Interference of UDP-glucuronyltransferase and beta-glucuronidase activity in rat liver microsomes at pH 7.5 with p-nitrophenol and p-nitrophenylglucuronide as substrates.

Microsomal fraction contains the whole of hepatic UDP-glucuronyltransferase as well as part of beta-glucuronidase. The activities of the two enzymes were assayed under identical conditions using untreated male rat liver microsomes at pH 7.5. In a 30-min incubation with p-nitrophenol and UPD-glucuronic acid, a net glucuronide formation of 0.010 mumol.min-1.g.liver-1 was measured. In the presence of saccharolactone at concentrations selectively blocking beta-glucuronidase, the glucuronidation rate was 0.015 mumol.min-1.g.liver-1. Using the kinetic parameters of beta-glucuronidase (Km = 0.06 mmol/l p-nitrophenylglucuronide, Vm = 0.075 mumol pNP formed.h-1.g.liver-1) determined in the absence of UDP-glucuronic acid, to correct for the beta-glucuronidase's interference on the glucuronidation process, a glucuronide formation of 0.011 mumol.min-1.g.liver-1 was calculated.     

Biosynthesis of beta-glucuronides of retinol and of retinoic acid in vivo and in vitro

After the intraportal injection of retinol-6,7-(14)C to rats, the O-ether derivative of retinol, retinyl -glucosiduronate, appears in the bile. Both retinoyl -glucuronide and retinyl -glucosiduronate are also synthesized in vitro when washed rat liver microsomes are incubated with uridine diphosphoglucuronic acid (UDPGA) and either retinoic acid or retinol, respectively. The synthesis of retinoyl -glucuronide was also demonstrated in microsomes of the kidney and in particulate fractions of the intestinal mucosa. The glucuronides were characterized by their UV absorption spectra, by their quenching of UV light or fluorescence under it, by their thin-layer chromatographic behavior in two solvent systems, and by the identification of products released during their hydrolysis by -glucuronidase. With retinoic acid as the substrate, the UDP glucuronyl transferase of rat liver microsomes had a pH optimum of 7.0, a temperature optimum of 38 degrees C, and a marked dependence on the concentrations of both retinoic acid and UDPGA, but was unaffected by a number of possible inhibitors, protective agents, and competitive substrates. The conversion of retinal to retinoic acid and the synthesis of retinoyl -glucuronide from retinoic acid could not be detected in whole homogenates, cell fractions, or outer segments of the bovine retina.     

Molecular and functional characterization of microsomal UDP-glucuronic acid uptake by members of the nucleotide sugar transporter (NST) family

Transport of the co-substrate UDPGA (UDP-glucuronic acid) into the lumen of the endoplasmic reticulum is an essential step in glucuronidation reactions due to the intraluminal location of the catalytic site of the enzyme UGT (UDP-glucuronosyltransferase). In the present study, we have characterized the function of several NSTs (nucleotide sugar transporters) and UGTs as potential carriers of UDPGA for glucuronidation reactions. UDPGlcNAc (UDP-N-acetylglucosamine)-dependent UDPGA uptake was found both in rat liver microsomes and in microsomes prepared from the rat hepatoma cell line H4IIE. The latency of UGT activity in microsomes derived from rat liver and V79 cells expressing UGT1A6 correlated well with mannose-6-phosphatase latency, confirming the UGT in the recombinant cells retained a physiology similar to rat liver microsomes. In the present study, four cDNAs coding for NSTs were obtained; two were previously reported (UGTrel1 and UGTrel7) and two newly identified (huYEA4 and huYEA4S). Localization of NSTs within the human genome sequence revealed that huYEA4S is an alternatively spliced form of huYEA4. All the cloned NSTs were stably expressed in V79 (Chinese hamster fibroblast) cells, and were able to transport UDPGA after preloading of isolated microsomal vesicles with UDPGlcNAc. The highest uptake was seen with UGTrel7, which displayed a V(max) approx. 1% of rat liver microsomes. Treatment of H4IIE cells with beta-naphthoflavone induced UGT protein expression but did not affect the rate of UDPGA uptake. Furthermore, microsomes from UGT1-deficient Gunn rat liver showed UDPGA uptake similar to those from control rats. These data show that NSTs can act as UDPGA transporters for glucuronidation reactions, and indicate that UGTs of the 1A family do not function as UDPGA carriers in microsomes. The cell line H4IIE is a useful model for the study of UDPGA transporters for glucuronidation reactions.     

