Microsomal Reductase Activity Research Articles

АНАЛИЗ АКТИВНОСТИ МИКРОСОМАЛЬНЫХ РЕДУКТАЗ ТКАНИ ЯИЧНИКОВ ПОСЛЕ КРИОКОНСЕРВАЦИИ МЕТОДОМ АКТИВИРОВАННОЙ ХЕМИЛЮМИНЕСЦЕНЦИИ

The aim of the study was to investigate the activity of NADH-dependent cytochrome b5 reductase (CYB5R) and NADPH-dependent cytochrome P450 reductase (CYPOR) in ovarian tissues after cryopreservation by lucigenin-enhanced chemiluminescence with NADH and NADPH stimulation, respectively. The results indicate that both mitochondrial and microsomal reductase activities are preserved in cryopreserved ovarian tissues. After cryopreservation, the level of production of superoxide anion radical by mitochondria drops by 3–10 times, while the presence or absence of chemotherapy has no effect, and this parameter is also not affected by the severity of the disease. Compared to the control group (patients with benign tumors), the activity of CYB5R and CYPOR of ovarian tissue in a malignant cancer process decreases 1.5–10 times depending on the disease severity, and the presence of chemotherapy dramatically affects these parameters — the activity of microsomal reductases decreases by 50–100 times in chemotherapy compared to the control group. Thus, microsomal reductase activity is a more informative parameter for assessing the functionality of cryopreserved ovarian tissue than superoxide-producing capacity of mitochondria, because, firstly, it depends on the stage of disease and prior chemotherapy, and secondly, the analytical signal of NADH/NADPH stimulated chemiluminescence is characterized by approximately 30 times higher intensity than mitochondrial chemiluminescence, which leads to higher analytical sensitivity of the technique.

Microsomal reductase activity in patients with thyroid neoplasms.

Cytochrome b5-reductase (CYB5R) and cytochrome P450 reductase (CYPOR) are important for cell metabolism; however, their role in thyroid hormonogenesis and carcinogenesis has not been elucidated yet. The activity of CYB5R correlates with the metastasis in breast cancer, but there are no similar studies for CYB5R and CYPOR for thyroid tumors. The aim of this study was to elucidate the activity of CYB5R and CYPOR changes in benign euthyroid and hyperthyroid neoplasms and in papillary thyroid cancer for their potential application as biomarkers for diagnosis and prognosis prediction of thyroid cancer. Thirty-six patients with thyroid diseases participated in the study. The control euthyroid nodular goiter group included ten patients; the thyrotoxic nodular or diffuse goiter group included 14 patients; the papillary thyroid cancer T1-2N0-1M0 (PTC) group included 12 patients. The activity of CYB5R and CYPOR was assessed with lucigenin-enhanced chemiluminescence stimulated by NADH and NADPH, respectively. Compared to the control euthyroid nodular goiter group, activity of CYB5R and CYPOR increased ~5 and 10 times, respectively, in toxic goiter, and 15 and 30 times, respectively, in half of cases of PTC. The change in activity of CYPOR was more pronounced compared to CYB5R. Within the PTC group, the subgroups with low and high activities of microsomal reductases were identified. Microsomal reductases in follicular adenoma was 2-4-fold less active compared to the euthyroid nodular goiter and the low-activity PTC group. Activity of tissue microsomal reductases varies in thyroid pathology and can be considered as a promising biomarker for differential diagnostics of benign and malignant thyroid tumors.

Coumarins in helianthus tuberosus : characterization, induced accumulation and biosynthesis

The biosynthesis of 7-hydroxylated coumarin phytoalexins has been examined in the tuber of Helianthus tuberosus. Ayapin and scopoletin where characterized by TLC, HPLC and GC-MS. Both compounds were excreted from sliced tuber tissues. They accumulated in response to treatment with chemical elicitors like CuCl 2 or sucrose, but not in response to MnCl 2. Scopoletin accumulation preceded ayapin build-up in the medium. Highest and earliest accumulation of both coumarins was measured after copper treatment. The formation of methylenedioxy bridges in several secondary metabolites is catalyzed by cyt P450 oxygenases. The changes in cyt P450 content, trans-cinnamate 4-hydroxylase and NADPH-cytochrome c reductase activities in microsomes from tuber tissues were, therefore, monitored for three days after wounding and elicitation. Cyt P450 content and NADPH-Cyt c reductase activity were not significantly modified in copper treated tissues, but increased three to five fold in response to sucrose. Copper elicitation resulted in a decrease in cinnamate-4-hydroxylase activity, while sucrose was a very strong and specific inducer of this Cyt P450 enzyme. No formation of ayapin was observed when radiolabelled scopoletin was incubated with NADPH and elicited plant microsomes, or fed to induced tuber tissues in vivo. No formation of methylenedioxy metabolite was obtained in vitro or in vivo from radiolabelled ferulate or feruloyl CoA.

