a group of microsomal mixed function oxidases of the cytochrome P-450 type. However, the physiological importance of this electron transfer pathway in drug metabolism is currently a subject of controversy in biochemical pharmacology. A few review articles that touch on the subject of cytochrome b5 have appeared, including those by C. F. Strittmatter (1961) and by P. Strittmatter (1963) dealing with early studies on this cytochrome, and by Schenkrnan et al. (1976) on the function of cytochrome bs, especially in relation to the drug metabolizing system. The aim of the present review is to delineate the global features of cytochrome b5 from a standpoint independent of the drug metabolizing enzyme system and, thus, the description is divided into sections concerning historical aspect, distribution, purification, membrane-binding property, biosynthesis and physiological functions. A detailed account of the acyl CoA AD-desaturase system is also provided in a separate section of this article, with the intention of emphasizing the presence of a group of unique oxidases by which a vital role of cytochrome b5 may be well substantiated. 2. HISTORICAL ASPECT Most living organisms were found to contain, besides hemoglobin, a hemochromelike substance which showed a spectrum with characteristic absorption bands, visible spectroscopically under reducing conditions. In 1925, Keilin demonstrated in his early study on cellular respiration, that the absorption bands of this substance, i.e. A band (615 n m - 593 nm), B band (567.5 n m - 561 nm), C band (554.5 n m - 546 nm) and D band (532 n m - 511 nm), did not arise from a single component but were composed of a- and B-bands of spectra belonging to three distinct pigments, a', b' and c'. He described these pigments under the name of cytochrome, signifying merely 'cellular pigment'. Vast numbers of microspectroscopic examinations were subsequently carried out, in a period between 1925 and 1955, to analyze the spectra of cytochromes. During the progress of these studies, many variations of absorption spectra were observed, especially in the region of the B band in various tissues, plants and microorganisms. The cytochromes responsible for these spectral variations were successively named cytochrome b (Keilin, 1925), cytochrome b~ (Keilin, 1934), cytochrome b2 (Bach et al., 1946; Appleby and Morton, 1954), cytochrome b3 (Martin and Morton, 1955) and cytochrome b4 (Egami et al., 1953). Sanbarn and Williams (1950) observed that Cecropia silkworm larvae contained a cytochrome with a broad absorption band extending from 551 nm to 562 nm with a maximum around 557 nm. They termed this pigment cytochrome x. In a later report, Pappenheimer and Williams (1954) felt this 477

478

N. OSH|NO

pigment to be of the b group of cytochrome and it was referred to as cytochrome bs. Chance and Williams (1954) examined the kinetics of the reduction by NADH of a cytochrome which was located in rat liver microsomes. They used in their report the name of cytochrome bs, simply because absorption maxima in the reduced minus oxidized difference spectrum of this cytochrome were observed at 426 nm and 556 nm, the spectrum being nearly identical with that measured for the Cecropia preparation. The microsomal cytochrome in rat liver had already been, by that time, named cytochrome rn by Strittmatter and Ball (1952); the new cytochrome was reducible by both NADH and NADPH and was distinct from the mitochondrial cytochromes. The microsomal cytochrome was found to occur widely in the liver of not only rat but also guinea pig, rabbit and calf (Strittmatter and Velick, 1956a). Strittmatter and Velick (1956a) succeeded in liberating and isolating it from rabbit liver microsomes by utilizing a pancreatic lipase fraction. Molecular properties of the isolated cytochrome were characterized, and it was confirmed to be one of the b type cytochromes which possess an iron protoporphyrin IX a s ' a prosthetic group (Strittmatter and Velick, 1956a) and to function in univalent oxidation-reduction reactions (Velick and Strittmatter, 1956). The microsomal cytochrome was also isolated from pig liver by Garfinkel (1957), from adrenal medulla of pig by Krish and Straudinger (1958) and from calf liver by Strittmatter (1960). Spectroscopic surveys of microsomal fractions revealed the presence of this cytochrome not only in liver and adrenal medulla, but also in pancreas (Palade and Siekevitz, 1956b), mammary gland, milk and intestinal mucosa (Bailie and Morton, 1955). Therefore, it was established with these studies that the microsomal cytochrome was one of the intrinsic components of endoplasmic reticulum, and, meanwhile, the name cytochrome b5 has become, by common usage, generally accepted in referring to spectrally similar cytochromes, not only from liver microsomes but also from other and often widely differing biological materials. In liver microsomes, cytochrome b5 is reduced easily by NADH and NADPH through NADH-cytochrome b5 reductase and NADPH-cytochrome c reductase, respectively. The reduced cytochrome in microsomes is oxidized only very slowly, either directly or indirectly through the auto-oxidation of other components, by molecular oxygen (Chance and Williams, 1954; Strittmatter and Velick, 1956a; Modirzadeh and Kamin, 1965). The cytochrome b5 purified after solubilization by i~ancreatic lipase- or protease-treatment showed a molecular weight of about 12,000 (Spatz and Strittmatter, 1971) and a standard redox potential of +0.02 V at pH 7.0 (Velick and Strittmatter, 1956). Cytochrome b5 is reduced fully or partially by chemical reagents such as potassium borohydride, sodium hydrosulfite, cysteine and ascorbate, and is oxidized by reagents having appropriate redox potential, such as potassium ferricyanide, ferric chloride, cytochrome c and menadione (Strittmatter and Velick, 1956a). The optical absorption spectrum of cytochrome b5 in the visible region shows a maximum at 413 nm for the oxidized form, and a-,/3- and ~,-peaks of the reduced form at 556 nm, 526 nm and 423 nm, respectively (Strittmatter and Velick, 1956a). A unique spectral property of cytochrome b5 is its broad and asymmetric a-band, which, at low temperature, is split into two absorption peaks at 553 nm and 557 nm (Garfinkel, 1957). Though molecular characterization of cytochrome b5 has been achieved rather early as described above, its physiological significance has remained obscure only until recently. This microsomal fraction was known already in 1949 to exhibit a NADHcytochrome c reductase activity (Hogeboom, 1949). Strittmatter and Velick (1956b) were able to solubilize and purify a NADH-cytochrome b5 reductase from rabbit liver microsomes. This reductase is a flavoprotein which is capable of catalyzing cytochrome b5 reduction by NADH and shows diaphorase activity with several redox dyes as electron acceptors. Though not capable of reducing cytochrome c directly, the reductase was demonstrated to show NADH-cytochrome c reductase activity in the presence of a catalytic amount of cytochrome b5 because of rapid reduction of cytochrome c by reduced cytochrome b5 under in vitro conditions. This reconstitution

Cytochrome b5and its physiologicalsignificance

479

of the microsomal NADH-cytochrome c reductase activity in vitro (Strittmatter and Velick, 1956b) is one of the early examples in biochemistry which elegantly substantiated the molecular constituents of a complex enzyme system. The microsomal NADH-cytochrome c reductase activity was distinguished clearly by its susceptibility to antimycin A, from a similar mitochondrial activity which plays a major role in cellular respiration (De Duve et al., 1955). In addition, cytochrome c (Beinert, 1951) and its oxidase (Schneider, 1946), which may act in vitro as an electron acceptor for the microsomal reductase system, are strictly confined to a separate cellular compartment, the mitochondrion. Therefore, it remained to establish a physiological meaning for the cytochrome bs-containing electron transfer system with respect to its ability to reduce cytochrome c. Its high content in liver implies important roles of cytochrome b5 in cellular function, yet, cytochrome b5 itself does not react with molecular oxygen and other chemical ligands such as CO and cyanide, suggesting that it functions only as univalent electron carrier. Strittmatter (1961) pointed out in his review article the following four possible roles for cytochrome bs: (i) to serve as a storage form of hemin or a precursor in the formation of other cytochromes or hemin compounds; (ii) to serve as a link in terminal electron transport to oxygen directly or via a mediator; (iii) to play an active role in biological oxidation-reduction reactions; and (iv) to funnel electrons available from NADH or NADPH to acceptors in synthetic processes that are reductive in character or which involve elements of both reduction and oxidation, such as hydroxylation reaction requiring both NADH or NADPH and molecular oxygen. This problem was approached from a variety of directions, as exemplified by a study in relation to the possible participation of cytochrome b5 in Na ÷-, K+-transport mechanisms (Siekevitz, 1965), and most attempts appeared to be without success. Staudinger and .his associates (Kersten et al., 1958; Krish and Staudinger, 1959) studied a possible role for cytochrome b5 in the ascorbate oxidation-reduction system and showed what might be one of the few positive results for the function of cytochrome b5 at that period. However, until the middle 1960s, a series of enzymological and kinetic studies on a purified system of cytochrome b5 and its reductase was continued successfully only by Strittmatter and his collaborators (Strittmatter, 1965), and the attention of researchers in the area was shifted to other subjects, such as to another microsomal component, cytochrome P-450. Such an impasse in the investigation of microsomal cytochrome b5 was broken by work carried out by a Japanese group in the late 1960s (Oshino et al., 1967; Ito and Sato, I968). An enzyme catalyzing the Ag-desaturation of long chain acyl CoA in microsomes was considered to be a mixed function oxidase which required NADPH and molecular oxygen (Bloomfield and Block, 1960; Stoffel, 1961; Marsh and James, 1962). Because of apparent similarity between their cofactor requirements, enzymatic properties of the desaturation activity were compared to those of aniline hydroxylation activity to assess the possible participation of the same microsomal electron transfer components in both activities. Oshino et al. (1966) found that, although the desaturase shares NADPH-cytochrome c reductase with the drug hydroxylation system, the desaturation enzyme contained a hitherto unknown terminal component, a cyanide-sensitive factor, which was distinct from cytochrome P-450 by its inability to react with CO. It was also found that, in addition to NADH and NADPH, ascorbate at high concentration served as a weak electron donor in the desaturation reaction. This finding led them to suspect a possible role of cytochrome b5 in the electron transfer mechanism of the desaturation system, because this was the only microsomal component known to be reducible by all three electron donors which support desaturation. Identification and characterization of the constituents of the desaturation system were first carried out mainly by analysis of reaction kinetics using intact microsomal membranes; Oshino et al. (1967) proposed that the overall desaturase system in rat liver microsomes consisted of at least four components, i.e. NADH-cytochrome b5 reductase, NADPH-cytochrome c reductase, cytochrome b5 and a terminal desa-

480

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turase, the cyanide sensitive factor. They considered cytochrome b5 to be an intermediate electron carrier which mediated reducing equivalents from NADH, NADPH and ascorbate to the terminal desaturase. Solubilization of microsomal electron transfer components was attempted with hydrolytic enzymes. However, Omura et al. (1967) and Ito and Sato (1969) came to a realization that the previously purified cytochrome bs, which had been solubilized by pancreatic lipase treatment, was not the intact form existing on the membrane but was a catalytically active peptide fragment, liberated from the membrane by proteolytic digestion. This was then substantiated by Ito and Sato (1968) who solubilized and purified the native form of cytochrome b5 with detergents. The cytochrome b5 thus purified possessed a larger molecular size and showed a strong hydrophobic character. As was revealed in subsequent investigations (Spatz and Strittmatter, 1971; Strittmatter et al., 1972), the native cytochrome b5 differed markedly from the hydrophilic segment of cytochrome b5 in regard to its capability of binding to the membrane and in reconstructing the various enzyme activities. These new developments with cytochrome b5 encouraged many new investigations in this field. They were followed by works concerning: (i) preparation of antibody against cytochrome b5 (Raftell and Orrenius, 1971; Oshino and Omura, 1973) and its use in demonstration of participation of this cytochrome in several microsomal activities (Sasame et al., 1974; Mannering et al., 1974; Paltauf et al., 1974; Jansson and Schenkman, 1977; Reddy et al., 1977); (ii) purifications of the native form of NADHcytochrome b5 reductase (Spatz and Strittmatter, 1973; Mihara and Sato, 1975) and of acYl CoA A9-desaturase (Strittmatter et al., 1974); and (iii) reconstruction of the complete desaturation enzyme system with these purified constituents into aggregates (Shimakata et al., 1972; Strittmatter et al., 1974), and on vesicles of synthetic phospholipids (Enoch et al., 1976). It is now established that at least one of the major roles of cytochrome b5 in microsomal membranes is to manipulate the supply of reducing equivalents from NADH and NADPH to a group of several desaturases which are key enzymes in biosynthetic pathways of unsaturated fatty acids and of two kinds of phospholipids. The knowledge obtained within the last 15 years will be the major topic of the following sections.

3. DISTRIBUTION OF CYTOCHROME b5 3.1. CYTOCHROME b5 IN LIVERS OF DIFFERENT ANIMALS The hemoprotein which shows the characteristic absorption spectrum of cytochrome b5 is detectable not only in tissues of most animals but also in insects (Okada and Okunuki, 1969; Capdevila et a l l 1975), plants (Potts et al., 1976) and yeast (Yoshida et al., 1974). The specific content of cytochrome b5 in microsomal fractions may show some variations, depending on its purity, degree of subfractionation and other conditions of preparation. In order to provide a rough guide for contents of cytochrome b~ and of cytochrome P-450 in ordinary microsomal fractions of livers from various animals, some of the results reported by Ichikawa and Yamano (1967) are cited in Table 1. There are species differences in the hepatic content of cytochrome bs, varying from 0.96nmolmg -~ of microsomal protein in sheep to 0.14 nmol mg -j of microsomal protein in carp. The hydrophilic and catalytically active segment of cytochrome b5 has been purified from eight species of vertebrate, i.e. human, monkey, calf, horse, pig, rabbit, rat and chick. Comparative studies of their primary structures showed that the cytochrome b5 of mammalian livers contained common structures for the major portions of their amino acid sequences (Tsugita et al., 1968, 1970; Ozols and Strittmatter, 1969; Ozols, 1970; Nobrega and Ozols, 1971; Ozols et al., 1976), and that some replacements of amino acids have arisen from a single mutation of a base pair in DNA (Tsugita et al., 1970). A somewhat different amino acid sequence was detected in chick liver cytochrome

Cytochrome b 5 and its physiological significance

481

TABLE 1. Contents of Cytochrome b5 and Cytochrome P-450 in Micro-

somal Fractions from Liver o[ Various Animal Species Species

Sex

Sheep Rabbit Calf Hamster Rat Horse Guinea pig Dog Mouse Pig Goose Hen Carp

female male -male male male female male male female female female --

Cytochrome b5 Cytochrome P-450 (nmol/mg of protein) 0.96 0.88 0.80 0.64 0.64 0.61 0.55 0.42 0.40 0.29 0.24 0.16 0.14

0.67 1.72 1.55 1.14 0.82 0.88 0.79 0.75 0.61 0.42 0.18 0.24 0.38

Some of the values reported by Ichikawa and Yamano (1967) were cited in this table. More information can be seen in the original report.