Hydrolysis of chyle cholesterol esters with cell-free preparations of rat liver.

1. The effect of pH on the hydrolysis of chylomicron and chylomicron remnant cholesterol ester with rat liver homogenate was examined. The hydrolysis had three pH optima, at pH 4.5, at pH 6.0-6.5 and at pH 8.5. At the two upper pH optima extensive cholesterol ester hydrolysis occurred without simultaneous degradation of the triacylglycerol portion. 2. Similarly, microsomes (at pH 6.5-8.0) and 100 000 X g supernatant (at pH 7.5-8.5) efficiently hydrolyzed the cholesterol ester but not the triacylglycerol of chylomicron remnants. 3. With the same substrate no enrichment of neutral cholesterol esterase activity was seen in isolated plasma membranes. 4. At pH 4.5 lysosomes efficiently hydrolyzed both the cholesterol ester and the triacylglycerol portion of chylomicron remnants. 5. Three conclusions are drawn: (a) the study provides evidence against the existence of a plasma membrane-bound enzyme-hydrolyzing chylomicron cholesterol ester before or during its penetration into the cell; (b) enzymes of the cell sap and possibly of the endoplasmic reticulum can degrade cholesterol ester of chylomicron remnants without preceeding hydrolysis of the triacylglycerol core; and (c) lysosomal enzymes can degrade both the cholesterol ester and the triacylglycerol portion of chylomicron remnants if these are taken up as whole particles by endocytosis.     

A unique multifunctional transporter translocates estradiol-17beta -glucuronide in rat liver microsomal vesicles

A wide array of drugs, xenobiotics, and endogenous compounds undergo detoxification by conjugation with glucuronic acid in the liver via the action of UDP-glucuronosyltransferases. The mechanism whereby glucuronides, generated by this enzyme system in the lumen of the endoplasmic reticulum (ER), are exported to the cytosol prior to excretion is unknown. We examined this process in purified rat liver microsomes using a rapid filtration technique and [(3)H]estradiol-17beta-d-glucuronide ([(3)H]E(2)17betaG) as model substrate. Time-dependent uptake of intact [(3)H]E(2)17betaG was observed and shrinkage of ER vesicles by raffinose lowered the steady-state level of [(3)H]E(2)17betaG accumulation. In addition, rapid efflux of [(3)H]E(2)17betaG from rat liver microsomal vesicles suggested that the transport process is bidirectional. Microsomal uptake was saturable with an apparent K(m) and V(max) of 3.29 +/- 0.58 microm and 0.19 +/- 0.02 nmol.min(-1).mg protein(-1), respectively. Transport of [(3)H]E(2)17betaG was inhibited by the anion transport inhibitors 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid and probenecid. Specificity of the transport process was investigated by studying the cis-inhibitory effect of anionic metabolites, as well as substrates of the plasma membrane multidrug resistance-associated proteins on the uptake of [(3)H]E(2)17betaG. Collectively, these data are indicative of a novel multifunctional and bidirectional protein carrier for E(2)17betaG and other anionic compounds in the hepatic ER. This intracellular membrane transporter may contribute to the phenomenon of multidrug resistance.     

Properties of microsomal phospholipases in rat liver and hepatoma.

Phospholipase A1, A2 and lysophospholipase activities in microsomes of Novikoff hepatoma host rat liver and regenerating rat liver were compared using 1-[9', 10'-3H2]palmitoyl-2-[1'-14C] linoleoyl-sn-glycero-3-phosphoethanolamine, 1-[1' -3H-]hexadecyl-2-acyl-sn-glycero-3-phosphoethanolamine, and 1-[9', 10'-3H2]palmitoyl-sn-glycero-3-phosphoethanolamine as substrates. 1. Microsomes of all three tissues showed two pH dependent peaks of hydrolytic activity, one at pH 7.5 and another at pH 9.5. 2. Phospholipid hydrolytic activity in microsomes from host liver and regenerating liver require Ca2+ for hydrolysis at pH 9.5, but not at pH 7.5. Hepatoma microsomes require Ca2+ for activity at both pH values. 3. Phospholipase A1 activity, stimulated by addition of Triton X-100 to the incubation mixtures, was detected in both host liver and regenerating liver microsomes. There was no evidence of phospholipase A1 activity in hepatoma microsomes. 4. Phospholipase A2 was detected in microsomes of all three tissues using 1-[1'-3H] hexadecyl-2-acyl-sn-glycero-3-phosphoethanolamine as a substrate. The activity required calcium and was inhibited by Triton X-100. 5. Lysophospholipase activity was evident in the microsomes from all three tissues. The activity was inhibited by both Ca2+ and Triton X-100. 6. Differences were also detected between host liver and hepatoma microsomal phospholipid hydrolase activities with respect to the effect of increasing protein concentration, apparent Michaelis-Menten constants, and time course of the reaction.     

