Androgen concentration and partial characterization of 5alpha reductase in the epididymis of the rhesus monkey.

The 5alpha reductase activity ofthe monkey epididymis was studied. The enzyme was found in particulate subcellular fractions, its distribution closely resembling that of the microsomal marker enzyme NADPH: cytochrome c reductase, suggesting an association of 5alpha reductase with membranes of the endoplasmic reticulum. Maximal enzyme activity was found at pH 5.4 and at 32--37 C. The crude nuclear preparation had a Km: 0.315 x 10(-6)M and Vmax: 168 pmoles/mg protein/h. The microsomal enzyme had a Km: 0.243 x 10(-6)M and Vmax: 828 pmoles/mg protein/h. Neither enzyme preparation was affected by addition to the incubation media of dihydrotestosterone (DHT) or 5alpha-androstane-3alpha,17beta-diol. The endogenous androgen concentration in the epididymides of 2 different monkeys, in ng/g wet weight was: DHT 20.81 +/- 1.98; T: 9.0L +/- 2.83; diol: 3.03 +/- 0.41.     

Ecdysterone biosynthesis: a microsomal cytochrome-P-450-linked ecdysone 20-monooxygenase from tissues of the African migratory locust.

Ecdysone 20-monooxygenase, an enzyme which converts ecdysone to ecdysterone (the major moulting hormone of insects) has been characterized in cell-free preparations of tissues from African migratory locust. The product of the reaction has been identified as ecdysterone on the basis of several microchemical derivatization and chromatographic methods. Ecdysone 20-monooxygenase activity is located primarily in the microsomal fraction which also carries NADPH cytochrome c reductase and cytochrome P-450, as shown by sucrose density gradient centrifugation. Optimal conditions for the ecdysone 20-monooxygenase assay have been determined. The enzyme has a Km for ecdysone of 2.7 x 10(-7) M and is competitvely inhibited by ecdysterone (Ki = 7.5 x 10(-7) M). Ecdysone 20-monooxygenase is a typical cytochrome P-450 linked monooxygenase: the reaction requires O2 and is inhibited by CO, an effect partially reversed by white light. The enzyme is effectively inhibited by several specific monooxygenase inhibitors and by sulfhydryl reagents, but not by cyanide ions. Ecdysone elicits a type I difference spectrum when added to oxidized microsomes. NADPH acts as preferential electron donor. The transfer of reducing equivalents proceeds through NADPH cytochrome c (P-450) reductase: ecdysone 20-monooxygenase is inhibited by cytochrome c. Both NADPH cytochrome c reductase and ecdysone 20-monooxygenase are inhibited by NADP+ and show a similar Km for NADPH. The Malpighian tubules have the highest specific activity of ecdysone 20-monooxygenase, while fat body contain most of the cytochrome P-450 and NADPH cytochrome c reductase.     

Electron-transport pathway of the NADH-dependent haem oxygenase system of rat liver microsomal fraction induced by cobalt chloride.

The hepatic microsomal haem oxygenase activity of rats treated with CoCl2 was studied kinetically by measuring biliverdin, the immediate product of the reaction. Biliverdin was extracted with diethyl ether/ethanol mixture, and was determined by the difference between A690 and A800. The apparent Km value for NADPH (at 50 microM-haematin) was about 0.2 microM when an NADPH-generating system was used, whereas that for NADH was about 630 microM. Essentially the same Vmax. values were obtained for both the NADH- and NADPH-dependent haem oxygenase reactions. No synergism was observed with NADH and NADPH. The NADH-dependent reaction was competitively inhibited by NADP+, with a Ki of about 10 microM. The inhibitoin of the NADH-dependent reaction by the antibody against rat liver microsomal NADPH-cytochrome c reductase was essentially complete, with a pattern similar to that of the NADPH-dependent reaction. The immunochemical experiment and the comparison of the kinetic values with the reported data on isolated NADH-cytochrome b5 reductase and NADPH--cytochrome c reductase indicated the involvement of the latter enzyme in NADH-dependent haem oxygenation by microsomal fraction in situ.     