Disruption of a Regulatory System Involving Cobalamin Distribution and Function in a Methionine-dependent Human Glioma Cell Line

Cobalamin metabolism and function were investigated at the levels from transcobalamin II (TCII) receptor to the cobalamin-dependent enzymes, methionine synthase and methylmalonyl-CoA mutase, in a methionine-dependent (P60) and a methionine-independent (P60H) glioma cell line. Using P60H as reference, the P60 cells cultured in a methionine medium had slightly lower TCII receptor activity and normal total cobalamin content, a moderately reduced microsomal and mitochondrial cobalamin(III) reductase activity but only trace amounts of the methylcobalamin and adenosylcobalamin cofactors. When transferred to a homocysteine medium without methionine, P60H cells showed a slightly enhanced TCII receptor activity, but the other cobalamin-related functions were essentially unchanged. In contrast, the methionine-dependent P60 cells responded to homocysteine medium with a nearly 6-fold enhancement of TCII receptor expression and a doubling of both the hydroxycobalamin content and the microsomal reductase activity. The mitochondrial reductase and the cobalamin-related processes further down the pathway did not change markedly. In both cell lines, TCII receptor activity was further increased when growth in homocysteine medium was combined with N2O exposure. These data suggest that low methionine and/or high homocysteine exert a positive feedback control on TCII receptor activity. The concurrent increase in hydroxycobalamin content and in microsomal reductase activity are either subjected to similar regulation or secondary to increased cobalamin transport. This regulatory network is most prominent in the methionine-dependent P60 cells harboring a disruption of the network in the proximity of cobalamin(III) reductase.

Metabolism of 14C-labelled 5-nitro-1,2,4-triazol-3-one by rat liver microsomes--evidence for the participation of cytochrome P-450.

In the present study, we synthesized 14C-labelled 5-nitro-1,2,4-triazol-3-one (NTO) and investigated its hepatic metabolism by dexamethasone-induced murine hepatic microsomes. Under the nitrogen atmosphere, 5-amino-1,2,4-triazol-3-one was the only detected metabolite of NTO. The microsomal nitroreductase activity was dependent on NADPH, totally inhibited by carbon monoxide and partially inhibited by oxygen. In aerobic conditions, beside a low amount of amine, the major metabolite formed is the 5-hydroxy-triazolone, urazole. This compound resulted from the oxidative denitrification of NTO, which produced equivalent amount of nitrite. This reaction, like the nitroreductase activity, was dependent on NADPH and totally inhibited by carbon monoxide. Both nitroreduction and oxidative denitrification were inhibited by imidazole-related inhibitors: miconazole and methimazole, and to a less extent by N-octylamine. The microsomal denitrification was induced by the treatment of rats with dexamethasone and phenobarbital. The microsomal reductase activity is present in untreated rat microsomes, and recovered with various inducers. The results of this study indicate the role played by cytochrome P-450 in the metabolism of NTO, supported by its transformation with reconstituted cytochrome P-450 systems.

Effects of propionic acid and pravastatin on HMG-CoA reductase activity in relation to forestomach lesions in the rat.