bs, yet it still retained the essential structure, which consisted of the peptide sequence commonly observed in cytochrome b5 of mammalian livers (Tsugita et al., 1970; Ozols et al., 1976). 3.2. INTRACELLULAR LOCALIZATION OF CYTOCHROME b5 IN LIVER Regarding its intracellular localization, cytochrome b5 was believed to be located specifically on hepatocyte endoplasmic reticulum (Strittmatter, 1963; Chance and Williams, 1955). However, because of an occasional morphological continuity of endoplasmic reticulum membrane to nuclear envelope, plasma membrane, outer mitochondrial membrane and Golgi apparatus, a possible presence of cytochrome b5 in these membrane fractions was carefully re-examined. Golgi membrane and nuclear membrane fractions from livers of calf, rat, rabbit and pig were found to contain a cytochrome reducible with NADH, the content being of order of 1-0.6 nmol mg -~ of protein in the former (Ichikawa and Yamano, 1970a; Fleischer et al., 1971) and of 0.3-0.5 nmol mg -j of protein in the latter (Franke et al., 1970; Fleischer et al., 1971). The cytochrome detected in these membranes was judged to be cytochrome bs, based on a reduced minus oxidized difference spectrum; the cytochrome showed a characteristic absorption spectrum of microsomal cytochrome b5 at room temperature, and its asymmetric a-peak was split into two peaks at 552 and 558nm under liquid nitrogen temperature (Ichikawa and Yamano, 1970a; Fleischer et al., 1971). By comparing this cytochrome to the contents of cytochrome P-450 and of other marker enzymes such as glucose 6-phosphatase and ATPase, it was determined that significant portions of the cytochrome found in these membrane fractions were the intrinsic component of these membrane structures. Plasma membrane fractions contained only a very small amount of the b type cytochrome, and it was ascribed to contaminating cytochrome b5 of microsomes (Ichikawa and Yamano, 1970a; Fleischer et al., 1971). Mitochondrial fractions of rat liver contain at least three kinds of cytochromes (Fukushima et al., 1972) which resemble spectroscopically microsomal cytochrome b5 and which have been often referred to erroneously as cytochrome bs. Two of them are now known to be the sulfite oxidase and probably its peptide fragment (Ito, 1971) located in the space between the outer and inner membranes of the mitochondrion (Ito, 1971; Fukushima et al., 1972). The third component is located specifically on the outer surface of the outer membrane; its content in the purified outer membrane of rat liver mitochondria was reported to be 0.54 nmol mg -~ of protein (Fukushima and Sato, 1973). This b-type cytochrome was almost indistinguishable from the microsomal cytochrome b5 not only because the two cytochromes were constituents of antimycin A-insensitive NADH-cytochrome c reductase activities (Sottocasa et al.,

482

N. OSHINO

1967), but also based on their chromatographic behavior after tryptic solubilization, molecular weights and absorption spectra (Fukushima and Sato, 1973). Only recently was it shown that the c y t o c h r o m e of the mitochondrial outer m e m b r a n e differed completely from microsomal c y t o c h r o m e b5 in regard to its amino acid sequence and inability to react with the specific antibody against microsomal c y t o c h r o m e b5 (Fukushima and Sato, 1973), indicating the different origins of these apparently identical c y t o c h r o m e s in the evolutionary process. A c y t o c h r o m e bs-like h e m o p r o t e i n having a molecular weight of about 100,000 was isolated f r o m a supernatant fraction of kidney h o m o g e n a t e s and was assumed to be an intact f o r m of c y t o c h r o m e b5 (Hangum et al., 1970). These investigators used hypotonic conditions in the process of subfractionation and, hence, their findings m a y be attributable to the intact f o r m of sulfite oxidase which is easily liberated from mitochondria under hypotonic conditions (Ito, 1971; F u k u s h i m a e t al., 1972). 3.3. DISTRIBUTIONIN VARIOUS ORGANS AND TISSUES S o m e reported values for contents of microsomal c y t o c h r o m e b5 in various tissues are listed in Table 2. These values should be regarded only as contents of the c y t o c h r o m e which were detected spectrophotometrically upon reduction with N A D H ; some of the c y t o c h r o m e m a y not be identical to hepatic c y t o c h r o m e b5 in its ability to be reduced by N A D H . C y t o c h r o m e b5 appears to be present in almost all tissues examined. Detection of c y t o c h r o m e b5 and its reductase in the m i c r o s o m e s f r o m epididymal adipose tissues, lung, m a m m a r y gland, intestinal m u c o s a and brain m a y be consistent with the o c c u r r e n c e in these tissues of various desaturation activities in which c y t o c h r o m e b5 is a participant (Oshino e t al., 1971; McDonald and Kinsella, 1973; Cook and Spencer, 1973a; M o n t g o m e r y , 1976). It is of interest to point out that, TABLE2. Cytochrome b~ Content in the Microsomal Fractions from Various Tissues Animals Rabbit

Organs Kidney Ovary Adrenal gland Skeletal muscle Heart Neutrophilic granule of leucocyte Erythrocyte

Pig

Thyroid Pancreas Adrenal cortex Adrenal medulla

Calf

Cerebellum Pituitary gland

Rat

Kidney Lung Epididymal adipose tissues Intestinal mucosa Testis

Cytochrome b5 (nmol/mg protein) 0.10 0.21 0.08 N.D. N.D. 0.03 3.3-2.3 (nmol/5 ml cell)

References Ichikawa and Yamano, 1967 Ichikawa and Yamano, 1967 Ichikawa and Yamano, 1967 Imai et al., 1966 Imai et al,, 1966 Shinagawa et al., 1966 Passon et al., 1972

0.03-0.04 0.06 0.24 0.25

Hosoya and Morrison, 1967 Ichikawa and Yamano, 1967 Ichikawa and Yamano, 1967 Ichikawa and Yamano, 1967

0.04 0.07

Ichikawa and Yamano, 1967 Ichikawa and Yamano, 1967

0.15 0.03--0.05

Oshino and Sato, 1971 Montgomery, 1976

0.07-0.13 0.13 0.03

Oshino and Sato, 1971 Takasue and Sato, 1968 Oshino and Sato, 1971

Calf

Thymus

Cow

Milk Mammary gland

0.05 (~mol/g total N) 1.8 (txmol/gtotal N)

Bailie and Morton, 1955 Bailie and Morton, 1955

Human

Erythrocyte

4.06 (nmol/5 ml cell)

Passon et al., 1972

N.D. = not detected.

0.04

Yamagata et al., 1966

Cytochrome b 5 and its physiological significance

483

in spite of a relatively high content of cytochrome bs, microsomes prepared from kidney did not show detectable activity of acyl CoA A9-desaturation (Oshino and Sato, 1971; Cinti and Montgomery, 1976). Though cytochrome b5 was not detected in microsomal fractions from skeletal and heart muscles, the activity of antimycin A-insensitive NADH-cytochrome c reductase was detected in the latter, suggesting the presence of small amounts of cytochrome b5 in the heart (Imai et al., 1966). However, the mitochondrial fraction from heart is known to contain sulfite oxidase (Maclead et al., 1961) and also the outer-membrane cytochrome (Fukushima et al., 1972) and, hence, identification of a small amount of the microsomal cytochrome b5 in these organs may not be feasible. Microsomes from tumor cells usually contain lower amounts of cytochrome bs, and a correlation appears to exist between the growth rate of tumors and the degree of decrease in contents of microsomal cytochromes (Sugimura et al., 1966; Hagihara et al., 1973). Contents of microsomal cytochrome b~ were found to be of the order of 0.1 nmolmg -~ of protein in relatively slow growing hepatomas such as in minimal deviation Morris hepatomas (Hagihara et al., 1973) and 3'-methyl-4-dimethylaminoazobenzene induced hepatomas (Oyanagui et al., 1974), those being comparable to a content of 0.09 nmol mg -1 of protein in embryonic liver of rat (Hagihara et al., 1973). Little or no microsomal cytochrome b5 was detectable in the rapidly growing hepatomas such as Ascites tumor cell (Sugimura et al., 1966; Sato and Hagihara, 1970). It is worth mentioning that, despite the remarkable decrease of the cytochrome b5 content in the hepatoma, the host liver during carcinogenesis maintained only a slightly less than normal level of cytochrome b5 (Hagihara et al., 1973; Oyanagui et al., 1974).

3.4. CYTOCHROME b5 IN ERYTHROCYTE Though the mammalian erythrocyte is considered to be devoid of endoplasmic reticulum, mitochondria and nuclei (Beams and Kessel, 1966), it has been reported to contain cytochrome bs (Passon et al., 1972). In contrast to the hepatic cytochrome b5 which binds tightly to membranous systems and is solubilized only by disruption of the membrane, the erythrocyte cytochrome was found in the supernatant fraction of red cell hemolysates and has been purified from human and rabbit erythrocytes (Passon et al., 1972). The purified cytochrome was reduced by NADH in the presence of either microsomal NADH-cytochrome b5 reductase or a NADH-dependent reductase of erythrocyte (Passon and Hultquist, 1972). The absorption spectrum of the reduced cytochrome showed an asymmetric a-peak at 556 nm identical to that of microsomal cytochrome bs. The molecular weight of the cytochrome was estimated to be 14,600 from sedimentation and diffusion measurements and 18,400 by gel filtration (Passon et al., 1972). The cytochrome, in combination with its reductase, exhibited methemoglobin reductase activity (Passon and Hultquist, 1972), and the antibody against microsomal cytochrome b5 inhibited this enzymic activity (Kuma et al., 1976), indicating that the erythrocyte cytochrome resembled closely the microsomal cytochrome bs. However, the erythrocyte cytochrome lacked amphipathic nature and appears to correspond to the hydrophilic segment of liver cytochrome bs; the possibility that this soluble form of the cytochrome was derived from proteolytic digestion in the process of preparation was ruled out in this study (Passon et al., 1972). Electron microscopic examination (Beams and Kessel, 1966) demonstrated that reticulocytes contain polysomes, mitochondria, micropinocytosis vesicles, tubules of varying size and other cellular structures. As maturation of the reticulocyte occurred, the cell gradually decreased in size and increased in density; concomitant with these changes was a progressive degradation of the cellular structure so that by the time the erythrocyte stage was reached, these materials had completely disappeared. It is of interest whether the erythrocyte soluble cytochrome bs exists as a soluble enzyme in the reticulocyte stage of development.

484

N. OSHINO

4. SOLUBILIZATION AND PURIFICATION OF CYTOCHROME b5 As will be described later, cytochrome b5 is a hemoprotein having an amphipathic nature, arising from the separate localization of hydrophobic and hydrophilic domains in the molecule. This amphipathic molecule is thought to bind on the outer surface of the • endoplasmic reticulum, embedding its hydrophobic moiety in the lipid layer and exposing the hydrophilic moiety to cytoplasm. Two methods are available for solubilization of this cytochrome: (i) the method in which the catalytically active hydrophilic segment of the molecule is liberated from the hydrophobic segment by protease treatment; and (ii) the method in which detergents are used to disrupt the hydrophobic interaction with the membrane to bring the amphipathic molecule into the aqueous medium. The former methods utilize pancreatic lipase (Strittmatter and Velick, 1956a), trypsin (Omura and Takesue, 1970) or other proteases (Kajihara and Hagihara, 1968), and the latter utilize nonionic and ionic detergents in the purification of native cytochrome b5 (Ito and Sato, 1968; Spatz and Strittmatter, 1971).

4.1. SOLUBILIZATION WITH PROTEASES Three microsomal electron transfer components, i.e. NADH-cytochrome b5 reductase (Mihara and Sato, 1972; Spatz and Strittmatter, 1973), NADPH-cytochrome c reductase (Vermilion and Coon, 1974) and cytochrome b5 (Spatz and Strittmatter, 1971) are known to be amphipathic proteins of single peptide composition. Catalytically active moieties of these molecules are liberated from the microsomal membranes by cleaving the peptide junction between hydrophilic and hydrophobic domains of the molecule. These molecules showed different sensitivities to proteolytic cleavage; for instance, Ito and Sato (1969) observed that while NADPH-cytochrome c reductase is solubilized easily at a very low concentration of trypsin, bacterial protease or chymotrypsin, liberation of cytochrome b5 from microsomes required relatively high concentrations of these proteases. NADH-cytochrome b5 reductase was liberated only by lysosomal acid-protease (Takesue and Omura, 1970a; Sargent et al., 1970), i.e. cathepsin D (Ito, 1974), under slightly acidic conditions. These solubilizations were rather specific, and no solubilization of other enzyme activities, such as ATPase, glucose-6-phosphatase (Ito and Sato, 1969) and esterase (Akao and Omura, 1972), as well as phospholipids and cholesterol (Ito and Sato, 1969), was obtained by these treatments.

4.2. PURIFICATION OF THE HYDROPHILIC SEGMENTS OF CYTOCHROME b5 Since the first success by Strittmatter and Velick (1956a), many other reports on solubilization and purification of cytochrome bs, using pancreatic lipase, have appeared. It was generally considered that the intact cytochrome b5 had been liberated upon digestion of membrane lipids by lipase because care was taken to remove and/or inhibit protease activity in the lipase fractions. Nevertheless, it now becomes clear that mild protease treatment of the intact form of cytochrome b5 yields hydrophilic segments which are in many respects identical to the cytochrome b5 obtained previously by lipase treatment (Nobrega and Ozols, 1971). Probably in the latter studies, cytochrome b5 was liberated from the membrane by proteolytic action of either a contaminative protease in the lipase fraction or by endogenous proteases activated by the lipase treatment. The process of purification of the hydrophilic segment of cytochrome b5 consists usually of a combination of the following three steps. (i) proteolytic cleavage; (ii) separation of components based on their molecular sizes by means of either Sephadex gel filtration or ammonium sulfate fractionation; and (iii) column chromatography on ion-exchange cellulose. Combinations of these steps should be selected depending on the amount of starting material, the purity desired, available times and equipment etc.

Cytochrome b5 and its physiological significance

485

SCHEME 1. Flow Sheet for Purification o.f the Hydrophilic Segment of Cytochrome b~ Step I.

Step 2.

Step 3.

Step 4.

