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References 1 Kell, D. G. (1982) Trends Biochem. Sci. 7, 1 2 Payens, T. A. J. (1983) Trends Biochem. Sci. 8,46 3 Welch, G., Somogyi, B. and Damjanovich, S. (1982) Prog. Biophys. Molec. BioL 39, 109-146 4 Stere, P. A. and Mosbach, K. (1974) Ann. Rev. Micro. 28, 61-83 5 Welch, G. (1977) Prog. Biophys. Molec. BioL 32, 103-191 6 Welch, G. and Keleti, T. (1981) J. Theor. Biol. 93,701-735 7 Mitchell, P. (1959) Annu. Rev. Mierobiol. 13, 407--440 8 Munkres, K. D. and Woodward, D. O. (1966) " Proc. Natl Acad. Sci. USA 55, 1217-1224 9 Kiehn, E. D. and Holland, J. J. (1970)Nature 226, 544--545 10 Wetlaufer, D. B. (1981)Adv. Prot. Chem. 34,

21 Zaiokar, M. (1960)Exptl Cell Res. 19, 114-132 61-92 22 Halper, L. H. and Srere, P. A. (1977) Arch. 11 Blake, C. (1983) Nature 306, 535-537 Biochem. Biophys. 184, 529-534 12 Remington, S,, Wiegand, G. and Huber, R. 23 Sumegi, B., Gyocsi, L. and Alkonyi, I. BIO(1982) J. Mol. Biol. 158, 111-152 chim. Biophys. Acta (in press) 13 Maugh, T. H. (1983) Science 221,351-354 14 Rose, G. D. and Wetlaufer, D. B. (1977) 24 Porpaezy, Z., Sumegi, B. and Alkonyi, I. (1983) Biochim. Biophys. Acta 749, 172-179 Nature 268, 769-770 15 Matthew, J. B., Weber, P. C., Salemme, F. R. 25 Beeckmans, S. and Kanarek, L. (1982)Eur. J. Biochem. 117, 527-535 and Richards, F. M. (1983)Nature 301,169-171 16 Wodak, S. J. and Janin, J. (1980) Proc. Natl 26 Fahien, L. A. and Kmiotek, E. (1983) Arch. Biochem. Biophys. 220, 386-397 Acad. Sci. USA 77, 1736-1740 17 Richards, F. M. (1977) Annu. Rev. Biophys. 27 D'Souza, S. F. and Srere, P. A. (1983)J. BioL Chem. 258, 4706-4709 Bioeng. 6, 151-176 18 Welch, G. R. and DeMoss, J. A. (1978) in 28 Minton, A. P. (1981) Biopolymers 20, 20932120 Mieroenvironments and Metabolic Compart. mentation (Srere, P. A. and Estabrook, 29 McConkey, E. H. (1982)Proc. NatlAcad. Sci. USA 79, 3236-3240 R. W., eds), pp. 323--344,Academic Press 19 Manney,T. R. (1970)J. Bacteriol. 102, 483--488 30 Srere, P. A. and Estabrook, R. (eds) (1978) Mieroenvironments and Metabolic Compart20 Kempner, E. S. and Miller, J. H. (1968) Exptl mentation, Academic Press Cell Res. 51,150-156

DNA polymerase holoenzymes Ulrich HObscher D N A polymerases responsible f o r chromosomal D N A replication o f prokaryotes and eukaryotes are multipolypeptide complexes. The functional forms o f D N A polymerases can be isolated and have been designated as D N A polymerase holoenzymes. They are required f o r efficient, processive and accurate in vitro replication o f naturally occurring genomes such as bacteriophage or viral D N A .

D N A replication and D N A repair require the concerted action of many proteins and enzymes either separately or in a complex 1. These include D N A polymerases, D N A polymerase accessory proteins, primase, topoisomerase, helicase, DNA-binding proteins, ribonuclease and ligase 1. Since the discovery of a D N A polymerase in Escherichia coil, 28 years ago 2, such enzymes have been found in all prokaryotic and eukaryotic organisms tested (see Ref. 3 for review).' With the discovery of several distinct D N A polymerases in a single cell, the search began for their different biological roles. In general, three types of DNA-polymerizing enzymes were identified in prokaryotes (I, II and III) and eukaryotes (a, 13 and ~/), and many (but not all) of their functions are now known 1'3 (Table I).

