Characterization of microsomal methyl sterol demethylase in two Morris hepatomas.

Previously, we reported that the rate of metabolism of methyl sterol intermediates of cholesterol biosynthesis by broken-cell preparations of Morriss hepatoma 7777 is very slow, whereas the intact tumors are known to synthesize cholesterol quite efficiently. Active preparations have now been obtained by substitution of pyrophosphate for phosphate buffer. Although substitution of pyrophosphate buffer markedly enhances microsomal methyl sterol demethylation rates 3- to 4-fold in hepatoma 7777, other microsomal enzymes and electron carriers in either liver or a more slowly growing hepatoma appear to be unaffected by pyrophosphate. Several properties of the active microsomal methyl sterol demethylase have now been compared for control rat liver, host liver, tumor 7777, and tumor 5123C. Conditions necessary for the assay of initial velocities of enzymic reactions in the tumor microsomes have been established with respect to the amount of protein, time-course, concentrations of cofactors and substrate, pH, and other variables. The K'm and the responses to the variables studied above are very similar for methyl sterol demethylase of microsomes isolated from control liver, host liver, tumor 5123C, and tumor 7777. The multienzymic demethylase in the various preparations has been found to be inhibited similarly by in vitro additions of cyanide, cytochrome c, and bile salts. Thus, the enzymes of the microsomal-bound 4-methyl sterol demethylase of cholesterol biosynthesis appear to be very similar in liver and these 2 Morris hepatomas. When xenobiotic inducers of microsomal oxidases, such as phenobarbital and methylcholanthrene, are administered to normal and tumor-bearing rats, elevated rates of methyl sterol demethylation are observed with isolated liver microsomes obtained from both normal and tumor-bearing rats. Similar increases are not observed in the tumors. Furthermore, daily administration of an intestinal bile acid sequestrant elevates hepatic methyl sterol demethylase, but statistically significant changes were not observed in tumors 7777 and 5123C. Since the enzymes of methyl sterol demethylase appear to be grossly similar in liver and these hepatomas, regulation of the activity of the multienzymic system contained in the tumors may be altered. On the other hand, these agents in vivo simply may not affect liver and the hepatomas similarly, due to a lack of uptake of the foreign substances by the tumor that has been transplanted to the thighs.

Investigation of the rate-determining microsomal reaction of cholesterol biosynthesis from lanosterol in Morris hepatomas and liver.

Previously, we reported that the properties or microsomal 4-methyl sterol demethylase isolated from liver and Morris hepatomas 5123C and 7777 are grossly similar. The individual enzymic steps of this multicomponent system have now been studied, and the rate-determining step has been determined and shown to be identical for liver and these hepatomas. The rates of microsomal oxidative attacks of the 4alpha-methyl, 4alpha-hydroxymethyl, and 4-aldehydic groups are similar for microsomes prepared from rat liver and hepatoma 7777. The rates of mixed-function oxidative attack appear to increase in the order;--CH3 less than --CH2OH less than --CHO. Furthermore, the hepatic and hepatoma NAD-dependent decarboxylase, which catalyzes the reaction following the three oxidative attacks is similar in properties and velocity. The fifth step, an NADPH-dependent reduction of the 3 ketosteroid that is produced by decarboxylation, is also similar. For both tissues, the latter two reactions, under in vitro conditions, proceed at rates that exceed the initial oxidative process. Thus, for elimination of both of the 4-methyl groups of lanosterol, the 10 individual reactions catalyzed in this multicomponent system are identical in liver and hepatoma 7777 microsomes, and the rate-determining stop for both liver and hepatoma is the inital oxidative attack on the 4alpha-methyl group of cholesterol procursors. When the rate-determining reaction of both liver and hepatoma 7777 microsomes is assayed at different temperatures, the same activation energies and the same characteristic breaks in the arrhenius plots are observed. Thus, for both liver and hepatoma, both the nature and the site of rate determination in this multienzymic system must be similar. Since the microsomal enzymes of liver nad hepatoma appear to be catalytically similar and rate determination appears to be similar, too, the characteristic lact of response of tumor microsomes to treatments in vivo that alter host liver microsomal demethylation activity suggests that the insensitivity of these tumors to dietary cholesterol should not be ascribed to alterations in the catalytic proteins. Evidence in this report suggests that the postmicrosomal supernatant fraction of both liver and hepatoma contains a cytosolic protein that may participate in the regulation of the rate-determining attack of 4alpha-methyl sterol substrates. Thus, either qualitative or quantitative differences between the postmicrosomal supernatant fractions obtained from liver and heptomas may account for the observed differences in rates of cholesterol biosynthesis.