Rat livers (600-700 g) -4 vol. of 0.15 M KCI Homogenate -differential centrifugations Microsomal pellet -suspended in 700 ml of 0.15 M KCI-10 mM EDTA (pH 7.0) -78,000 × g, 60 min, centrifugation Pellet -suspended in 700 ml of 0.1 M potassium phosphate buffer (pH 7.0) -78,000 × g, 60 min, centrifugation Washed microsomes -suspended in 0.1 M phosphate buffer (15-20 mg protein/ml) -add 1/10 vol. of trypsin (3 mg ml-~ of 2 mM HCl) -0°C for 14-16 hr -78,000 × g, 120 rain, centrifugation Clean red supernatant -lyophilization Freeze-dried extract -dissolved in 100 ml of 10mM phosphate buffer pH 7.5 -Sephadex G-100 column (5 × 80 cm) equilibrated with 10 mM phosphate buffer pH 7.5 -eluted with the same buffer (flow rate 30-40 ml hr -~) Eluate containing cytochrome b5 -applied on DEAE-cellulose column (2 x 25 cm) equilibrated with 10 mM phosphate buffer (pH 7.5) -washed with 50 ml of 50 mM phosphate buffer pH 7.5 -linear concentration gradient of KCI from 0 to 0.35 M in 50 mM phosphate buffer pH 7.5 (total vol, of 350 ml, flow rate 20-30 ml hr -~) -cytochrome b5 was eluted with about 0.2 M KCI Cytochrome bs-rich fraction -diluted with 200 ml of H20 -applied on a DEAE-cellulose column (2 x 3 cm, equilibrated with 10 mM phosphate buffer) -washed with 20 ml of 10 mM phosphate buffer -eluted with 0.2 M KCI in 50 mM phosphate buffer Concentrated cytochrome b5 (5-7 ml)

Criteria for purity of rat liver cytochrome b5 hydrophilic segment: Protoheme content=8081 nmol mg-~ of protein; molecular weight = 12,500. Absorption maxima in the visible spectrum: reduced form: 423nm, 526nm, 555 nm, 559nm (shoulder); oxidized form: 414nm, 530nm, 560nm. The ratio A555nm(~cduced)/A2e0,~,~oxidizca) = 1.28--1.32. Method: Omura and Takesue, 1970. T h e r e f o r e , o n l y t h e p r i n c i p a l p o i n t s o f p u r i f i c a t i o n will b e d e s c r i b e d , as e x e m p l i f i e d b y t h e m e t h o d o f O m u r a a n d T a k e s u e (1970) ( S c h e m e 1). S t e p 1: Preparation o f ' Washed M i c r o s o m e s ' It is o f u t m o s t i m p o r t a n c e in p u r i f i c a t i o n to refine the s t a r t i n g m a t e r i a l s as m u c h as p o s s i b l e . I n this p r o c e d u r e , m i c r o s o m e s a r e w a s h e d first w i t h 0.15M K C I - 1 0 m M E D T A s o l u t i o n a n d t h e n w i t h 0.I t,i p h o s p h a t e buffer. M i c r o s o m e s a r e s t r i p p e d o f ribosomes and of non-membranous materials, particularly of hemoglobin, by these w a s h i n g s ( O m u r a et al., 1967). P r o t e i n s t h u s r e m o v e d c o r r e s p o n d to m o r e t h a n 30 p e r c e n t o f t h e o r i g i n a l l e v e l ( D a l l n e r , 1974). If t h e s e p r o c e s s e s a r e o m i t t e d , i n c u b a t i o n w i t h p r o t e a s e s m a y c a u s e n o t o n l y l i b e r a t i o n o f c y t o c h r o m e b5 b u t also a c t i v a t i o n of l a t e n t r i b o s o m a l R N a s e (Ito a n d S a t o , 1969), p r o d u c i n g v a r i o u s l e n g t h s o f d e g r a d e d R N A . B e c a u s e o f the w i d e d i s t r i b u t i o n o f m o l e c u l a r size a n d p o l y i o n i c n a t u r e , the degradation products of RNA interfere with the separation of protein components by gel filtration a n d also b y ion e x c h a n g e c o l u m n c h r o m a t o g r a p h y . S t e p 2: Tryptic Digestion W a s h e d m i c r o s o m e s a r e i n c u b a t e d w i t h t r y p s i n ( a b o u t 5 / z g m g -~ o f m i c r o s o m a l p r o t e i n ) at 0°C, o v e r n i g h t . T r y p s i n s h o u l d b e p r e - a c t i v a t e d w i t h 2 m ~ H C I s o l u t i o n . C y t o c h r o m e b5 (70-80 p e r c e n t ) is u s u a l l y r e c o v e r e d in t h e c l e a r r e d s u p e r n a t a n t f r a c t i o n b y u l t r a c e n t r i f u g a t i o n a t 78,000 x g f o r 120 min. T h e s u p e r n a t a n t is l y o p h i l i z e d , a n d the l y o p h i l i z e d e x t r a c t c a n be s t o r e d in a f r e e z e r w i t h o u t d e t e c t a b l e loss o f c y t o c h r o m e b5 f o r s e v e r a l m o n t h s .

486

N. OSHINO

Step 3: Sephadex Gel Filtration The lyophilized extract is dissolved in a minimal volume of 10 mu phosphate buffer, followed by gel filtration on Sephadex G-75. Clear separation of a yellow b~ind of NADPH-cytochrome c reductase (molecular weight = 40,000) from a red band of cytochrome b5 (molecular weight = 12,500) is seen on the column. Since no clear separation of these two components is expected on DEAE-cellulose column chromatography (Phillips and Langdon, 1962), ammonium sulfate fractionation should be used if the process of Sephadex gel filtration is omitted. Step 4- Chromatography on DEAE-Cellulose Cytochrome bs-rich fractions of the Sephadex eluate may be directly applied to a column of DEAE-cellulose, and a deep red band of cytochrome b5 is eluted with an increasing linear concentration gradient of KCI from 0 to 0.35 u in 50 mu phosphate buffer. The cytochrome b5 in the eluate may be concentrated by a simple adsorptionelution method on a small column of DEAE-cellulose. Highly pure cytochrome b5 is obtainable with a yield of 35--45 per cent. This method is convenient, reliable and applicable not only to large scale preparation but also to purification on a very small scale, such as that with two rat livers (Omura et al., 1967; Kuriyama et al., 1969). Several purification methods for cytochrome b5 have been reported. Among these, the methods of Strittmatter (1967) and Kajihara and Hagihara (1968) are still currently used with slight modifications. Cytochrome b5 purified by these methods is, in general, a mixture of a few different hydrophilic segments (Strittmatter and Ozols, 1966; Kajihara and Hagihara, 1968), containing some 85 amino acid residues (Spatz and Strittmatter, 1971). Tryptic hydrolysis of intact cytochrome b5 from horse liver occurred at lysyl residues 6 and 90 of the peptide (Ozols et al., 1976). The obtained segments contain the noncovalently bound heme group and show some cytochrome b5 catalytic activities. Trypsin-cleaved cytochrome b5 has been crystallized (Kajihara and Hagihara, 1968) and used for many purposes such as in X-ray crystallography (Mathews et al., 1971, 1972), preparation of antibodies and physicochemical studies on the heme-environment. 4.3. PURIFICATION OF NATIVE CYTOCHROME b5 Purification of the native form of cytochrome b5 was first achieved by Ito and Sato in 1968. To bring an amphipathic protein from membrane into aqueous solution without denaturation or its aggregation with other components, it was necessary to utilize nonionic and ionic detergents and chaotrophic agent and also to remove lipids from the molecule. Thus, the method reported by Ito and Sato (1968) consisted of four steps: (i) solubilizatio/a by a mixture ~f deoxycholate and Triton X-100; (ii) column chromatography on DEAE-Sephadex in the presence of the non-ionic detergent, Triton X-100; (iii) removal of lipid and detergent by cold acetone treatment; and (iv) Sephadex gel filtration in the presence of a chaotrophic agent, 4.5 u urea. This procedure included vigorous treatments of steps 3 and 4 in the later stage of purification and, hence, the purified preparation appeared to contain certain amounts of apo-cytochrome b5 produced by removal of heme during these treatments. Spatz and Strittmatter (1971) developed a milder method which is currently used with satisfactory reliability. A flow-sheet of this method is shown in Scheme 2 and the principal points are enumerated below. Step 1: Refinement of Microsomal Fraction Though it is often omitted, refinement of starting material is in many cases one of the more critical steps in purification. It should be noted that this procedure also includes a step of repeated washings for removal of non-membranous materials. Liver of rabbit is perfused in situ with cold sucrose-buffer medium to remove blood. Crude microsomai fractions are prepared by an ordinary procedure and then subjected to a

Cytochrome b~ and its physiological significance

487

SCHEME 2. Flow Sheet for the Purification of Native Cytochrome bs Step I.

Step 2.

Step 3.

Step 4.

Rabbit liver (perfused in situ with cold 0.25 M sucrose--0.01 M tris-acetate-I mM EDTA pH 8.1) -9 vol. of the buffer-sucrose medium Homogenate -differential centrifugations (18,000 × g, 15 min × 2; 120,000× g, 60 min) Crude microsomes -suspended in 0.1 M tris-acetate-I mM EDTA (pH 8.1) (buffer medium) -added solid NaC1 to a final concentration of 1 M -sonication for 2 min -120,000 x g, 90 min Pellet -resuspended in 0.1 M tris-acetate-1 mM EDTA (pH 8.1) -sonication and centrifugation -repeat the above washing process Washed microsomes -suspended in an equal vol. of the buffer medium to the original wet weight of the tissue -added glycerol (25 ml/100 ml suspension) -NADH (0.05 mg ml-~) -added to a 10 vol. of cold acetone at -5°C -stirred for 30 min -centrifugation Protein precipitate -suspended in 100 ml of the buffer medium -stirred for 1 hr -48,000 × g, 20 min Residue -suspended in 100 ml of 1.5 per cent Triton X-100 in the buffer medium -extract overnight -48,000 x g, 20 min Supernatant -applied to a DEAE-cellulose column (equilibrated with the buffer medium) -NADH-cytochrome b5 reductase passed through without retention -washed with the buffer solution -eluted the red band with 0.25 Mthiocyanate--0.25 per cent deoxycholate in the buffer medium Cytochrome bs-containing fraction -applied to Sephadex G-75 column (equilibrated with 0.4 per cent deoxycholate in the buffer medium) Cytochrome b~ fraction (dimer) -passed through a Sephadex G-25 column equilibrated with the buffer medium Cytochrome b5 (free from detergent, octamer)

Criteria for purity of native rat liver cytochrome bs: protoheme content= 55-60 nmol/mg-~ protein; molecular weight= 16,000; absorption maximum in the visible region is the same as the hydrophylic segment, A4t3= 117 mM-J cm-~ for the ferric enzyme. Method: Spatz and Strittmatter, 1971. v i g o r o u s e x t r a c t i o n with 1 M NaCI in 0.1 M t r i s - a c e t a t e - I mM E D T A solution, f o l l o w e d b y twice w a s h i n g with the tris-buffer in o r d e r to r e m o v e r e s i d u a l NaCI. T h e salt e x t r a c t i o n u n d e r t h e s e c o n d i t i o n s r e s u l t s in r e m o v a l of a b o u t 30 per c e n t of the p r o t e i n s f r o m the c r u d e m i c r o s o m a l f r a c t i o n , with o n l y m i n i m a l loss of c y t o c h r o m e bs.

Step 2: A c e t o n e T r e a t m e n t a n d S o l u b i l i z a t i o n W a s h e d m i c r o s o m e s are s u s p e n d e d in 20 per c e n t glycerol s o l u t i o n i n c l u d i n g N A D H , a n d , in o r d e r to r e m o v e lipid, t h e y are s u b j e c t e d to cold a c e t o n e t r e a t m e n t u n d e r c o n d i t i o n s in w h i c h m i c r o s o m a l r e d o x c o m p o n e n t s are m a i n t a i n e d in the r e d u c e d state. T h e p r e c i p i t a t e of d e f a t t e d p r o t e i n s is c o l l e c t e d , w a s h e d o n c e b y using c e n t r i f u g a t i o n a n d t h e n s o l u b i l i z e d with 1.5 per c e n t T r i t o n X-100 in the tris-buffer. C y t o c h r o m e b5 a n d N A D H - c y t o c h r o m e b5 r e d u c t a s e are b r o u g h t into a q u e o u s solution b y i n t e r a c t i o n of their h y d r o p h o b i c d o m a i n s with T r i t o n X-100, w h e r e a s o t h e r p r o t e i n s with s t r o n g h y d r o p h o b i c i t y s u c h as s t e a r y l C o A d e s a t u r a s e r e m a i n in the i n s o l u b l e r e s i d u e ( S t r i t t m a t t e r et al., 1974).

Step 3: C o l u m n C h r o m a t o g r a p h y on D E A E - C e l l u l o s e T h e e x t r a c t is a p p l i e d to a D E A E - c e l l u l o s e c o l u m n . C y t o c h r o m e b5 is r e t a i n e d while the r e d u c t a s e p a s s e s t h r o u g h the c o l u m n . T h e c y t o c h r o m e b5 r e t a i n e d is eluted JPTAVol.2, No. 3--D

488

N. OSH1NO

with a mixture of a chaotrophic agent and an anionic detergent, i.e. 0.25 M thiocyanate and 0.25 per cent deoxycholate, in the tris-buffer solution. Step 4: Gel Filtration on S e p h a d e x C o l u m n Further purification of cytochrome b5 is achieved by gel filtration on Sephadex G-75 in the presence of 0.4 per cent deoxycholate in the buffer solution. Detergent in the purified cytochrome b5 can be removed by passage through a Sephadex G-25 column equilibrated with the buffer solution. Cytochrome b5 in 0.4 per cent deoxycholate solution exists predominantly as a dimer (molecular weight of about 35,000), whereas it easily polymerizes to an octamer (molecular weight of about 120,000) upon removal of the detergent (Spatz and Strittmatter, 1971). The purified preparation of native cytochrome b5 from rabbit liver contains a small amount of phospholipids and was shown to be homogeneous by SDS-gel electrophoresis. A modification of this purification procedure was reported by Ozols (1974). 5. CYTOCHROME b5 ON MICROSOMAL MEMBRANES 5.1. CHARACTERISTICS OF CYTOCHROME b5 MOLECULES

A brief description will be given here concerning differences between the native cytochrome b5 and its hydrophilic, catalytically active peptide segment. Detailed information on the structural and physicochemical properties will be presented in a separate article by Hagihara. According to Spatz and Strittmatter (1971), native cytochrome b5 of rabbit liver is a single peptide consisting of 141 amino acid residues, among which are 44 residues in addition to those found in the hydrophilic segment. Molecular weights calculated from amino acid composition are 16,000 and 11,100 for the native cytochrome b5 and its hydrophilic segment, respectively. Although the former molecular weight was first reported to be about 25,000 (Ito and Sato, 1968), the high value is now believed to be an overestimation ascribable to the presence of apoprotein in the preparation. Visible absorption spectra of the native cytochrome b5 and its hydrophilic segment were identical, and some extra increments in absorbance in a near-ultraviolet region were entirely accounted for by the contribution of the additional three tryptophan and two tyrosine residues present in the native cytochrome b5 (Spatz and Strittmatter, 1971; Ozols, 1974). X-ray crystallography of the hydrophilic segment revealed that the protein was globular, with a high percentage of its surface covered by hydrophilic amino acid residues (Mathews et al., 1971). A crevice for heme binding is located on a side opposite to both ends of the single peptide (Mathews et al., 1971, 1972). Residues 49-valine, 50-1eucine and 65-valine in the amino acid sequence are involved in the hydrophobic interaction between the heme and protein (Keller and Wiitrich, 1972). About 60 per cent of the cytochrome b5 amino acid residues are hydrophobic in character, and 40 of these residues are located in the hydrophobic domain as a peptide linked to the C-terminus of the hydrophilic segment (Ozols, 1974; Ozols et al., 1976). In circular dichroism studies of the denaturation process of cytochrome bs, the hydrophilic segment was found to undergo, with concomitant detachment of heme, a one-stage denaturation at a guanidine concentration of 2.9 M (Schnellbacker and Lumper, 1971; Tajima et al., 1976). On the other hand, denaturation of the native cytochrome b5 by guanidine proceeded in a two-stage process (Tajima et al., 1976); the denaturation of the hydrophilic domain occurred at a concentration of 2.6 M, whereas the unfolding of the hydrophobic domain was observed at a higher concentration of guanidine (5-5.5 M). In addition, t h e removal of heme from the native cytochrome b5 rendered the hydrophilic domain unstable, but it did not affect the susceptibility to guanidine of the hydrophobic domain. It was concluded from this and other physicochemical studies (Dehlinger et al., 1974; Vissen et al., 1975; Robinson

