Effects of diffusible products of peroxidation of rat liver microsomal lipids

The effects on cellular structures of products of peroxidation of rat liver microsomal lipids were investigated. A system containing actively peroxidizing liver microsomal fraction was separated from a revealing or target system by a dialysis membrane. The target system, contained in the dialysis tube, consisted of either intact cells (erythrocytes) or subcellular fractions (liver microsomal fraction). When liver microsomal fractions were incubated with NADPH (or an NADPH-generating system), lipid peroxidation, as measured by the amount of malonaldehyde formed, occurred very rapidly. The malon-aldehyde concentration tended to equilibrate across the dialysis membrane. When the target system consisted of erythrocytes, haemolysis occurred abruptly after a lag phase. The lysis was greatly accelerated when erythrocytes from vitamin E-deficient rats were used, but no haemolysis was observed when erythrocytes from vitamin E-treated rats were used. When, in the same system, freshly prepared liver microsomal fractions were exposed to diffusible factors produced by lipid peroxidation, the glucose 6-phosphatase activity markedly decreased. A similar decrease in glucose 6-phosphatase activity, as well as a smaller but significant decrease in cytochrome P-450, was observed when the target microsomal fractions were exposed to diffusible factors derived from the peroxidation of liver microsomal lipids in a separate preincubation step. These and additional experiments indicated that the toxicological activity is relatively stable. Experiments in which the hepatic microsomal fractions destined for lipid peroxidation contained radioactively labelled arachidonic acid, previously incorporated into the membranes, showed that part of the radioactivity released from the microsomal fraction into the incubation medium entered the dialysis tube and was recovered bound to the constituents of the microsomal fractions of the target system. These results indicate that during the course of the peroxidation of liver microsomal lipids toxic products are formed that are able to induce pathological effects at distant loci.     

Activation of hypolipidaemic drugs to acyl-coenzyme A thioesters.

Compounds possessing the characteristics of CoA thioesters of the hypolipidaemic peroxisome proliferators clofibric acid, nafenopin and ciprofibrate were formed on incubation of the drugs with rat liver microsomal fractions, ATP and CoA. The reactivity of the drugs correlated with their pharmacological potency. It is proposed that the active species of these compounds are their acyl-CoA thioesters.     

Formation of (4R)- and (4S)-4-hydroxyochratoxin A from ochratoxin A by liver microsomes from various species.

Two metabolic products were formed from ochratoxin A by human, pig, and rat liver microsomal fractions in the presence of reduced nicotinamide adenine dinucleotide phosphate. They were isolated from the incubation mixture in the presence of pig liver microsomes by extraction, thin-layer chromatography, and high-pressure liquid chromatography Their structures are suggested to be (4R)- and (4S)-4-hydroxyochratoxin A on the basis of mass and nuclear magnetic resonance spectroscopy. Km and the maximum velocity for the formation of the two metabolites by human, pig, and rat microsomes were determined. Their formation was inhibited by carbon monoxide and metyrapone. The results indicate that the microsomal hydroxylation system is a cytochrome P-450 and that different species are involved in the formation of the two epimeric forms of 4-hydroxyochratoxin A.     

UDP-glucose: ceramide glucosyltransferase of rat brain: an association with smooth microsomes and requirement of an intact membrane for enzyme activity

Subcellular distribution of rat brain UDP-glucose:ceramide glucosyltransferase, the enzyme which catalyses the first step during the sequential addition of carbohydrate moieties for ganglioside biosynthesis, was studied. The activity of the enzyme was highest in the fraction rich in microsomes. Subfractionation of crude microsomal fractions resulted in a further enrichment of the enzyme activity in the fraction which contained smooth microsomes, thus suggesting that the enzyme is associated with microsomal membranes. The enzyme does not appear to be associated with synaptosomes or myelin. Treatment of the microsomal fraction with phospholipase A and C or detergents resulted in the loss of enzyme activity. Preincubation of the microsomal fraction at 37 °C also resulted in a loss of enzyme activity. These results suggest the requirement of specific membrane structure for the activity of the enzyme UDP-glucose:ceramide glucosyltransferase of rat brain. The amount of the enzyme activity lost during preincubation was dependent on the composition of the incubation medium and the age of the rats from which microsomal fractions were obtained.     