Effect of lipid composition on the transfer of sterols mediated by non-specific lipid transfer protein (sterol carrier protein2).

The rate of non-specific lipid transfer protein (nsLTP)-mediated exchange is independent of structure for dissimilar sterols: cholesterol, lanosterol, sitosterol and vitamin D-3. Conversely, the nsLTP-mediated exchange of cholesterol is markedly affected by the phospholipid composition of the donor liposome. Negatively charged phosphatidylglycerols strikingly increase cholesterol exchange in the presence of nsLTP while not altering the exchange in the absence of nsLTP. The presence of unsaturated acyl chains in the phospholipid enhances exchange. Sphingomyelin drastically decreases cholesterol exchange, as does di-O-alkylphosphatidylcholine. Decreased exchange produced by these substitutions can be reversed by addition of phosphatidylcholine. The presence of an acyl group and a negative charge in the phospholipid are critical for the nsLTP-mediated transfer of cholesterol. In addition to these studies on composition of the donor membrane, the charge on the membrane also appears critical. Maximal exchange rates accompany optimization of potential interaction of negatively charged surface and the basic nsLTP. The nsLTP also mediates an approximately equal rate of exchange of cholesterol and phosphatidylcholine. However, approaching equilibrium, only half of the phospholipid can be exchanged while there is exchange of about 90% of cholesterol. Thus, it appears that only the phospholipid in an outer membrane layer may be available whereas cholesterol is fully available. Therefore, in contrast to a 'carrier' model we suggest that nsLTP facilitates exchange by binding to the membranes, and binding is highly dependent upon lipid composition. Once bound, the protein functions as a bridge between membranes, thus, facilitating exchange.

Cytosolic modulators of activities of microsomal enzymes of cholesterol biosynthesis. Purification and characterization of a non-specific lipid-transfer protein.

Rat liver cytosol contains proteins which in the presence of low-molecular-weight metabolites modulate activities of membrane-bound enzymes of cholesterol biosynthesis. In the preceding paper, we identified Z-protein as a mediator in fatty acyl-CoA modulation of microsomal cholesterol synthetic and metabolizing enzymes. In this communication, we describe a second cytosolic protein which displays cholesterol-exchange activity. Purification of the protein to over 10000-fold and homogeneity has been achieved by gel permeation HPLC on an analytical Spherogel TSK-2000 SW column. Elution of both a single peak of active protein and one SDS-polyacrylamide gel electrophoresis species upon HPLC-purification suggests that homogeneous protein aggregates, with loss of exchange activity. In addition to stimulating microsomal enzymes of sterol synthesis, incubations of microsomes with cholesterol-containing liposomes and the protein consistently yields a 2-3-fold stimulation of microsomal acyl CoA: cholesterol acyltransferase activity. Under similar incubation conditions the protein enhances only slightly the extent of inhibition of microsomal hydroxymethylglutaryl-CoA reductase by liposomal cholesterol. The protein also catalyzes net transfer of cholesterol between membranes of different cholesterol content. The lipid-transfer protein and another cytosolic protein, also implicated in the regulation of sterol synthetic enzymes, appear identical. Regulation of activities of several membrane-bound enzymes of cholesterol metabolism in which the lipid-transfer protein and cytosolic Z-protein modulate uptake of lower-molecular-weight water-insoluble and water-soluble effectors, respectively, is discussed.