Alterations in enzyme and cytochrome profiles of Rana catesbeiana liver organelles during thyroxine-induced metamorphosis. Changes in membrane-localized phosphohydrolases, oxidoreductases, and cytochrome levels in response to in vivo thyroxine administration.

A primary objective of the present study has been to determine the changes which occur in Rana catesbeiana liver organelle membranes during thyroxine-induced metamorphosis. To this end, enzyme and cytochrome profiles were determined for mitochondria, microsomes, and nuclear membrane fractions isolated from livers of R. catesbeiana tadpoles which had been fasted for 6 days at 15 +/- 0.5 degrees and then immersed in thyroxine, 2.6 X 10(-8) M, for periods of up to 12 days at 23.5 +/- 0.4 degrees. The ratio of total succinate-cytochrome c reductase activity in the initial homogenate fraction to the total activity of this mitochondrial "marker" enzyme recovered in the final mitochondrial fraction remained constant, approximately 0.5, throughout the course of thyroxine treatment; however, after a 3- to 4-day latency the mitochondrial protein mass recovered per unit mass of initial homogenate protein was found to increase significantly (approximately 2-fold by Day 10 of thyroxine treatment). A similar increase was also observed in the yield of microsomal, but not nuclear membrane, protein mass as a function of thyroxine treatment. Prolonged thyroxine treatment (12 days) resulted in approximately 50% decreases in tadpole liver homogenate and microsomal NADH-cytochrome c reductase specific activities; in contrast, mitochondrial and nuclear membrane NADH-cytochrome c reductase specific activities were not altered under the same conditions. In addition, homogenate and microsomal NADPH-cytochrome c reductase specific activities were found to have increased significantly after 12 days of thyroxine treatment; however, the specific activity of NADPH-cytochrome c reductase in the mitochondrial fraction was unchanged. It was also observed that thyroxine treatment resulted in increases in homogenate and microsomal glucose-6-phosphatase specific activities, whereas the mitochondrial as well as nuclear membrane glucose-6-phosphatase specific activities remained unchanged. Furthermore, in contrast to homogenate and mitochondrial monoamine oxidase specific activities, which decreased 30 and 40%, respectively, as a consequence of thyroxine treatment (12 days), the succinate-cytochrome c reductase and oligomycin-sensitive Mg2+ ATPase specific activities determined for these fractions increased significantly. In all instances, changes as a result of thyroxine treatment in membrane-localized homogenate or organelle enzyme specific activities were apparent only after a 3- to 4-day initial latent period. The in vitro effects of thyroxine (10(-10) - 10(-5) M) on the membrane-localized enzyme activities examined in this study were either negligible or, as in the case of mitochondrial succinate-cytochrome c reductase and microsomal NADH-cytochrome c reductase, opposite to the changes observed in response to in vivo thyroxine treatment, with the exception of microsomal NADPH-cytochrome c reductase activity which was enhanced approximately 2-fold by 10(-5) M thyroxine...     

Characterization and modulation by drugs of sheep liver microsomal flavin monooxygenase activity

The flavin monooxygenases (FMO) catalyse the NADPH and oxygen-dependent oxidation of a wide range of nucleophilic nitrogen-, sulfur-, phosphorus-, and selenium heteroatom-containing chemicals, drugs, and agricultural agents. In the present study, sheep liver microsomal FMO activity was determined by measuring the S-oxidation rate of methimazole and the average specific activity obtained from different microsomal preparations was found to be 3.8 +/- 1.5 nmol methimazole oxidized min(-1) mg(-1) microsomal protein (mean +/- SE, n = 7). The presence of 0.1% Triton X-100 in the reaction mixture caused an increase of specific sheep liver microsomal FMO activity towards methimazole to 6.1 +/- 1.4 nmol methimazole oxidized min(-1) mg(-1) microsomal protein (mean +/- SE, n = 6). Metabolism of imipramine and chlorpromazine was measured by following the oxidation of cofactor NADPH spectrophotometrically at 340 nm. Sheep liver microsomal FMO activity towards imipramine and chlorpromazine was found to be 10.7 and 12.3 nmol NADPH oxidized min(-1) mg(-1) microsomal protein, respectively. Characterization of sheep liver enzyme was carried out using methimazole as substrate and the maximum FMO enzyme activity was detected at 37 degrees C and at pH 8.0. The apparent K(m) value of sheep liver microsomal FMO for methimazole was 0.118 mM. Effects of the detergents Triton X-100, Cholate, and Emulgen 913, on FMO activity were determined and FMO activity was found to increase with the addition of detergents to the reaction medium. Sheep liver microsomal FMO-catalysed methimazole oxidation was inhibited by imipramine and chlorpromazine when these drugs were used at high concentrations. Western blot-immunochemical analysis revealed the presence of FMO3 in sheep liver microsomes.     

