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Supernatant Protein Relevant to the Activity of Membrane-Bound Enzymes: Studies on Lathosterol 5-Desaturase
Biochemical and Biophysical Research Communications 292, 1293–1298 (2002) doi:10.1006/bbrc.2002.2012, available online at http://www.idealibrary.com on
Supernatant Protein Relevant to the Activity of Membrane-Bound Enzymes: Studies on Lathosterol 5-Desaturase Teruo Ishibashi 1 Department of Biochemistry, Hokkaido University School of Medicine, N15 W7, Sapporo 060-8638, Japan
EARLY WORK IN BLOCH’S LABORATORY All the enzymes required for the synthesis of cholesterol from squalene, the first sterol intermediate, are bound to the endoplasmic reticulum (1), which can be isolated from cell-free homogenates as microsomes. Water-insoluble intermediates are generated in the membrane and metabolized by membranebound enzymes without diffusion of steroid from the membrane (1). The final oxygenase in cholesterol synthesis, lathosterol 5-desaturase (EC 1.3.3.2), catalyzes insertion of the C-5 double bond into lathosterol (5␣-cholest7-en-3-ol) to yield 7-dehydrocholesterol (cholesta5,7-dien-3-ol) (2– 4). This enzyme system consists of NADH-cytochrome b5 reductase, cytochrome b5, and the terminal desaturase (5, 6), and requires molecular oxygen (7, 8), reduced pyridine nucleotide (5, 7), and the 105,000g supernatant (2– 4) for activity. The 5-desaturase system is closely associated with the microsomal membranes, as is its lipid substrate, lathosterol. The striking overall efficiency of the process suggests that these enzymes may exist as an organized system in the microsomal membrane. However, published reports do not include any information about clustering of these membrane components, or about the possible existence of different phases in membrane preparations. INSIGHTS INTO MECHANISM(S) OF FACILITATION BY SUPERNATANT PROTEIN OF MEMBRANEBOUND ENZYMES The enzymes involved in hepatic cholesterol biosynthesis from squalene are located in microsomes (1), and several steps of the sequence are stimulated by soluble, presumably cytoplasmic, proteins (9 –22). The lathosterol 5-desaturase activity was stimulated by purified “squalene and sterol carrier protein” (SCP) (3). SCP has been shown to stimulate the conversion of lathosterol to 7-dehydrocholesterol by influencing events within the endoplasmic reticu1
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lum, e.g., directing the substrate to its specific enzyme site (4). This study (4) was my project in Bloch’s laboratory. I had a very hard time preparing the radioactive substrate ([H 3 ]lathosterol), which was not commercially available at that time. Dr. Bloch took great interest in this work, because he was originally an organic chemist. SCP seems to act by raising the substrate concentration at the enzyme active site rather than by stimulating enzyme activity. Prerequisites for such a mechanism would be firstly an interaction of SCP with the microsomes, and secondly, binding of SCP to free or membrane-associated lathosterol. However, whether SCP acts as a carrier for the lathosterol substrate or products within the microsomal membrane is not clear, because there is no direct evidence for an interaction of SCP with either microsomes or the substrate (4). When the integrity of microsomal membranes was perturbed by treatment with a detergent such as 0.2% Triton or 0.05% sodium deoxycholate, or by phospholipase A 2, the stimulatory effect by SCP was abolished, indicating that an intact membrane system is required for the response to SCP (4). Furthermore, the effect of SCP on the enzymatic desaturation of lathosterol previously incorporated into microsomes was significantly greater than that on the conversion of exogenous substrate. Kinetically, SCP did not increase initial velocity, but maintained it for a much longer period of time. These findings make it unlikely that SCP functions as a carrier protein in the conventional sense, i.e., serving as a vehicle for transporting substrate from the aqueous medium into the microsomal membrane where the enzyme resides. We have questioned this type of carrier mechanism because all lipophilic intermediates in the pathway from squalene to cholesterol are formed and transformed in the endoplasmic reticulum. Presumably, these intermediates remain associated with microsomes throughout (1). The alternative possibility that proteins like “supernatant protein factor” (SPF), named by Konrad Bloch (17), and SCP promote intramembrane events, e.g., translocating substrate from one enzyme site to another, or from an inactive to an active pool, has re-