DISTRIBUTION OF ENZYMES CLEAVING PYRIDINE NUCLEOTIDES IN ANIMAL TISSUES

1. The distribution of DPN and DPNH pyrophosphatases and DPNase in centrifugally prepared fractions of organs of several species of animals is reported. 2. A DPNH pyrophosphatase was found in the soluble fraction of pigeon and of rabbit liver. This enzyme did not split DPN but accounted for over 50 per cent of the DPNH pyrophosphatase activity of the whole homogenates. 3. All the organs tested, including the pigeon liver and rabbit liver, contained a microsomal pyrophosphatase that attacked both DPNH and DPN. This microsomal enzyme split DPNH faster than DPN in all cases. 4. DPN pyrophosphatase and DPNase activity were generally concentrated in the microsomal fraction of liver, of kidney, and of brain. 5. The DPNase of hamster liver was virtually inactive at pH 7.5 but was optimally active at pH 5.5. Considerable difference was found with respect to pH on the activity of DPNase from organs of different animals. 6. The inhibition of mitochondrial and microsomal DPNH oxidation by nicotinamide was noted during the course of these experiments. 7. The significance of some of the distribution patterns is discussed.     

In vitro study of the aromatic hydroxylation of 1-methyltetrahydro-beta-carboline (methtryptoline) in rat

The incubation of 1-methyltetrahydro-beta-carboline (1-MeTHBC) with hepatocytes isolated from 3-methylcholanthrene-treated rats led to formation of the 5-, 6- and 7-hydroxylated products. The hydroxylating activity was associated with the microsomal fraction as indicated by testing different subcellular fractions. The highest activity for hydroxylating 1-MeTHBC was found in liver which was about ten times as active as lung. Only a trace amount of hydroxylating activity was present in brain and kidney tissue. Analysis using chiral gas chromatography revealed an unequal abundance of enantiomers in all three products. The formation of the 5-, 6- and 7-hydroxylated products was confirmed in vivo by analysis of 24 h urine samples after intraperitoneal administration of 1-MeTHBC to 3-methylcholanthrene-treated rats.     

Enzymatic sulfation of exogenous high molecular weight glycopeptides by microsomal fraction of the rabbit uterine endometrium

Incorporation of radioactive sulfate into exogenous glycopeptides and glycoproteins from adenosine 3'-phosphate 5'-phospho[35S]sulfate was studied with the microsomal fraction of the uterine endometrium of rabbits. A high molecular weight (Mr greater than 750,000) glycopeptide fraction from rat adenocarcinoma was found to be active as substrate, while several other glycoproteins were not. The rate of incorporation of sulfate was almost proportional to the concentration of glycopeptide substrate, the quantity of microsomal fraction, and the length of the incubation period. Cellulose acetate membrane electrophoresis demonstrated that radioactivity was incorporated into the glycopeptide fraction. The radioactive glycopeptide was excluded from a Sephadex G-50 column, but the 35S radioactivity and oligosaccharides were found in the retarded fractions after treatment with alkali in the absence of sodium borohydride. These observations indicated the presence of an enzyme which catalyzes the transfer of sulfate residue from adenosine 3'-phosphate 5'-phosphosulfate into the carbohydrate units attached to the polypeptide via O-glycosidic linkage. The sulfotransferase activity was measured with the microsomal fractions from the animals which had been treated with female sex hormones. As a result, it was found that estrogen enhances the activity and that progesterone suppresses the effect of estrogen.     

Protein kinases of rat liver endoplasmic reticulum. Solubilisation, partial characterisation and comparison with protein kinases of rat liver cytosol.