Cytochrome b 5 and its physiological significance

489

and Tanford, 1975) that the two domains of the cytochrome b5 molecule are rather independent and are linked to each other by a flexible interdomain peptide of about 15 amino acid residues. 5.2. DISTRIBUTION ON MICROSOMAL MEMBRANE Almost all cytochrome bs, as well as NADH-cytochrome b5 reductase and NADPHcytochrome c reductase, could be liberated from microsomal membranes by incubation with various proteases. These treatments also caused the removal of ribosomes from the outer surface of rough microsomal membranes (Lust and Drochmans, 1962), whereas proteases did not solubilize esterase (Akao and Omura, 1972) and nucleosidase (Kuriyama, 1972), both of which are located on the inside surface of closed microsomal vesicles. Similarly, these conditions did not solubilize ATPase and glucose-6-phosphatase (G6erlich and Heise, 1962), which are probably buried in the membrane structure. Digestion of microsomal membranes by proteases always proceeded to a stage at which about 30 per cent of the total protein had been solubilized (Ito and Sato, 1969). Electron microscopic studies (Lust and Drochmans, 1962; Omura et al., 1967) demonstrated that trypsin-treated microsomes still retained a closed vesicular structure of triple-layer appearance, indicative of the persistence of a unit membrane structure. Density-gradient experiments in sucrose and dextran media confirmed this fact and further indicated that the treated vesicles retained their impermeability to macromolecules such as dextran and probably proteases as well (Ito and Sato, 1969). Since immunoglobulin is a hydrophilic macromolecule with a molecular weight of about 160,000, it should be unable to penetrate the lipoprotein membrane of intact microsomes. Nevertheless, an immunoglobulin fraction prepared again.st cytochrome b5 was capable of inhibiting almost completely the microsomal activities of NADHcytochrome c reductase and stearyl CoA desaturase, in which involvement of cytochrome b5 had been established (Oshino and Omura, 1973).* Conversion of endoplasmic reticulum into microsomal vesicles occurs upon homogenization of tissues (Palade and Siekevitz, 1956a), and there is evidence that the original insideoutside relationship of the membrane is preserved in the microsomal vesicles (Wallach and Kamat, 1964). Therefore, it is concluded that cytochrome b5 is located on the outer surface of endoplasmic reticulum, its hydrophilic domain sticking out into the cytoplasm. Delineation of the distribution of cytochrome b5 on the membrane surface may differ considerably, depending on the nature of studies, static or dynamic. Remacle et al. (1974) prepared a hybrid molecule of antibodies prepared respectively against cytochrome b5 and ferritin and an antigen-antibody complex between ferritin and [he hybrid antibodies. This complex was able to bind microsomal cytochrome bs, and ferritin grains were distributed homogeneously on almost all profiles in the purified microsomal fraction viewed microscopically, indicating a homogeneous distribution of cytochrome b5 throughout the endoplasmic reticulum. On the other hand, some differences in contents of cytochrome bs as well as of other microsomal components were detected, on the basis of both protein and phospholipid, in a series of subfractionations of microsomal membrane fragments, prepared by sonic disruption (Dallner et al., 1968; Dallman et al., 1969). This was regarded as an indication of the heterogeneous distribution of cytochrome b5 on the microsomal membrane. Schulze et al. (1972) attempted to separate sonicated microsomal fragments by affinity chromatography, using Sephadex linked covalently to a specific antibody against N A D H cytochrome b5 reductase. It was found that, whereas microsomal enzyme components on large vesicles sediment in an identical pattern, as vesicle size was decreased by sonication, fractions containing NADH-cytochrome b5 reductase and cytochrome b5 tended to separate. It is possible that some artificial rearrangement of localization may *Other studies have suggested that only part of microsomal cytochrome b5 is accessible to the antibodies (Jansson and Schenkman, 1977).

490

N. OsrtINO

occur upon fragmentation of microsomal membrane. Therefore, it may be concluded that cytochrome bs, rather than being confined to a particular area of endoplasmic reticulum, is distributed homogeneously throughout the membrane surface. However, this problem is not settled. Rogers and Strittmatter (1974a) demonstrated that added native cytochrome b5 molecules were randomly incorporated on the microsomal membrane in vitro and that the kinetics of reduction of the extra cytochrome b5 by NADH were indistinguishable from those of endogenous cytochrome b5 on the membrane. These results emphasized a tendency of random distribution of cytochrome b5 on the microsomal membrane, compatible with a recent concept of the fluid-like structure of biological membrane (Singer and Nicolson, 1972). Digestion with cathepsin D causes selective liberation of NADH-cytochrome b5 reductase from microsomes (Takesue and Omura, 1968, 1970a) and thus, it is feasible to prepare microsomes with various molecular ratios of cytochrome b5 and its reductase. By using such microsomal preparations, reduction kinetics of cytochrome b5 by NADH were analyzed in detail (Ito, 1974). The data was interpreted to indicate the presence of an enzyme assembly in which a few molecules of NADH-cytochrome b5 reductase are associated with about ten times as many molecules of cytochrome bs. The specific protein-protein interactions between cytochrome b5 and its reductase and also between cytochrome b5 and desaturases may favor the association of these components, leading to the formation of multimolecular electron transfer complexes of varying size on the membrane, whereas the freedom of lateral movement of these components tends to dissociate these molecular aggregates. The actual situation may be, therefore, between the two extreme situations; the cytochrome molecule may be in a dynamic state of association with and dissociation from other components in the membrane. 5.3. BINDING TO THE MEMBRANE STRUCTURE The mitochondrial respiratory chain is composed of a unique, sequential arrangement of Complexes I, II, III and IV, each of which contains its own composition of electron carriers such as flavoproteins, ubiquinone, non-heine iron and several types of cytochrome. The presence of a structural protein was proposed in the membrane, which assures a proper position for each component (Criddle et al., 1964). This concept was once popular and considered as one of the possible models of biological membrane structure. Accordingly, when Ito and Sato (1968) purified native cytochrome b5 with hydrophobic character, they suggested a property whereby the cytochrome b5 molecule has a 'built-in' structural protein, responsible for its attachment to the microsomal membrane. The significance of the hydrophobic moiety of the molecule was substantiated by subsequent studies as described in the following. When incubated with microsomes in vitro, the native cytochrome b5 was incorporated into the membrane and separated from the unbound cytochrome by Sephadex gel filtration (Strittmatter et al., 1972). The process of binding was limited by temperature-dependent dissociation rate of monomeric cytochrome b5 from the octamer (Enomoto and Sato, 1973; Calabro et al., 1976). The binding of additional cytochrome b5 to the microsomal vesicles reached an apparent saturation at 10-11 fold molar excess of cytochrome b5 (Strittmatter et al., 1972). Repeated washing with various aqueous media such as high salt solution (Strittmatter et al., 1972) and 10 mM EDTA solution (Enomoto and Sato, 1973) and mechanical disruption by sonication (Strittmatter et al., 1972) failed to remove the extra-bound cytochrome b5 from the membrane, indicating the nature of the binding to be nonionic. The extra-bound cytochrome b5 was indistinguishable from endogenous cytochrome b5 as an electron acceptor from NADH-cytochrome b5 reductase and as an electron donor for external cytochrome c or rat liver stearyl CoA desaturase (Strittmatter et al., 1972; Rogers and Strittmatter, 1974a). The native cytochrome b5 purified from rabbit liver is able to bind not only to microsomes from rabbit liver but also to those from other species. Of interest is an

Cytochrome b~ and its physiologicalsignificance

491

observation that, in spite of a considerable variation in the content of endogenous cytochrome bs, i.e. 0.5, 0.9 and 1.2nmol cytochrome b5 per mg of protein in microsomes, prepared from livers of rat, calf and rabbit, respectively, saturation in binding of the purified cytochrome to these microsomes occurred practically at the same level of 11-13 nmol per mg of protein. The bound cytochrome b5 at 12 nmol per mg of protein is a very significant part, representing nearly 20 per cent of the total microsomal protein. Incubation of microsomes with the amphipathic form of NADH-cytochrome b5 reductase also resulted in binding of a maximum of 7.2 nmol of the reductase per mg of protein, which represented a 100 fold increase over endogeneous enzyme level of 0.07 nmol per mg of protein (Rogers and Strittmatter, 1974b). When extra-bound, cytochrome b5 occupied the membrane to 80 per cent of its maximal binding and binding capacity of the reductase was inhibited by more than 50 per cent. Conversely, a nearly maximal amount of extra-bound reductase caused an inhibition of cytochrome b5 binding by only about 35 per cent of the maximum (Rogers and Strittmatter, 1974b). As indicated by these results, these extra-bound components compete for binding sites and tend to exclude each other from the membrane; the mode of binding of these constituents was observed to be reversible. Specificity of the binding of cytochrome b5 is not restricted to the microsomal membrane; cytochrome b5 binds to both the outer and inner membranes of the mitochondria at amounts of 5.9 and 2.0nmol per mg of protein, respectively (Strittmatter et al., 1972). Binding of cytochrome b5 was also observable, though to a lesser extent with erythrocyte ghost membranes, but this binding was limited to the inner surface of the membrane (Enomoto and Sato, 1977). This asymmetric binding was ascribed to preferential localization of cholesterol on the outer surface of the erythrocyte membrane. Rogers and Strittmatter (1975) studied the binding of cytochrome b5 on liposomal vesicles prepared with phosphatidyl choline and found 244nmol of cytochrome b5 per vesicle at saturation of binding. This number corresponded to 1 mol of cytochrome b5 per 11 mol of phospholipid, and the data was interpreted to represent that, under these conditions, about 80 per cent of the surface of liposome vesicle is occupied by cytochrome bs. It should also be noted that both cytochrome b5 and NADH-cytochrome b5 reductase bind to liposome and that the bound components are fully reduced by NADH (Rogers and Strittmatter, 1975). A variety of physicochemical studies have been reported with regard to the nature of interaction between cytochrome b5 and lipophilic substances such as detergents and lipids (Dehlinger et al., 1974; Holloway and Katz, 1975; Robinson and Tanford, 1975). A conclusion has been drawn from these studies that the ability to insert a part of the molecule into any available hydrophobic environment is derived from the hydrophobic domain of cytochrome bs; this domain has a diffuse hydrophobic surface that acts as a nonspecific nucleus for the formation of a micelle with a variety of amphipathic substances. It is of interest that, in spite of ability for nonspecific binding, cytochrome b5 is located mainly on the endoplasmic reticulum system in vivo. 6. BIOSYNTHESIS AND DEGRADATION OF CYTOCHROME b5 The liver microsomes of rat fetus contain small amounts of the constitutive enzyme components of the electron transfer system. Each of these components appears in the microsomes with a time course different from the others, and reaches adult level during the development of the rat, from a few days before birth to a few days after birth (Dallner et al., 1966). This observation, together with the induction phenomenon of microsomal drug metabolizing enzymes (Orrenius et al., 1965), provided evidence that, rather than being constant in its constituents, the endoplasmic reticulum is a dynamic structure whose constituents and enzyme activities are continuously changing. The overall renewal process of cytochrome bs, i.e. its biosynthesis, incorporation into the membrane and degradation, may be studied by isolating cytochrome b5 from

492

N. OSHINO

liver microsomes at appropriate intervals after intravenous injection to animals of a radioactive amino acid as a pulse and by measuring specific radioactivity in the purified cytochrome. Based on this principle, the overall renewal processes of cytochrome b5 and NADPH-cytochrome c reductase were investigated by using taC-nL-leucine (Omura et al., 1967; Kuriyama et al., 1969). Half-lives for cytochrome b5 and NADPH-cytochrome c reductase were found to be 100-120 hr and 72-84hr, respectively. Unfortunately, however, it became obvious that analyses of synthesis of the protein cytochrome b5 by the pulse labeling method may be hampered by: (i) alteration in intracellular pool size for free amino acids; (ii) a slow conversion of D- to L-leucine and subsequent incorporation into proteins; and (iii) transient formation of laC-apocytochrome bs, especially in an initial phase of protein synthesis. Sargent and Vadlamudi (1968) investigated biosynthesis of cytochrome b5 by incubating an isolated microsomal fraction of rat liver with ~4C-lysine in vitro. The cytochrome b5 from the incubated microsomes contained a small amount of the radioactivity. Among eleven peptide fragments prepared by tryptic digestion of the purified cytochrome bsi all seven peptides which should contain lysine were labeled by this radioisotope. This indicated the biosynthesis of cytochrome b5 in this system in vitro, and suggested that at least a portion of cytochrome b5 was synthesized on membrane-bound ribosomes. Assuming a sequence in which cytochrome b5 in vivo is synthesized on free polysomes, liberated into a pool in the cytoplasm and then incorporated simultaneously into rough and smooth microsomal membranes, we may expect a random distribution of newly synthesized cytochrome b5 between rough and smooth microsomal fractions. On the other hand, if we assume that cytochrome b5 is synthesized on membrane-bound ribosomes and incorporated exclusively into nearby membranes, preferential localization of the labeled cytochrome b5 in rough microsomal membranes should be expected, especially during an early stage of labeling. Omura and Kuriyama (1971) compared the time course of J4C-leucine incorporation into cytochrome b5 to that into NADPH-cytochrome c reductase by the pulse labeling method Shortly after an injection of ~4C-leucine, labeled NADPH-cytochrome c reductase molecules appeared predominantly in the rough membrane fraction. The specific radioactivity of the reductase in rough microsomes attained a peak value at l0 min and then decreased gradually, whereas that of the reductase in smooth microsomes increased steadily until these two values became equal at 120 min after pulse labeling. Thus, the newly synthesized reductase is incorporated preferentially into rough membrane and moved subsequently from rough to smooth membrane's to attain an equilibrated partition between these two membrane systems. In contrast to the appearance of a transient peak in labeling of the reductase, the specific radioactivity of cytochrome b5 in rough membrane increased rather slowly and, within 120 min after the pulse labeling, reached a level found also in the cytochrome b5 of smooth membranes. The specific radioactivities were, however, 1.5 to 2 fold higher in rough microsomes than in smooth microsomes within 30 min after the pulse labeling, indicating that cytochrome b5 is synthesized on membrane-bound ribosomes and incorporated into the nearby rough membrane. The delay observed in the appearance of newly synthesized cytochrome b5 in rough microsomal fraction may be attributable to the production of apo-cytochrome b5 in an early stage of the synthesis and also to slow supply of 14C-L-leucine from 14C-D-leucine isomer which is biologically inactive. Because of its susceptibility to protease (Strittmatter and Ozols, 1966), apo-cytochrome b5 is not recovered in the purified preparation of hydrophilic cytochrome b~. Therefore, microsomal fractions from the rat administered with 14C-leucine were incubated with protohemin in order to convert apo-cytochrome b5 into the holo-form. This pretreatment resulted in significant increases in the specific radioactivities of the purified cytochrome b5 preparations, indicating the presence of apo-cytochrome b5 in microsomal fractions (Negishi and Omura, 1970). The ratios of radioactivity found in apo- and holo-cytochrome b5 were 2:1 at 10min and 2:3 at 30min after the pulse labeling. No radioactivity was detected in the apo-cytochrome b5 of microsomes