Administration of 4% propionic acid in powdered diet to rats for 12 weeks induces severe hyperplastic lesions in the forestomach mucosa. The mechanisms underlying this damage are not yet clear. Several lipophilic 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors such as lovastatin and simvastatin produce forestomach lesions similar to propionic acid after oral administration and the degree of alterations is correlated with their in vitro inhibitory potency (Kloss et al. 1991). Therefore it is possible, that sustained inhibition of HMG-CoA reductase and induction of hyperplasias may be somehow connected. For that reason we investigated, whether or not propionic acid has any influence on HMG-CoA reductase activity in vitro and in vivo, because propionic acid has been suggested to suppress liver cholesterol synthesis, and also whether or not pravastatin, a more polar HMG-CoA reductase inhibitor than lovastatin displays similar effects on forestomach mucosa. In untreated forestomach microsomes in vitro, propionic acid at a concentration of 51 mM (pH 5.7) inhibited HMG-CoA reductase activity by 51 + or - 10%, but at pH 7.2 no inhibition of the enzyme could be detected. Furthermore 4% propionic acid-treatment did not lower serum cholesterol. In contrast to lovastatin (Kloss et al. 1991), oral administration of pravastatin (up to 25% in the diet) did not produce any forestomach lesions in the rat. On the other hand, pretreatment with pravastatin revealed that HMG-CoA reductase activity in microsomes exceeded the activity of control forestomach and liver microsomes by 4.9 fold and 6.7 fold respectively, whereas no induction of this enzyme (neither liver nor forestomach) could be observed by pretreatment with 4% propionic acid for 12 weeks. Despite increased hepatic HMG-CoA reductase activity, pravastatin-treatment significantly lowered serum cholesterol levels of rats. These results show that sustained inhibition of HMG-CoA reductase activity in forestomach microsomes is not strongly connected with hyperplasia development.

Ontogeny and dietary modulation of 3-hydroxy-3-methylglutaryl-CoA reductase activities in neonatal pigs.

The development of hepatic and ileal -hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase was studied in three types of young pigs crossbred, obese, and lean pigs). Pigs were fed one of two diets: a high-fat (coconut oil), high-cholesterol 1.0%; designated HC) diet or a high-fat, noncholesterol (designated NC) diet from postpartum d 3 to d 13, 25, and 42 (crossbred only). There were four pigs per age per diet group (except for obese pigs). Liver and ileal mucosal microsomal reductase activities were determined by the conversion of [14C]HMG-CoA to mevalonic acid followed by lactonization of the product. The samples were analyzed by thin layer chromatography and liquid scintillation spectrometry. Hepatic reductase activity (1 unit of specific activity = 1 pmol.min-1.mg protein-1) was < 20 units on d 3 in all groups. By d 13, the activity was 40 to 46 units in all groups of pigs fed HC and approximately 50 to 80 units in pigs fed NC. Reductase activity then decreased at d 25 to 18 to 40 units in pigs fed NC and to < 14 units in pigs fed HC. The d 42 reductase values (crossbred only) were approximately 14 units for pigs fed both HC and NC diets. Intestinal reductase activity was not affected (P > .1) by either age or diet. The data suggest that dietary cholesterol suppressed hepatic reductase activity in young pigs (d 13 and 25) from divergent genetic backgrounds. The data also suggest that the stage of development is a dominant factor in regulating porcine hepatic HMG-CoA reductase activity, which was considerably increased at d 13, even in pigs fed HC diets. The relatively modest increase in plasma cholesterol, even in pigs fed cholesterol during the suckling period, provides evidence that both dietary and endogenously synthesized cholesterol are probably used predominantly for tissue building in very young pigs (d 13).

Cytochrome P450 and associated monooxygenase activities in the rat and human spinal cord: induction, immunological characterization and immunocytochemical localization.

We have discovered cytochrome P450 and associated monooxygenase activities in microsomes prepared from spinal cord tissues from rats and a human. Cytochrome P450 levels and nicotinamide adenine dinucleotide phosphate cytochrome c reductase activities in microsomes from rat spinal cord were similar to those observed from the whole brain. However, certain monooxygenase activities were significantly lower in the rat spinal cord microsomes as compared to the corresponding activities observed in the whole brain. Cytochrome P450-mediated monooxygenase activities were also detectable in microsomes prepared from human spinal cord. Immunoblot analyses of rat and human spinal cord microsomes using antisera to various forms of hepatic cytochrome P450 namely (2B1 + 2B2), 1A1, 1A2 and 2E1 revealed the presence of immunologically similar forms. The spinal cord microsomes also cross-reacted with the antiserum to the phenobarbital-inducible form of rat brain cytochrome P450. Immunocytochemical stain was predominant in the gray horns of the rat spinal cord. At the cervical level, lamina 1 and 2 representing the substantia gelatinosa were intensely stained. In the ventral horns, lamina 7, 8 and 9 containing the large motor neurons were strongly labelled, while small neurons revealed variable staining. In the white matter, the glial cells were stained but the axons remained non-reactive.(ABSTRACT TRUNCATED AT 250 WORDS)

Stable Expression of Mouse NADPH-Cytochrome P450 Reductase and Monkey P4501A1 cDNAs in Chinese Hamster Cells: Establishment of Cell Lines Highly Sensitive to Aflatoxin B1