Analysis of debromination of 1,2-dibromoethane by cytochrome P-450-linked hydroxylation systems as observed by bromide electrode

Debromination of 1,2-dibromoethane (DBE) by a rabbit liver microsomal preparation and a reconstituted cytochrome P-450 enzyme system was investigated. The reaction was performed in our newly constructed reaction vessel, in which a bromide electrode was installed. During the reaction, the liberated bromide ion was continuously measured by the bromide electrode, and the amount was recorded. In the microsomal preparation, the DBE-debromination rate per nmol cytochrome P-450 was enhanced by phenobarbital-pretreatment of rabbits compared with the untreated microsomes, whereas it was diminished by 3-methylcholanthrene-pretreatment. The debromination reaction was reconstituted in a purified enzyme system containing phenobarbital-inducible rabbit liver microsomal cytochrome P-450 (P-450PB), NADPH-cytochrome P-450 reductase, and NADPH. The optimum conditions required the presence of dilauroylphosphatidylcholine and cytochrome b5. Cytochrome b5 was found not to be an obligatory component for the DBE-debromination in the reconstituted system, but it stimulated the activity about 3.4-fold. Preincubation of the reconstituted mixture with guinea pig anti-cytochrome P-450PB antiserum markedly inhibited the debromination reaction.     

Assay of microsomal oxysterol 7alpha-hydroxylase activity in the hamster liver by a sensitive method: in vitro modulation by oxysterols

A method of assaying hepatic cytochrome P-450, oxysterol 7alpha-hydroxylase (CYP7B), was developed by combining the use of 25-[26,27-(3)H]hydroxycholesterol as a substrate and hydroxypropyl-beta-cyclodextrin as a substrate vehicle. When these assay conditions were tested, an undesirable transformation was observed of the reaction product, 7alpha,25-dihydroxycholesterol, into 3-oxo-7alpha,25-dihydroxy-4-cholesten by the activity of 3beta-hydroxy-Delta(5)-C(27) steroid oxydoreductase, a microsomal NAD(+) and NADP(+) dependent enzyme of bile acid metabolism. A great improvement was reached by using a continuous NADPH generating system which constantly re-transforms NADP(+) into NADPH, thus inhibiting this activity. This improved CYP7B assay, comparable to our previously described assay for cholesterol 7alpha-hydroxylase (CYP7A), allowed a 3-fold increase of the apparent enzyme activity. The possibility to simultaneously measure CYP7A and CYP7B activities on the same microsomal preparation was investigated. A marked decrease (-33%) in the CYP7B activity was noticed, while that of CYP7A remained unchanged. The CYP7B activity was observed to be inhibited by cholesterol (-30%) and also by the oxysterols 7alpha-hydroxycholesterol (-21%), 7beta-hydroxycholesterol (-25%) and epicoprostanol (-20%), and by cyclosporin A (-26%). It can be concluded that this sensible and easy to perform CYP7B assay allows to observe, at least in vitro, a modulation of the enzyme activity by oxysterols.     