Protein kinase associated with rat liver microsomes was only partly extracted by treatment with 1.5 M KCl. The enzyme was solubilised by Triton X-100 or sodium deoxycholate at the same or slightly higher detergent concentrations than microsomal marker components. The enzyme activity increased 2-3 fold upon solubilisation. Three peaks with protein kinase activity (fractions MI, MII and MIII) were resolved on DEAE-cellulose chromatography. Fraction MIII but not fractions MI or MII was activated by adenosine 3':5'-monophosphate (cyclic AMP). All fractions catalysed the phosphorylation of protamine and histones but not that of casein or phosvitin. Fractions MI and MIII had a similar substrate specificity and phosphorylated histones at a relatively much higher rate than did fraction MII. The isoelectric points were 8.1 for fraction MI, 5.5 for fraction MII and 4.9 for fraction MIII. On incubation of fraction MIII with cyclic AMP it was split into two catalytically active components with pI 8.1 and 7.35. The component with pI 8.1 was predominant and corresponded to fraction MI. Five protein kinase peaks were resolved from rat liver cytosol by DEAE-cellulose chromatography. Three of them (fractions CIa, CIIb and CIII) had the same properties as each of the microsomal kinase fractions. A forth fraction (CIIa) was cyclic-AMP-dependent and had the same substrate specificity as fractions MI and MIII. Its pI was 5.1, and it was split into two components by cyclic AMP (pI 8.1 and 7.35). In binding studies fraction CIIb bound more efficiently to microsomes than fraction CIII, while fractions CIa, CIIa and the microsomal protein kinase fractions did not bind appreciably. When microsomes were treated with trypsin exposed protein kinase was inactivated and the latency of the remaining enzyme increased substantially. Most of fraction MII was inactivated by trypsin while fraction MIII was resistant. The possible orientation of protein kinase fractions MII and MIII in the microsomal membrane is discussed.     

The activity of beta-hydroxy-beta-methylglutaryl-CoA reductase in the soluble fraction of rat liver]

Assay conditions are worked out for determination of activity of beta-hydroxy-beta-methylglutaryl-CoA reductase (HMG-CoA reductase) in 140.000 g supernatant fraction of the rat liver. Some kinetic properties of the enzyme are studied: the activity dependency on the incubation time, protein concentration, pH, glutathione, dithiothreitol and HMG-CoA contents in the incubation medium. The effect of Triton WR 1339 on the activity of HMG-CoA reductase in the liver 140.000 g supernatant and microsomal fractions is comparatively studied. Diurnal activity variations of soluble and microsomal enzymes are also investigated. It is suggested that the rat liver HMG-CoA reductase in the 140.000 g supernatant fraction is not identical to the enzyme located in the microsomal fraction.     

Characterization of phytol-phytanate conversion activity in rat liver

The enzymatic conversion of phytol to phytanic acid was investigated in rat liver postnuclear and other subcellular fractions using [1-3H]phytol as the substrate. The assay method involved incubation of the substrate with appropriate cofactors and the enzyme source, followed by subjecting the mixture to Folch partition and measuring the radioactivity in the upper layer. The phytol-phytanate conversion activity was present in mitochondrial and microsomal fractions. Cytosol had no activity. In mitochondrial fraction, investigation of cofactor requirements indicated that only NAD was required for activity. Other pyridine nucleotides supported the activity to a lesser extent when compared with NAD. FAD at 1 mM concentration did not support the activity. Bovine serum albumin (0.4 mg/ml) stimulated the activity. The reaction did not require molecular oxygen. From substrate kinetic studies, an apparent Km of 14.3 and 11.1 microM was calculated for phytol in mitochondrial and microsomal fractions, respectively. The amount of tritiated water produced from incubation increased linearly up to 7-8 min. The activity was linear with the amount of mitochondrial and microsomal protein up to 200 and 40 micrograms, respectively. Among the various rat tissue homogenates tested, liver had the highest activity. Spleen and kidney had 8-9% of the activity of liver. Brain possessed negligible activity. Both ethanol and pyrazole had no inhibitory effect on phytol-phytanate conversion. This observation and the absence of activity in cytosol suggests that alcohol dehydrogenase may not be involved in phytol-phytanate conversion.     