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ceived strong support from studies with the SPFpromoted squalene epoxidase system and squalenelanosterol cyclase (19 –21). Both SPF and SCP may interact with the respective steroid intermediates either within the microsomal membrane or at the membrane–water interface. The latter seems more likely, since there is no evidence that SPF and SCP can enter the microsomal space. As for SPF, its binding to microsomes has shown to be very weak (22). Much is still unknown about the roles of soluble factors in the microsomal lathosterol 5-desaturase system. MICROHETEROGENEITY OF SCP BY VIRTUE OF THE FATTY ACIDS CONTENT SCP, in contrast to SPF, appears to play a more general role in lipid metabolism, as Dempsey et al. have suggested (3). Not only is it a major liver protein, but it appears to be identical with the hepatic fatty acid binding protein (FABP) isolated by several laboratories (23, 24) and also with the phospholipid exchange protein of Bloj and Zilversmit (25). FABP is an abundant, low-molecular-weight (around 14,000) cytosolic protein and contains long-chain fatty acids with K d values of 1– 4 M (26 –29). FABP in amounts up to 5 to 8% of cytosolic proteins was found in rat liver (3, 30), yet an astonishing array of conflicting data with regard to isoelectric points, molecular masses, stability, and binding specificities have been reported. For example, isoelectric focusing of binding proteins yielded several subfractions still capable of complexing fatty acids (23, 28, 30), a phenomenon that could be explained by heterogeneity, or multiplicity, or concomitant occurrence of liganded and free forms of the binding proteins. As a result of these uncertainties, the true cellular function of FABP has yet to be determined. In the course of our studies on fatty acid–protein interactions (31, 32), we observed the occurrence of many discrete bands upon gel electrofocusing of FABP in rat liver cytosol, partly in accordance with other findings (33–35). Then, we aimed to define the exact nature of the isoform occurrence in view of the possibility that bound fatty acids might make an important contribution to the observed isoforms (36). Rat liver FABP was purified to homogeneity by procedures including Sephadex G-100 and DEAEcellulose column chromatographies. FABP was resolved into two major peaks, A and B, by the first DEAE-cellulose column chromatography. Each of these fractions exhibited apparent homogeneity upon polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate with a molecular weight of 14,000 Da and amino acid analysis of these fractions revealed that they are virtually identical or at least closely resemble each other. However, their fatty acid
contents were significantly different and heterogeneity was clearly demonstrated in the isoelectric focusing patterns. In that study (36), a single isoform (pI 5.0) from peak of B FABP was further purified by successive DEAEcellulose column chromatography and used as the final preparation. When the final FABP was partly freed of fatty acids by a mild delipidation technique using Lipidex 1,000, the pI shifted upward from 5.0 to 7.0. However, the pI of the delipidated FABP returned to its original pI of 5.0 after recombining with fatty acids. These in vitro manipulations of bound fatty acid content clarified the possible cause of the microheterogeneity of FABP. In addition, when the final FABP (pI 5.0) was partly freed of fatty acids by mild delipidation, the secondary and tertiary structures were significantly changed, as demonstrated by circular dichroism and one- and twodimensional nuclear magnetic resonance spectroscopy (37). After delipidation, FABP’s ␣-helix content was significantly increased and the -sheet content was decreased. The structural properties of the delipidated FABP, however, could be restored to nearly the original condition by recombining fatty acids. The findings suggest that the weakly bound fatty acids determine the functional capacity of FABP by changing the protein conformation. MODEL OF INTERACTION BETWEEN ENZYME AND SUBSTRATE WITHIN THE SAME MEMBRANE Although kinetic studies of membrane enzymes are generally difficult because the usual kinetic formalism refers to nonaggregated homogenous solutions, a major goal of our laboratory is to define the molecular mechanism(s) by which alterations in membrane-bound substrate contents affect the enzyme activity in the same membrane. Since the endoplasmic reticulum of rat liver is poor in both the substrate (lathosterol) and reaction product (7-dehydrocholesterol) of the 5-desaturase, this step in cholesterol synthesis could be a suitable model to study enzyme kinetics in the membrane. The enzyme kinetics obtained with lipophilic substrates often deviate from the Michaelis–Menten-type reaction, because the enzyme-substrate interaction proceeds within individual membranes (38, 39). As suggested by Scheel et al. (39), the classical Michaelis– Menten theory, which is applicable to water-soluble (or solubilized) enzymes and substrates, may not be applicable to membrane-bound enzymes, particularly if the substrate is also in the membrane. This is due in part to geometric restrictions when both the enzyme and the substrate are confined to the plane of the membrane. This is presumably the case in vivo and in studies using transfer protein.