Purification and characterization of 5 beta-cholestane-3 alpha,7 alpha,12 alpha-triol 27-hydroxylase from female rat liver mitochondria

5 beta-Cholestane-3 alpha,7 alpha,12 alpha-triol 27-hydroxylase (5 beta-cholestane-3 alpha,7 alpha,12 alpha-triol, NADPH:oxygen oxidoreductase (26-hydroxylating), EC 1.14.13.15) was purified from female rat liver mitochondria based on its catalytic activity. The final preparation of the enzyme showed a single major band on the sodium dodecyl sulfate-polyacrylamide gel electrophoretogram. The content of purified enzyme was 12 nmol/mg of protein, and the specific activity was 431 nmol/min/mg of protein. The molecular weight of the enzyme was determined from sodium dodecyl sulfate-polyacrylamide gel electrophoresis as 52,500. The absorption spectra of the purified enzyme and that of the dithionite-reduced CO complex showed peaks at 417 and 450 nm, respectively, indicating the enzyme belongs to the cytochrome P-450 family. Upon reconstitution with the electron-transferring system of the adrenal (adrenodoxin and NADPH-adrenodoxin reductase), the enzyme showed high activity hydroxylating 5 beta-cholestane-3 alpha,7 alpha-12-triol at position 27 with a turnover number of 35.5 min-1 and Km of 6.3 microM. The enzyme activity was completely lost when the electron-transferring system was replaced by that of microsomes (NADPH-cytochrome P-450 reductase purified from rat liver microsomes), confirming that the P-450 enzyme was of the mitochondrial type, but not of the microsomal. The omission of cytochrome P-450, adrenodoxin, or NADPH-adrenodoxin reductase resulted in complete loss of enzyme activity. The specific activity toward 5 beta-cholestane-3 alpha, 7 alpha-diol was less than one-half that toward cholestanetriol and that toward cholesterol was about one-fiftieth. The enzyme showed no activity toward xenobiotics such as benzphetamine, 7-ethoxycoumarin, and benzo[a]pyrene. Its activity was not inhibited by metyrapone and slightly inhibited by aminoglutethimide. The enzyme activity was markedly lowered in an atmosphere of CO/O2/N2, 40/20/40.     

Hydroxymethylglutaryl coenzyme A reductase activity of adult Hymenolepis diminuta

The occurrence of hydroxymethylglutaryl coenzyme A (HMG-CoA) reductase in adult Hymenolepis diminuta was demonstrated. This activity was negligible in the cestode's cytosolic fraction but was noted when the mitochondrial or microsomal fraction served as the enzyme source. The predominant localization of HMG-CoA reductase activity was with the microsomal fraction. This fraction did not contain appreciable mitochondrial contamination based on the distribution of marker enzymes. The enzymatic nature of HMG-CoA conversion to mevalonic acid by either fraction was apparent because the reaction was heat labile and responded linearly to time of assay and protein content. The enzymatic reduction of HMG-CoA absolutely required NADPH when either fraction was assayed. The lesser activity of the mitochondrial fraction was membrane-associated. The predominant localization of HMG-CoA reductase activity with microsomal membranes and its separation with the membranous component of the mitochondrial fraction suggest that mitochondrial activity reflects the presence of microsomal membranes. In its predominant localization and pyridine nucleotide requirement, the cestode's HMG-CoA reductase activity resembles that of mammalian systems. The finding of HMG-CoA reductase provides an enzymatic mechanism for the intermediate conversion of HMG-CoA to mevalonic acid that would be needed for acetate-dependent isoprenoid lipid synthesis by adult H. diminuta.     

Purification and properties of biliverdin reductases from pig spleen and rat liver.

Biliverdin reductase was purified from pig spleen soluble fraction to a purity of more than 90% as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme was a monomer protein with a molecular weight of about 34,000. Its isoelectric point was at 6.1-6.2. The enzyme was strictly specific to biliverdin and no other oxiodoreductase activities could be detected in the purified enzyme preparation. The purified enzyme could utilize both NADPH and NADH as electron donors for the reduction of biliverdin. However, there were considerable differences in the kinetic properties of the NADPH-dependent and the NADH-dependent biliverdin reductase activities: Km for NADPH was below 5 microM while that for NADH was 1.5-2 mM; the pH optimum of the reaction with NADPH was 8.5 whereas that of the reaction with NADH was 6.9; Km for biliverdin in the NADPH system was 0.3 microM whereas that in the NADH system was 1-2 microM. In addition, both the NADPH-dependent and NADH-dependent activities were inhibited by excess biliverdin, but this inhibition was far more pronounced in the NADPH system than in the NADH system. IX alpha-biliverdin was the most effective substrate among the four biliverdin isomers, and the dimethylester of IX alpha-biliverdin could not serve as a substrate. Biliverdin reductase was also purified about 300-fold from rat liver soluble fraction. The hepatic enzyme was also a monomer protein with a molecular weight of 34,000 and showed properties quite similar to those of the splenic enzyme as regards the biliverdin reductase reaction. The isoelectric point of the hepatic enzyme, however, was about 5.4. It was assumed that NADPH rather than NADH is the physiological electron donor in the intracellular reduction of IX alpha-biliverdin. The stimulatory effects of bovine and human serum albumins on the biliverdin reductase reactions were also examined.     