Cytosolic modulators of activities of microsomal enzymes of cholesterol biosynthesis. Effects of Acyl-CoA inhibition and cytosolic Z-protein.

Physiological concentrations of long-chain fatty acyl-CoAs have now been shown to inhibit microsomal methyl sterol oxidase. Acyl-CoA inhibition of hydroxymethylglutaryl-CoA reductase as well as methyl sterol oxidase can be either prevented or reversed by the addition of purified Z-protein (fatty acid-binding protein). Concomitantly, Z-protein addition decreases the extent of binding of radioactively labeled oleoyl-CoA to microsomal membranes. Free heme also inhibits hydroxymethylglutaryl-CoA reductase, and Z-protein reverses the extent of observed inhibition by binding heme analogous to the effect observed with acyl-CoAs. Similarly, Z-protein reverses substrate inhibition of acyl-CoA:cholesterol acyltransferase at high concentrations of acyl-CoA substrate. All these observations are consistent with the suggestion that, by binding acyl-CoAs and other enzyme effectors such as free heme, Z-protein modulates the effects of fluctuations of concentrations of major cellular metabolites. Furthermore, because the concentration of Z-protein is very low in rapidly growing hepatomas, such tumors may be very poorly buffered against the effects of acyl-CoAs, free fatty acids, heme and other effectors that may vary markedly by either altered metabolism or release of metabolites from necrotic tumor tissue.

Cholesterol biosynthesis from lanosterol: regulation and purification of rat hepatic sterol 14-reductase.

We have previously characterized the membrane-bound sterol 14-reductase (14-reductase) that catalyzes anaerobically NADPH-dependent reduction of the 14-double bond of delta 8,14-diene or delta 7,14-diene sterols that are sterol intermediates in cholesterol biosynthesis in mammals (Paik et al. (1984) J. Biol. Chem. 259, 13413-13423). To elucidate the regulatory mechanism as well as molecular characteristics of the 14-reductase, we extended our investigation on the consequences of alteration of the enzymic activity under various physiological conditions. The enzymic activity of rat hepatic sterol 14-reductase was induced more than 11-fold by feeding 5% cholestyramine plus 0.1% lovastatin (the CL-diet) for 7 days but was severely suppressed by feeding 5% cholesterol or 0.01% AY-9944 (an inhibitor of 14-reductase) for the same period. The increase or decrease in the 14-reductase activity also parallels the same change in the cholesterol synthetic rate in hepatocytes from rats that had been fed either the CL-diet or 0.01% AY-9944. In vitro inhibition studies revealed that AY-9944 acts as a competitive inhibitor of the 14-reductase (Ki = 0.26 microM). A diurnal variation was observed for the 14-reductase with peak activity near the middle of the dark cycle (10 p.m.), which was abolished by administration of cycloheximide. With induced enzyme conditions 14-reductase has been further purified with chromatographic procedures to near homogeneity. Purified 14-reductase appears to be a M(r) = 70,000 protein that is composed of two equally-sized subunits having a M(r) = 38,000. All properties of the purified 14-reductase suggest that the solubilized enzyme is the principal 14-reductase of microsomes. Taken together, our results provide the first evidence in support of a previously unknown regulatory role for the 14-reductase in the overall cholesterol synthetic pathway.

Regulation of microsomal stearoyl-coenzyme A desaturase. Purification of a non-substrate-binding protein that stimulates activity.