Characterization of vitamin K-dependent carboxylase from the livers from the adult ox and dicoumarol-treated calf.

The properties of the microsomal vitamin K-dependent carboxylase from the livers of the adult ox and dicoumarol-treated calf were investigated. The enzymes from both sources utilized glutamic residues of synthetic peptides as substrates and could be solubilized with Triton X-100 similarly to the enzyme from vitamin K-deficient rat liver. Under the optimal assay conditions, the microsomes from calf liver had peptide carboxylase activity comparable with that of the rat liver microsomes and 6.5-fold that of adult ox liver microsomes. The apparent Km for reduced vitamin K and the ionic strength optima of the calf and adult ox enzyme clearly differ from those of the rat enzyme. Pyridoxal phosphate activated the adult ox carboxylase only slightly, whereas the calf enzyme was activated by pyridoxal phosphate as effectively as was the enzyme from the vitamin K-deficient rat. Mn2+ activated the adult ox enzyme 9-fold and calf enzyme 22-fold under optimal conditions (no KCl). Three other divalent metal cations (Ca2+, Ba2+, and Mg2+) activated the adult ox and calf enzymes to about half the extent caused by Mn2+, KCl inhibited this activation. The vitamin K-dependent carboxylase from the dicoumarol-treated calf is apparently more tightly bound to the microsomal membrane than is the adult ox enzyme. In many other respects (pH optimum), temperature optimum, Km values for peptide substrate, substrate specificity, inhibitor effects), the properties of the adult ox and calf enzymes resemble closely those of the rat enzyme.     

Characterization of proteolytic systems in human and rat urine

Activities of proteolytic enzymes were detected in rat and human urine by using [125 l] iodo-insulin B chain as a substrate. The pH optimum of human urine activity was in the acidic range (pH 2.0) whereas the rat urine had two pH optima, one at the acidic range similar to human urine and another at pH 7.5. The activities were linear with time and amount of enzyme. Study with various proteinase inhibitors revealed that the acidic pH activities of human and rat urine were apparently of carboxyl endopeptidases since they were totally inhibited by pepstatin 10-8M. The neutral pH proteolysis of rat urine was inhibited by chelating agents and therefore it was considered as a metalloendopeptidase activity. These findings show the difference between the content of urinary proteolytic enzymes in humans and in rats by using a sensitive and simple radioactive assay.     

Metabolism of halazepam by rat liver microsomes: stereoselective formation and N-dealkylation of 3-hydroxyhalazepam

Metabolism of halazepam [7-chloro-1,3-dihydro-5-phenyl-1-(2,2,2-trifluoroethyl)-2H-1,4-benzod iazepin- 2-one, HZ] was studied by incubation with liver microsomes prepared from untreated, phenobarbital (PB)-treated, and 3-methylcholanthrene (3MC)-treated male Sprague-Dawley rats. Metabolites of HZ were separated by normal-phase HPLC. Relative rates of HZ metabolism by liver microsomes prepared from untreated and treated rats were PB-treated much greater than untreated greater than 3MC-treated at low concentration of microsomal enzymes (0.25 mg protein per ml of incubation mixture) and PB-treated much greater than 3MC-treated approximately untreated at high concentration of microsomal enzymes (2 mg protein per ml of incubation mixture). The relative amounts of major metabolites were found to be 3-hydroxy-HZ (3-OH-HZ) greater than N-desalkylhalazepam (NDZ, also known as N-desmethyldiazepam and nordiazepam) much greater than oxazepam (OX) for all three rat liver microsomal preparations and the distribution of metabolites was independent of microsomal enzyme concentrations. Enantiomers of 3-OH-HZ were resolved by HPLC on a Chiralcel OC column (cellulose trisphenylcarbamate coated on silica gel, particle size 10 microns). 3-OH-HZ enantiomeres have racemization half-lives of approximately 150 min in pH 4, 7.5, and 10 aqueous solutions. 3-OH-HZ formed in the metabolism of HZ by liver microsomes prepared from untreated and treated rats were found to have 3R/3S enantiomer ratios of 37/63 (untreated), 55/45 (PB-treated), and 36/64 (3MC-treated), respectively. N-dealkylation of 3-OH-HZ by liver microsomes from PB-treated rats was substrate enantioselective; the 3R-enantiomer was N-dealkylated faster than 3S-enantiomer.(ABSTRACT TRUNCATED AT 250 WORDS)     

