Thiol-protein disulphide oxidoreductases. Assay of microsomal membrane-bound glutathione-insulin transhydrogenase and comparison with protein disulphide-isomerase.

1. Inhibition of endogenous microsomal NADPH oxidase by CO enables membrane-bound glutathione-insulin transhydrogenase (EC 1.8.4.2) to be assayed conveniently by a linked assay involving NADPH and glutathione reductase (EC 1.6.4.2). 2. The specific activity of the enzyme in rat liver microsomal preparations is of the order of 1 nmol of oxidized glutathione formed/min per mg of membrane protein. 3. The specific activity of the enzyme is comparable in rough and smooth microsomal fractions, and the activity is not affected by treatment with EDTA and the removal of ribosomes from rough microsomal fractions. 4. Membrane-bound glutathione-insulin transhydrogenase is not affected by concentrations of deoxycholate up to 0.5%, whereas protein disulphide-isomerase (EC 5.3.4.1) is drastically inhibited. 5. On these grounds it is concluded that, in rat liver microsomal fractions, glutathione-insulin transhydrogenase and protein disulphide-isomerase activities are not both catalysed by a single enzyme species.     

Regulation of expression of male-specific rat liver microsomal 3 beta-hydroxysteroid dehydrogenase

In the steroidogenic pathways present in the gonads and adrenal cortex, 3 beta-hydroxysteroid dehydrogenase isomerase (3 beta HSD) is a key enzyme which controls the formation of delta 4-3-ketosteroids from delta 5-3 beta-hydroxysteroids. Herein, we used an antibody against human placental 3 beta HSD and a rat testicular 3 beta HSD cDNA probe to study the expression of rat liver 3 beta HSD mRNA and protein. Rat liver microsomal 3 beta HSD activity has been previously reported to exhibit a significant sex difference, with much higher activity in the male. We have shown an age-dependent increase in levels of immunoreactive 3 beta HSD through the time of maturation of the male rat. The immunoreactive protein, of similar molecular size to the human placental and rat testicular 3 beta HSD, was localized to the microsomal fraction of liver and was concentrated in pericentral locations. Immunoreactive protein was not detected in liver of immature (before 25 days of age) rats of either sex or in adult female liver. Northern blot analysis of liver and testicular RNA with a rat testicular 3 beta HSD cDNA probe revealed the presence of a 1.6-kilobase mRNA species in addition to the major 2.1-kilobase mRNA species in adult male liver, neither of which was detected in immature or adult female liver RNA. Hypophysectomy of female rats or treatment with testosterone implants caused induction of liver 3 beta HSD protein, while continuous infusion of GH to male rats decreased the level of 3 beta HSD protein. Similarly, the levels of the mRNA species were decreased after GH treatment. Using [3 alpha-3H]dehydroepiandrosterone as substrate for 3 beta HSD activity, we determined the apparent Km for liver microsomal NAD(+)-dependent 3 beta HSD activity to be 20 microM in both adult male and female liver and was much greater than the Km of rat Leydig tumor 3 beta HSD activity (0.2 microM). Liver 3 beta HSD activity was inhibited by trilostane, a proven inhibitor of gonadal and adrenal 3 beta HSD activity. A rat liver 3 beta HSD cDNA was isolated from a male liver cDNA library that was closely related to the type II 3 beta HSD form of rat ovary but different from type III liver 3 beta HSD. The enzyme obtained upon expression of this cDNA had properties characteristic of male-specific NAD(+)-dependent liver microsomal 3 beta HSD (i.e. high apparent Km for dehydroepiandrosterone) and distinct from those of the high affinity gonadal type I 3 beta HSD.(ABSTRACT TRUNCATED AT 400 WORDS)     

Human liver microsomal steroid metabolism: identification of the major microsomal steroid hormone 6 beta-hydroxylase cytochrome P-450 enzyme