Crude cytosolic fraction from rat liver was examined for proteins that may be involved in regulation of microsomal stearoyl-CoA desaturase activity. Gel filtration revealed the presence of several components that either stimulate or inhibit this enzyme. In addition, other components bind the acyl-CoA substrate, thus affecting observed activities in vitro. A protein that stimulates stearoyl-CoA desaturase but does not bind substrate was purified approx. 1100-fold. The purified protein had no visible absorption spectrum and an approximate mol.wt. of 26500. Maximal stimulation of desaturase activity occurred with less than 2 micrometer purified protein. The protein was heat-labile and exhibited neither catalase nor glutathione peroxidase activity. Addition of the cytosolic protein produced no effect on the desaturase reaction stoicheiometry; the proportions O2 consumed/NADH oxidized/stearoyl-CoA desaturated remained 1:1:1. Because the Km' for stearoyl-CoA was also unchanged by addition of the cytosolic protein, no change in substrate affinity was suggested. Furthermore addition of the cytosolic protein also produced no effect on desaturase inhibition by oleoyl-CoA, which suggested that the protein does not simply relieve apparent product inhibition. These results indicate that, in analogy to other cytosolic proteins that stimulate microsomal oxidase activities, the protein may have a regulatory function, perhaps related to activity modulation via organization of the multienzymic desaturase in the membrane.

Cytosolic modulators of activities of microsomal enzyme of cholesterol biosynthesis. Role of a cytosolic protein with properties similar to Z-protein (fatty acid-binding protein).

Rat liver cytosol contains proteins and smaller molecules that affect activities of the microsomal enzymes of cholesterol biosynthesis. One protein (mr = 12,000) has been purified by chromatography, gel filtration, and preparative isoelectric focusing. Properties of the protein on sodium dodecyl sulfate-, and anodic-, and cathodic-disc gel electrophoresis are reported in addition to gel filtration and electrofocusing that were used for purification. By comparison of these properties, amino acid compositions, ligand binding, and the abundance in various tissues the protein appears to be very similar to Z-protein that has been shown by others to be identical with hepatic fatty-acid binding protein. The same properties also appear to be very similar to sterol carrier protein. A cytosolic metabolite, heme, which stimulates 4-methyl sterol oxidase, is bound to the protein during purification. When endogeneous heme is removed, both protein and heme are required for maximal stimulation of microsomal 4-methyl sterol oxidase activity. Hemoglobin produces an equal extent of stimulation presumably by heme group exchange, but the nonexchangeable heme group of cytochrome c is ineffective. Thus, the cytosolic protein may promote uptake and retention of relatively more water-soluble substances into the hydrophobic environment of the membrane-bound enzymes of cholesterol biosynthesis. Z-protein exhibits affinity for an exceptionally wide variety of ligands (e.g. fatty acids, azodyes, organic anions, bile pigments, etc) that may either stimulate or inhibit the microsomal enzymes. Accordingly, with the suggestion that the cytosolic protein is Z-protein, the wide variety of potential interactions of both endogenous metabolites and exogenous xenobiotics may account, in part, for facile modulations of activities of the rate-limiting microsomal synthetic enzymes in various physiological and nutritional states.

Characterization of the microsomal steroid-8-ene isomerase of cholesterol biosynthesis.

Rat liver microsomes contain an enzyme that catalyzes the isomerization of the nuclear double bond of steroids from the 8(9) position to the 7(8) position. The enzyme is most active with zymosterol, 5alpha-cholesta-8,24-dien-3beta-ol, which is a precursor of cholesterol. Properties of the microsomal isomerase have now been studied, and preliminary data are reported on both regulation of enzymic activity and first steps in the solubilization of the enzyme from membranes. After a brief lag period, the velocity of isomerase is relatively constant for about 5 min of incubation, and then isomerization subsides. The apparent Michaelis constant (52-70 micro M) is difficult to determine accurately, due to these complex kinetic changes. V(max) is 4.0-4.7 nmol/min per mg of microsomal protein. The apparent specific activity is more than ten times that of liver microsomal methyl sterol oxidase. The maximal specific activity of microsomal isomerase is approximately doubled when rats are fed an intestinal bile acid sequestrant, cholestyramine. Changes in specific activity of isomerase parallel changes in activities of other microsomal enzymes of cholesterol biosynthesis, such as 3-hydroxy-3-methylglutaryl-CoA reductase and 4-methyl sterol oxidase. Isomerase activity is destroyed by phospholipase A digestion, high concentration of bile salts, and solvent extraction, all of which are known either to remove phospholipid or to alter microsomal membrane integrity. On the other hand, isomerase remains active in the presence of a mild, nonionic detergent, Triton WR-1339; thus, solubilization with nonionic detergents is under study.