Inter-tissue and inter-species comparison of butyrylcholinesterases

Butyrylcholinesterase (BuChE) occurs in a multiple molecular forms whose catalytic activity depends on tissue distribution and species. The hypothesis led us to the study of BuChE catalytic properties focused on the inter-tissue and inter-species level with benzoylcholine and N-alkyl derivates of benzoylcholine (BCHn) as substrates. These compounds are soft disinfectants easily biodegradable to biologically inactive hydrolytic products, substituted choline and benzoic acid. Different sources of BuChE were used: rabbit and rat liver microsomal fraction (membrane-anchored enzyme) and serum (soluble form). Hydrolytic activity of both these BuChE forms was compared to rat recombinant BuChE (rBuChE). Hydrolytic product (benzoic acid) formation was recorded as function of time, and hydrolytic rate was determined. Tissue distribution of BuChE plays an important role in hydrolysis of BCHn. High BuChE activity was observed in a serum of both studied species rat and rabbit and was significantly dependent on a structure of substrates. Activity of soluble serum forms was the same as that for the rBuChE. Significant change of BuChE activity was recorded on the inter-species level in the microsomal fractions. It is because the rabbit microsomal BuChE activity had absolutely different course for all substrates as compared to rat microsomes. Inhibitory studies of BCHn enzymatic hydrolysis of all BuChE forms were performed to determine the level of BuChE participation in BCHn hydrolysis. It can be concluded that short-chain BCHn substrates are exclusively hydrolyzed by BuChE from all studied sources except for the rabbit liver microsomal fraction. Rabbit seems to have different enzymes involved in the hydrolysis of all studied BCHn compounds.     

Metabolism of DHEA by cytochromes P450 in rat and human liver microsomal fractions

Administration of dehydroepiandrosterone (DHEA) to rodents produces many unique biological responses, some of which may be due to metabolism of DHEA to more biologically active products. In the current study, DHEA metabolism was studied using human and rat liver microsomal fractions. In both species, DHEA was extensively metabolized to multiple products; formation of these products was potently inhibited in both species by miconazole, demonstrating a principal role for cytochrome P450. In the rat, use of P450 form-selective inhibitors suggested the participation of P4501A and 3A forms in DHEA metabolism. Human liver samples displayed interindividual differences in that one of five subjects metabolized DHEA to a much greater extent than the others. This difference correlated with the level of P4503A activity present in the human liver samples. For one subject, troleandomycin inhibited hepatic microsomal metabolism of DHEA by 78%, compared to 81% inhibition by miconazole, suggesting the importance of P4503A in these reactions. Form-selective inhibitors of P4502D6 and P4502E1 had a modest inhibitory effect, suggesting that these forms may also contribute to metabolism of DHEA in humans. Metabolites identified by LC-MS in both species included 16alpha-hydroxy-DHEA, 7alpha-hydroxy-DHEA, and 7-oxo-DHEA. While 16alpha-hydroxy-DHEA appeared to be the major metabolite produced in rat, the major metabolite produced in humans was a mono-hydroxylated DHEA species, whose position of hydroxylation is unknown.     

Phosphatidylinositol kinase,phosphatidylinositol-4-phosphate kinase and diacylglycerol kinase activities in rat brain subcellular fractions

Subcellular fractions isolated and purified from rat brain cerebral cortices were assayed for phosphatidylinositol (PI-), phosphatidylinositol-4-phosphate (PIP-), and diacylglycerol (DG-) kinase activities in the presence of endogenous or exogenously added lipid substrates and [γ-³²P]ATP. Measurable amounts of all three kinase activities were observed in each subcellular fraction, including the cytosol. However, their subcellular profiles were uniquely distinct. In the absence of exogenous lipid substrates, PI-kinase specific activity was greatest in the microsomal and non-synaptic plasma membrane fractions (150–200 pmol/min per mg protein), whereas PIP-kinase was predominantly active in the synaptosomal fraction (136 pmol/min per mg protein). Based on percentage of total protein, total recovered PI-kinase activity was most abundant in the cytosolic, synaptosomal, microsomal and mitochondrial fractions (4–11 nmol/min). With the exception of the microsomal fraction, a similar profile was observed for PIP-kinase activity when assayed in the presence of exogenous PIP (4 nmol/20 mg protein in a final assay volume of 0.1 ml). Exogenous PIP (4 nmol/20 mg protein) inhibited PI-kinase activity in most fractions by 40–70%, while enhancing PIP-kinase activity. PI- and PIP-kinase activities were observed in the cytosolic fraction when assayed in the presence of exogenously added PI or PIP, respectively, but not in heat-inactivated membranes containing these substrates. When subcellular fractions were assayed for DG-kinase activity using heat-inactivated DG-enriched membranes as substrate, DG-kinase specific activity was predominantly present in the cytosol. However, incubation of subcellular fractions in the presence of deoxycholate resulted in a striking enhancement of DG-kinase activities in all membrane fractions. These findings demonstrate a bimodal distribution between particulate and soluble fractions of all three lipid kinases, with each exhibiting its own unique subcellular topography. The preferential expression of PIP-kinase specific activity in the synaptic membranes is suggestive of the involvement of PIP₂ in synaptic function, while the expression of PI-kinase specific activity in the microsomal fraction suggests additional, yet unknown, functions for PIP in these membranes.     