Properties of liver microsomal cholesterol 5,6-oxide hydrolase

Cholestane 3 beta,5 alpha, 6 beta-triol has been identified as the exclusive product formed on hydration of cholesterol 5,6 alpha- and 5,6 beta-oxide catalyzed by cholesterol oxide hydrolase in liver microsomes obtained from five mammalian species. Highest activities were present in microsomes from rats and humans. Both acid- and base-catalyzed hydrolysis of the two epoxides also produce this product, presumably due to preference for pseudo-axial opening of the oxirane ring to form product with a trans-AB ring junction. Although the beta-oxide is more reactive than the alpha-oxide upon acid-catalyzed hydration, the alpha-oxide is a 4.5-fold better substrate than the beta-oxide as indicated by values of Vmax/Km. The kinetic parameters Vmax and Km for the reaction catalyzed by rat liver microsomes are 1.68 +/- 0.15 X 10(-7) M min-1 and 10.6 +/- 1.5 microM for the alpha-oxide and 1.32 +/- 0.11 X 10(-7) M min-1 and 37.2 +/- 5.5 microM for the beta-oxide at 0.35 mg protein/ml, pH 7.4, 6.35% (v/v) CH3CN, and 37 degrees C. Several imino compounds are competitive inhibitors for the enzyme from rat liver. The most effective of these is 5,6 alpha-iminocholestanol (Ki = 0.085 microM) which was known to be a good inhibitor from previous studies. Inhibition by aziridines is consistent with the participation of acid catalysis in the mechanism of action of the enzyme. Cholesterol oxide hydrolase is a distinct enzyme from oxidosqualene cyclase as well as microsomal epoxide hydrolase (EC 3.3.2.3) and the recently reported mouse hepatic microsomal epoxide hydrolase that catalyzes the hydration of trans-stilbene oxide.     

Inhibition of hepatic 17 beta- and 3 alpha-hydroxysteroid dehydrogenases by antiinflammatory drugs and nonsteroidal estrogens

Antiinflammatory agents and estrogens have been tested as inhibitors of two isozymes of guinea pig liver testosterone 17 beta-dehydrogenase (NADP) 1.1.1.64) and rat liver 3 alpha-hydroxysteroid dehydrogenase (EC 1.1.1.50). Antiinflammatory steroids and estradiols were highly inhibitory to 3 alpha-hydroxysteroid dehydrogenase and one isozyme of testosterone 17 beta-dehydrogenase, respectively, but nonsteroidal antiinflammatory agents and nonsteroidal estrogens such as hexestrol, dienstrol, diethylstilbestrol and zearalenone showed potent inhibitions on all the enzymes. Although the inhibitory potency of indomethacin for one isozymes of testosterone 17 beta-dehydrogenase and 3 alpha-hydroxysteroid dehydrogenase decreased with changing pH from 9.7 to 7.0, that of the nonsteroidal estrogens for all the enzymes was little affected by pH. No additive effect in double inhibitor experiments with indomethacin and the nonsteroidal estrogens was observed, and the compounds were all competitive inhibitors with respect to steroidal substrate. The results suggest that there is a very similar region in substrate binding sites of the enzymes.     

Flavin-containing monooxygenase activity in camel tissues: comparison with rat and human liver enzymes

We previously reported the occurrence of multiple forms of drug metabolizing enzymes in camel tissues. In this study, we demonstrated for the first time, flavin-containing monooxygenase (FMO)-dependent metabolism of two model substrates methimazole (MEM) and N,N'-dimethylaniline (DMA) by camel liver, kidney, brain and intestine. FMO-catalyzed metabolism in the microsomes of camel tissues was independent of cytochrome P450 (CYP) activity and exhibited a pH and temperature dependence characteristic of FMO enzymes. Use of inhibitors of CYP activities, SKF525A, octylamine or antibody against NADPH-P450 reductase, did not significantly alter the FMO-dependent substrate metabolism. Using MEM as a model substrate for FMO activity, we show that camel liver has an activity similar to that in rat and human livers. MEM metabolism in extrahepatic tissues in camels was significantly lower (60%-80%) than that in liver. Our results suggest occurrence of FMO in camel tissues, with catalytic properties similar to those in rat and human livers. These results may help in better understanding the effects of pharmacologically and toxicologically active compounds administered to camels.     