DNA polymerases in chromosomal DNA replication

(1) The enzyme that provides the basic machinery for polymerization is designated as the D N A polymerase core. It can polymerize very short D N A stretches (a few bases only) on any templates that have been treated with DNase to create artificial gaps.

of the genetic material, which is accom(2) A D N A polymerase that is fully plished through the cooperation of operative in vitro on defined genomes auxiliary proteins. The high accuracy of such as those of bacteriophages or these replicative D N A polymerase comviruses is termed a holoenzyme. This plexes ensures the overall conservation D N A polymerase form replicates these of a species. On the other hand, their chromosomes by starting at a defined very limited n u m b e r of mistakes may be primer terminus and polymerizes long one of the sources of evolutionary variD N A stretches (several hundred to ability. A functional replicative D N A several thousand bases). The holopolymerase has several other roles enzyme possesses, in addition to the besides polymerization, including (1) D N A polymerase core, several auxiliary participation in the unwinding process proteins that build a complex with the ahead of the continuously-synthesized D N A polymerase. leading strand; (2) interaction with (3) This in vitro functioning holomultiple primers in advance of the discontinuously-synthesized lagging strand; enzyme may be part of an even higher (3) the processive translocation of the order structure at the replication fork, D N A polymerase along the D N A strand; fitting i n t o / n vivo functioning replication and (4) the implementation of an complexes, called primosomes or repliextremely high accuracy of polymeri- somes 1, and even into the intact replicating chromatin. zation. Table L Different prokaryotic and eukaryotic DNA polymerases and their main biologicalfunction

DNA polymerase Replication of chromosomal D N A requires D N A polymerase 111 in E. coli 1, Prokaryotes and D N A polymerase et or a D N A I polymerase a-like enzyme in eukaryotes 3. II The common universal role of these in Eukaryotes enzymes is to produce an accurate copy

Ulrich Hiibscher is at the Department of Pharmacology and Biochemistry, University of Ziirichlrchel, Winterthurerstrasse 190, CH-8057 Ziirich, Switzerland.

A common hierarchy of structures for replicative D N A polymerases is proposed. Three structural levels can be distinguished:

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a Minor role.

1 ~4 , FAscvier Science Publishers B.V., Amslexdam 0376 - 5067/84¢$02.00

Main biologicalfunction DNA repair (DNA replication)a Unknown DNA replication (DNA repair)a DNA replication(DNA repair)~ in nucleus DNA repair in nucleus DNA replicationin mitochondria

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weight for the catalytic polymerizing subunit of 110 000-185 000; (4) If insufBiological function ficient precautions against proteolysis were taken, M r of less than 105 were Polymerization, 3'-5' exonuclease obtained for the catalytic polymerizing Initiation complex formation, processivity subunit; (5) Different forms of D N A Processivity, fidefity, ATP activation polymerase et were described; (6) Processivity, fidefity, ATP activation Regulation of proofreading Factors and proteins were isolated that Unknown interact with the polymerase in a positive ATP activation, dimerization of hole,enzyme or negative fashion. By analogy with the E. coli replicative D N A polymerase III one can now define and isolate two functional forms of polymerase IIl holoenzyme minus the 13 D N A polymerase a. Using the criteria subunit) to form a tight initiation comto distinguish between a pure DNA plex with a primed D N A template 11'12 polymerase et core enzyme (operating and renders the holoenzymes more proon nuclease-treated D N A only) and a cessive if present in large excess 13. The functional D N A polymerase ct holoenthree other subunits ~, ~ and "r are coded zyme form (operating on naturally for by the genes dnaZ t4, dnaX 15 and occurring templates) a holoenzyme dnaX-dnaZ t6, respectively. Whether ~/ form can be isolated from freshly harand 8 are processed from -r is specuvested calf thymus and can be dislative7. All these subunits contribute to tinguished from a corresponding, apparATP activation H and to the processivitya7 ently homogeneous core enzyme by of the holoenzyme. Finally, the intact functional, biochemical and physicoholoenzyme replicates a ~bX174 D N A chemical parameters ~9. This eukaryotic with a fidelity two to three orders of holoenzyme, but not its core, can replimagnitude higher than a homogeneous cate in vivo-like templates such as (1) D N A polymerase III core or D N A single-stranded parvoviral D N A possesspolymerase I (Ref. 18). ing a short hairpin at the 3' end to serve as a defined primer or (2) a singleThe replicase of eukaryotes: DNA stranded M13 D N A that has been RNApolymerase c~ primed at the origin of replication, D N A polymerase et, the major repliThe functionally best characterized case in eukaryotes, has been isolated eukaryotic replicative D N A polymerase and characterized in many laboratories 'complex' is the a-polymerase from the (see Ref. 3 for review). (1) The poly- fruit fly Drosophila melanogaster 2°'21 merases have mostly been assayed with This complex consists of at least three nuclease-treated D N A and therefore polypeptides: c~ (Mr 182 (~)0L 13 (Mr should only be classed as a core enzyme 60 000) and ~ (Mr 50 0(X)). A fourth (see above); (2) The isolated enzymes polypeptide of Mr 73 000 routinely coconsist of two and more polypeptides; purifies with the complex but is not (3) They have an estimated molecular antigenically related to the others 2~. The c~ subunit is the polymerizing core enzyme, while 13 and ~/ appear to enhance the processivity of the polymerase 2°. When a single-stranded M13 D N A template that has been primed with a short R N A piece at the origin ,of replication is elongated with this D N A polymerase c~, the polymerizing rate of the entire complex is several orders of magnitude higher than that of the ct subunit "alone, indicating that these additional subunits might be essential for elongation. However, the enzyme has not been isolated by monitoring for Fig. 1. SDS-polyacrylamide gel electrophoresis of function on an in vivo-like chromoprokaryotic and eukaryotic DNA-polymerase core some, as has been performed with E. and holoenzyme. A: E. coli D N A polymerase 111 coli ~'4 and more recently with calf core enzyme; B: E. coli D N A polymerase I11 holothymus t9. This D. melanogaster comenzyme; C: calf thymus DNA polymerase a core plex might therefore only be a fragmentenzyme; D: calf thymus DNA polymerase ta holoary complex of a holoenzyme. The holoenzyme. The arrows indicate the catalytic DNA enzyme isolated from calf thymus by polymerizing subunits (see Refs 8 and 23 for explanations of how the catalytic subunits were using naturally occurring chromosome determined). probes appears to have a more complex