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To study the kinetics and properties of a membrane enzyme where one of the substrates is also an integral component of the membrane, it is necessary to alter the amount of substrate in the membrane while minimizing changes in the membrane environment or in the conformation of the enzyme (40). Thus, exogenous lathosterol substrate was preincorporated into the microsomal membranes by the use of liposomes in the presence of cytosolic protein, thereby eliminating the influence of the insertion kinetics of the lipophilic substrate. The inserted portion of the substrate ought to reach the enzyme by lateral diffusion within the plane of the membrane. The lathosterol 5-desaturation reaction occurred in two distinct phases (40). That is, there was an initial burst of product formation over an approximate time scale of 5 min that fell off thereafter to a steady-state rate for over 30 min. The latter steady-state phase was slower than the burst phase, because lateral diffusion of the lathosterol substrate must occur before the reaction can continue. The total amount of the burst, which may be obtained by extrapolating the linear part of the curve in the steady-state phase back to zero time, provides a means of obtaining the enzyme concentration in terms of functional active sites. Using this approach, the active enzyme concentration in the microsomal membranes was calculated to be approximately 0.2 nmol/mg of microsomal proteins. Furthermore, it was found that the interaction between enzyme and substrate within the same membrane followed the usual kinetic formalism of a Michaelis–Menten-type reaction as in nonaggregated homogenous solution. Further examples, however, must be tested to establish the applicability and limits of the suggested model. DIFFERENTIAL KINETIC PROPERTIES BETWEEN INITIAL BURST AND THE SUBSEQUENT STEADY STATE In general, the activity of integral membrane enzymes is often sensitive to the lipid dynamics, composition and state. During our investigations of the lathosterol 5-desaturase system we have noticed that the reaction rate is highly dependent on the physical state of the membrane because treatment of microsomes with low concentrations of detergents and phospholipases alters the catalytic activity of the enzyme (4). Analysis of the role of diffusion is especially strengthened when studies of biophysical structural and functional relationships can be correlated directly with enzyme kinetics in a particular membrane (41). Recent detailed experiments and theoretical work have made is possible to test various descriptions of the lateral diffusion of lipids and proteins in lipid bilayers (42).