Cytochrome P-450-dependent steroid hormone metabolism was studied in isolated human liver microsomal fractions. 6 beta hydroxylation was shown to be the major route of NADPH-dependent oxidative metabolism (greater than or equal to 75% of total hydroxylated metabolites) with each of three steroid substrates, testosterone, androstenedione, and progesterone. With testosterone, 2 beta and 15 beta hydroxylation also occurred, proceeding at approximately 10% and 3-4% the rate of microsomal 6 beta hydroxylation, respectively, in each of the liver samples examined. Rates for the three steroid 6 beta-hydroxylase activities were highly correlated with each other (r = 0.95-0.97 for 25 individual microsomal preparations), suggesting that a single human liver P-450 enzyme is the principal microsomal 6 beta-hydroxylase catalyst with all three steroid substrates. Steroid 6 beta-hydroxylase rates correlated well with the specific content of human P-450NF (r = 0.69-0.83) and with its associated nifedipine oxidase activity (r = 0.80), but not with the rates for debrisoquine 4-hydroxylase, phenacetin O-deethylase, or S-mephenytoin 4-hydroxylase activities or the specific contents of their respective associated P-450 forms in these same liver microsomes (r less than 0.2). These correlative observations were supported by the selective inhibition of human liver microsomal 6 beta hydroxylation by antibody raised to either human P-450NF or a rat homolog, P-450 PB-2a. Anti-P-450NF also inhibited human microsomal testosterone 2 beta and 15 beta hydroxylation in parallel to the 6 beta-hydroxylation reaction. This antibody also inhibited rat P-450 2a-dependent steroid hormone 6 beta hydroxylation in uninduced adult male rat liver microsomes but not the steroid 2 alpha, 16 alpha, or 7 alpha hydroxylation reactions catalyzed by other rat P-450 forms. Finally, steroid 6 beta hydroxylation catalyzed by either human or rat liver microsomes was selectively inhibited by NADPH-dependent complexation of the macrolide antibiotic triacetyloleandomycin, a reaction that is characteristic of members of the P-450NF gene subfamily (P-450 IIIA subfamily). These observations establish that P-450NF or a closely related enzyme is the major catalyst of steroid hormone 6 beta hydroxylation in human liver microsomes, and furthermore suggest that steroid 6 beta hydroxylation may provide a useful, noninvasive monitor for the monooxygenase activity of this hepatic P-450 form.     

Inhibitory monoclonal antibodies against rat liver alcohol dehydrogenase

Eleven hybridoma clones which secrete monoclonal antibodies against purified rat liver alcohol dehydrogenase (EC 1.1.1.1) were isolated. Antibodies (R-1-R-11) were identified by their ability to bind to immobilized pure alcohol dehydrogenase in an enzyme-linked immunoadsorbent assay, in which antibody R-9 showed the highest binding capacity. Except for R-1 and R-7, all antibodies inhibited catalytic activity of the enzyme isolated from inbred (Fischer-344) or outbred (Sprague-Dawley) strains (R-11 greater than R-9 greater than R-4 greater than R-6 greater than R-10 greater than R-8 greater than R-2 = R-3 = R-5). The inhibition of enzyme activity by antibodies was noncompetitive for ethanol and NAD+, and was dependent on antibody concentration and incubation time. Antibodies R-4, R-9, and R-11 were most effective when enzyme activity was assayed below pH 7.7-7.8, a condition thought to protonate the enzyme's active center. These three antibodies did not inhibit horse liver alcohol dehydrogenase activity, indicating their species specificity. Such antibodies will be useful to delineate structural and functional roles of rat liver alcohol dehydrogenase.     

Microassay of heme oxygenase by high-performance liquid chromatography: Application to assay of needle biopsies of human liver

We developed a microassay for heme oxygenase, in which bilirubin (BR) production was measured by HPLC, and compared it to previously reported spectrophotometric methods. The microassay required as little as 5 mg wet human, rat, or chick embryo liver. Using the HPLC assay, values for heme oxygenase activity in extracts (10,000 g supernatant) of normal human liver obtained by needle biopsies were 44 +/- 7 (pmol BR.min-1.mg protein-1). Spectrophotometric assays of homogenates of human liver resulted in low values for heme oxygenase, due to unknown sources of interference. Comparative values of microsomal heme oxygenase activity were 294 +/- 25, 95 +/- 3, and 87 +/- 9 pmol BR.min-1.mg protein-1 for chick, rat, and human livers, respectively.     