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.     

Insulin degradation V. Unmasking of glutathione-insulin transhydrogenase in rat liver microsomal membrane

Glutathione-insulin transhydrogenase (glutathione:protein disulfide oxidoreductase, EC 1.8.4.2) inactivates insulin by cleaving its disulfide bonds. The distribution of GSH-insulin transhydrogenase in subcellular fractions of rat liver homogenates has been studied. From the distribution of insulin-degrading activity and marker enzymes (glucose-6-phosphatase and succinate-INT reductase) (INT, 2-p-iodophenyl-3-p-nitrophenyl-5-phenyl tetrazolium chloride) after cell fractionation by differential centrifugation, the immunological analysis of the isolated subcellular fractions with antibody to purified rat liver GSH-insulin transhydrogenase, and chromatographic analysis (on a column of Sephadex G-75 in 50% acetic acid) of the products formed from ¹²⁵I-labelled insulin after incubation with the isolated subcellular fractions, it is concluded that GSH-insulin transhydrogenase is located primarily in the microsomal fraction of rat liver homogenate. An enzyme(s) that further degrades insulin by proteolysis is located mainly in the soluble fraction; a significant amount of the protease(s) activity is also present in the mitochondrial fraction. The possibility has been discussed that the protease(s) acts upon the intermediate product of insulin degradation, A and B chains of insulin, rather than upon the intact insulin molecule itself.The GSH-insulin transhydrogenase in intact microsomes occurs in a latent state; it is readily released from the microsomal membrane and its activity is greatly increased by treatments which affect the lipoprotein membrane structure of microsomal vesicles. There include homogenization with a Polytron homogenizer, sonication, freezing and thawing, alkaline pH, the nonionic detergent Triton X-100, and phospholipases A and C.     

Brain processing of hemorphin-7 peptides in various subcellular fractions from rats

Hemorphins are multifunctional peptides derived from hemoglobin or blood processing. They have been found at high levels within the central nervous system where they have a direct effect on neuronal cells via peptidergic receptors. As relatively few studies have examined their metabolic stability in the brain, such investigation was performed to locate the cellular distribution of enzymatic activity against these peptides. High-performance liquid chromatography (HPLC) combined with electrospray ionisation mass spectrometry (ESI-MS) allows identification of degradation products resulting from incubation of hemorphin-7 peptides (LVV-hemorphin-7, VV-hemorphin-7 and hemorphin-7) with subcellular fractions isolated from rat brain tissue. Metabolic activities were found against the three peptides in brain homogenate and subcellular fractions with the highest metabolic activity (<3% peptide remaining after 10 min) observed in the microsomal fraction which processed hemorphin-7 peptides mainly into N-terminal fragments (giving LVVH5) suggesting action of brain-membrane enzymes with C-terminal specificity. Incubation of the ACE inhibitor captopril (0.2 μM) with microsomal fraction, together with LVVH7, decreased the processing of LVVH7 to form LVVH5 by 85%.     

Subcellular localization of 3 alpha, 7 alpha-dihydroxy- and 3 alpha,7 alpha,12 alpha-trihydroxy-5 beta-cholestanoyl-coenzyme A ligase(s) in rat liver

Liver peroxisomes from both rat and humans have previously been shown to contain enzymes that catalyze the oxidative cleavage of the C27-steroid side chain in the formation of bile acids. It has not been clear, however, whether the initial step, formation of the CoA-esters of the 5 beta-cholestanoic acids, also occurs in these organelles. In the present work the subcellular localization of 3 alpha,7 alpha,12 alpha-trihydroxy-5 beta-cholestanoyl-CoA (THCA-CoA) ligase (THCA-CoA synthetase) and of 3 alpha,7 alpha-dihydroxy-5 beta-cholestanoyl-CoA (DHCA-CoA) ligase in rat liver has been investigated. Main subcellular fractions and peroxisome-rich density gradient fractions from rat liver were incubated with THCA or DHCA, CoA, ATP, and Mg2+. Formation of THCA-CoA and DHCA-CoA was determined after high pressure liquid chromatography of the incubation extracts. The microsomal fraction contained the highest specific (and also relative specific) activity both for the formation of THCA-CoA and DHCA-CoA. The rates of THCA-CoA formation were further increased from 124-159 nmol/mg.hr-1 in crude microsomal fractions to 184-220 nmol/mg.hr-1 when studied in purified rough endoplasmic reticulum fractions. Formation of THCA-CoA in peroxisomal fractions prepared in Nycodenz density gradients could be accounted for by a small contamination (3-7%) by microsomal protein. The distribution of THCA-CoA ligase was different from that of palmitoyl-CoA ligase that was found to be localized also to the peroxisomal fractions.     