Although there is only limited direct experimental proof to substantiate lateral diffusion of lathosterol, an intact microsomal membrane is needed to observe stimulation by noncatalytic protein during enzyme activity (4, 6). As mentioned above, we presented evidence that the lipophilic substrate and enzyme interact mainly within the plane of the membrane, presumably by lateral diffusion (40). One hypothesis that has been proposed to explain the biphasic nature of the reaction is diffusion control of the lipophilic substrate. Therefore, we reexamined the lathosterol desaturation reaction with a more detailed kinetic analysis (43). The effects of temperature on bilayer structures are of special importance, since the sensitivity of membrane enzymes to structural changes of the surrounding lipid phase has been demonstrated by temperature-change studies. Thus, changes in the structure and function of the membrane as a function of temperature were investigated using fluorescence anisotropy (44) and measurement of the Arrhenius activation energy. At the burst phase, there was a lack of discontinuity in the Arrhenius plots at the presumed phase transition temperature for the microsomal membrane. However, the plots of the activities in the steady state showed breaks at around 17 and 32°C. It was concluded that phospholipid phase transition affects the steady-state phase, but not the burst phase. Furthermore, treatment of microsomes with low concentrations of deoxycholate, known to perturb the membrane integrity, resulted in a break of the activation energy of the burst phase. These results represent further evidence for our model of interaction between the substrate and enzyme within the microsomal membrane via lateral diffusion. We are unaware of other reports dealing with the detailed kinetics of these membrane-associated events. The study of these phenomena of membrane enzymology should yield a better understanding of membrane structure and the turnover and function of membrane components. CLONING AND SEQUENCING OF THE 5-DESATURASE Recently, we have cloned and sequenced human and mouse 5-desaturase cDNA from liver libraries by utilizing DNA database-registered partial sequences homologous to the yeast desaturase sequence (45). The open reading frame-encoded protein complemented desaturase deficiency of yeast mutant cells. Mammalian 5-desaturase appears to be an integral membrane protein containing histidine residues, which are also implicated in an active center of acyl-CoA desaturase (46). Hydropathy analysis (47) led us to propose that
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5-desaturase is an integral membrane protein with 4 –5 membrane-spanning regions. Histidine residues are conserved in 10 positions among the enzymes of mammals, yeast (48, 49) and plant (50). Possibly the enzyme holds non-heme iron(s) in the catalytic center containing histidine residues because, first, the enzyme can be inhibited by cyanide ions and Tiron (6), and second, acyl-CoA desaturase holds non-heme iron in the active center and also contains phylogenetically conserved histidine residues (46).
place close interest in my progress, and to offer suggestions and comments. One of his letters is reproduced below. May 8, 1985 Dear Teruo: I was very pleased to read your recent fine paper on lathosterol desaturase in J. Biochem. (6). Please send me a reprint. Of special interest to me are two of your findings. The similarity of the lathosterol desaturase to stearyl-CoA desaturase with respect to the b5 requirement and secondly the inability of SCP to stimulate the reconstituted system. You may have seen the paper by Jean Chin in J. Biol. Chem. (21) showing that reconstituted squalene epoxidase also fails to respond to cytosolic protein factor, in this case SPF. With best regards, Sincerely, Konrad Bloch
FUTURE PROSPECTS Activities of many intrinsic membrane enzymes are affected by the physical state and composition of the lipids immediately surrounding them (51, 52) as well as by the rate of lateral diffusion of these lipids. Membrane-bound enzymes in many cases require lipids for activity, or their activity is modulated by lipids. However, in spite of the high lipid mobility in most biological membranes, little free lipid may be present. The lipid shell solvating the protein has been termed the “boundary lipid” or “lipid annulus.” Geometric considerations, lipid transition data, and electron spin resonance measurements have indicated that the boundary lipid layer typically comprises 30 –100 lipid molecules (53). It has been demonstrated that changes in the lipid environment of a membrane can change the conformation of a membrane protein (54). The necessity of understanding the microarchitecture of biological membranes in order to understand how they perform their various functions is now universally acknowledged. However, significant experimental obstacles intrinsic to membrane-associated enzymes have impeded progress in these studies. Accordingly, further examples of substrate– enzyme interaction within membranes must be investigated to establish the applicability and the limits of our translational diffusion model (40). Another aspect of this problem is the identification of the rate-limiting step of enzyme catalysis. It is proposed that the viscosity of the membrane can influence the rate-limiting step of the enzyme reaction, which is the rate of transition over the energy barrier leading to product formation. However, this idea requires still confirmation. In order to ascertain whether the phenomena so far described are of a general nature, studies of many other membrane-bound enzyme systems are needed. CORRESPONDENCE FROM DR. BLOCH After I returned to Japan, I continued the work that I had started in Dr. Bloch’s laboratory. It was characteristic of Konrad Bloch that he continued to take a
ACKNOWLEDGMENT I thank Dr. Richard Steele for critically reading the manuscript.