3-Hydroxy-3-methylglutaryl-coenzyme A reductase. A comparison of the modulation in vitro by phosphorylation and dephosphorylation to modulation of enzyme activity by feeding cholesterol- or cholestyramine-supplemented diets

The activity of 3-hydroxy-3-methylglutaryl-coenzyme A reductase (hydroxymethylglutaryl-CoA reductase) was considerably inhibited during incubation with ATP+Mg²⁺. The inactivated enzyme was reactivated on further incubation with partially purified cytosolic phosphoprotein phosphatase. The inactivation was associated with a decrease in the apparent K_m of the reductase for hydroxymethylglutaryl-CoA, and this was reversed on reactivation. The slight increase in activity observed during incubation of microsomal fraction without ATP was not associated with a change in apparent K_m and, unlike the effect of the phosphatase, was not inhibited by NaF. Liver microsomal fraction from rats given cholesterol exhibited a low activity of hydroxymethylglutaryl-CoA reductase with a low apparent K_m for hydroxymethylglutaryl-CoA. Mícrosomal fraction from rats fed cholestyramine exhibited a high activity with a high K_m. To discover whether these changes had resulted from phosphorylation and dephosphorylation of the reductase, microsomal fraction from rats fed the supplemented diets and the standard diet were inactivated with ATP and reactivated with phosphoprotein phosphatase. Inactivation reduced the maximal activity of the reductase in each microsomal preparation and also reduced the apparent K_m for hydroxymethylglutaryl-CoA. There was no difference between the preparations in the degree of inactivation produced by ATP. Treatment with phosphatase restored both the maximal activity and the apparent K_m of each preparation, but never significantly increased the activity above that observed with untreated microsomal fraction. It is concluded that hydroxymethylglutaryl-CoA reductase in microsomal fraction prepared by standard procedures is almost entirely in the dephosphorylated form, and that the difference in kinetic properties in untreated microsomal fraction from rats fed the three diets cannot be explained by differences in the degree of phosphorylation of the enzyme.     

Pituitary progesterone 5 alpha-reductase: solubilization and partial characterization

The microsomal progesterone 5 alpha-reductase activity from female rat anterior pituitary has been solubilized and partially characterized with regard to some of its kinetic and physical properties. The solubilization of progesterone 5 alpha-reductase has been achieved through the use of either an n-octyl glucoside (OG)-KCl- or a digitonin-KCl-extraction. The total yield and specific activity of solubilized enzyme activity is greater using the OG-KCl method. Kinetic analyses of microsomal and OG-KCl-solubilized progesterone 5 alpha-reductase have indicated that both of these preparations exhibit a similar apparent Km for progesterone (microsomal Km = 117 +/- 12 nM; solubilized Km = 123 +/- 11 nM), suggesting that the solubilization procedure does not appreciably alter the kinetic behavior of this enzyme activity. The OG-KCl-extracted progesterone 5 alpha-reductase activity also appears quite stable, since essentially no enzyme activity is lost following dialysis at 4 degrees C for 22 h. In addition, the activity of the solubilized-dialyzed enzyme preparation can be slightly stimulated via the addition of phospholipids. Studies on the properties of the microsomal enzyme activity have indicated that this preparation is unaffected by metal chelators (EDTA or EGTA) but can be completely inhibited by the powerful sulfhydryl blocking agent p-chloromercuribenzoic acid. An evaluation of the possible role of flavins (as a hydride carrier between NADPH and the steroid) has shown that progesterone 5 alpha-reduction is inhibited by high levels of flavins and flavin analogs.     