Cytochrome b5 and its physiologicalsignificance

493

prepared at 180 min after the pulse labeling. The occurrence of apo-cytochrome b5 could be confirmed in an observation that about 7.5 per cent of cytochrome b5 was labeled with radioisotope when an isolated microsomal fraction was incubated with ~4C-hemin in vitro (Hara and Minakami, 1970). In this system, a spontaneous exchange-reaction of heme between cytochrome b5 and free hemin proceeded slowly (Hara et al., 1970), and, hence, the amount of ~4C-hemin found in cytochrome b5 should be higher than that representing the amount of apo-cytochrome b5 in microsomal fraction. It may be concluded from the above-mentioned results that apocytochrome b5 is synthesized on the membrane-bound ribosome, attaches to the rough endoplasmic reticulum and then diffuses by translational movement into the smooth endoplasmic reticulum with a concomitant insertion of hemin into the molecule. Two hypotheses were considered concerning the mechanism of distribution of the newly synthesized cytochrome b5 on the microsomal membrane: (i) the translational movement of the molecule and the resulting uniform distribution on the membrane; and (ii) direct interconversion between rough and smooth membranes by detachment and attachment processes of ribosomes. The first mechanism is generally accepted since the half-life of the cytochrome b5 in rough membrane is indistinguishable from that of the cytochrome b5 in smooth membrane (Kuriyama et al., 1969), a fact indicating an attainment of dynamic equilibrium in regard to the distribution of the new cytochrome b5 between these areas of continuing membrane structure. A half-life of the overall renewal process of cytochrome b5 was reported to be 100-120 hr in the liver of rat under normal conditions (Omura et al., 1967; Kuriyama et al., 1969). This number obtained with ~4C-DL-leucine was confirmed in subsequent studies (Kuriyama and Omura, 1971), by using ~4C-L-leucine and t4C-L-arginine. A half-life of about 55 hr was reported for the heme moiety of the cytochrome b5 molecule, which was determined by using ~4C-8-aminolevulinic acid (Druyan et al., 1969; Greim et al., 1970) or 59Fe (Hara et al., 1970). Thus, the heme moiety of cytochrome b5 is renewed independently from the protein, probably due to the occurrence of the heme-exchange reaction in vivo. The half-lives for total microsomal proteins, NADPH-cytochrome c reductase and microsomal phospholipids were also determined in the liver of rat to be about 80, 80 and 30-40 hr, respectively, these being shorter than that of cytochrome b5 (Omura et al., 1967; Kuriyama et al., 1969). An interesting problem is the mechanism by which a newly synthesized protein is transferred from ribosome to rough membrane and is stabilized as a constituent attaching firmly to the membrane structure. Negishi and Omura (1972) investigated in detail the initial phase of the time course of ~4C-L-leucine incorporation into microsomal proteins. Ten minutes after pulse-labeling with ~4C-L-leucine, specific activities in both the total microsomal proteins and NADPH-cytochrome c reductase had attained their maximal values; however, about two-thirds of the incorporated radioactivity disappeared subsequently within 60-120 min with a decay half-time of about 1 hr. A second phase was seen, in which the labeled protein which remains was degraded with a half-life of about 80 hr as described above. In the rat pretreated with phenobarbital, the fast phase in the decay curve of the labeled NADPH-cytochrome c reductase selectively disappeared, whereas the decay curve for the total microsomal protein was still biphasic (Negishi and Omura, 1972). These observations concerning the initial decay of the incorporated radioactivity are regarded as representing the actual behavior of microsomal components in the process of stabilization on the membrane and imply the presence of a certain regulatory mechanism for this process. The same event may also occur in the process of biosynthesis of cytochrome bs, although because of the formation of apo-cytochrome bs, the event has so far not been observed. The half-life of the messenger RNA for the bulk of rat liver proteins is about 5 hr (Tominaga et al., 1971). The rate of a protein synthesis is known to be proportional to the amount of messenger RNA for the protein in the cell (Moat et al., 1971). Therefore, it may be feasible to estimate a half-life of the messenger RNA for cytochrome b5 by measuring the time course of changes in the rate of cytochrome b5

494

N. OSH1NO

synthesis in the presence of actinomycin D, an inhibitor for the DNA-directed messenger RNA synthesis (Reich et al., 1962). Thus, rats were injected intraperitoneally with actifiomycin D at a dose of 0.15 m g k g -~ of body weight, which resulted in 80-90 per cent inhibition of total RNA synthesis in rat liver. 3H-L-lysine was subsequently injected intravenously as a pulse at either 1, 3 or 5 hr after the actinomycin D treatment. Cytochrome b5 was purified from the liver 40 min after pulse labeling. It was revealed in this study (Matsumura and Omura, 1973) that the time course of appearance and disappearance of injected 3H-L-lysine in the intracellular amino acid pool of rat liver was greatly affected by treatment with actinomycin D. Therefore, the specific radioactivity of the purified cytochrome b5 should be corrected for the changes in the radioactivity of lysine in the intracellular free-amino acid pool. As a result of this correction, a half-life of about 3 hr was detected for the messenger R N A responsible for c y t o c h r o m e b5 synthesis (Matsumura and Omura, 1973). It has been repeatedly shown that soon after phenobarbital injection there is a proliferation of smooth endoplasmic reticulum membrane (Remmer and Merker, 1963) with an increase in the net synthesis of c y t o c h r o m e P-450 and N A D P H - c y t o c h r o m e c reductase (Orrenius et al., 1965; Conney, 1967). The content of c y t o c h r o m e b5 in the microsornal membrane was not altered by a single injection of the drug, though repeated phenobarbital doses caused a moderate increase in c y t o c h r o m e b5 (Kuriyama and Omura, 1971). The rate of synthesis of the protein moiety of cytochrome b5 was increased to an insignificant extent under the latter condition, and the moderate increase in the c y t o c h r o m e b5 content was attributed to a reduction in the rate of degradation through an as yet unknown mechanism. 7. C O N S T I T U E N T S OF E L E C T R O N T R A N S F E R S Y S T E M AND ITS P H Y S I O L O G I C A L F U N C T I O N C y t o c h r o m e b5 is a c o m p o n e n t of a unique electron transfer system in combination with either of two flavoproteins, N A D H - c y t o c h r o m e b5 reductase or N A D P H c y t o c h r o m e c reductase and distributes reducing equivalents from N A D H and N A D P H to a number of acceptors, such as those catalyzing various types of fatty acid desaturation reactions. The pathways of electron transfer in this multicomponent system are illustrated schematically in Fig. 1 and a brief description for each c o m p o n e n t is given in the following. 4-Methyl sterol oxidase ( /~ :,/

NADH

• FpD

. N-Hydroxyaminereductase

/ / • Cyt b 5 ~

~

Acyl CoA &9-deseturase AcYi CoA A6-desei"urase Acyl CoA &5-desatumse Alkyl ocyl GPE desoturose

NADPH

• Fpr

- Cyt P450 I Drug oxidetion /

Orgenic hydroperoxide peroxidase

FIG. 1. The cytochrome bs-dependent pathway of electron transfer in microsomes. FpD: NADH-cytochrome bs reductase; Fpx: NADPH-cytochrome c reductase; Alkylacyl GPE: 1-Alkyl-2-acyl-sn-glycero-3-phosphorylethanolamine.

7.1. NADH-CYTOCHROME b5 REDUCTASE N A D H - c y t o c h r o m e b5 reductase is a flavoprotein containing FAD as a prosthetic group (Strittmatter and Velick, 1956b). The reductase purified by Strittmatter and Velick (1956b) was recently demonstrated to be, as in the case of c y t o c h r o m e bs, a

Cytochrome b5 and its physiological significance

495

catalytically active hydrophilic segment which was liberated from the native molecule by action of a lysosomal protease (Takesue and Omura, 1968, 1970a,b). The native reductase of amphipathic character was purified by Spatz and Strittmatter (1973) and Mihara and Sato (1972, 1975). The molecular weight of the native reductase was estimated to be about 43,000 from its amino acid analysis (Spatz and Strittmatter, 1973) and about 33,000 by a gel filtration study (Mihara and Sato, 1975). The molecule is capable of binding to several membrane structures, including not only microsomes (Spatz and Strittmatter, 1973) but also mitochondria, erythrocytes (Mihara and Sato, 1975) and phospholipid iiposomes (Rogers and Strittmatter, 1973, 1975). This ability to bind to the membrane was abolished by proteolytic conversion of the molecule into its hydrophilic segment (Spatz and Strittmatter, 1973; Mihara and Sato, 1975). Both the native reductase and its hydrophilic segment are able to interact with the cytochrome b5 in the native and hydrophilic forms and to reduce them in the presence of NADH in vitro. The rate of electron transfer between the native forms of reductase and cytochrome b5 was significantly accelerated under conditions optimal for proteinprotein interaction, such as those obtained by the addition of appropriate amounts of detergent and/or phospholipids (Rogers and Strittmatter, 1973; Mihara and Sato, 1975). Therefore, a favorable interaction would be expected between these native components on the microsomal membrane. The direct electron acceptor from NADHcytochrome b5 reductase in microsomal membrane seems to be only cytochrome bs. Although an existence of an electron transfer pathway which did not include cytochrome b5 was proposed for the NADH-dependent cytochrome P-450 reductase reaction (Ichikawa and Yamano, 1970b; Ichikawa and Loehr, 1972), it will be difficult to substantiate this pathway since electron transfer from cytochrome b5 to cytochrome P-450 has been well established (Sasame et al., 1974; Lu et al., 1974; Mannering et al., 1974). However, NADH-dependent lipid peroxidation was shown to use the reductase without cytochrome b5 (Jansson and Schenkman, 1977). 7.2. NADPH-CYTOCHROME c REDUCTASE

NADPH-cytochrome c reductase is a flavoprotein containing one molecule each of FAD and FMN as a prosthetic group (Iyanagi and Mason, 1973; Van der Hoeber and Coon, 1974). The reductase is also known as one of the rare flavoproteins which are directly oxidized by cytochrome c in vitro. In the microsomal membrane, it acts as a reductase responsible for a supply of reducing equivalent from NADPH to such terminal oxidases as cytochromes P-450 and P-488 of microsomal drug metabolizing systems. The reductase in microsomes is also able to donate electrons to cytochrome bs, but because of a lack of reactivity between the purified preparations of the reductase and cytochrome b5 (Modirzadeh and Kamin, 1965), the presence of an unknown mediator was postulated in the electron transfer pathway between NADPHcytochrome c reductase and cytochrome bs in microsomal membrane. In that study, the reductase was a catalytically active peptide segment prepared by proteolytic digestion. The native reductase purified with detergents exhibited the NADPHcytochrome b5 reductase activity with the native form of cytochrome b5 under appropriate conditions (Bilimoria and Kamin, 1973; Prough and Masters, 1974), indicating that direct reduction of cytochrome b5 by NADPH-cytochrome c reductase is possible on the intact microsomal membrane. Because of this reactivity with cytochrome bs, NADPH-cytochrome c reductase may serve as an electron donor for all reactions in which cytochrome b5 is involved as an electron carrier. In fact, its participation in the NADPH-dependent stearyl CoA desaturation was demonstrated with isolated microsomal fraction (Oshino et al., 1966, 1971). However, the physiological significance of these results is in doubt. Cytochrome b5 in hepatocyte (Chance, 1961) and in perfused rat liver (Scholz and Biicher, 1966) is maintained in an almost fully reduced state, probably by a potent activity of NADH-cytochrome b5 reductase, supported by an abundant supply of NADH from various metabolic reactions. Therefore, an active contribution of NADPH-cytochrome c reductase in the cytochrome bs-dependent reactions is not expected in intact organs in vivo.

496

N. OSHINO 7.3. CYTOCHROME b50XIDASES

A group of enzyme desaturases is found in microsomal fractions, which, with a supply of reducing equivalents from cytochrome bs, catalyze the biosynthetic reactions of unsaturated fatty acids. These desaturases can be classified based on their substrate specificities, into three categories, i.e. acyl CoA desaturase, alkylacylglycerophosphoryl-ethanolamine desaturase and phospholipid desaturase. These desaturases appear to be metallo-proteins containing an iron atom as a catalytic centre and catalyze a unique cis-elimination of hydrogen from fatty acid chains by a simultaneous concerted-removal mechanism in the presence of activated oxygen in the desaturase molecule (Morris et al., 1968; Stoffel and LeKim, 1971). Besides these physiologically important cytochrome b5 oxidases, N-hydroxyamine reductase is also reported to be one of the cytochrome b5 oxidases in liver and kidney microsomes. Circumstantial evidence seems to suggest that 4-methyl sterol oxidase of liver microsomes, which is an enzyme in the path of lanosterol conversion to cholesterol, may be one of the cytochrome b5 oxidases (see discussion by Seifried and Gaylor, 1976). However, the data available are rather inconsistent (Gaylor and Mason, 1968; Gaylor et al., 1970; Bechtold et al., 1972) and thus, the assumption still remains to be proved. In addition, a desaturation step in the biosynthesis of cholesterol appears to be mediated by cytochrome b5 (Reddy et al., 1977). A number of reports appeared concerning evaluation of the physiological meanings of the electron transfer pathway from cytochrome b5 to cytochromes P-450 and P-448 (see review by Schenkman et al., 1976). It is now obvious that cytochrome P-450 acts as one of the cytochrome b5 oxidases in the isolated microsomal membrane under the conditions in vitro. However, it is Still difficult to answer the question as to whether the activity has any physiological meaning. Therefore, topics regarding the cytochrome P-450-catalyzed oxidation of cytochrome bs, which also include organic peroxide peroxidase reactions of cytochrome P-450 (Hrycay and O'Brien, 1974; Hrycay and Prough, 1974; Hrycay et al., 1975), are omitted from this article. 7.3.1. F a t t y A c y l CoA D e s a t u r a s e s At least three kinds of desaturation reactions specific for CoA derivatives of long chain fatty acids, namely, A9-, A6- and ALdesaturation, have been found in the microsomal fractions from liver (Stoffel, 1961; Marsh and James, 1962), lung (Montgomery, 1976). adipose tissues (Gellhorn and Benjamin, 1964), mammary gland (Brett et al., 1971; McDonald and Kinsella, 1973), brain (Cook and Spencer, 1973a), testis (Ayala et al., 1973) and other organs (Brenner, 1973). A9-desaturation is a reaction in which a double bond is introduced between carbon atoms 9 and l0 of the saturated acyl chain. A6-desaturation is the first step of the biosynthetic pathway of essential fatty acids, in which an additional double bond is introduced between carbon atoms 6 and 7 of the acyl chain possessing, at least, a double bond at the carbon atoms 9 and 10 such as those of oleyl CoA (18: 1), linoleyl CoA (18:2) and a-linolenyl CoA (18:3). The C~8-polyunsaturated fatty acids produced in A6-desaturation undergo a chain elongation reaction to form C20-polyunsaturated fatty acids which are then desaturated between carbon atoms 5 and 6. The resulting products are a series of polyunsaturated fatty acids essential to the biological system. All of the desaturation reactions require molecular oxygen and NADH or NADPH as an electron donor, and are probably catalyzed by microsomal multicomponent systems which consist of NADH-cytochrome b5 reductase, NADPH-cytochrome c reductase, cytochrome b5 and one of the terminal enzyme desaturases. Several lines of evidence which demonstrate cytochrome b5 to be an essential constituent in the desaturation enzyme system will be described in detail in a separate section, and, thus, the molecular properties of the terminal desaturase component as a cytochrome b5 oxidase are the major topics in the following discussion. The presence of a common terminal constituent in the NADH-, NADPH- and ascorbate-dependent Ag-desaturation of stearyl CoA in rat liver microsomes was first