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Supernatant Protein Relevant to the Activity of Membrane-Bound Enzymes: Studies on Lathosterol 5-Desaturase Teruo Ishibashi 1 Department of Biochemistry, Hokkaido University School of Medicine, N15 W7, Sapporo 060-8638, Japan
EARLY WORK IN BLOCH’S LABORATORY All the enzymes required for the synthesis of cholesterol from squalene, the first sterol intermediate, are bound to the endoplasmic reticulum (1), which can be isolated from cell-free homogenates as microsomes. Water-insoluble intermediates are generated in the membrane and metabolized by membranebound enzymes without diffusion of steroid from the membrane (1). The final oxygenase in cholesterol synthesis, lathosterol 5-desaturase (EC 1.3.3.2), catalyzes insertion of the C-5 double bond into lathosterol (5␣-cholest7-en-3-ol) to yield 7-dehydrocholesterol (cholesta5,7-dien-3-ol) (2– 4). This enzyme system consists of NADH-cytochrome b5 reductase, cytochrome b5, and the terminal desaturase (5, 6), and requires molecular oxygen (7, 8), reduced pyridine nucleotide (5, 7), and the 105,000g supernatant (2– 4) for activity. The 5-desaturase system is closely associated with the microsomal membranes, as is its lipid substrate, lathosterol. The striking overall efficiency of the process suggests that these enzymes may exist as an organized system in the microsomal membrane. However, published reports do not include any information about clustering of these membrane components, or about the possible existence of different phases in membrane preparations. INSIGHTS INTO MECHANISM(S) OF FACILITATION BY SUPERNATANT PROTEIN OF MEMBRANEBOUND ENZYMES The enzymes involved in hepatic cholesterol biosynthesis from squalene are located in microsomes (1), and several steps of the sequence are stimulated by soluble, presumably cytoplasmic, proteins (9 –22). The lathosterol 5-desaturase activity was stimulated by purified “squalene and sterol carrier protein” (SCP) (3). SCP has been shown to stimulate the conversion of lathosterol to 7-dehydrocholesterol by influencing events within the endoplasmic reticu1
Fax: 81-11-706-5169. E-mail: [email protected].
lum, e.g., directing the substrate to its specific enzyme site (4). This study (4) was my project in Bloch’s laboratory. I had a very hard time preparing the radioactive substrate ([H 3 ]lathosterol), which was not commercially available at that time. Dr. Bloch took great interest in this work, because he was originally an organic chemist. SCP seems to act by raising the substrate concentration at the enzyme active site rather than by stimulating enzyme activity. Prerequisites for such a mechanism would be firstly an interaction of SCP with the microsomes, and secondly, binding of SCP to free or membrane-associated lathosterol. However, whether SCP acts as a carrier for the lathosterol substrate or products within the microsomal membrane is not clear, because there is no direct evidence for an interaction of SCP with either microsomes or the substrate (4). When the integrity of microsomal membranes was perturbed by treatment with a detergent such as 0.2% Triton or 0.05% sodium deoxycholate, or by phospholipase A 2, the stimulatory effect by SCP was abolished, indicating that an intact membrane system is required for the response to SCP (4). Furthermore, the effect of SCP on the enzymatic desaturation of lathosterol previously incorporated into microsomes was significantly greater than that on the conversion of exogenous substrate. Kinetically, SCP did not increase initial velocity, but maintained it for a much longer period of time. These findings make it unlikely that SCP functions as a carrier protein in the conventional sense, i.e., serving as a vehicle for transporting substrate from the aqueous medium into the microsomal membrane where the enzyme resides. We have questioned this type of carrier mechanism because all lipophilic intermediates in the pathway from squalene to cholesterol are formed and transformed in the endoplasmic reticulum. Presumably, these intermediates remain associated with microsomes throughout (1). The alternative possibility that proteins like “supernatant protein factor” (SPF), named by Konrad Bloch (17), and SCP promote intramembrane events, e.g., translocating substrate from one enzyme site to another, or from an inactive to an active pool, has re-