Specificity of purified monoacylglycerol lipase, palmitoyl-CoA hydrolase, palmitoyl-carnitine hydrolase, and nonspecific carboxylesterase from rat liver microsomes

The comparative substrate specificities of five purified serine hydrolases from rat liver microsomes have been investigated, especially their action upon natural lipoids. All enzymes had high carboxylesterase activities with simple aliphatic and aromatic esters and thioesters. The broad pH optima were in the range of pH 6-10. Synthetic amides were less potent substrates. The hydrolytic activities towards palmitoyl-CoA and monoacyl glycerols were generally high, whereas phospholipids and palmitoyl carnitine were cleaved at moderate rates. Acetyl-CoA, acetyl carnitine, and ceramides were not cleaved at all. The closely related hydrolases with the highest isoelectric points (pI 6.2 and 6.4) were most active with palmitoyl-CoA and palmitoyl glycerol. One of these enzymes might also be responsible for the low cholesterol oleate-hydrolyzing capacity of rat liver microsomes. Among the other hydrolases, that with pI 6.0 showed significant activities with simple butyric acid esters, 1-octanoyl glycerol, and octanoylamide. The esterase with pI 5.6 had the relatively highest activities with palmitoyl carnitine and lysophospholipids. The purified enzyme with pI 5.2 showed some features of the esterase pI 5.6, but generally had lower specific activities, except with 4-nitrophenyl acetate. The lipoid substrates competitively inhibited the arylesterase activity of the enzymes. The varying activities of the individual hydrolases were influenced in parallel by a variety of inhibitors, indicating that the purified hydrolases possessed a relatively broad specificity and were not mixtures of more specific enzymes. The nomenclature of the purified hydrolases is discussed.     

Effect of albumin on the in vitro conjugation of bilirubin by rat liver microsomes

These studies were carried out to determine whether bovine serum albumin (BSA), which is usually included in the incubation mixture for the in vitro determination of bilirubin-UDP-glucuronyl transferase (GT) activity, affects GT activity. Using bilirubin as substrate, addition of BSA to the enzyme reaction mixture at concentrations varying from 2 to 30 mg/ml resulted in a dose-related inhibition of "native" GT activity of rat liver microsomes. When detergent-activated enzyme was employed, increasing concentrations of BSA also required higher concentrations of deoxycholate, digitonin, or Triton X-100 to produce maximal bilirubin conjugation. Low BSA concentrations (2 mg/ml) prevented enzyme activation by both detergents and UDP-N-acetyl glucosamine. When BSA was omitted and bilirubin dissolved in dimethyl sulfoxide, UDP-N-acetyl glucosamine failed to enhance GT activity, and activation by detergents was only 15-25% of that observed in the presence of optimal concentrations of BSA. When rat albumin was substituted for BSA, a similar dose-related inhibition of in vitro bilirubin conjugation by untreated microsomes was observed, although at any given albumin concentration, GT activity was lower with rat than with bovine albumin. Additionally, both detergents and UDP-N-acetyl glucosamine produced similar GT activation regardless of the rat albumin concentration. Finally, these effects of BSA and rat albumin could not be reproduced when beta-lactoglobulin was employed and/or when p-nitrophenol was the acceptor substrate of GT. These findings indicate that albumin, in particular BSA, profoundly and selectively influences the in vitro activity of microsomal GT toward bilirubin as the acceptor substrate.     

Demethylation of radiolabelled dextromethorphan in rat microsomes and intact hepatocytes.