Genetically determined differences in liver alcohol dehydrogenase activity in male rats

Alcohol dehydrogenase (EC 1.1.1.1) activity was measured in liver extracts from one outbred and three inbred strains of rats. Strain-specific differences in enzyme activity were observed in the adult male rats. The differences appeared as the animals reached puberty. Studies on the enzyme purified from Sprague-Dawley and ACI rats indicate that the enzymes in these strains are identical and that the difference in activity found in liver extracts is due to differences in the amount of enzyme present. Genetic crosses between Sprague-Dawley and ACI rats suggest that the liver content of alcohol dehydrogenase is controlled by an autosomal regulatory locus with the characteristics of a temporal gene.     

Purification and characterization of microsomal and lysosomal beta-glucuronidase from rat liver by use of immunoaffinity chromatography.

Rat liver beta-glucuronidase (EC 3.2.1.31), both from microsomal and lysosomal fractions, were purified about 9500-fold over the homogenate with high yield using affinity chromatography prepared by coupling purified specific immunoglobulin G against rat preputial gland beta-glucuronidase to Sepharose 2B and isoelectric focusing. The purified enzymes appeared homogeneous on electrophoresis in polyacrylamide gel and had a molecular weight of approximately 310000. In dodecylsulfate polyacrylamide gel electrophoresis, the microsomal beta-glucuronidase showed a single band corresponding to a molecular weight of 79000, while the lysosomal beta-glucuronidase had three distinct bands which consisted of one major and two minor bands corresponding to molecular weight of 79000, 74000, and 70000, respectively. A broad pH activity curve with a single optimum at pH 4.4 was observed in both the microsomal and the lysosomal beta-glucuronidases. Immunological gel diffusion technique with rabbit antiserum against rat liver lysosomal beta-glucuronidase revealed that both enzymes had the same or quite similar antigenic determinants.     

Hydroxymethylglutaryl CoA reductase and the modulation of microsomal cholesterol content by the nonspecific lipid transfer protein

The influence of membrane cholesterol content on 3-hydroxy-3-methylglutaryl CoA reductase (HMG-CoA reductase, EC 1.1.1.34) in rat liver microsomes was investigated. Microsomes were enriched in cholesterol by incubation with egg phosphatidylcholine-cholesterol vesicles and the nonspecific lipid transfer protein from rat liver. By this method, the microsomal cholesterol content was 2.5-fold enhanced up to final concentrations of 140 nmol cholesterol per mg microsomal protein. In another experiment, microsomes isolated from rats fed a cholesterol-rich diet were depleted of cholesterol by incubation with egg phosphatidylcholine vesicles and the transfer protein. Both cholesterol enrichment and depletion had virtually no effect on the microsomal HMG-CoA reductase activity. In another set of experiments, normal rat liver microsomes were incubated with human serum, resulting in a rise of microsomal cholesterol content. This was reflected in an increase of acyl-CoA:cholesterol acyltransferase activity but failed to have an effect on HMG-CoA reductase.     

Structure and organization of the microsomal xenobiotic epoxide hydrolase gene

The gene for the microsomal xenobiotic rat liver epoxide hydrolase has been isolated and characterized. Clones were obtained from a Wistar Furth Charon 35 genomic library by hybridization with a full-length epoxide hydrolase cDNA. The gene for the xenobiotic epoxide hydrolase is approximately 16 kilobases in length and consists of 9 exons ranging in size from 109 to 420 base pairs and 8 intervening sequences, the largest of which is 3.2 kilobases. S1-nuclease mapping, primer extension studies, and sequence analysis were used to determine the 5' cap site and the size of the first exon (170 base pairs). Regulatory sequences analogous to TATA, CCAAT, and core enhancer sequences were noted in the 5'-flanking region of the gene. The cDNA and gene for epoxide hydrolase displayed nucleotide sequence identity although they were isolated from different rat strains. Also, Southern blot analysis of restricted liver DNA from inbred Fischer 344 and Wistar Furth rat strains, and outbred Sprague-Dawley rats indicated a high degree of structural similarity for the epoxide hydrolase gene within these three strains. Only a single functional epoxide hydrolase gene was identified and no evidence of hybridization to the genes for the microsomal cholesterol epoxide hydrolase or the cytosolic epoxide hydrolase was observed. However, a pseudogene for the microsomal xenobiotic epoxide hydrolase was isolated and characterized from the genomic library.     