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ceived strong support from studies with the SPFpromoted squalene epoxidase system and squalenelanosterol cyclase (19 –21). Both SPF and SCP may interact with the respective steroid intermediates either within the microsomal membrane or at the membrane–water interface. The latter seems more likely, since there is no evidence that SPF and SCP can enter the microsomal space. As for SPF, its binding to microsomes has shown to be very weak (22). Much is still unknown about the roles of soluble factors in the microsomal lathosterol 5-desaturase system. MICROHETEROGENEITY OF SCP BY VIRTUE OF THE FATTY ACIDS CONTENT SCP, in contrast to SPF, appears to play a more general role in lipid metabolism, as Dempsey et al. have suggested (3). Not only is it a major liver protein, but it appears to be identical with the hepatic fatty acid binding protein (FABP) isolated by several laboratories (23, 24) and also with the phospholipid exchange protein of Bloj and Zilversmit (25). FABP is an abundant, low-molecular-weight (around 14,000) cytosolic protein and contains long-chain fatty acids with K d values of 1– 4 M (26 –29). FABP in amounts up to 5 to 8% of cytosolic proteins was found in rat liver (3, 30), yet an astonishing array of conflicting data with regard to isoelectric points, molecular masses, stability, and binding specificities have been reported. For example, isoelectric focusing of binding proteins yielded several subfractions still capable of complexing fatty acids (23, 28, 30), a phenomenon that could be explained by heterogeneity, or multiplicity, or concomitant occurrence of liganded and free forms of the binding proteins. As a result of these uncertainties, the true cellular function of FABP has yet to be determined. In the course of our studies on fatty acid–protein interactions (31, 32), we observed the occurrence of many discrete bands upon gel electrofocusing of FABP in rat liver cytosol, partly in accordance with other findings (33–35). Then, we aimed to define the exact nature of the isoform occurrence in view of the possibility that bound fatty acids might make an important contribution to the observed isoforms (36). Rat liver FABP was purified to homogeneity by procedures including Sephadex G-100 and DEAEcellulose column chromatographies. FABP was resolved into two major peaks, A and B, by the first DEAE-cellulose column chromatography. Each of these fractions exhibited apparent homogeneity upon polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate with a molecular weight of 14,000 Da and amino acid analysis of these fractions revealed that they are virtually identical or at least closely resemble each other. However, their fatty acid
contents were significantly different and heterogeneity was clearly demonstrated in the isoelectric focusing patterns. In that study (36), a single isoform (pI 5.0) from peak of B FABP was further purified by successive DEAEcellulose column chromatography and used as the final preparation. When the final FABP was partly freed of fatty acids by a mild delipidation technique using Lipidex 1,000, the pI shifted upward from 5.0 to 7.0. However, the pI of the delipidated FABP returned to its original pI of 5.0 after recombining with fatty acids. These in vitro manipulations of bound fatty acid content clarified the possible cause of the microheterogeneity of FABP. In addition, when the final FABP (pI 5.0) was partly freed of fatty acids by mild delipidation, the secondary and tertiary structures were significantly changed, as demonstrated by circular dichroism and one- and twodimensional nuclear magnetic resonance spectroscopy (37). After delipidation, FABP’s ␣-helix content was significantly increased and the -sheet content was decreased. The structural properties of the delipidated FABP, however, could be restored to nearly the original condition by recombining fatty acids. The findings suggest that the weakly bound fatty acids determine the functional capacity of FABP by changing the protein conformation. MODEL OF INTERACTION BETWEEN ENZYME AND SUBSTRATE WITHIN THE SAME MEMBRANE Although kinetic studies of membrane enzymes are generally difficult because the usual kinetic formalism refers to nonaggregated homogenous solutions, a major goal of our laboratory is to define the molecular mechanism(s) by which alterations in membrane-bound substrate contents affect the enzyme activity in the same membrane. Since the endoplasmic reticulum of rat liver is poor in both the substrate (lathosterol) and reaction product (7-dehydrocholesterol) of the 5-desaturase, this step in cholesterol synthesis could be a suitable model to study enzyme kinetics in the membrane. The enzyme kinetics obtained with lipophilic substrates often deviate from the Michaelis–Menten-type reaction, because the enzyme-substrate interaction proceeds within individual membranes (38, 39). As suggested by Scheel et al. (39), the classical Michaelis– Menten theory, which is applicable to water-soluble (or solubilized) enzymes and substrates, may not be applicable to membrane-bound enzymes, particularly if the substrate is also in the membrane. This is due in part to geometric restrictions when both the enzyme and the substrate are confined to the plane of the membrane. This is presumably the case in vivo and in studies using transfer protein.