Cytochrome b5 and its physiologicalsignificance

497

recognized by the same susceptibility of the activity to cyanide: this component was referred to as the cyanide sensitive factor (CSF) (Oshino et al., 1966). Besides acyl CoA, a number of phenols such as p-cresol, p-aminophenol and p-chlorophenol interact with the cyanide sensitive factor in microsomes and thereby cause an increased utilization by oxygen of reducing equivalents from cytochrome b5 (Oshino and Sato, 1971). Since its activity was inhibited by cyanide and azide and not by CO, the cyanide sensitive factor was thought of as a metallo-protein which acts as a site for 02 activation (Oshino et al., 1966). Later the CSF was identified as the A9desaturase itself (Shimakata et al., 1972). Purification of hg-desaturase from rat liver microsomes has been achieved by Strittmatter et al. (1974). They treated microsomes with increasing concentrations of deoxycholate, in a stepwise fashion, to remove various components. From the extremely hydrophobic residues remaining after these pretreatments, Ag-desaturase was solubilized with an aqueous mixture of 2.5 per cent Triton, 0.2 per cent deoxycholate, 15mM CaC12 and 25 per cent propylene glycol. 120 to 130 fold purification was achieved subsequently by column chromatography on DEAE-cellulose and following gel filtration of Sephadex G-75. The purified desaturase is a single peptide consisting of 456 amino acid residues, among which 62 per cent are non-polar residues. Therefore, the protein is extremely hydrophobic and has a strong tendency to form inactive aggregates in aqueous solution. The desaturase activity of the protein is unstable, particularly in detergents, and undergoes inactivation with a concurrent liberation of non-heme iron from the molecule. The absorption spectrum of the purified desaturase is composed of a major absorption band at 280 nm, with a 290 nm shoulder, indicative of a high tryptophan content in the molecule. A signal of high spin iron could not be detected in electron paramagnetic resonance spectroscopy measured at 100 K, but chemical analysis has shown that the desaturase molecule contained 18nmol of iron per mg of the protein. This iron content provided a minimal molecular weight of the desaturase of about 55,000, comparable to a value of 53,000 determined by SDS-gel electrophoresis. The NADH-dependent A%desaturation system was reconstituted on phospholipid liposome, by using purified preparations of Ag-desaturase, cytochrome b5 and NADHcytochrome b5 reductase (Enoch et al., 1976). The reconstituted system was able to catalyze a conversion of saturated acyl CoA into monoenoyl CoA in the presence of both NADH and molecular oxygen. The activity reconstituted on liposomes was inhibited by bathophenanthroline, a chelator specific for ferrous iron, in a particular manner; inhibition took place only in the presence of NADH, concurrently with removal of the iron atom from the molecule (Strittmatter et al., 1974). Similarly, cyanide inhibition of the microsomal desaturase occurred only in the presence of both NADH and a substrate, either acyl CoA or phenol; cyanide was accessible to the active site of desaturase in the membrane only during its oxidation-reduction cycle (Oshino et al., 1971; Tamura et al., 1976). In this manner, a catalytic function for the non-heme iron in the desaturase was deduced. Though the cyanide-sensitivity is a convenient criterion for a group of desaturases in microsomes, Hiwatashi et al. (1975) reported that Ag-desaturase activities in certain organs were not sensitive even to very high concentrations of cyanide, indicating that the cyanide sensitivity was not a common characteristic of Ag-desaturases. However, the analytical methods employed in this study were unsuitable for assay of the very slow rates of desaturation in those organs. It should also be borne in mind that the inhibition by cyanide proceeded only while the desaturation reaction was taking place (Oshino et al., 1971). Therefore, a slow rate of desaturation may result in an incomplete inhibition by cyanide. By using a different method, the unique kinetics of cyanide inhibition were confirmed in the desaturation activity of yeast microsomes, which had previously been thought to be cyanide-insensitive (Tamura et al., 1976). With regard to the molecular properties of A6- and ALdesaturases, little has been shown, except that these activities are also sensitive to cyanide and azide (Stoffer and Schiefer, 1966). The three desaturases differ from each other, in respect of their tissue

498

N. OSHtNO

distribution and also induction of activity by nutritional alteration of animals (Inkpen et al., 1969; Brenner, 1974). 7.3.2. Alkylacylglycerophosphoryl-ethanolamine

Desaturase

l-O-Alk-l'-enyl-2-acyl-sn -glycerophosphoryl-ethanolamine (ethanolamine plasmalogen) is a kind of alk-l-enyl glycerolipid which is found in a high content in myeline phospholipid fraction from certain mammalian species (Harrocks, 1968). Tumor cells such as Morris hepatomas (Snyder et al., 1969) and Fischer sarcomas (Snyder and Wood, 1968) also contain relatively high amounts of this lipid. Ethanolamine plasmalogens are synthesized from long chain fatty alcohols and dihydroxyacetone phosphate, and the last step of the biosynthetic pathway is an oxidative introduction of a double bond between carbon atoms 1 and 2 of the O-alkyl moiety in l-O-alkyl-2-acyl-sn-glycerophosphoryl ethanolamine (Wykle et al., 1972; Paltauf and Holasek, 1973). An enzyme which catalyzes this reaction has been found in the microsomal fractions or post-mitochondrial supernatant fraction of myelinating rat brain (Blank et al., 1972), intestinal mucosa of hamster (Paltauf and Holasek, 1973), pig spleen (Paltauf et al., 1974), Fisher sarcoma R-3259 (Wykle et al., 1972), Ehrlich ascites and preputial gland tumor (Synder et al., 1971). The reaction requires molecular oxygen and reduced pyridine nucleotides (Wykle et al., 1972; Paltauf and Holasek, 1973; Wykle and Schremmer-Lackmiller, 1975) and is strongly inhibited by cyanide. The reaction resembles in many respects that of the mammalian acyl CoA A9-desaturase system; the analogies between these two systems include, in addition to their cofactor requirements, susceptibility to inhibition by sulfhydryl blocking agents, menadione, metal chelators, azide (Paltauf and Holasek, 1973) and inhibition by a specific antibody to microsomal cytochrome b5 (Paltauf et al., 1974). Therefore, NADH-cytochrome b5 reductase, NADPH-cytochrome c reductase and cytochrome b5 were considered to participate in supplying reducing equivalents from NADH and NADPH to the terminal cyanide-sensitive desaturase specific for alkylacylglycerophosphoryl-ethanolamine as a substrate. The molecular properties of the desaturase have not been characterized. From its cyanide-sensitivity, the desaturase may contain a non-heme iron, as the catalytic center for the oxidative abstraction of cis-hydrogens from hydrocarbon chains. In contrast to A9-desaturase activity, which is significantly affected by nutritional alteration, the desaturation activity of alkylacylglycerophosphoryl-ethanolamine in various tissues remains unaltered under these conditions (Lee et al., 1973). 7.3.3. Phospholipid Desaturase The first evidence suggesting the possibility of direct desaturation of an acyl moiety esterified to the phospholipid molecule was presented by Gurr et al. (1969), using the chloroplast fraction of Chlorella vulgaris, and by Baker and Lynen (1971), using the microsomal fraction of Neurospore crassa. Subsequently the operation of such an enzyme system requiring reduced pyridine nucleotides and molecular oxygen as essential cofactors was demonstrated in yeast cells (Talamo et al., 1973; Pugh and Kates, 1975). Microsomal fractions of rat liver were also found to catalyze the oxidative desaturation of l~acyl-2-[~4C]-eicosatrienoyl-sn-glycerophosphoryl-choline to 1-acyl-2-[~4C]-arachidonyl-sn-glycerophosphoryl-choline (Pugh and Kates, 1977). NADH was a better electron donor for the system, although both NADH and NADPH could serve as electron sources; this property is a characteristic of the systems in which microsomal cytochrome b5 acts as a common electron carrier between the two flavoprotein reductases and a terminal enzyme component. The reaction was inhibited by cyanide but not by carbon monoxide (Pugh and Kates, 1977), indicating that the terminal desaturase may belong to the group of non-heineiron proteins in which acyl CoA desaturase and the desaturase in plasmalogen synthesis are classified. The activity was induced by the refeeding of starved animals, a response distinct from the desaturase in plasmalogen synthesis which was not inducible by nutritional alterations.

Cytochrome b 5 and its physiological significance

499

7.3.4. N - h y d r o x y a m i n e R e d u c t a s e Microsomal fractions of liver and kidney, and, to a lesser extent, lung microsomes, contain, besides an NADPH- and oxygen-dependent oxidase that catalyzes the oxidation of N,N'-disubstituted hydroxyamine to the corresponding nitrones, a multicomponent enzyme system that catalyzes the reduction of hydroxyamine and its mono- and di-substituted derivatives to the parent amine (Kadlubar et al., 1973). NADH was a preferential electron donor in the system. The reaction did not require molecular oxygen and was insensitive to both cyanide and carbon monoxide. Three protein fractions which were required to reconstitute the NADH-dependent hydroxyamine reductase activity were isolated from pig liver microsomes by means of detergent solubilization and subsequent purification by column chromatography on DEAE-cellulose and Sephadex gel-filtration (Kadlubar and Ziegler, 1974). Two of the protein fractions were identified as the native forms of cytochrome b5 and NADHcytochrome b5 reductase, respectively. In the reconstitution experiment, the native cytochrome b5 could not be replaced with its hydrophilic segment, suggesting that the terminal component was able to catalyze the reduction of hydroxyamine only by coupling to the oxidation of cytochrome b5 in a proper protein-protein interaction of hydrophobic environments. However, the terminal component differed in many respects from the previously identified cytochrome b5 oxidases such as Ag-desaturase and cytochrome P-450. The isolated terminal component was a protein of an apparent molecular weight of 45,000-65,000 (Kadlubar and Ziegler, 1974). The protein contained no detectable chromophore which absorbed in the visible region of the spectrum, and a metal analysis failed to detect the presence of significant amounts of non-heme iron, copper or molybdenum. The reaction' mechanism of the hydroxyamine reduction by this system seems to be different from that observed in the cytochrome b5 oxidase activity of Ag-desaturase with aniline or hydroxyamine as unphysiological substrates (Oshino and Sato, 1971), although further investigation of these activities will be needed. 8. ACYL CoA Ag-DESATURATION ENZYME SYSTEM Cytochrome b5 is a univalent electron carrier and, hence, its physiological importance could be evaluated only in terms of the physiological functions in which cytochrome b5 serves as an electron carrier. Acyl CoA Ag-desaturase is only one of the cytochrome bs-containing systems on which biochemical and physiological characterizations have been extensively conducted. Therefore, a detailed account will be given in this section of the h9-desaturase system, focusing attention on various experimental means by which features of the desaturase system were substantiated. The phenomenon of dietary induction of the desaturation enzyme system will also be described, with the intention that a unique character of microsomal cytochrome b5 be grasped through an understanding of the events occurring during the process of dietary induction of the activity. 8.1. BACKGROUND

It was in 1959 that desaturation of fatty acid was recognized to be an oxidative process and not a simple dehydrogenation reaction; Bernhard et al. (1959) demonstrated for the first time that the conversion of stearic acid to oleic acid proceeded in a postmitochondrial fraction of rat liver under aerobic conditions. This observation was further expanded to include the obligatory requirement of NADPH and molecular oxygen for the conversions of palmitic acid to palmitoleic acid by yeast microsomes (Bloomfield and Bloch, 1960) and of eicosatrienoic acid to arachidonic acid by liver microsomes (Stoffel, 1961). The cofactor requirement of reduced pyridine nucleotides (NADH or NADPH) and molecular oxygen was confirmed further in the desaturation of linoleic acid to 3,-linolenic acid (Nugteren, 1962), of oleic acid to octadecadienoic acid (Holloway et al., 1963) and of stearic acid to oleic acid (Marsh and James, 1962). Bloomfield and Bloch (1960) had suggested that fatty acid desaturation might involve

500

N. OSH1NO

the participation of a hydroxylated intermediate. An enzyme system which catalyzes the oxidation of NADPH, accompanied by the activation of oxygen for substrate hydroxylation, has been called a mixed function oxidase (Mason, 1957a,b) or a mono-oxygenase (Hayaishi, 1961, 1964). Though the occurrence of a hydroxylated intermediate in a process of desaturation could not be substantiated by subsequent work (Marsh and James, 1962), it has generally been assumed that the fatty acyl CoA desaturase is a microsomal mixed function oxidase. Consequently, a possible alternative physiological role of cytochrome P-450, a mixed function oxidase known to participate in hepatic drug and carcinogen metabolism, was explored in these desaturation reactions. It was soon revealed that the h9-desaturase reaction in microsomes of yeast (Omura et al., 1966) and of rat liver (Oshino et al., 1966) was not inhibited by strong ligands for cytochrome P-450, i.e. carbon monoxide and ethyl-isocyanide, instead, it was inhibited by cyanide and azide under the conditions where drug hydroxylation was unaffected (Oshino et al., 1966). The following characteristics of the desaturation system were clarified in the study by Oshino et al. (1966). In contrast to drug hydroxylation in which NADH served as a very poor electron donor, Ag-desaturase activity was supported by NADH even more effectively than NADPH, further confirming that cytochrome P-450 does not function as a desaturase. However, the presence of menadione or cytochrome c together with microsomes in the reaction mixture resulted in a dose-dependent inhibition of both NADPH-dependent desaturation and aniline hydroxylation activities. Therefore, these two activities share the NADPH-cytochrome c reductase as a common source of the reducing equivalents available from NADPH. Nevertheless, the desaturation activity observed in the presence of both NADH and N A D PH was the same as that obtained with NADH alone, indicating that although the reducing equivalents from these donors entered the pathways through separate reductases, they were transferred to a common enzyme responsible for the desaturation reaction. In addition to these observations, it was noticed that ascorbate at a relatively high concentration served as a weak electron donor and exhibited about 20 per cent of the maximal activity. This activity was increased at alkaline pH and showed an optimal pH around 7.8 which differed from the optimal pH of 7.0 found in the NADH- and NADPH-dependent desaturation activities. The increased activity of the ascorbate-dependent desaturation was accompanied by a parallel increase in the extent of reduction of cytochrome b5 by 10 mM ascorbate; while with ascorbate about 15 per cent of the cytochrome was in the reduced form at pH 7.0, the reduced level was increased to almost 40 per cent at pH 7.8 (N. Oshino, unpublished observation). Since cytochrome b5 was the only microsomal component which was reducible by all three electron donors, a hypothesis to elucidate all the above-mentioned observations was presented, which assumed the involvement of cytochrome b5 in the electron transfer pathway from these donors to a cyanide-sensitive A9-desaturase (Oshino.et al., 1967). 8.2. EVIDENCE FOR THE PARTICIPATION OF CYTOCHROME b5 8.2.1. Analysis Using Intact M i c r o s o m e s The sites of action of various inhibitors, treatments and artificial electron acceptors in the microsomal electron transfer system are illustrated in Fig. 2. When microsomes are used for the analysis of cytochrome bs-containing systems, special attention should be focused on the rate-limiting step of the overall reaction, which often presents a serious pitfall in interpreting experimental data. The desaturation activity of stearyl CoA in microsomal preparations is only of the order of 0.3 nmol oleate formed per min per mg of microsomal protein, which is far slower than the maximal NADPH-cytochrome c reductase activity (100nmol cytochrome c reduced per min per mg of protein) or NADH-cytochrome b5 reductase activity (about 1000nmol cytochrome c reduced per rain per mg of protein). Furthermore, there has been an empirical assumption that the enzyme system may be rigid in composition. This possibility differs greatly from the actual situation in microsomes, where the molecu-

Cytochrome b 5 and its physiological significance

Antibody PCMS NEM, NADH

Dyes

Antibody

. Ferricyan=de ,~

[I Cyt c II /'~

- - ~ a - ~ treatment =- t F p ~ - - I I - - ~ - - ~ " Cathepsin D

Trypsin •

Ascorb~ ~

NADPH----~ ,Fp.~, NADP ÷ HgCI 2 Mersalyl Antibody NEM PCMS

-.