Table I1. Subunit structure of Escherichia coil DNA polymerase Ill holoenzyme a Subunit

M~ × 10 ~

Genetic locus

a [3 ~,

140 37 52 32 25 10 78

dnaE dnaN dnaZ dnaX dnaQ ? dna X-dna Z

0 "r

~For explanations and references see text.

The replicase of prokaryotes: DNA polymerase m The introduction of small bacteriophage DNAs, e.g. ~bX174, G4 and M13, as E. coli model replicons led to the discovery that multienzyme systems are involved in bacterial replication 1. D N A polymerase III, among many other enzymes and factors, was found to be necessary for elongation. Soon it became evident that, in vitro, the D N A elongation step itself needed a complex form of D N A polymerase III. Such a multipolypeptide complex was isolated by using natural D N A templates to assay the D N A polymerase and was called D N A polymerase III holoenzyme 4. On the other hand, the D N A polymerase III that was purified to homogeneity by using a D N A that had been treated with DNase to create gaps of random sizes, was unable to replicate these natural DNAs. It was designated as the core of the holoenzyme, or D N A polymerase III core 5. The term holoenzyme was henceforth used for the complex comprising the D N A polymerase III core and several auxiliary proteins. The D N A polymerase III holoenzyme consists of at least seven distinct polypeptides although more than eleven polypeptides are evident after rigorous purification of the enzyme 6'7 (Fig. 1B). These seven polypeptides (Table II) are called ~ (M~ 140 (KI0), 13 (37 000), ~ ( M r 52 000), g (M~ 32 000), ~ (Mr 25 000), 0 (M~ 10 000) and r (Mr 78 1300). Three of these polypeptides, a, ~ and 0, constitute the core enzyme (Fig. 1A). The a subunit, encoded by the dnaE gene 8, is responsible for polymerization and possesses a 3'-5' proofreading exonuclease 5. The e subunit, the dnaQ gene product, has a regulatory function in proofreading 9. Neither a genetic locus nor a functional task has so far been found for 0. The four proteins 13, % g and -r are the additional auxiliary proteins that enable the core enzyme to operate with an extremely high efficiency, fidelity and processivity. The [3 subunit, the dnaN gene ~°, acts as a dimer molecule and enables the DNA polymerase III* (DNA