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To study the kinetics and properties of a membrane enzyme where one of the substrates is also an integral component of the membrane, it is necessary to alter the amount of substrate in the membrane while minimizing changes in the membrane environment or in the conformation of the enzyme (40). Thus, exogenous lathosterol substrate was preincorporated into the microsomal membranes by the use of liposomes in the presence of cytosolic protein, thereby eliminating the influence of the insertion kinetics of the lipophilic substrate. The inserted portion of the substrate ought to reach the enzyme by lateral diffusion within the plane of the membrane. The lathosterol 5-desaturation reaction occurred in two distinct phases (40). That is, there was an initial burst of product formation over an approximate time scale of 5 min that fell off thereafter to a steady-state rate for over 30 min. The latter steady-state phase was slower than the burst phase, because lateral diffusion of the lathosterol substrate must occur before the reaction can continue. The total amount of the burst, which may be obtained by extrapolating the linear part of the curve in the steady-state phase back to zero time, provides a means of obtaining the enzyme concentration in terms of functional active sites. Using this approach, the active enzyme concentration in the microsomal membranes was calculated to be approximately 0.2 nmol/mg of microsomal proteins. Furthermore, it was found that the interaction between enzyme and substrate within the same membrane followed the usual kinetic formalism of a Michaelis–Menten-type reaction as in nonaggregated homogenous solution. Further examples, however, must be tested to establish the applicability and limits of the suggested model. DIFFERENTIAL KINETIC PROPERTIES BETWEEN INITIAL BURST AND THE SUBSEQUENT STEADY STATE In general, the activity of integral membrane enzymes is often sensitive to the lipid dynamics, composition and state. During our investigations of the lathosterol 5-desaturase system we have noticed that the reaction rate is highly dependent on the physical state of the membrane because treatment of microsomes with low concentrations of detergents and phospholipases alters the catalytic activity of the enzyme (4). Analysis of the role of diffusion is especially strengthened when studies of biophysical structural and functional relationships can be correlated directly with enzyme kinetics in a particular membrane (41). Recent detailed experiments and theoretical work have made is possible to test various descriptions of the lateral diffusion of lipids and proteins in lipid bilayers (42).