Enzymic formation of esters of methyl sterol precursors of cholesterol.

For investigation of the reactions of cholesterol biosynthesis, a number of workers use the 10,000 g supernatant fraction (or similar preparations) obtained from cell-free homogenates of rat liver. We have found that esters of methyl sterol biosynthetic intermediates are formed by this crude source of enzymes. Esters of C(30)-, C(29)-, C(28)-, and C(27)-sterol intermediates have been isolated by silicic acid chromatography of an acetone extract of incubation mixtures. Competition between ester formation and demethylation of the C(28)-sterol intermediate has been demonstrated. With 4alpha-methyl-5alpha-cholest-7-en-3beta-ol as substrate, maximal velocities of ester formation (0.36 nmole/30 min per mg of protein) were almost equivalent to maximal velocities of demethylation (0.45 nmole/30 min per mg of protein). Ester formation may be eliminated by carrying out incubations with microsomal preparations; ester formation may be restored completely upon addition (to the microsomes) of either coenzyme A and ATP or the supernatant fraction resulting from centrifugation at 105,000 g. Ester formation has been examined similarly with broken-cell preparations of rat skin. With $$Word$$ as substrate, the rate of ester formation was more than six times the rate of methyl sterol demethylation. The very significant competition between esterification and demethylation of methyl sterol intermediates of skin suggests that sterol intermediates accumulate in rat skin because of the rapid formation of esters that may not be further metabolized.

Kinetic investigation of rat liver microsomal electron transport from NADH to cytochrome P-450.

NADH-dependent reduction of microsomal cytochrome P-450 has been analyzed kinetically by observing formation of the ferrous-carbonyl complex. Reduction is best described by two exponential equations with apparent first-order rate constants of 1.49 +/- 0.20 and 0.077 +/- 0.015 min-1. By either selective removal or inhibition of specific electron carriers, the kinetic data of minimally altered microsomes are further resolved into a third slow phase. Either addition of anti-cytochrome b5 immune globulin or reduction of cytochrome b5 with ascorbic acid markedly diminishes only the third phase. In reconstitution of purified flavoproteins, phospholipids, and a single isozymic form of cytochrome P-450, without cytochrome b5 only biphasic reduction of cytochrome P-450 is observed. Thus, microsomal cytochrome P-450 appears to be reduced via two independent pathways of electron transport from NADH; the biphasic reduction occurs via cytochrome P-450 reductase while the slower monophasic reduction occurs via cytochrome b5. Multiphasic reduction occurs via cytochrome b5. Multiphasic kinetics are not altered by in vivo inductions of different isozymes of cytochrome P-450. Accordingly, the rates of reduction appear to be an intrinsic property of the electron transport process and not directed by the heterogeneity of the isozymic mixture of ultimate electron acceptors.

Purification of a soluble rat liver protein that stimulates microsomal 4-methyl sterol oxidase activity.

A soluble rat liver protein that stimulates microsomal methyl sterol oxidase activity has been isolated and purified to homogeneity by salt fractionation, differential heat inactivation, two calcium phosphate gel association, and Sephadex filtration. The protein, as isolated, may be dissociated with detergent into subunits with a molecular weight of approximately 10,300, as determined electrophoretically. In addition to this stimulatory protein, postmicrosomal supernatant fraction of rat liver contains a low molecular weight methyl sterol oxidase inhibitor, possibly cholesterol ester, which is removed during protein purification. Purified soluble protein enhances the observed rate of oxidative attack of the methyl sterol substrates, 4, 4-dimethyl-5alpha-cholest-7-en-3beta-ol and 4alpha-methyl-5alpha-cholest-7-en-3beta-ol. Addition of increasing amounts of either partially purified or homogeneous soluble protein yields hyperbolic stimulation, from which a K'm of about 7 muM has been calculated (based on a monomeric molecular weight of 10,300). Sequential additions of the soluble protein yield equal increments of stimulation. These results are consistent with the suggestion that the soluble protein may be a reactant in the oxidative process. Methyl sterol oxidase is inhitited in vitro by cholesterol, several oxygenated sterols, and cholesterol esters. The extent of inhibition is much greater when the soluble protein is present in the incubation. The inhibition is competitive with respect to methyl sterol substrate; cholesterol succinate, a water-soluble ester, is strongly inhibitory, K'i (ester)/K'm(substrate) approximately 0.2. Since end product inhibition of methyl sterol oxidase may be produced by accumulation of cholesterol or cholesterol metabolites, the soluble protein may participate in regulation of the activity of some microsomal enzymes of cholesterol biosynthesis.