To the cell line (A-15) expressing monkey P4501A1, which was developed from a Chinese hamster fibroblast cell line (CHL), a mouse cDNA encoding NADPH-cytochrome P450 reductase was introduced and the transformants (designated AR-10, AR-13, and AR-18) stably expressing the reductase of increased level were established. The high-level expression of the reductase was confirmed by Northern and Western blot analyses and by measurement of the reductase activity in microsomes. The transfected clones showed 4- to 11-fold higher activity of the reductase than the parental cells. These cells showed about 9-fold higher sensitivity toward aflatoxin B1 (AFB1) cytotoxicity than A-15 cells and about 330-fold more than parental CHL cells. No difference in sensitivity was observed between CHL and the CHL-derived clones (CR-50, CR-52, and CR-68) expressing high levels of the reductase alone. Increased sensitivity of AR-b cells was also observed with respect to sterigmatocystin cytotoxicity. Furthermore, in the assay for the induction of chromosomal aberrations, these clones were highly sensitive to AFB1. These results indicate that the level of the P450 reductase existing in parental CHL cells is not sufficient for maximal activity of monkey P4501A1.

Effect of boron on plasma membrane proton extrusion and redox activity in sunflower cells

The effect of boron on H + extrusion and FeCN reduction in sunflower cell suspensions and on vanadate-sensitive H +-ATPase and NADH-FeCN reductase activities of isolated microsomes was investigated. Boron deficiency inhibited FeCN-dependent H + extrusion and FeCN reduction in cells grown in darkness or at a light irradiance of 30 μmol · m −2 · s −1. In addition, an inhibition of FeCN-independent H + extrusion by boron deficiency was detected when cells were maintained at a light irradiance of 30 μmol · m −2 · s −1. FeCN-dependent and FeCN-independent H + extrusion were stimulated by FC and inhibited by vanadate and DCCD. However, FeCN reduction was not affected either by FC or by vanadate. In isolated microsomes, boron deficiency inhibited the vanadate-sensitive H +-ATPase and NADH-FeCN reductase activities. The presence of 2,4-D in the assay medium provoked an inhibition of H + extrusion and FeCN reduction in cells grown with 0.1 mM H 3BO 3; in the same way, the addition of 2,4-D to the reaction medium inhibited the NADH-FeCN reductase activity of microsomes isolated from cells grown in media with boron. These data support the existence of a relationship between boron and auxins. Likewise, the present results suggest that boron could regulate H + extrusion in sunflower cells through an effect on plasma membrane H +-ATPase and redox systems.

Regulation of 3-hydroxy-3-methylglutaryl-coenzyme A reductase activity in murine epidermis. Modulation of enzyme content and activation state by barrier requirements.

Epidermal cholesterol biosynthesis is regulated by barrier function. We quantitated the amount and activation state (phosphorylation-dephosphorylation) of the rate-limiting enzyme, 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase, in epidermis before and after barrier disruption. In murine epidermis we found high enzyme activity (1.75 +/- 0.02 nmol/min per mg protein). After acute barrier disruption, enzyme activity began to increase after 1.5 h, reaching a maximum increase by 2.5 h, and returned to normal by 15 h. Chronic barrier disruption increased total enzyme activity by 83%. In normal epidermis, measurement of HMG CoA reductase activity in microsomes isolated in NaF- vs. NaCl-containing buffers demonstrated that 46 +/- 2% of the enzyme was in the active form. After acute or chronic barrier disruption, a marked increase in the percentage of HMG CoA reductase in the active form was observed. Acute disruption increased enzyme activation state as early as 15 min, reaching a maximum after 2.5 h, with an increase still present at 15 h, indicating that changes in activation state had a close temporal relationship with barrier function. Increases in total HMG CoA reductase activity occurred only after profound barrier disruption, whereas changes in activation state occur with lesser degrees of barrier disruption. Artificial correction of barrier function prevented the increase in total HMG CoA reductase activity, and partially prevented the increase in enzyme activation. These results show that barrier requirements regulate epidermal cholesterol synthesis by modulating both the HMG CoA reductase amount and activation state.