Although there is only limited direct experimental proof to substantiate lateral diffusion of lathosterol, an intact microsomal membrane is needed to observe stimulation by noncatalytic protein during enzyme activity (4, 6). As mentioned above, we presented evidence that the lipophilic substrate and enzyme interact mainly within the plane of the membrane, presumably by lateral diffusion (40). One hypothesis that has been proposed to explain the biphasic nature of the reaction is diffusion control of the lipophilic substrate. Therefore, we reexamined the lathosterol desaturation reaction with a more detailed kinetic analysis (43). The effects of temperature on bilayer structures are of special importance, since the sensitivity of membrane enzymes to structural changes of the surrounding lipid phase has been demonstrated by temperature-change studies. Thus, changes in the structure and function of the membrane as a function of temperature were investigated using fluorescence anisotropy (44) and measurement of the Arrhenius activation energy. At the burst phase, there was a lack of discontinuity in the Arrhenius plots at the presumed phase transition temperature for the microsomal membrane. However, the plots of the activities in the steady state showed breaks at around 17 and 32°C. It was concluded that phospholipid phase transition affects the steady-state phase, but not the burst phase. Furthermore, treatment of microsomes with low concentrations of deoxycholate, known to perturb the membrane integrity, resulted in a break of the activation energy of the burst phase. These results represent further evidence for our model of interaction between the substrate and enzyme within the microsomal membrane via lateral diffusion. We are unaware of other reports dealing with the detailed kinetics of these membrane-associated events. The study of these phenomena of membrane enzymology should yield a better understanding of membrane structure and the turnover and function of membrane components. CLONING AND SEQUENCING OF THE 5-DESATURASE Recently, we have cloned and sequenced human and mouse 5-desaturase cDNA from liver libraries by utilizing DNA database-registered partial sequences homologous to the yeast desaturase sequence (45). The open reading frame-encoded protein complemented desaturase deficiency of yeast mutant cells. Mammalian 5-desaturase appears to be an integral membrane protein containing histidine residues, which are also implicated in an active center of acyl-CoA desaturase (46). Hydropathy analysis (47) led us to propose that
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5-desaturase is an integral membrane protein with 4 –5 membrane-spanning regions. Histidine residues are conserved in 10 positions among the enzymes of mammals, yeast (48, 49) and plant (50). Possibly the enzyme holds non-heme iron(s) in the catalytic center containing histidine residues because, first, the enzyme can be inhibited by cyanide ions and Tiron (6), and second, acyl-CoA desaturase holds non-heme iron in the active center and also contains phylogenetically conserved histidine residues (46).
place close interest in my progress, and to offer suggestions and comments. One of his letters is reproduced below. May 8, 1985 Dear Teruo: I was very pleased to read your recent fine paper on lathosterol desaturase in J. Biochem. (6). Please send me a reprint. Of special interest to me are two of your findings. The similarity of the lathosterol desaturase to stearyl-CoA desaturase with respect to the b5 requirement and secondly the inability of SCP to stimulate the reconstituted system. You may have seen the paper by Jean Chin in J. Biol. Chem. (21) showing that reconstituted squalene epoxidase also fails to respond to cytosolic protein factor, in this case SPF. With best regards, Sincerely, Konrad Bloch
FUTURE PROSPECTS Activities of many intrinsic membrane enzymes are affected by the physical state and composition of the lipids immediately surrounding them (51, 52) as well as by the rate of lateral diffusion of these lipids. Membrane-bound enzymes in many cases require lipids for activity, or their activity is modulated by lipids. However, in spite of the high lipid mobility in most biological membranes, little free lipid may be present. The lipid shell solvating the protein has been termed the “boundary lipid” or “lipid annulus.” Geometric considerations, lipid transition data, and electron spin resonance measurements have indicated that the boundary lipid layer typically comprises 30 –100 lipid molecules (53). It has been demonstrated that changes in the lipid environment of a membrane can change the conformation of a membrane protein (54). The necessity of understanding the microarchitecture of biological membranes in order to understand how they perform their various functions is now universally acknowledged. However, significant experimental obstacles intrinsic to membrane-associated enzymes have impeded progress in these studies. Accordingly, further examples of substrate– enzyme interaction within membranes must be investigated to establish the applicability and the limits of our translational diffusion model (40). Another aspect of this problem is the identification of the rate-limiting step of enzyme catalysis. It is proposed that the viscosity of the membrane can influence the rate-limiting step of the enzyme reaction, which is the rate of transition over the energy barrier leading to product formation. However, this idea requires still confirmation. In order to ascertain whether the phenomena so far described are of a general nature, studies of many other membrane-bound enzyme systems are needed. CORRESPONDENCE FROM DR. BLOCH After I returned to Japan, I continued the work that I had started in Dr. Bloch’s laboratory. It was characteristic of Konrad Bloch that he continued to take a
ACKNOWLEDGMENT I thank Dr. Richard Steele for critically reading the manuscript.
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