392 polypeptide structure than the fruit fly enzyme, because it possesses at least 10-12 different proteins as judged by SDS-polyacrylamide gel electrophoresis (Fig. 1D). In contrast, the complexity of the core enzyme resembles that of the Drosophila enzyme (Fig. 1C). Priming the template with short pieces of RNA is a prerequisite for the synthesis of Okazaki fragments. The priming enzyme has first been detected in E. coli and has been named primase 1. This special form of RNA polymerase is associated with the DNA polymerase III holoenzyme but can be completely separated from the latter TM. Lehman's group has described a primase in D. melanogaster that is tightly associated with DNA polymerase ct (Ref. 21) and appears to be part of the [3 and/or -/subunit 22. This is in contrast to the calf thymus c~ polymerase that appears to possess the polymerase and the primase in the same polypeptide23. In any case, it is tempting to speculate that a primasepolymerase entity exists that guarantees an extremely efficient way to synthesize Okazaki pieces on the lagging-strand of the replication fork. Another approach towards a functional replicase is to purify factors that stimulate an extensively purified DNA polymerase a. Several factors can stimulate the polymerase on long, singlestranded DNA, and such proteins have been characterized (see, for example, Refs 24-26). It will be interesting to see whether these factors are similar to the polypeptides in the holoenzymes which elongate naturally occurring DNA templates.

Coordinated replication of leading and tagging strands How do these replicase complexes act in vivo? They might be part of an even higher order structure such as the replisome or the intact chromatin 1. An important question is: what strategy do DNA polymerase holoenzymes use to replicate the leading and lagging strand in an efficient way? Since all DNA polymerases synthesize DNA in 5'-3' direction, one strand (the leading) is replicated in a continuous and the other (the lagging) in a discontinuous fashion1. Sinha et al. 27 and Kornberg I have proposed models in which both strands could be synthesized coordinately. Figure 2 demonstrates a simplified illustration of these proposals. Recent data might support the concept of replication of both strands by dimeric replicase forms:

TIBS - September 1984

yielded results which suggest that they might possess a form of dimeric replicase analogous to that in E. coli. A n early purification stage of calf thymus DNA polymerase ot holoenzyme can be separated into different forms by chromotography on DEAE-ceUulose. Six different enzyme activities involved in DNA replication copurify with the DNA polymerase ot (Ottiger, H.-P. and Hiibscher, U., Proc. Natl Acad. Sci. USA, 81 (in press)). On the one hand, copurification of a double-stranded DNA-dependent ATPase, 3'-5' exonuclease and DNA topoisomerase type II with the DNA polymerase et is suggestive of a leading-strand replicase, while on the other, copurification of primase, ribonuclease H, 3'-5' exonuclease, DNA topoisomerase type II and DNA methylase is indicative of a lagging-strand replicase. 31

= 113'

Fig. 2. Model for a coordinated function of leading and lagging strand DNA polymerase holoenzymes at the replication fork. Recent data from studies

with prokaryotic and raanvnalian replicases support the concept of replication of both strands by dimeric replicase forms. For details see text.

(1) A new form of an intermediate holoenzyme was isolated in E. coli and designated as DNA polymerase III'. It contains the three core subunits (c~, e, 0) and the "r protein6 (see above). The big difference between this form of the enzyme and the core DNA polymerase III is its dimeric structure 6"28. The "r subunit holds two polymerase molecules together. Furthermore, two other holoenzyme subunits, 13 and % when purified to homogeneity, form dimers14'2s. Initiation complex formation of holoenzyme with a primed DNA is dependent on ATP 11't2 or on its analogue adenosine 5'-O-(3-triphosphate)2s. Using these two nucleosidetriphosphates it was possible to distinguish two populations of holoenzymes. A two-fold difference was observed, however, in both the extent of initiation complex formation and in the dissociation of initiation complexes once formed 2a. These results suggested that DNA polymerase III holoenzyme consists of two asymmetric halves. This asymmetry could have a structural basis because either one haft of the polymerase might contain an extra subunit or a subunit with a covalent modification2s. Complete stoichiometry of the entire holoenzyme is needed to support this attractive, economic model. (2) A completely different experimental strategy for eukaryotes has

Conclusions Replicases from prokaryotes and eukaryotes exist as multipolypeptide complexes. Their structures are not yet solved in detail, but appear to be more complex than those of well characterized replicases of bacteriophages T7 (Ref. 29) and T4 (Ref. 30). Three structural levels can be distinguished; first, the core polymerase which is responsible for the basic polymerization step; second, the holoenzyme, composed of the core polymerase and associated accessory proteins, a complex which is fully active on naturally occurring DNA templates; and third, a holoenzyme embedded in a higher order structure, such as an asymmetric dimer or other complex which acts in concert with other known replication enzymes. Acknowledgements The author thanks C. C. Kuenzle, C. S. McHenry and S. Spadari for critical reading of the manuscript. Part of the work in the author's laboratory was supported by the Swiss National Science Foundation, Grant 3.0064).81 and by the Canton of Zfirich. References TIBS requires a short refet~ence list designed to direct those who are interested to further literature. I apologize to authors whose papers are not cited.