Endothelial beta2 adrenergic signaling to AKT: role of Gi and SRC

We have recently demonstrated that endothelial beta(2) adrenergic receptors (beta(2)AR) regulate eNOS activity and consequently vascular tone, through means of PKB/AKT. In this work we explored the signal transduction pathway leading to AKT/eNOS activation in endothelial cells (EC). Using pharmacological and molecular inhibitors both in cultured EC cells and in ex vivo rat carotid preparations, we found that G(i) coupling of the beta(2)AR is needed for AKT activation and vasorelaxation. Since endothelial activation is sensitive to pertussis toxin but not to G(ibetagamma) inhibition by betaARKct, we conclude that G(alphai) mediates betaAR induced AKT activation. Downstream, betaAR signalling requires the soluble tyrosine kinase SRC, as both in cultured EC and rat carotid, the mutant dominant negative of SRC prevent beta(2)AR induced endothelial activation and vasodilation. In EC, G(alphai) directly interacts with SRC and this interaction leads to SRC activation and phosphorylation in a manner that is regulated by beta(2)AR stimulation. We propose a novel signal transduction pathway for beta(2)AR stimulation trough G(alphai) and SRC, leading to activation of AKT.     

Synthesis of phosphatidylcholine and phosphatidylglycerol in rat lung mitochondria

Summary The mitochondrial fraction of adult rat lung contains choline phosphotransferase (EC 2.7.8.2) activity which can not be explained by microsomal contamination estimated on the basis of marker enzyme distribution. Mitochondrial (¹⁴C)glycerol-3-phosphate incorporation into PC (phosphatidylcholine) can be distinguished from the microsomal incorporation by different sensitivity to N-ethylmaleimide inhibition. The data indicate that rat lung mitochondria have the intrinsic capability to synthesize PC. Both synthesis of PC and PG (phosphatidylglycerol) are susceptible to isotonic tryptic attack against the cytoplasmic face of isolated rat lung mitochondria, suggesting the outer membrane location of crucial activities involved in the formation of these phospholipids. Rat liver mitochondria are different from rat lung mitochondria with respect to their capability to synthesize PC, their rate of (¹⁴C)glycerol-3-phosphate incorporation into PG as well as the submitochondrial site of PG formation.Abbreviations PC Phosphatidylcholine - PG Phosphatidylglycerol - PA Phosphatidic Acid - DPG Diphosphatidylglycerol (cardiolipin) - CPT Choline Phosphotransferase (EC 2.7.8.2) - SEM Standard Error of Mean     

Human microsomal glutathione S-transferase. Its involvement in the conjugation of hexachlorobuta-1,3-diene with glutathione.

A microsomal glutathione S-transferase (GST) was purified from human liver. This enzyme was shown to have characteristics similar to those of the rat microsomal GST described by Morgenstern & De Pierre [(1983) Eur. J. Biochem. 134, 591-597]. The specific activity of human microsomal GST towards 1-chloro-2,4-dinitrobenzene or cumene hydroperoxide can be stimulated by treating the enzyme with N-ethylmaleimide. This enhancement of activity is accompanied by increased sensitivity to inhibition by haematin and cholic acid. The subunit Mr values of the rat and human enzymes are similar (approx. 17,300), and the proteins are immunologically related. During purification, both human and rat microsomal GST enzymes are the only hepatic proteins obtained from Triton X-100-solubilized microsomal fractions that show activity towards the nephrotoxin hexachlorobuta-1,3-diene. The involvement of microsomal GST in toxification reactions is discussed.     

Endogenous esterification of bilirubin by liver microsomes. Evidence for an intramicrosomal pool of UDP-glucose and lumenal orientation of bilirubin UDP-glycosyltransferase