Investigation of microsomal oxygenases of biosynthetic processes. Stearyl-CoA desaturase of adipose tissue and liver.

Porcine and rat microsomal stearyl-CoA desaturases require reduced pyridine nucleotide and oxygen, are cyanide sensitive, and are insensitive to carbon monoxide. The Km for stearyl-CoA is somewhat larger for liver than for the adipose desaturases, but, in general, assay conditions are quite similar. Adipose tissue microsomes contain cytochromes b5 and P-450, as well as the NADH- and NADPH-specific cytochrome reductases. Compared to liver, the specific contents and activities of electron carriers are much lower in adipose tissue, and activities of 4-methyl sterol oxidase of cholesterol biosynthesis, as well as the cytochrome P-450-dependent aminopyrene demethylase and benzypyrene hydroxylase, are negligible in adipose tissue microsomes. Furthermore, unlike hepatic desaturase, administration of insulin stimulates the adipose desaturase 3-fold without affecting either the amounts or activities of microsomal oxidation-reduction proteins; the changes in desaturase activities produced either by altering dietary fat or by fasting and/or fasting followed by refeeding are, in general, both more extensive and more permanent in adipose compared to liver microsomes. The effects produced by isotopic hydrogen substitution both in stearyl-CoA and in the medium (2H2O) are similar with microsomes from both tissues. The rate-determining step of desaturase appears to be similar in both tissues. The primary isotope effect, k H/Tr, observed with [9,10-3H2]stearyl-CoA is relatively small, 2.88. Since little, if any, primary isotope effect is associated with methyl sterol oxidase, these two mixed function oxidases of biosynthetic processes also appear to share this property in common.

Total enzymic synthesis of cholesterol from lanosterol. Cytochrome b5-dependence of 4-methyl sterol oxidase.

Methyl sterol oxidase of microsomal synthesis of cholesterol from lanosterol is a mixed-function oxidase that is dependent upon reduced pyridine nucleotide. The methyl sterol oxidase, as well as NADH-cytochrome c reductase, in intact rat liver microsomes are inhibited by anti-cytochrome b5 immunoglobulin, but NADPH-cytochrome c reductase is not affected. There is a decreased time lag prior to onset of reoxidation of steady state levels of reduced cytochrome b5 when 4-methyl sterol oxidase substrates are present. Trypsin treatment of microsomes destroys cytochrome b5 with loss of methyl sterol oxidase activity. Activity is restored by addition of purified cytochrome b5 to trypsin-treated microsomes. Initial attempts to solubilize and purify 4-methyl sterol oxidase have been only partially successful due to the extreme lability of the oxidase. However, DEAE-cellulose column chromatography of a detergent extract of microsomes yields a fraction that contains the oxidase, lipids, and NADH-cytochrome b5 reductase but is free of cytochrome b5. Oxidation of 4 alpha [30-3H] methyl-5 alpha-cholest-7-en-3 beta-ol by methyl sterol oxidase in this isolated fraction can be fully restored by the addition of purified liver microsomal cytochrome b5. These results strongly support the suggestion that membrane-bound cytochrome b5 of rat liver microsomes is an obligatory electron carrier from NADH to 4-methyl sterol oxidase.