Liver microsomal preparations are routinely used to predict drug interactions that can occur in vivo as a result of inhibition of cytochrome P450 (CYP)-mediated metabolism. However, the concentration of free drug (substrate and inhibitor) at its intrahepatic site of action, a variable that cannot be directly measured, may be significantly different from that in microsomal incubation systems. Intact cells more closely reflect the environment to which CYP substrates and inhibitors are exposed in the liver, and it may therefore be desirable to assess the potential of a drug to cause CYP inhibition in isolated hepatocytes. The objective of this study was to compare the inhibitory potencies of a series of CYP2D inhibitors in rat liver microsomes and hepatocytes. For this, we developed an assay suitable for rapid analysis of CYP-mediated drug interactions in both systems, using radiolabelled dextromethorphan, a well-characterized probe substrate for enzymes of the CYP2D family. Dextromethorphan demethylation exhibited saturable kinetics in rat microsomes and hepatocytes, with apparent Km and Vmax values of 2.1 vs. 2.8 microM and 0.74 nM x min(-1) per mg microsomal protein vs. 0.11 nM x min(-1) per mg cellular protein, respectively. Quinine, quinidine, pyrilamine, propafenone, verapamil, ketoconazole and terfenadine inhibited dextromethorphan O-demethylation in rat liver microsomes and hepatocytes with IC50 values in the low micromolar range. Some of these compounds exhibited biphasic inhibition kinetics, indicative of interaction with more than one CYP2D isoform. Even though no important differences in inhibitory potencies were observed between the two systems, most inhibitors, including quinine and quinidine, displayed 2-3-fold lower IC50 in hepatocytes than in microsomes. The cell-associated concentrations of quinine and quinidine were found to be significantly higher than those in the extracellular medium, suggesting that intracellular accumulation may potentiate the effect of these compounds. Studies of CYP inhibition in intact hepatocytes may be warranted for compounds that concentrate in the liver as the result of cellular transport.     

Characterization of CYP1A enzyme in Adélie penguin liver

As CYP1A enzymes are induced by certain contaminants, their induction pattern has been used as a biomarker for exposure of certain pollutants. Ethoxyresorufin O-deethylase (EROD) activities are widely used in environmental assessments of polychlorinated biphenyls in many wildlife species. The EROD activity, a typical probe for CYP1A enzyme was studied in liver microsomes prepared from Adélie penguins (Pygoscelis adeliae) (n=10). Penguin liver microsomes (0.5 mg/mL) were incubated with the substrate ethoxyresorufin and NADPH at 37 degrees C for 10 min, and the reaction was terminated by addition of methanol. The formation of the metabolite resorufin was assayed by an HPLC method. EROD activity was present in all liver samples studied. Penguin liver microsomal fraction exhibits typical Michaelis-Menten kinetics in the O-deethylation of ethoxyresorufin. The data were best described by a biphasic kinetic model, which could be interpreted in terms of two populations of CYP enzyme. Mean (+/-S.D.) K(m) values for high- and low-affinity components of EROD were 51+/-109 (range: 0.16 to 358) and 872+/-703 (range: 303 to 2450) nM, respectively. The corresponding mean V(max) values for the high- and low-affinity enzyme activities were 1.8+/-1.4 (range: 0.21 to 5.1) and 9.6+/-3.7 (range: 6.0 to 18.3) pmol/min/mg. The EROD activity in penguin liver microsomes was inhibited by CYP1A inhibitors (phenacetin, 7-ethoxycoumarin and proportional variant-naphthoflavone), whereas other CYP inhibitors for CYP2C9 (tolbutamide), 2C19 (mephenytoin), 2D6 (debrisoquin) and 2E1 (diethyldithiocarbamate) had no effect. These results suggest that CYP1A-like enzymes are present in penguin livers. The activity of this enzyme may be a useful biomarker for assessing the environmental impact of pollutants on Antarctic wildlife.     

Effect of pH on substrate and inhibitor kinetic constants of human liver alanine aminopeptidase. Evidence for two ionizable active center groups.