Conjugation of natural bilirubin (BR) depends on a hepatic microsomal UDP-glycosyltransferase using UDP-Glc, UDP-xylose, and predominantly UDP-GlcA. We found that esterification of BR occurred when washed intact microsomes derived from rat or guinea pig liver were incubated with BR in the absence of added UDP-sugar. This endogenous esterification was shown to lead predominantly to formation of the two positional isomers of BR monoglucoside and displayed the same regioselectivity as found for the BR monoglucosides formed by microsomes incubated with a saturating concentration of added UDP-Glc. This finding and absence of endogenous esterification in liver microsomes from mutant rats lacking BR UDP-glycosyltransferase activities demonstrated that endogenous esterification depended on UDP-glycosyltransferase and indicated, therefore, that UDP-Glc was present in the intact microsomal vesicles. With UDP-Glc added to the extramicrosomal incubation medium, BR glucosidation was markedly enhanced when the membrane permeability barrier was disrupted by pretreatment of the microsomes with detergent, sonication, or Staphylococcus aureus alpha-toxin. In contrast, such membrane disruption resulted in abolishment of endogenous esterification of BR, and a direct relationship was found between impairment of endogenous esterification and degree of vesicle disruption, suggesting that the UDP-Glc on which endogenous esterification depended was present in the lumenal space of the microsomes. Kinetic evidence and absence of an effect of increasing the microsomal concentration of dolichol-P-Glc (Dol-P-Glc) on endogenous esterification excluded direct or indirect involvement of Dol-P-Glc in the endogenous esterification reaction. Preincubation of intact microsomes with UDP-Glc or UDP-xylose at 37 degrees C, but not at 0 degrees C, led to expansion of the microsomal UDP-sugar pool on which endogenous esterification depended, suggesting that both UDP-sugars can enter the microsomal vesicles by a temperature-dependent mechanism. In contrast to these findings, no increase of BR esterification was detected when the microsomes had been preincubated at 37 degrees C with UDP-GlcA. We conclude that native, intact microsomes contain a lumenal pool of endogenous UDP-Glc and that BR UDP-glucosyltransferase and UDP-xylosyltransferase, by virtue of a lumenal orientation, have direct access to the postulated intramicrosomal pool of nucleotide sugar.     

Effects of phenobarbitone on hepatic microsomal enzyme activity and liver blood flow in spontaneously hypertensive rats.

Hepatic microsomal enzyme activity, liver blood flow and pentobarbitone sleeping time were determined in spontaneously hypertensive rats (SHR) and normotensive Wistar rats (NR) after pretreatment with saline or phenobarbitone. In NR and SHR the increases in total liver blood flow produced by phenobarbitone were sufficient to maintain liver perfusion despite the increase in liver weight and in both strains of rat the increase was entirely due to increased portal venous return. Saline pretreated SHR had shorter pentobarbitone sleeping times than control NR and their livers had greater total cytochrome c reductase activities and total microsomal protein than those of NR but cytochrome P-450 contents were not significantly different. Phenobarbitone significantly shortened sleeping times in both strains but NR still slept longer than SHR. Total microsomal protein, cytochrome P-450 content and cytochrome c reductase activity were increased by phenobarbitone in both SHR and NR but the increases in cytochrome P-450 and cytochrome c reductase were greater in the hypertensive rats.     

Studies of microsomal glucose-6-phosphatase on liver of irradiated rats

The activity of microsomal glucose-6-phosphatase (EC 3.1.3.9) on male rat liver was measured 1-9 days after whole-body gamma-irradiation. A marked fall of activity, expressed per whole liver, was observed reaching a minimum on the 4th day following irradiation. The enzyme activity is partially and momentarily restored (on day 7), before a new decrease occurred. Furthermore, when the results are expressed per milligram of microsomal proteins, there was no change. Cysteamine, when injected in vivo, kept up the glucose-6-phosphatase of whole liver. On day 4, a histochemical demonstration of the enzyme in liver cells is in accordance with enzyme measures. These observations suggested that the enzyme quantity was altered during the acute radiation syndrome in the rat.     

Regulation of human leukocyte microsomal hydroxymethylglutaryl-CoA reductase activity by a phosphorylation and dephosphorylation mechanism