Characterization of human neutrophil leukotriene B4 omega-hydroxylase as a system involving a unique cytochrome P-450 and NADPH-cytochrome P-450 reductase

Leukotriene B4 (LTB4), a potent chemotactic agent, was catabolized to 20-hydroxyleukotriene B4 (20-OH-LTB4) by the 150,000 x g pellet (microsomal fraction) of human neutrophil sonicate. The reaction required molecular oxygen and NADPH, and was significantly inhibited by carbon monoxide, suggesting that a cytochrome P-450 is involved. The neutrophil microsomal fraction showed a carbon monoxide difference spectrum with a peak at 450 nm in the presence of NADPH or dithionite, indicating the presence of a cytochrome P-450. The addition of LTB4 to the microsomal fraction gave a type-I spectral change with a peak at around 390 nm and a trough at 422 nm, indicating a direct interaction of LTB4 with the cytochrome P-450. The dissociation constant of LTB4, determined from the difference spectra, is 0.40 microM, in agreement with the kinetically determined apparent Km value for LTB4 (0.30 microM). Such a spectral change was not observed with prostaglandins A1, E1 and F2 alpha or lauric acid, none of which inhibited the LTB4 omega-hydroxylation. The inhibition of the LTB4 omega-hydroxylation by carbon monoxide was effectively reversed by irradiation with monochromatic light of 450 nm wavelength. The photochemical action spectrum of the light reversal of the inhibition corresponded remarkably well with the carbon monoxide difference spectrum. These observations provide direct evidence that the oxygen-activating component of the LTB4 omega-hydroxylase system is a cytochrome P-450. Ferricytochrome c inhibited the hydroxylation of LTB4 and the inhibition was fortified by cytochrome oxidase. An antibody raised against rat liver NADPH-cytochrome-P-450 reductase inhibited both LTB4 omega-hydroxylase activity and the NADPH-cytochrome-c reductase activity of human neutrophil microsomal fraction. These observations indicate that NADPH-cytochrome-P-450 reductase acts as an electron carrier in LTB4 omega-hydroxylase. On the other hand, an antibody raised against rat liver microsomal cytochrome b5 inhibited the NADH-cytochrome-c reductase activity but not the LTB4 omega-hydroxylase activity of human neutrophil microsomal fraction, suggesting that cytochrome b5 does not participate in the LTB4-hydroxylating system. These characteristics indicate that the isoenzyme of cytochrome P-450 in human neutrophils, LTB4 omega-hydroxylase, is different from the ones reported to be involved in omega-hydroxylation reactions of prostaglandins and fatty acids.     

Induction and characterization of a microsomal flavonoid 3'-hydroxylase from parsley cell cultures

A microsomal preparation from irradiated parsley cell cultures catalyses the NADPH and dioxygen-dependent hydroxylation of (S)-naringenin [(S)-5, 7, 4'-trihydroxyflavanone] to eriodictyol (5, 7, 3', 4'-tetrahydroxyflavanone). Dihydrokaempferol, kaempferol, and apigenin were also substrates for the 3'-hydroxylase reaction. In contrast prunin (naringenin 7-O-beta-glucoside) was not converted by the enzyme. The microsomal preparation, which also contains cinnamate 4-hydroxylase, did not catalyse hydroxylation of 4-coumaric acid to caffeic acid. 3'-Hydroxylase activity is partially inhibited by carbon monoxide in the presence of oxygen as well as by cytochrome c and NADP+. These properties suggest that the enzyme is a cytochrome P-450-dependent flavonoid 3'-monooxygenase. Pronounced differences in the inhibition of flavonoid 3'-hydroxylase and cinnamate 4-hydroxylase were found with EDTA, potassium cyanide and N-ethylmaleimide. Irradiation of the cell cultures led to increase of flavonoid 3'-hydroxylase activity with a maximum at about 23 h after onset of irradiation and subsequent decrease. This is similar to light-induction of phenylalanine ammonialyase and cinnamate 4-hydroxylase. In contrast, treatment of the cell cultures with a glucan elicitor from Phytophthora megasperma f. sp. glycinea did not induce flavonoid 3'-hydroxylase nor chalcone isomerase but caused a strong increase in the activities of phenylalanine ammonia-lyase, cinnamate 4-hydroxylase, and NADPH--cytochrome reductase. The results prove that flavonoid 3'-hydroxylase and cinnamate 4-hydroxylase are two different microsomal monooxygenases.     