The presence of at least two ionizable active center groups has been detected by a study of the effect of pH upon catalysis of hydrolysis of L-alanyl-beta-naphthylamide by human liver alanine aminopeptidase and upon the inhibition of hydrolysis by inhibitors and substrate analogs. Octanoic acid, octylamine, and peptide inhibitors have been found to be competitive inhibitors and are therefore thought to bind the active center. L-Phe was previously shown to bind the active center since it was found to be a competitive inhibitor of the hydrolysis of tripeptide substrates (Garner, C. W., and Behal, F. J. (1975), Biochemistry 14, 3208). A plot of pKm vs. pH for the substrate L-Ala-beta-naphthylamide showed that binding decreased below pH 5.9 and above 7.5, the points at which the theoretical curve undergoes an integral change in slope. These points are interpreted as the pKa either of substrate ionizable groups or binding-dependent enzyme active center groups. Similar plots of pKm vs. pH for L-alanyl-p-nitroanilide (as substrate) and pKi vs. pH for L-Leu-L-Leu-L-Leu and D-Leu-L-Tyr (as inhibitors) gave pairs fo pKa values of 5.8 and 7.4, 6.0 and 7.5, and 5.7 and 7.5, respectively. All the above substrates (and D-Leu-L-Tyr) have pKa values near 7.5; therefore, the binding-dependent group with a pKa value near 7.5 is possibly this substrate group. Similar plots of pKi vs. pH for the inhibitors L-Phe, L-Met, L-Leu, octylamine, and octanoic acid had only one bending point at 7.7, 7.6, 7.4, 6.3, and 5.9, respectively. Amino acid inhibitors, octylamine, and octanoic acid have no groups with pKa values between 5 and 9. These data indicate that there are two active center ionizable groups with pKa values of approximately 6.0 and 7.5 which are involved in substrate binding or inhibitory amino acid binding but not in catalysis since Vmax was constant at all pH values tested.     

A comparison of microbody membranes with microsomes and mitochondria from plant and animal tissue

Microbodies (peroxisomes and glyoxysomes), mitochondria, and microsomes from rat liver, dog kidney, spinach leaves sunflower cotyledons, and castor bean endosperm were isolated by sucrose density-gradient centrifugation. The microbody-limiting membrane and microsomes each contained NADH-cytochrome c reductase and had a similar phospholipid composition. NADH-cytochrome c reductase from plant and animal microbodies and microsomes was insensitive to antimycin A, which inhibited the activity in the mitochondrial fractions. The pH optima of cytochrome c reductase in plant microbodies and microsomes was 7.5–9.0, which was 2 pH units higher than the optima for the mitochondrial form of the enzyme. The activity in animal organelles exhibited a broad pH optimum between pH 6 and 9. Rat liver peroxisomes retained cytochrome c reductase activity, when diluted with water, KCl, or EDTA solutions and reisolated. Cytochrome c reductase activity of microbodies was lost upon disruption by digitonin or Triton X-100, but other peroxisomal enzymes of the matrix were not destroyed. The microbody fraction from each tissue also contained a small amount of NADH-cytochrome b₅ reductase activity. Peroxisomes from spinach leaves were broken by osmotic shock and particles from rat liver by diluting in alkaline pyrophosphate. Upon recentrifugation liver peroxisomes yielded a core fraction containing urate oxidase at a sucrose gradient density of 1.23 g × cm⁻³, a membrane fraction at 1.17 g × cm⁻³ containing NADH-cytochrome c reductase, and soluble matrix enzymes at the top of the gradient.     

The subcellular localization of neutral sphingomyelinase in rat liver.

The subcellular distribution of neutral sphingomyelinase activity has been determined in rat liver. Neutral sphingomyelinase is present in the plasma membrane. This enzyme requires either Mg2+ or Mn2+ for full activity; these cations cannot be replaced by Co2+ or Ca2+. The plasma membrane sphingomyelinase is strongly inhibited by Hg2+. A small amount of neutral spingomyelinase activity appears to be present in microsomes. No neutral sphingomyelinase activity is present in liver mitochondria or bytosol. Lysosomal sphingomyelinase is fully active at pH 4.4--4.8 without added divalent cations. However, between pH 5.0 and 7.5 lysosomal sphingomyelinase activity is stimulated by Mg2+, Mn2+, Co2+, and Ca2+. Below pH 4.8, Mg2+ inhibits the reaction. In contrast to the results obtained with the neutral sphingomyelinase activity of plasma membranes and microsomes, lysosomal sphingomyelinase is unaffected by sulfhydryl inhibitors.     

Purification and properties of arginase of rat kidney

l-Arginase from rat kidney was partially purified and some properties were compared with those of l-arginase of rat liver. The kidney enzyme was firmly bound to the mitochondrial fraction and after solubilization required arginine or an unknown factor in tissue extracts for stabilization after dialysis. The two enzymes differed also in stability with respect to acetone treatment, heating or freezing. In further contrast with liver arginase, arginase from kidney was not adsorbed to CM-cellulose at pH7.5 and its activity was not increased by incubation with Mn(2+). Other differences were seen in relative specificities for substrates, ratio of hydrolysis rates with high and low concentrations of arginine and effects of certain inhibitors. Antisera prepared to pure liver arginase did not cross-react with partially purified kidney arginase.