The activity of microsomal 3-hydroxy-3-methylglutaryl coenzyme A reductase (EC 1.1.1.34), obtained from cultured human IM-9 lymphoid cells or freshly isolated human peripheral blood leukocytes, is modulated by a phosphorylation/dephosphorylation mechanism. Addition of MgATP + ADP to IM-9 cell microsomal reductase leads to a time-dependent loss of enzyme activity. Inactivated reductase is reactivated by rat liver reductase phosphatase. Kinase-dependent IM-9 cell microsomal reductase, prepared by heating IM-9 microsomes for 15 min at 50°C, is inactivated in the presence of MgATP and ADP only after addition of cytosolic reductase kinase from either IM-9 cells, freshly isolated leukocytes or rat liver. Inactivation is time-dependent and dependent on the cytosolic protein concentration. Inactivated reductase is reactivated by rat liver reductase phosphatase. For cultured IM-9 cells and freshly isolated leukocytes incubated with culture medium for 2 h, the ratios of active (unphosphorylated) to total (phosphorylated + unphosphorylated) reductase activity are 0.22 and 0.43, respectively. Thus, in addition to its regulation by changes in the amount of total enzyme protein, human leukocyte reductase activity is also modulated by a phosphorylation/dephosphorylation mechanism.     

AM1, a glycoprotein from the submandibular glands of the mouse, has esterolytic and amidolytic activities

The activity of microsomal 3-hydroxy-3-methylglutaryl coenzyme A reductase (EC 1.1.1.34), obtained from cultured human IM-9 lymphoid cells or freshly isolated human peripheral blood leukocytes, is modulated by a phosphorylation/dephosphorylation mechanism. Addition of MgATP + ADP to IM-9 cell microsomal reductase leads to a time-dependent loss of enzyme activity. Inactivated reductase is reactivated by rat liver reductase phosphatase. Kinase-dependent IM-9 cell microsomal reductase, prepared by heating IM-9 microsomes for 15 min at 50 degrees C, is inactivated in the presence of MgATP and ADP only after addition of cytosolic reductase kinase from either IM-9 cells, freshly isolated leukocytes or rat liver. Inactivation is time-dependent and dependent on the cytosolic protein concentration. Inactivated reductase is reactivated by rat liver reductase phosphatase. For cultured IM-9 cells and freshly isolated leukocytes incubated with culture medium for 2 h, the ratios of active (unphosphorylated) to total (phosphorylated + unphosphorylated) reductase activity are 0.22 and 0.43, respectively. Thus, in addition to its regulation by changes in the amount of total enzyme protein, human leukocyte reductase activity is also modulated by a phosphorylation/dephosphorylation mechanism.     

Characteristics and subcellular localization of pristanoyl-CoA synthetase in rat liver.

We have investigated the activation of pristanic acid to its CoA-ester in rat liver. The results show that peroxisomes, mitochondria as well as microsomes contain pristanoyl-CoA synthetase activity. On the basis of competition experiments and immunoprecipitation studies using antibodies raised against rat liver microsomal long-chain fatty acyl-CoA synthetase (EC 6.2.1.3) we conclude that pristanic acid is activated by the same enzyme which activates long-chain fatty acids, i.e., long-chain fatty acyl-CoA synthetase.     

The cryptic nature of hepatic microsomal sulphite oxidase

1. The activity of rat liver microsomal sulphite oxidase (EC 1.8.3.1) was increased several-fold on aging of microsomes, on delipidation by extraction with acetone or on solubilization with deoxycholate, suggesting its existence in a cryptic state. 2. In rat liver most of the sulphite oxidase was present in the nuclear fraction and only a small portion in the microsomes. 3. Microsomal sulphite oxidase activity was low in the developing embryo and increased rapidly after birth.     

Subcellular distribution of hepatic bile acid-conjugating enzymes.

1. The subcellular location of enzymes conjugating bile acids with glycine or taurine was investigated by centrifugation of rat liver homogenates. 2. [14C]Cholic acid-conjugating activity was predominantly associated with the soluble-microsomal region of the gradient after centrifugation in a Ti-15 zonal rotor but the bulk of the conjugating activity sedimented with mitochondrial-lysosomal fractions in differential pelleting experiments. 3. Cholate: CoA ligase (EC 6.2.1.7) and cholyltransferase (EC 2.3.1) were not enriched in purified Golgi or plasma-membrane fractions. Cholate: CoA ligase was distributed evenly between rough- and smooth-surfaced microsomal subfractions but cholyltransferase showed a dual soluble-rough microsomal activity distribution. 4. Sedimentation of cholyltransferase in mitochondria-enriched fractions prepared by differential centrifugation appears to be an artefact of sedimentation of rough microsomal membranes in mitochondrial fractions. 5. The subcellular distribution of bile acid-conjugating enzymes is discussed with reference to hepatic processing of bile acids.