Characterization of oxidative and reductive metabolism in vitro of nitrofluoranthenes by rat liver enzymes

Nitrofluoranthenes (NFs) are mutagenic and carcinogenic environmental pollutants found in incomplete combustion products and urban air particulate. We have studied both oxidative and reductive metabolism in vitro of different NF isomers mediated by subcellular rat liver fractions. Under aerobic conditions only ring hydroxylation of NFs by rat liver microsomes occurred and the isomeric position of the nitro group affected both the amount and the type of phenolic metabolites formed. Liver microsomes from 3-methylcholanthrene-induced rats were most effective in giving ring hydroxylated 7- and 8-nitrofluoranthene, whereas liver microsomes from phenobarbital-pretreated rats were the most active in metabolizing 1- and 3-nitrofluoranthene. Under anaerobic conditions, only reduction of NFs mediated by both cytosolic and microsomal rat liver enzymes occurred. Cofactor requirements and inhibition experiments indicated that the reductase activity in rat liver cytosolic fractions could be ascribed to DT-diaphorase, aldehyde oxidase and/or other unknown enzymes. The microsomal reductase activity was inhibited by oxygen, carbon monoxide, 2-diethylaminoethyl-2,2-diphenylvalerate hydrochloride and n-octylamine, and slightly by cytochrome c; flavin mononucleotide greatly enhanced this activity. 3-Nitrofluoranthene microsomal nitroreductase activity was increased by phenobarbital rat pretreatment and this increment correlated well with the content of cytochrome P450. These results indicate a participation of cytochrome P450 in the reductive metabolism of NFs by rat liver microsomes.

Warfarin resistance: Vitamin K epoxide reductase of scottish resistance genes is not irreversibly blocked by warfarin

The dithiothreitol-dependent vitamin K 2,3 epoxide (vitamin KO) reductase activity in liver microsomes of Scottish-derived warfarin-resistant Wistar rats (Tolworth Laboratory) was compared to that of susceptible Wistar rats. Under the test conditions, reductase activities in liver homogenates and in liver microsomes were comparable for both strains. The in vitro 150 of S-warfarin for microsomal reductase activity was 1 to 2 μM in both strains. The effect of in vivo S-warfarin was investigated after single doses, i.e. 0.2 and 1mg/kg for the susceptible rats, and 1 and 5mg/kg for the resistant rats. At 20 hr following the warfarin administrations in the susceptible strain, microsomal reductase was suppressed to about 30% of control. Microsomal reductase activity in the resistant strain was not reduced. Tissue and microsomal warfarin concentrations, however, were comparable in both strains. Wash experiments with microsomes which were treated in vitro with S-warfarin showed that vitamin KO reductase of the warfarin-resistant strain was not irreversibly inactivated by warfarin. The reactivation was mediated by DTT. The results suggest the following characteristic of the vitamin KO reductase of the Scottish resistance gene: contrary to the ◂normal” enzyme, the tight complex between the inhibitor and the resistant enzyme is liable to reactivation by reduction of the disulfide bridge in the active centre of the enzyme. This property explains the resistance for 4-hydroxycoumarin anticlotting activity.

Adrenocorticotropin and the time of day both influence the amount and activity of 3-hydroxy-3-methylglutaryl coenzyme-A reductase in the hamster adrenal.

We studied the effect of ACTH, alone or in combination with cycloheximide, on hamster adrenal 3-hydroxy-3-methylglutaryl coenzyme-A (HMG-CoA) reductase protein content (mass) and activity. Immunoblotting was performed on both homogenate and microsomal preparations, using a rabbit antirat liver reductase antibody and 125I-labeled protein A. A dose-dependent increase in HMG-CoA reductase protein content and activity was produced by ACTH. A time-course study revealed a latency of 60 min, followed by a significant increase at 120 and 180 min, in this response. At 180 min, microsomal reductase activity was significantly increased 4.7-fold, whereas the reductase protein content of microsomes and homogenate preparations was enhanced 4.4- and 3.1-fold, respectively. We calculated that only 40-50% of the reductase protein content was microsomal, indicating that more than one pool of the enzyme is present in adrenals. The coadministration of cycloheximide with ACTH not only prevented the hormonal effect on the protein content and activity of the reductase, but also produced, 180 min posttreatment, 58-69% and 65-86% decreases in reductase protein and activity, respectively. Also, both reductase activity and protein content fluctuated in parallel during the day. In the presence of proteolytic enzyme inhibitors and iodoacetamide, a major band in the area of 102K mol wt was evidenced by immunoblotting. These results indicate that ACTH and other conditions that provoke a change in adrenal HMG-CoA reductase activity also induce, in parallel, a change in the reductase protein content. The basic unit of the reductase has an apparent mol wt of about 102K.