~ Ferricyanide Dyes Menodione Cyt c

V v, Cyt b=; I

\\, ,,"

I :" Oz J. /"

501

Bothophenanthroline

Azide

Cyanide v ~ CSF (Desaturase) p'~-Cresol Phenols

-.~ Cyt P 450 =:;--~ Organic hydroperoxides Drugs II CO

Antibody

FIG. 2. Electron donors and acceptors, inhibitors and various treatments which were utilized in the studies on microsomal electron transfer system. 7_3 : solubilization; ~ : inhibition; ): electron donor; ~ = = : unphysiological acceptor; , , - - - - - : slow auto-oxidation. FpD: NADH-cytochrome b5 reductase; Fpr: NADPH-cytocbrome c reductase; NEM: N-ethylmaleimide; PCMS: p-chloromercuribenzene sulfonate; CSF: cyanide-sensitive factor (desa-

turase). lar ratio of cytochrome b5 to its reductase appears to be 14 in the hepatic microsomes of rabbit (Rogers and Strittmatter, 1974b). The difficulty which arose from the low desaturation activity was solved by the finding that the activity in rat liver was induced profoundly by refeeding the starved rat on a fat-free carbohydrate diet (Oshino et al., 1971; Oshino and Sato, 1972). With this dietary pretreatment, it became feasible to consistently obtain a microsomal preparation which possesses desaturation activity of the order of 3-6nmol oleate formed per min per mg of microsomal protein. The first indirect evidence linking cytochrome b5 to the Ag-desaturation system was obtained by analysis of the steady-state reduced-level of cytochrome b5 in the microsome with an induced desaturation activity (Oshino et al., 1971). With NADPH as an electron donor, microsomal cytochrome b5 was reduced to about 70-80 per cent of maximum. Initiation of the desaturation reaction by addition of stearyl CoA caused an enhanced utilization of reducing equivalent from cytochrome b5 and consequently resulted in an instant decrease in the steady-state concentration of the reduced cytochrome bs. The newly established steady-state persisted for a while and returned toward the original level when the added stearyl CoA was consumed. The extent of the decrease observed in the concentration of the reduced cytochrome depended on both the desaturation activity (i.e. the rate of cytochrome b5 oxidation by desaturase) and the ability of cytochrome b~ to be reduced by NADPH-cytochrome c reductase in microsomes; partial inhibition of the reductase by low concentrations of HgC12 or specific antibody to this flavoprotein was accompanied by proportional increases in the extent of the stearyl CoA-induced shift in the steady-state reduced-level of cytochrome bs. Microsomes possess a very potent NADH-cytochrome b5 reductase activity, which is sufficient to maintain, with NADH, a nearly full reduction of cytochrome b5 despite its enhanced oxidation by the induced desaturation reaction. Thus, stearyl CoA did not produce a detectable decrease in the NADH-supported level of reduced cytochrome b5 under ordinary conditions. However, since this reductase was sensitive to p-chloromercuribenzene sulfonate (PCMS) and also to a specific antibody against this reductase, it was possible to demonstrate the stearyl CoA-induced decrease in the steady-state level of cytochrome b5 reduced with NADH when the reductase activity was inhibited by more than 99 per cent by one of these inhibitors (Oshino et al., 1971). It should be noted that, because of large differences between their reaction rates, the

502

N. OSHIr~O

degree of the inhibition of these reductases could not by any means correlate directly with the extent of the inhibition of the desaturation activity; the latter reaction was still supported with an abundant supply of reducing equivalents, even under the above mentioned conditions, where the steady-state reduced-level of cytochrome be was lowered significantly by the addition of stearyl CoA. This method of steady-state analysis has been used in the study of the Ag-desaturation system in yeast microsomes (Tamura et al., 1976) and may be of use for the demonstration of the participation of these electron transfer components in a certain system in microsomes. One of the simplest methods for examining the possibility of participation of cytochrome b5 in a given microsomal reaction is to measure the effect of specific substrates on the oxidation rate of reduced cytochrome be in microsomes. When NADH is added to a microsomal suspension in a final concentration of a few /xM, cytochrome be is reduced instantaneously and is maintained for a while in an almost fully reduced state. Upon consumption of the added NADH, a decrease in the level of the reduced cytochrome b5 begins. The process of reduction and reoxidation can be followed easily by dual-wavelength spectrophotometry, using the absorbance difference between 424 nm and 409 nm. Approximately 70-80 per cent of the oxidation can be measured without significant disturbance by the reduction process. If oxidation by contaminating mitochondrial fragments is prevented by addition of 0.15 mM sodium sulfide, the auto-oxidation process of microsomal cytochrome be in general obeys first-order kinetics with a rate constant (k) between 1 and 3 per min at 30°C (Oshino and Sato, 1971). Assuming reducing equivalents from cytochrome be to be essential in the desaturation reaction, the presence of either stearyl CoA or p-cresol in the reaction mixture must cause an enhanced oxidation of cytochrome bs. In fact, it was demonstrated that the first-order rate constant was increased up to 40 per min in a microsomal preparation with an induced desaturation activity of nearly 10nmol oleate formed per min per mg of protein (Oshino and Sato, 1971). Cyanide inhibited the stimulatory action of acyl CoA and phenols on the oxidation of cytochrome be. A correlation between the increment in the first-order rate constant of cytochrome bs oxidation by substrates and the desaturation activity has been established by using not only microsomes of liver, adipose tissues (Oshino and Sato, 1971; Oshino, 1972) and yeast (Tamura et al., 1976) but also various microsomal preparations prepared at different stages of development of chick liver after hatching (Wilson et al., 1976). This method has sufficient sensitivity to detect an increment in the rate of cytochrome be oxidation caused by substrate oxidation, with a specific activity of more than about 0.5 nmol cytochrome bs oxidized per min per mg of microsomal protein. It should be noted that NADPH is an unsuitable electron donor in this method; NADP ~, which is accumulated during the reaction inhibits competitively the interaction between NADPH and NADPH-cytochrome c reductase (Philips and Langdon, 1962; Modirzadeh and Kamin, 1965), and hence the reduction rate of cytochrome b~ is progressively decreased, resulting in an appearance of the oxidation phase at a certain stage where significant amounts of NADPH still remain in the system. The oxidation process observed under this condition provides false kinetics due to concurrent oxidation and reduction of cytochrome bs. The most conclusive evidence for the operation of cytochrome b5 in the microsomal desaturation system is the fact that an antibody specific to cytochrome b5 caused the same concentration-dependent inhibition of both NADH- and NADPH-dependent desaturation reactions without producing any effect on the corresponding two reductase activities (Oshino and Omura, 1973; Jansson and Schenkman, 1977). Binding of the antibody to cytochrome be was shown to disturb both reduction of cytochrome b5 by its reductase and oxidation of reduced cytochrome be by A9-desaturase. It was also reported recently that an antibody to cytochrome b5 was able to inhibit microsomal A%desaturation of fatty acids (Lee et al., 1977): Ascorbate was able to produce only a partial reduction of cytochrome be in microsomes. The rate of ascorbate-dependent desaturation appeared to be proportional to the steady-state concentration of reduced cytochrome b5 and, hence, a partial

Cytochrome b5 and its physiologicalsignificance

503

removal of cytochrome b5 by proteolytic digestion resulted in a loss of the ascorbatedependent desaturation activity, the extent of which was inversely proportional to the amount of cytochrome b5 remaining in the protease-pretreated microsomal membrane (Oshino et al., 1971). An unexpected result in this study was the observation that, in spite of the removal not only of cytochrome b5 but also of more than 90 per cent NADPH-cytochrome c reductase, the protease-pretreated microsomes still exhibited almost full activity for both NADH- and NADPH-dependent desaturation (Oshino et al., 1971). This result can now be interpreted to indicate that, rather than being in a rigid enzyme complex in which a loss of a particular component is directly accompanied by a loss of overall enzyme activity, electron carriers in the microsomal system distribute randomly to allow free rearrangement for interaction between the remaining constituents, whereby the relatively slow rate of desaturation persists without apparent decrease. This consideration is supported by the following result. The microsomal membranes, from which more than 90 per cent of the cytochrome b5 had been removed by tryptic digestion, still retained almost maximal activity for the NADH-dependent desaturation of stearyl CoA, but th.e similarly treated desaturation induced microsomes became very susceptible to the inhibitory action of an antibody against cytochrome bs; the antibody at a concentration of 2.1 mg 3,-globulin per mg of microsomal protein caused 87 per cent inhibition of the desaturation activity in the pretreated microsomes, whereas the same amount of the antibody produced practically no inhibition in the corresponding amount of the original untreated microsomes (Oshino and Omura, 1973). It may be deduced from this result that less than 10 per cent of the cytochrome b5 on the microsomal membrane is sufficient to maintain maximal A9-desaturation activity. However, this does not mean that the cytochromes which have survived proteolytic digestion are the only ones normally comprising the desaturation system in the intact microsomes; instead it may be that all of the cytochrome b5 molecules are equally capable of donating reducing equivalent to the desaturase molecules in microsomes. Because the kinetics of component interaction may be further confused by problems arising from the rate-limiting step of the reaction, a clear correlation between increases in the specific activity of the overall reaction and in a specific content of cytochrome b5 may never be obtained, even after purification of the enzyme system. 8.2.2. Fractionation and Reconstitution of the A9-Desaturase S y s t e m In contrast to the works of Oshino et al., in which analyses of the A9-desaturase of the intact membrane system were performed, Wakil and Holloway's group elected from the beginning to study the enzyme system by removal from its membranous surroundings. Based on their experience of the susceptibility of NADH-cytochrome c reductase activity to lipid extraction, Jones et al. (1968) treated microsomes with cold 90 per cent aqueous acetone and found a concurrent inactivation of the activities of NADHcytochrome c reductase and stearyl CoA desaturation. Both the activities could be restored by re-addition of the lipid mixture to the treated microsomes. Since the obligatory requirement for phospholipid in the reconstitution of the NADH-cytochrome c reductase activity could be confirmed later with the purified forms of intact cytochrome b5 and its reductase (Rogers and Strittmatter, 1973), this observation of Jones et al. (1968) has been often cited as evidence indicating the requirement for phospholipids by the desaturase system at a specific site in the electron transfer pathway from NADH to cytochrome bs. The term 'phospholipid requirement' might imply a possible action on the part of phospholipid which is able to affect the overall activity, and, therefore, the meaning of the term requires clarification. In the study of Jones et al. (1968), the removal of phospholipid from microsomal membranes resulted in a simultaneous inactivation of the activities of not only NADH-cytochrome c reductase and NADH-dependent desaturation but also NADPH-dependent desaturation, and phospholipids were able to restore the NADPH-dependent desaturation as well. Since this latter activity does not utilize J P T A VoL 2, No. 3 - - E

504

N. OSHINO

NADH-cytochrome b5 reductase, the result has to be interpreted to indicate that the removal of phospholipid did affect, beside the NADH-cytochrome c reductase system, a certain site common in the NADH- and NADPH-dependent desaturation pathways. In addition to this, it is obvious that, though the acetone-treatment produced a profound decrease in the NADH-cytochrome c reductase activity, the treated microsomes still possessed activity as high as 29nmol cytochrome c reduced per min per mg of microsomal protein, which is more than sufficient to support the reported desaturation activity of less than 0.3 nmol oleate formed per min per mg of microsomal protein in their microsomal preparations. Therefore, in spite of an apparent parallel observed in the inactivation and reactivation of desaturation and NADH-cytochrome c reductase activities, the changes in the desaturation activity by the acetone-treatment cannot be ascribed to a phospholipid requirement in the electron transfer pathway between N A D H and cytochrome bs. In fact, Holloway and Katz (1972) later noticed in their recombination experiment that the removal of lipid had affected also a site in the electron transfer pathway between cytochrome b5 and molecular oxygen. The rate-limiting step in the overall desaturation reaction is that proceeding in the A9-desaturase itself because the maximal rate of cytochrome b5 oxidation observed with unphysiological substrates for the desaturase, i.e. p-cresol and p-chlorophenol, was faster than the rate of stearyl CoA desaturation (Oshino and Sato, 1971). A conclusion that hydrogen abstraction from the acyl moiety is the rate-limiting step of the enzymic desaturation was also drawn from the studies on isotope effects in the rate of desaturation (Enoch et al., 1976; Seifried and Gaylor, 1976). Thus, the possibility remains that phospholipid may act directly on the desaturase itself. This possibility was examined in a reconstitution study by using purified desaturase and intact cytochrome bs, together with various compositions of phosphatidyl choline and oleate (Holloway and Holloway, 1975). The reconstituted desaturation activity was not affected by the composition of lipids, although the lipid was demonstrated to be essential for the reconstitution process itself. An appropriate concentration of detergents could replace phospholipids in this system. As described in a previous section, all the constituents of the desaturation enzyme system are amphipathic proteins showing a strong tendency to aggregate with each other in aqueous media. In order to maintain an optimal configuration for interaction with other components, it is necessary to provide a stable hydrophobic environment around the hydrophobic domain of the protein. This is an essential requirement for interaction between the membrane proteins which contain both hydrophilic and hydrophobic regions. It was demonstrated that acyl CoA molecules become utilizable by the desaturase system, reconstituted on liposomes, by binding to the phospholipid bilayer of liposome (Enoch et al., 1976). The term 'phospholipid requirement' should, therefore, be regarded as signifying, rather than that for a particular site, a necessity of the membrane structure as a ground for the interaction between these amphipathic proteins and substrates. Holloway and Wakil (1970), in their attempts to fractionate the desaturase system, treated hen liver microsomes with a mixture of sodium deoxycholate and glycerol and obtained a particle fraction in which NADH-dependent desaturation activity was increased four fold. This particle fraction, which still contained the three constituents of the desaturase system, i.e., NA~DH-cytochrome b5 reductase, cytochrome b5 and desaturase, was then treated with a sulfhydryl blocking agent, N-ethylmaleimide, in order to inactivate NADH-cytochrome b5 reductase completely. Thus, the resulting particle fraction (N-particle fraction) showed neither NADH-cytochrome c reductase activity nor NADH-dependent desaturation activity. Combination of the N-particle with a partially purified NADH-cytochrome b5 reductase fraction or, even better, with the hydrophilic segment of the reductase, could restore the hg-desaturation activity. In a further development of this line of investigation, cytochrome b5 was split from the N-particle by additional detergent treatments and was purified to a purity of about 8 per cent (Holloway and Katz, 1972). This cytochrome preparation was demonstrated to restore the NADH-dependent desaturation activity in the presence of the hydro-