Cytosolic and microsomal epoxide hydrolases are immunologically distinguishable from each other in the rat and mouse

Antibodies raised to homogeneous rat liver microsomal epoxide hydrolase were used to distinguish microsomal epoxide hydrolase from epoxide hydrolase of cytosolic origin in mice and rats. Using double diffusion analysis in agarose gels, we show that anti-rat liver microsomal epoxide hydrolase forms a single precipitin line with solubilized microsomes from rat and mouse liver, but no reaction is seen with the corresponding cytosolic fractions. Rat or mouse microsomal epoxide hydrolase activity (using benzo[a]pyrene 4,5-oxide as substrate) can be completely precipitated out of solubilized preparations by the antibody, which is equipotent against rat and mouse microsomal epoxide hydrolase. No precipitation of cytosolic hydrolase activity (using trans-beta-ethyl styrene oxide as substrate) is seen with any concentration of the antibody tested. Thus, in the case of microsomal epoxide hydrolase, extensive immunological cross-reactivity exists between the two species, rat and mouse. In contrast, no cross-reactivity is detectable between cytosolic and microsomal epoxide hydrolase, even when enzymes from the same species are compared. We conclude that microsomal and cytosolic epoxide hydrolase activities represent distinct and immunologically non-cross-reactive protein species.     

Arylamidases of rat liver and chemically induced hepatomas 1. Subcellular distribution of l-leucine 2. Naphthylamidase-active antigens

The subcellular distribution of arylamidase-active antigens in rat liver and in two chemically induced hepatomas (D23 and D33) was investigated. Soluble antigens or detergent-solubilized membrane antigens from isolated subcellular fractions were tested in fused rocket immunoelectrophoresis against antisera prepared against each of the fractions. The arylamidase active antigens were identified by means of a zymogram technique using l-leucine 2-naphthylamide as substrate.Two arylamidase-active antigens were shown to be shared between plasma membranes, microsomes, lysosomal membranes and lysosomal content of the hepatocytes. One of these occurred predominantly in the plasma membranes (the plasma membrane arylamidase) while the other was preferentially found in the lysosomal content (the lysosomal content arylamidase). Also a third arylamidase-active antigen was identified and was shown to be restricted to the microsomes and the lysosomal membranes (the microsomal/lysosomal arylamidase).The rat liver plasma membrane arylamidase-active antigen was also present in plasma membrane, microsomal an cell-sap fractions of both the hepatomas. However, in the hepatomas this antigen occurred predominantly in the microsomal fraction. The plasma membrane arylamidase was the only arylamidase-active antigen found in the hepatoma D33 while the plasma membrane and microsomal fractions of hepatoma D23 also contained another antigen with this activity. Neither the lysosomal content arylamidase nor the microsomal/lysosomal arylamidase could be detected in any of the hepatoma fractions.     

Effect of mineralocorticoids on the Na, K-ATPase activity of the rat brain

The effect of desoxycorticosterone (DOC) on Na, K-ATPase activity was studied in vivo and in vitro on microsomal rat brain fractions. An hour after intramuscular administration of DOC a noticeable increase in the enzyme activity was observed. Preincubation of microsomal brain fractions with 5 and 15 mkg/ml of DOC caused a decrease in Na, K-ATPase activity, with the results evident 3-5 minutes after the addition of the hormone into the incubation medium. The idea of a two-phase hormonal effect is suggested. It is likely that desoxycorticosterone effect is realized both by the direct influence, on Na, K-ATPase of the brain plasma membrane and by the influence on the biosynthesis.