Inhibition of human leukocyte 3-hydroxy-3-methylglutaryl coenzyme A reductase activity by ascorbic acid. An effect mediated by the free radical monodehydroascorbate.

3-Hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase activity in microsomes isolated from cultured lymphoid (IM-9) cells or freshly isolated human leukocytes was markedly decreased by either ascorbic acid or its oxidized derivative, dehydroascorbate. Inhibition of IM-9 leukocyte HMG-CoA reductase activity was log linear between 0.01 and 10 mM ascorbic acid (25 and 81% inhibition, respectively) and 0.1 and 10 mM dehydroascorbate (5 and 75% inhibition, respectively). Inhibition was noncompetitive with respect to HMG-CoA (Km = 10.2 microM (RS); ascorbic acid, Ki = 6.4 mM; dehydroascorbate, Ki = 15 mM) and competitive with respect to NADPH (Km = 16.3 microM; acetic acid, Ki = 6.3 mM; dehydroascorbate, Ki = 3.1 mM). Ascorbic acid and dehydroascorbate are interconverted through the free radical intermediate monodehydroascorbate. Reducing agents are required to convert dehydroascorbate to monodehydroascorbate, but prevent formation of the free radical from ascorbate. In microsomes from IM-9 cells, the reducing agent, dithiothreitol, abolished HMG-CoA reductase inhibition by ascorbate but enhanced inhibition by dehydroascorbate. In addition, the concentration of monodehydroascorbate present in ascorbate solutions was directly proportional to the degree of HMG-CoA reductase inhibition by 1.0 mM ascorbate. Fifty per cent inhibition of enzyme activity occurred at a monodehydroascorbate concentration of 14 microM. These data indicate that monodehydroascorbate mediates inhibition of HMG-CoA reductase by both ascorbate and dehydroascorbate. This effect does not appear to be due to free radical-induced membrane lipid modification, however, since both ascorbate and dehydroascorbate inhibited the protease-solubilized, partially purified human liver enzyme. Since inhibition of HMG-CoA reductase occurs at physiological concentrations of ascorbic acid in the human leukocyte (0.2-1.72 mM), this vitamin may be important in the regulation of endogenous cholesterol synthesis in man.

Effects of sodium metallochlorophyllins on the activity and components of the microsomal drug-metabolizing enzyme system in rat liver.

Several sodium metallochlorophyllins (Me-Chl-Na), i.e., sodium copper chlorophyllin (Cu-Chl-Na), sodium cobalt chlorophyllin and sodium iron chlorophyllin, decreased the activities of aminopyrine N-demethylase and aniline hydroxylase and the content of cytochrome P-450 in liver microsomes when given intraperitoneally to rats. The lowest levels of these variables were observed 24h after dosing. Me-Chl-Na administration did not modify the substrate-induced spectral changes of cytochrome P-450. Of the kinetic parameters studied in aminopyrine N-demethylation and aniline hydroxylation, only the Vmax values were markedly decreased by Cu-Chl-Na, indicating that the enzyme inhibition was non-competitive. Me-Chl-Na also reduced cytochrome b5 and total heme contents and NADPH-cytochrome c reductase activity in microsomes. Preincubation of liver microsomes from control rats with Cu-Chl-Na did not cause any reduction in the aminopyrine-and aniline-metabolizing enzyme activities and in the cytochrome P-450 content.These findings strongly suggest that the decreases in the activities of the hepatic microsomal drug-metabolizing enzyme system caused by Me-Chl-Na are correlated with the reduction in the contents of microsomal cytochromes P-450 and b5, and are brought about by primary action(s) of Me-Chl-Na on the control mechanism of microsomal hemoprotein levels, not by direct action of Me-Chl-Na on the system.

Characterization of 3-hydroxy-3-methylglutaryl coenzyme A reductase in human adrenal cortex.