1 Kornberg, A. (1980, supplement 1982) DNA Replication, W. H. Freeman 2 Komberg, A., Lehman, I. R., Bessmann, M. J. and Simms, E. S. (1956) Biochim. Biophys. Acta 21,197-198 3 Hfibscher, U. (1983) Experientia (Review) 39, 1-25 4 McHenry, C. and Kornberg, A. (1977)J. BioL Chem. 252, 6478--6484 5 McHenry, C. S. and Crow, W. (1979) J. Biol.

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T I B S - September 1984 Chem. 254, 1748-1753 6 McHenry, C. S. (1982) Z Biol. Chem. 257, 2657-2663 7 Kornberg, A. in Proteins involved in D N A replication (Hiibscher, U. and Spadari, S., eds), Plenum Press (in press) 8 Welch, M. M. and McHenry, C. S. (1982) J. Bacteriol. 152, 351-356 9 Scheuermann, R., Tam, S., Burgers, P. M. J., Lu, C. and Echols, H. (1983) Proc. NatlAcad. Sci. USA 80, 7085-7089 10 Burgers, P . M . J . , Kornberg, A. and Sakakibara, Y. (1981) Proc. Natl Acad. Sci. USA 78, 5391-5395 11 Burgers, P. M. J. and Kornberg, A. (1982) J. Biol. Chem. 257, 11468-11478 12 Johanson, K. O. and McHenry, C. S. (1982) J. Biol. Chem. 257, 12310-12315 13 Crute, J. J., La Duca, R. J., Johanson, K. O., McHenry, C. S. and Bambara, R. A. (1983) J. Biol. Chem. 258, 11344-11349

14 Hiibscher, U. and Komberg, A. (1980) Z Biol. Chem. 255, 11698--11703 15 Hiibscher,U. and Komberg, A. (1979)Proc. Natl Acad. Sei. USA 76, 6284--6288 16 Kodaira, M., Biswas, S. B. and Kornberg,A. (1983) Molec. Gen. Genet. 192, 80-86 17 Fay, P. J., Johanson, K. O., McHenry,C. S. and Bambara, R. A. (1982) J. Biol. Chem. 257, 5692-5699 18 Fersht, A. (1979) Proc. Natl Acad. Sci. USA 77, 4946-4950 19 Hiibscher,U., Gerschwiler,P. and McMaster, G. K. (1982) Eur. Mol. Biol. Org. J. 1, 1513-1519 20 Villani,G., Fay, P. J., Bambara, R. A. and Lehman, I. R. (1981) J. Biol. Chem. 256, 8202-8207 21 Kaguni, L. S., Rossignol. J.-M., Conway, R. C. and Lehman, I. R. (1983) Proc. Nail Acad. Sci. USA 80, 2221-2225 22 Kaguni, L. S., Rossignol, J.-M., Conway,

The role of cytochromes P-450 in adrenal steroidogenesis Shigeki Takemori and Shiro Kominami Steroid hormones from adrenal glands are synthesized from cholesterol via several monooxygenase reactions catalysed by different species of cytochrome P-450. P450s¢c and P-45011~ are located in the mitochondria and P-450c21 and P-45017~,tya~ are located in the endoplasmic reticulum. There are different electron transfer pathways from N A D P H to cytochrome P-450 in the two organelles. The metabolic intermediates move back and forth between the organelles during the synthetic process. Recently, steroidogenic electron transfer systems have been reconstituted and well characterized.