Sexual difference in properties of 5 alpha-reductase in microsome of rat submandibular glands

The properties of 5 alpha-reductase activity in the submandibular glands of rats were investigated using microsomal fraction in the presence of NADPH, and we found a sexual difference in these properties. The formation of 5 alpha-dihydrotestosterone and 5 alpha-androstane-3 alpha, 17 beta-diol from testosterone demonstrated 5 alpha-reductase in the rat microsomal fraction of this tissue sample. Apparent michaelis constants (Km) of the 5 alpha-reductase activity for testosterone was 1.02 x 10(-6) M for the female tissue. Microsomal fraction of submandibular glands of male rats had two Kms and were determined as 1.12 x 10(-6) M and 1.01 x 10(-5) M. The lower Km of 5 alpha-reductase for male rats was similar to for the females.     

Properties of 4-ene-5 alpha-reductase and studies on its solubilization from porcine testicular microsomes

The activity of 4-ene-5 alpha-reductase was assayed in porcine testis homogenates and subcellular fractions, using testosterone as substrate. 'Marker' enzyme activities were utilized to indicate the purity of the subcellular fractions. 4-Ene-5 alpha-reductase activity was associated with the microsomal fraction; there was no activity in the purified nuclear fraction. Enzyme activity was higher in the testes of 6 week old pigs than those of 3 and 17 week old animals, and a range of activity was found. The enzyme was unstable when stored at -20 degrees C but the addition of albumin (0.1%, w/v) or glycerol (20%, v/v) to the buffer and storage at -70 degrees C or in liquid nitrogen ensured that maximal activity was retained for at least 35 days. In addition to 5 alpha-DHT, other 5 alpha-reduced metabolites and 4-androstenedione were formed in this reaction; NADPH was the preferred cofactor, but 40% of the 4-ene-5 alpha-reductase activity was retained when NADH was used. Solubilization of the microsomal enzyme was achieved using sodium citrate (0.1 M); 4-ene-5 alpha-reductase activity was enhanced to greater than 120% and 60% of this activity was in the soluble fraction. The optimum pH and temperature for both soluble and membrane-bound 4-ene-5 alpha-reductase were 6.9 and 32 degrees C, respectively. The mean apparent Km and Vmax were 0.6 mumol/l and 158 pmol/min/mg microsomal protein for the microsomal enzyme and 1.42 mumol/l and 212.0 pmol/min/mg soluble protein for the solubilized 4-ene-5 alpha-reductase. The estimated sedimentation coefficient was 11.6.     

Modulation of 3-hydroxy-3-methylglutaryl-CoA reductase and of acyl-CoA–cholesterol acyltransferase by the transfer of non-esterified cholesterol to rat liver microsomal vesicles

The incubation of rat liver microsomal fraction with a serum preparation followed by the re-isolation of the microsomal membranes has resulted in an increase in the concentration of non-esterified cholesterol, a considerable decrease in the activity of 3-hydroxy-3-methylglutaryl-CoA reductase and in an increase in the activity of acyl-CoA–cholesterol acyltransferase in the treated microsomal preparation. These effects were related to the concentration of serum in the incubation mixture and to the duration of the incubation. The transfer of non-esterified cholesterol was specific in that the content of protein and the total phospholipids were similar in the original microsomal fraction and the serum-treated microsomal preparation. The incubation of the microsomal fraction with lipoprotein-deficient serum or with no serum resulted in both cases in small changes in the non-esterified cholesterol, the esterified cholesterol and the total phospholipid content in the treated preparations compared with these concentrations in the original microsomal fraction, whereas the activity of acyl-CoA–cholesterol acyltransferase and of 3-hydroxy-3-methylglutaryl-CoA reductase was similar in the lipoprotein-deficient-serum-treated and the buffer-treated microsomal preparations. The activity of 3-hydroxy-3-methylglutaryl-CoA reductase was lower and the activity of acyl-CoA–cholesterol acyltransferase was higher in the lipoprotein-deficient-serum-treated and the buffer-treated microsomal preparations as compared with these activities in the original microsomal fraction. However, the serum-treated microsomal preparation had considerably lower activity of 3-hydroxy-3-methylglutaryl-CoA reductase and considerably higher activity of acyl-CoA–cholesterol acyltransferase than these activities in buffer-treated and in lipoprotein-deficient-serum-treated microsomal preparations.     