Our study compares the properties of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase from a human metastatic virilizing carcinoma and that from normal adrenal glands obtained from kidney donors. Optimal conditions for enzyme assay were obtained when a 50-mM imidazole buffer (pH 7.2) containing 5 mM EDTA, 250 mM NaCl, 1 mM phenyl-methylsulfonylfluoride, 0.1 mM leupeptin, and 5.5 mM dithiothreitol (DTT) was used. A 30-min preincubation period preceding addition of substrates enhanced reductase activity by 1.75-fold. In crude microsomal preparations, Km values were similar for both tumor and normal tissues and varied between 4 and 5 microM (S)HMG-CoA. The presence of NaF in homogenization and incubation media decreased the maximum velocity, but not the Km. A partially purified rat liver phosphorylase phosphatase preparation or a similar preparation from the carcinoma restored to maximal levels the reductase activity of microsomes prepared in the presence of NaF. A Km of 96 microM NADP was found for the carcinoma microsomal preparation. Preincubation of microsomes in the presence of monothioglycerol or DTT resulted in an increased reductase activity, suggesting a possible inactive enzyme precursor(s) consisting of disulfide-linked units. Reactions of the DTT-activated enzyme incubated in the presence of increasing amounts of NADPH showed sigmoidal kinetics. Under reducing conditions, sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting revealed the presence of a 92.5K mol wt protein band that reacted with a rat antireductase antibody. Reductase activity in different regions of the carcinoma varied from 679-1763 pmol/mg protein, with an average of 1146 pmol. In three normal adrenal glands we found values of 23.4, 48.1, and 36 pmol. We concluded that the expression of HMG-CoA reductase activity was elevated in human adrenal carcinoma.

Measurement of human leukocyte microsomal HMG-CoA reductase activity.

Methods were developed for determination of microsomal HMG-CoA reductase activity from freshly isolated human lymphocytes, monocytes, and granulocytes or cultured human lymphoid cells. Reductase activity in monocytes is approximately twice that in lymphocytes or granulocytes. The activity in cultured cells is approximately 34-fold greater than that in freshly isolated cells. Assay conditions were such as to preclude formation of HMG-CoA cleavage products. Leukocyte reductase activity was inhibited by dichloroacetate, a noncompetitive inhibitor of rat liver reductase and a serum cholesterol-lowering agent in man. Measurement of microsomal reductase activity from freshly isolated leukocytes may prove useful in assessing in vivo regulation of cholesterol synthesis in man.

Topography of rat liver microsomal 3-hydroxy-3-methylglutaryl coenzyme A reductase

The orientation of 3-hydroxy-3-methylglutaryl coenzyme A reductase within the endoplasmic reticulum was investigated. Microsomal reductase activity was not latent, as addition of various detergents failed to activate the enzyme. Reductase activity was readily inhibited in intact microsomes by impermeable inhibitors such as trypsin, mercury dextran and anti-reductase IgG. Under the conditions used, these agents did not affect the intactness of microsomes as determined by latency of mannose-6-phosphate phosphohydrolase activity. The sensitivity to these inhibitors was not increased in disrupted microsomes. It is concluded that the domain containing the active site of the reductase is situated on the cytosolic surface of the endoplasmic reticulum.

Effects of transient anaerobic stress on fatty acid desaturation and electron-transport system in Tetrahymena microsomes

A transient anaerobic stress of Tetrahymena pyriformis NT-1 for 1 h resulted in slight decreases of unsaturated fatty acids in overall lipid composition. The rates of incorporation of [ 3H]palmitic acid plus sodium [ 14C]acetate into unsaturated fatty acids (palmitoleate, 16:1 Δ 9; oleate, 18:1 Δ 9; linoleate, 18:2 Δ 9,12; γ-linolenate, 18:3 Δ 6,9,12) were drastically reduced by exposing cells to anaerobic conditions for 1 h (anaerobic cells), and thereafter restored to the original levels at 4 h after subsequent reaeration. Activities of Δ 9- and Δ 12-desaturases (palmitoyl-CoA, stearoyl-CoA, oleoyl-CoA) and the terminal component of the desaturation system (cyanide-sensitive factor) decreased to 25–30% in microsomes from anaerobic cells, compared to those from control and reaerobic cells. Furthermore, changes induced by the anaerobic shock in the activities of microsomal reductases and the content of cytochrome b 5 were observed to behave comparably to changes in activities of fatty acyl-CoA desaturases. During the anaerobic state, the half-lives of microsomal enzyme activities (desaturases, cyanide-sensitive factor, reductases) and cytochrome b 5 content were diminished to 30–50% of those of control microsomes. But the K m values of these enzymes did not differ between microsomes from either anaerobic or control cells. These results suggest that the decreased activities of desaturases during anaerobic stress result principally from the reduction of cyanide-sensitive factor content, which would be caused by its instability and repressed synthesis.