Adrenal glands secrete three kinds of steroid hormone: mineralocorticoids, glucocorticoids and androgens. They are responsible for the regulation of electrolytes and water concentration, for the regulation of metabolism of protein, carbohydrate and lipid and for the secondary sex characteristics, respectively. A hemoprotein, cytochrome P-450 plays an important role in the process of adrenal steroidogenesis. Cytochrome P-450 catalyses the monooxygenase reaction expressed by the following equation: S + 02+NADPH+H +--~ S O + H 2 0 + NADP + where S denotes a steroid substrate. The adrenal mitochondria contain at least two types of cytochrome P-450, one (P-450~) catalysing the side-chain cleavage of cholesterol and another (p-450Ht3) catalysing 1113-hydroxylation of steroids 1. The endoplasmic reticulum also contains two species of cytochrome P-450:P-450c21 S. Takemori and S. Kominami are at the Department o f Environmental Sciences, Faculty of Integrated Arts and Sciences, Hiroshima University, Hiroshima 730, Japan.

which catalyses 21-hydroxylation of steroids 2 and P-45017a,lyas e which catalyses both l%t-hydroxylation and C-17--C-20 bond cleavage of steroids 3. These species of cytochrome P-450 as well as the other electron transfer components have been highly purified from adrenal cortex and the steroidogenic electron transfer systems have been reconstituted using the purified proteins. Biosynthesis of adrenal steroid hormones Figure 1 is a scheme for the biosynthetic pathway of steroid hormones in adrenal cortex cells. Cholesterol, the starting material for steroid hormone production, is transferred from blood and largely stored as cholesterol ester in lipid droplets in the adrenal gland. When required, it is released by the action of cholesterol esterase and passes into the mitochondria, where P-450~ cleaves the C-20-C-22 bond to form pregnenolone. This is the rate-determining step in steroidogenesis. The pregnenolone passes out of the mitochondria into the endoplasmic reticulum. Various reactions, which are catalysed by P--450c21 and P-45017a,lyase, take place here. The

23 24

25 26

27 28 29 30

R. C., Banks, G. R. and Lehman, I. R. (1983) at. Biol. Chem. 258, 9037-9039 Hiibseher, U. (1983) Eur. Mol. Biol. Org. Z 2, 133-136 Lamothe, P., Baril, B., Chi, A., Lee, L. and Baril, E. (1981) Proc. Natl Acad. Sci. USA 78, 4723-4727 Pritschard, C. G. and De Pamphilis, M. L. (1983) J. Biol. Chem. 258, 9801-9809 Yagura, T., Kozu, T., Seno, T., Saneyoshi, M., Hiraga, S. and Nagano, H. (1983) J. Biol. Chem. 258, 13070-13075 Sinha, N. K., Morris, C. F. and Alberts, B. M. (1980) J. Biol. Chem. 255, 4290-4303 Johanson, K. O. and McHenry, C. S. (1984) J. Biol. Chem. 259, 4589-4595 Richardson, C. C. (1983) Cell 33, 315-317 Alberts, B. M., Barry, J., Bedinger, P., Formosa, T., Jongeneel, C. V. and Kreuzer, K. N. (1983) Cold Spring Harbor Syrup. Quant. Biol. 47, 655-668

pathway which metabolizes pregnenolone in the endoplasmic reticulum is modulated by the relative activities of 313-hydroxy-AS-steroid dehydrogenaseA5-isomerase and 17~t-hydroxylase (P-45017c~,lyase). When the activity of the former is greater than that of the latter, pregnenolone is mainly oxidized and isomerized into progesterone. Progesterone is hydroxylated at the 21-position to give ll-deoxycorticosterone when 21-hydroxylase activity (P-450c21) is greater than l%t-hydroxylase activity (P-45017a,lyase). O t h e r w i s e , progesterone is hydroxylated at the l%t-position. Some of the l%t-hydroxyprogesterone so generated is converted into andro~tenedione by C-17,20-1yase action (P-45017a.lyase) a n d the rest is hydroxylated at the 21-position to give ll-deoxycortisol. Furthermore, the transference of ll-deoxycortisol and ll-deoxycorticosterone back into the mitochondria is necessary for 1113hydroxylation catalysed by P-45011a in the synthesis of cortisoi and corticosterone, respectively, and for 18-hydroxylation in the synthesis of aldosterone. Therefore, the metabolic intermediates must move back and forth between subcellular organelles during the series of reactions. Finally, steroid hormones such as corticosterone, cortisol, aldosterone or androgen pass from these organelles to the bloodL The mitoehondrial electron transfer system The electron transfer system of the adrenal mitochondria, which is composed of NADPH-adrenodoxin reductase, adrenodoxin and cytochrome P-450 (P450~¢ and P-45011a) 5, is localized on the matrix side of the mitochondrial inner membrane 6. Both adrenodoxin reductase

1984,~sev~ SoenoePublishersB.V.,Amslerdam 0376- 5067/84/$02.00