Cytochrome b5and its physiologicalsignificance

505

philic segment of NADH-cytochrome b5 reductase, lipid and a desaturase-rich fraction obtained after splitting cytochrome b5 from the N-particle. This cytochrome b5 fraction could not be replaced with a highly purified hydrophilic segment of cytochrome bs, leading us to recognize the importance of the hydrophobic domain of the native protein in reconstitution experiments. Ito and Sato (1968) achieved purification of the native form of cytochrome bs. This was followed by an improvement in the purification method by Spatz and Strittmatter (1971). Subsequently, purification of NADH-cytochrome b5 reductase was achieved by Spatz and Strittmatter (1973) and later by Mihara and Sato (1975). Along with progress in purification of the desaturase constituents, it was demonstrated that purified native cytochrome b5 (Strittmatter et al., 1972; Enomoto and Sato, 1973) and its native reductase (Rogers and Strittmatter, 1974b) could be incorporated into the microsomal membrane in a manner indistinguishable from the endogenous components. Shimakata et al. (1972) reported partial purification of the desaturase which, upon incubation with purified preparations of cytochrome b5 and its reductase, reconstructed the NADH-dependent stearyl CoA desaturation system. This preparation of desaturase could exhibit desaturation activity without exogenous cytochrome b5 when the hydrophilic segment of the reductase was employed. This result was attributed to artificial activity due to protolytic modification of the reductase. In agreement, Strittmatter et al. (1974) later reported that the purified desaturase could utilize only the native forms of cytochrome b5 and its reductase in the reconstitution of the system. With regard to the nature of the Ag-desaturase molecule, Gaylor et al. (1970) isolated a hemoprotein capable of binding cyanide and proposed it to be a monooxygenase probably functioning in the desaturation reaction. However, the purified desaturase was a non-heine protein capable of forming an NADH-dependent desaturase system with the native forms of both cytochrome b5 and its reductase (Strittmatter et al., 1974). Enoch et al. (1976) used dimyristyl-L-a-lecithin together with the three purified proteins, NADH-cytochrome b5 reductase, cytochrome b5 and desaturase, and prepared liposomal vesicles of 250-600 .~ diameter containing all three proteins required for desaturation activity. The reductase and cytochrome b5 in these liposomes were found to be located exclusively on the outer surface of the liposome. Location of the desaturase was also at or near the outer surface of the liposome since all the iron in the desaturase could be reduced by ascorbate, and formed a complex with bathophenanthroline sulfonate; both agents are known not to permeate the liposome. The minimal molar ratio of NADH-cytochrome b5 reductase, cytochrome bs, desaturase and phospholipid required for optimal desaturation activity in this system was 1 : 0.2 : 4 : 200. All the reconstituted systems cited above (Shimakata et al., 1972; Strittmatter et al., 1974; Enoch et al., 1976) showed, in many respects, the characteristics of the NADH-dependent desaturation system observed in the intact microsomes, such as 02 requirement, substrate specificity, susceptibility to inhibition by cyanide, reaction products and stimulation of cytochrome oxidation by p-cresol. Therefore, it is concluded that results drawn from the reconstituted system are equivalent to results drawn from analysis of the intact desaturase system in microsomes. When compared on the basis of cytochrome bs, the specific activity obtained in the reconstituted system is negligible compared with that in intact microsomes (12nmol oleate per min) or that in trypsin-treated microsomal membranes (120nmol oleate per min) (Oshino and Omura, 1973). This implies that an adequate environment for the interaction between desaturase and cytochrome b5 was not provided in these reconstituted systems. Catal~i et al. (1975) and Jeffcoate et al. (1976) reported the presence of protein factors required for maximal desaturase activity. These proteins appeared to affect the interaction between the desaturase and acyl CoA, suggesting that the discrepancy between the specific activities in the intact and reconstituted systems may be ascribable to a lack of an as yet uncharacterized protein factor in the latter system.

506

N. OSH|NO

8.3. INDUCTION AND REGULATION OF A°-DESATURASE ACTIVITY

In contrast to the relatively constant activity of the microsomal electron transfer system containing cytochrome bs, the desaturase activities in different organs vary, depending on physiological conditions in the rat, such as maturation (Peluffo et al., 1970; Cook and Spencer, 1973b; Wilson et al., 1976), diabetes (Imai, 1961; Gellhorn and Benjamin, 1964), hormonal status (Gellhorn and Benjamin, 1966; Ayala et al., 1973; deG6mez-Dumm et al., 1975), nutritional alterations (Inkpen et al., 1969; Oshino and Sato, 1972; Jansson and Schenkman, 1975; also see a review by Brenner, 1974) and drug administration (Montgomery and Holtzman, 1975; Jansson and Schenkman, 1975). The microsomal fraction from liver of alloxian-diabetic rat exhibited little A9-desaturation activity (Imai, 1961; Gellhorn and Benjamin, 1964). The activity was increased, in a dose-dependent fashion, by insulin administration; the increase continued for a duration of over 72 hr and thereafter decreased abruptly to the initial level (Gellhorn and Benjamin, 1966). This insulin-induced restoration of hepatic desaturation activity was inhibited by pretreatment of animal with either actinomycin D or puromycin, indicative of an involvement of DNA-directed protein synthesis in this induction. The newly induced activity appeared to last only shortly in the microsomes since it underwent rapid decay with a half-life of 3-4 hr when protein synthesis was inhibited by puromycin (Gellhorn and Benjamin, 1966). It was also reported that glucose induced an increase in the desaturation activity of adipose tissue which was preceded by synthesis of messenger RNA with a half-life of about 17 hr (Gellhorn and Benjamin, 1966). With regard to this feature of the enzyme induction, questions may arise as to: (i) whether the induction is accompanied by a simultaneous synthesis of all three constituents or of only a single component in the desaturase system; (ii) whether free or bound polysomes are the site of protein synthesis for the desaturase; and (iii) how the newly synthesized components are incorporated with other constituents of the desaturase system in the pre-existing microsomal membrane. In studies of Oshino and Sato (1972), starved rats were given a fat-free carbohydrate diet for only 1 hr. An increase in liver-glyc0gen content started to occur immediately after this limited amount of food-intake, followed after a slight delay by a parallel increase of the hepatic desaturation activity. Within 12 hr the specific activity of the desaturase increased from a value of below 0.1 nmol oleate formed per min per mg of protein to more than 3 nmol oleate formed per min per mg of protein. It then returned to the initial low activity as glycogen disappeared from the liver. An inverse correlation between serum-free fatty acid level and hepatic desaturation activity was also observed during alteration of activity under various nutritional conditions (Montgomery and Holtzman, 1975). Throughout the whole period of this reversible alteration in the desaturation activity, no change was detected in the activity or content of NADH-cytochrome b5 reductase, NADPH-cytochrome c reductase, cytochrome b5 or cytochrome P-450 (Oshino and Sato, 1972). When starved rats were allowed free access to the diet, the hepatic desaturase activity increased linearly and within a day reached the extraordinarily high activity of 8-10 nmol oleate formed per min per mg of protein. Within the following few days the activity declined to an ordinary steady-state level observable in regularly fed animals. Throughout this induction process the content of cytochrome b5 and the activity of NADH-cytochrome b5 reductase were elevated only moderately. In addition, the increase of A9-desaturation of refeeding was prevented completely when the rat was pretreated with either actinomycin D or cycloheximide, and, hence, it was concluded that the increased activity of A9-desaturation was caused by a net synthesis of only the A9-desaturase molecules in this system (Oshino and Sato, 1972). A half-life of the newly synthesized desaturase was about 3 hr, as seen with insulin-induced desaturase (Gellhorn and Benjamin, 1966). Strittmatter et al. (1974) were able to isolate A9-desaturase from the microsomal fractions of fed and starved-refed rats, but no protein band corresponding to the A9-desaturase was detected in SDS-gel electrophoresis when microsomes from starved rats were examined. This result confirmed a lack of the desaturase enzyme in the latter microsomal membrane.

Cytochrome bs and its physiologicalsignificance

507

In a course of dietary induction, Ag-desaturase appeared at first in the rough microsomal membrane and its content increased continuously. The activity in the smooth microsomal fraction, on the other hand, started to show a significant increase only after a 12 hr lag period. Based on this clear transition of the induced activity from the rough to the smooth membranes, the site of protein synthesis for the desaturase was determined to be polysomes bound to the endoplasmic reticulum (Oshino and Sato, 1972). Administration of actinomycin D to the rat 12 hr after the initiation of refeeding greatly depressed the increase of the activity in rough microsomes, yet the activity in smooth microsomes continued to increase, within 6 hr, at a rate similar to that observed without the drug treatment. Ten hours after administration of actinomycin D the desaturase activities in both types of the membrane tended to equilibrate. Since protein synthesis could be maintained by the already synthesized messenger RNA for at least several hours after administration of actinomycin D, the results were interpreted to indicate transverse movement of the newly synthesized desaturase from the rough to the smooth membranes (Oshino and Sato, 1972). Of interest would be more detailed information as to the process by which the newly synthesized desaturase molecule comes into contact with the pre-existing microsomal electron transfer components and exhibits the overall desaturation activity in the membrane. The half-lives of cytochrome b5 and total microsomal proteins were about 100 and 80hr, respectively (Omura et al., 1967; Omura and Kuriyama, 1971), and hence renewal of these components during about 20 hr of the dietary induction of the desaturase may be assumed to be insignificant. According to Ito (1974), each microsomal vesicle contained, as a mean, 1.5 of the enzyme assembly units, in which several molecules of NADH-cytochrome b~ reductase are associated with about ten times as many molecules of cytochrome bs. The assemblies appear to distribute uniformly throughout the endoplasmic reticulum. Incorporation of a newly synthesized desaturase molecule into this assembly may result in a stimulated oxidation of the cytochrome b5 by the desaturase, and the extent of the stimulation may be proportional to the number of desaturase molecules in the assembly. Since crossreactions between the electron transfer components located on different microsomal vesicles did not occur (Oshino and Sato, 1971), the kinetics of cytochrome b5 oxidation observable in a microsomal fraction represent a simple summation of the independent kinetics in each microsomal vesicle. Representative reoxidation patterns of microsomal cytochrome b5 in the presence of p-cresol are shown in a semilogarithmic plot in Fig. 3. The microsomal preparations in this example were those prepared after 12 hr of dietary induction and exhibited desaturation activities of 1.0, 1.7 and 0.4nmol oleate formed per min per mg of protein in total, rough and smooth microsomal fractions, respectively. As is evident from these traces for total and smooth microsomes, the oxidation patterns were biphasic in character and composed of two distinct components of 'functional' and 'non-functional' phases, both of which obeyed first-order kinetics. Of importance is the fact that the first-order rate constant of the 'non-functional' phase of the oxidation pattern is identical to the rate constant observed for the auto-oxidation of cytochrome b5 in the absence of p-cresol, indicating an existence of the cytochrome b5 assembly which does not contain desaturase at this moment. In this particular instance, 14 per cent and 30 per cent of the cytochrome b5 in total and smooth microsomes, respectively, were determined to be 'non-functional' in the desaturation system. In contrast to the biphasic character in smooth microsomes, the oxidation of cytochrome b5 in rough microsomes was enhanced homogeneously by p-cresol, indicating a random insertion of the newly synthesized desaturase molecule into all cytochrome b5 assemblies on the rough endoplasmic reticulum. As to the biochemical events occurring in a process of induction of the overall desaturation system in the endoplasmic reticulum in vivo: (1) The oxidation patterns of cytochrome b5 in the rough microsomal membranes were monophasic throughout the induction period, and the increments in the firstorder rate constant coincided with the increases in the desaturation activity. This is

508

N. OSHINO

Totalmicrosomes

k g

o 0.5 ~5 0.3 o.z

'~

5

~

3

>"

2

~Functionol~ p h a s e ~10sec,

r-microsomes

s-microsomes

~ Original a~ 0.5

0.3 0.2 IOsec OI

phase

Functional phase I0sec

FIG. 3. Biphasic nature of cytochrome b5 oxidation in total, rough and smooth microsomal fractions. The microsomal fractions used were prepared from the rats refed on a highcarbohydrate diet for 12 hr. The oxidation of cytochrome b5 was measured in the presence of 1.0 mM p-cresol as a substrate for the desaturase. For detailed accounts, see Oshino, 1973. The terms 'functional' and 'non-functional' are arbitrarily applied to the biphasic plots; as indicated in the text, the rate constants for the 'non-functional' phases are about equal to the values before addition of p-cresol.

distinct evidence that, rather than being incorporated into a restricted area of a concurrently synthesized membrane structure, the desaturase is incorporated uniformly over the pre-existing rough endoplasmic reticulum. (2) In contrast, the reoxidation pattern of cytochrome b5 in the smooth membrane showed clear biphasicity in the early period of induction, and percentages of the 'functional' cytochrome b5 were found to be 26, 40, 70 and more than 85 per cent after 4, 7, 12 and 15 hr of the induction period, respectively. This delayed increase in the amount of 'functional' cytochrome b5 was accompanied by a gradual increase in the first-order rate constant of the oxidation of 'functional' cytochrome bs. These results demonstrated that, as the concentration of desaturase in the rough membrane of endoplasmic reticulum was increased, the molecule started to diffuse, according to the concentration gradient, into the area of smooth endoplasmic reticulum, resulting, within about 21 hr, in an equilibrated distribution of the desaturase throughout the membrane. (3) Inhibition of protein synthesis by cycloheximide resulted in a rapid disappearance of the desaturase activity from microsomes with a half decay time of about 3 hr (Oshino and Sato, 1972). Analysis of the oxidation pattern of cytochrome b5 in this decay process revealed a monophasic character of the cytochrome b5 oxidation, which, established in the dietary induction, was continued throughout the process of decay of the activity, indicating that the number of desaturase molecules in each assembly decreased uniformly without positional preference on the membrane. An important conclusion drawn in this study is that lateral movement of desaturase molecules is continued even after reconstruction of the overall desaturation-enzyme system in an assembly with cytochrome bs, and emphasizes the dynamic state of the electron transfer assembly on the microsomal membrane. Developmental studies have also been performed on the appearance of A9-desaturase activity after hatching of the chick (Donaldson, 1973; Wilson et al., 1976).

Cytochrome b 5 and its physiological significance

509

A c c o r d i n g to W i l s o n et al. (1976), l i v e r m i c r o s o m e s o f c h i c k e m b r y o s s h o w e d v e r y l o w d e s a t u r a t i o n a c t i v i t y . A f t e r a lag p h a s e o f 2 4 h r a f t e r h a t c h i n g , t h e a c t i v i t y i n c r e a s e d l i n e a r l y to a v a l u e 100 t i m e s a b o v e t h e s t a r t i n g l e v e l an d a f t e r w a r d s d e c l i n e d to a n o r m a l a d u l t s t e a d y - s t a t e l e v e l , w i t h i n t h e f o l l o w i n g 6 - 7 d a y s . T h i s p r o c e s s w a s i n h i b i t e d b y a c t i n o m y c i n D an d c y c l o h e x i m i d e . S m a l l i n c r e a s e s in t h e c o n t e n t o f c y t o c h r o m e b5 a n d N A D H - c y t o c h r o m e b5 r e d u c t a s e a c t i v i t y w e r e o b s e r v e d in this p r o c e s s and w e r e n o t c o m p a r a b l e to t h e h i g h l y i n c r e a s e d d e s a t u r a t i o n a c t i v i t y ; it w a s c o n c l u d e d t h a t t h e i n c r e a s e w a s d u e to n e t s y n t h e s i s o f d e s a t u r a s e molecules after hatching. The transverse m o v e m e n t of the newly synthesized desat u r a s e f r o m r o u g h to s m o o t h m i c r o s o m a l m e m b r a n e s w a s d e m o n s t r a t e d b y a n a l y s i s o f t h e r e o x i d a t i o n p a t t e r n o f c y t o c h r o m e bs.

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