The effects of unsaturated fatty acids on hepatic microsomal drug metabolism and cytochrome P-450

1. The effects of unsaturated fatty acids on drug-metabolizing enzymes in vitro were measured by using rat and rabbit hepatic 9000g supernatant fractions. 2. Unsaturated fatty acids inhibited the hepatic microsomal metabolism of ;type I' drugs with inhibition increasing with unsaturation: arachidonic acid>linolenic acid>linoleic acid>oleic acid. Inhibition was independent of lipid peroxidation. Linoleic acid competitively inhibited the microsomal O-demethylation of p-nitroanisole and the N-demethylation of (+)-benzphetamine. 3. The hepatic microsomal metabolism of ;type II' substrates, aniline and (-)-amphetamine, was not affected by unsaturated fatty acids. 4. The rate of reduction of p-nitrobenzoic acid and Neoprontosil was accelerated by unsaturated fatty acids. 5. Linoleic acid up to 3.5mm did not decelerate the generation of NADPH by rat liver soluble fraction, nor the activity of NADPH-cytochrome c reductase of rat liver microsomes. Hepatic microsomal NADPH oxidase activity was slightly enhanced by added linoleic acid. 6. No measurable disappearance of exogenously added linoleic acid occurred when this fatty acid was incubated with rat liver microsomes and an NADPH source. 7. The unsaturated fatty acids used in this study produced type I spectra when added to rat liver microsomes, and affected several microsomal enzyme activities in a manner characteristic of type I ligands.     

Activation of detergent-solubilized rat hepatic 5'-iodothyronine deiodinase by NADPH and nonglutathione cytosolic components

An investigation was made of the possible role of the hepatic microsomal membrane in the activation of 5'-iodothyronine deiodinase (5'-DI) by a cytosolic activating system consisting of fraction A (relative mass (Mr) greater than 60,000), fraction B (Mr, approximately 13,000), and NADPH. Activation of 5'-DI in washed microsomes was compared with that of a microsome extract prepared by solubilization with 3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulphonate and further purification by fractional precipitation with polyethylene glycol and by DEAE-Sephacel chromatography. All 5'-DI preparations exhibited qualitatively similar dependence upon NADPH and cytosolic factors in fractions A and B for 5'-DI activation and were relatively unresponsive to NADH. Activation of solubilized preparations, unlike that of intact microsomes, was more readily inhibited by low concentrations of detergent and not inhibited by NADPH concentrations above 0.25 mM. Attempted purification of 5'-DI failed to produce a substantial increase in specific activity of the enzyme. It is concluded that, while glutathione-independent cytosolic factors and NADPH can activate 5'-DI in the absence of an intact microsomal membrane, some membrane constituents removed during solubilization and purification of the enzyme are required for maximal activation.     

Kinetic and structural properties of biocatalytically active sheep lung microsomal NADPH-cytochrome c reductase

NADPH-cytochrome c reductase was purified to electrophoretic homogeneity from detergent solubilized sheep lung microsomes. The specific activity of the purified enzyme ranged from 56 to 67 mumol cytochrome c reduced/min/mg protein and the yield was 48-52% of the initial activity in lung microsomes. The reductase had Mr of 78,000 and contained 1 mol each of FAD and FMN. Km values obtained in 0.3 M phosphate buffer, pH 7.8 at 37 degrees C for NADPH and cytochrome c were 11.1 +/- 0.70 microM and 20.0 +/- 2.15 microM. Lung reductase was inhibited by its substrate, cytochrome c when its concentration was above 160 microM. The lung reductase exhibited a ping-pong type kinetic mechanism for NADPH mediated cytochrome c reduction. Purified lung reductase was biocatalytically active in supporting benzo(a)pyrene hydroxylation reaction when coupled with lung cytochrome P-450 and lipid.