Comparison of hepatic drug metabolizing enzyme activities in several agricultural species

1. Several pathways of drug metabolizing enzyme activity were measured in hepatic fractions of cattle, sheep, goats, chickens, turkeys, ducks, rabbits and rats. The pathways examined included the O-demethylation of p-nitrophenol, microsomal ester hydrolysis of procaine and glucuronidation of p-nitrophenol, and the cytosolic acetylation of sulfamethazine and sulfation of 2-naphthol. 2. For most enzymatic pathways measured, goats were more similar to sheep (wether) than to cattle (steers). The exception was UDP-glucuronyltransferase activity, which was significantly higher for the goat than for any other species studied. 3. Within the avian subset, the chicken and turkey were usually the most similar species. 4. The activities of arylsulfotransferase isozymes III and IV were particularly low for the duck compared to the chicken and turkey. 5. N-acetyltransferase activity was very high for rabbits and very low for sheep and goats.     

The phospholipid-dependence of uridine diphosphate glucuronyltransferase. Temperature-dependence of microsomal enzyme activity and thermotropic changes in membrane structure.

Arrhenius plots of the non-latent UDP-glucuronyltransferase (p-nitrophenol acceptor) activity of guinea-pig microsomal membranes prepared with 154 mM-KCl were linear from 5 to 40 degrees C. Arrhenius plots for other microsomal preparations from guinea pig and rat liver that show various degrees of transferase latency, exhibited two linear regions intersecting at a sharp transition point near 20-25 degrees C. This discontinuity was abolished or greatly decreased when transferase latency was removed by treating the membranes with perturbants of phospholipid bilayer strucutre. The fluorescent probe N-phenyl-1-naphthyl-amine detected a thermotropic change in the fluidity of the phospholipid acyl chains of all the microsomal membrane preparations studied, at temperatures close to those of the Arrhenius-plot transitions. It is concluded that the thermotropic change in the structure of the membrane bilayer probably is a 'phase separation' or clustering of phospholipids, which affects a permeability barrier that restricts access of substrate to the transferase molecules.     

Studies of mammalian glucoside conjugation

The mammalian glucoside-conjugation pathway was studied by using p-nitrophenol as the model substrate and mouse liver microsomal preparations as the source of enzyme. The microsomal preparations supplemented with UDP-glucose glucosylated p-nitrophenol; p-nitrophenyl glucoside was identified by chromatography in six solvent systems. The unsolubilized glucosyltransferase of fresh microsomal preparations did not follow the usual Michaelis-Menten kinetics and was easily inhibited by many steroids. All the steroids tested inhibited glucosylation of p-nitrophenol to a greater degree than glucuronidation of p-nitrophenol when assayed in the same microsomal preparations. The steroids inhibited glucosylation with the following decreasing effectiveness: pregnan-3alpha-ol-20beta-one (3alpha-hydroxypregnan-20-beta-one)>oestradiol-17beta 3-methyl ether>oestradiol-17beta>oestriol>pregnane-3alpha,20beta-diol>oestrone. Pregnan-3alpha-ol-20beta-one, pregnane-3alpha,20beta-diol and oestrone had negligible effect on glucuronidation.     

Monooxygenase activity of rat liver microsomes immobilized by entrapment in a crosslinked prepolymerized polyacrylamide hydrazide

Rat liver microsomes were immobilized by entrapment in a chemically crosslinked synthetic gel obtained by crosslinking prepolymerized polyacrylamide-hydrazide with glyoxal. Approximately 88% of the microsomal fraction was entrapped in the gel. The specific rate of O-demethylation of p-nitroanisole was used to assay the microsomal cytochrome P-450 activity of the immobilized microsomal preparations. The gel entrapped microsomes showed monooxygenase activity at 37 degrees C of Vmax = 2.3 nmol p-nitrophenol/min per nmol cytochrome P-450, similar to that of microsomes in suspension. The Km value for the p-nitroanisole-immobilized microsomal cytochrome P-450 system (1.2 X 10(-5) M) was rather close to that of microsomes in suspension (0.8 X 10(-5) M). Under the experimental conditions used the pH activity curve of the immobilized preparation was shifted towards more alkaline values by approx. 0.5 pH unit in comparison with microsomes in suspension. The rate of cytochrome c reduction by the immobilized microsomal system (11.7 nmol/min per mg protein) at 25 degrees C was considerably lower than that of the control (microsomes in suspension, 78 nmol/min per mg protein). Enzyme activity in both preparations showed the same temperature dependence at the temperature range of 10 to 37 degrees C. The immobilized microsomal monooxygenase system could be operated continuously for several hours at 37 degrees C provided that adequate amounts of an NADPH-generating system were added periodically. Under similar conditions a control microsomal suspension lost its enzymic activity within 90 min.     

Strain differences in purified rat hepatic 3 alpha-hydroxysteroid UDP-glucuronosyltransferase.

Qualitative and quantitative differences of purified hepatic 3 alpha-hydroxysteroid UDP-glucuronosyltransferase were investigated in Wistar and Sprague-Dawley rats. Individual differences in the glucuronidation rate of androsterone and chenodeoxycholic acid were observed in hepatic microsomal fractions from Wistar but not Sprague-Dawley rats. No individual variation was observed in the glucuronidation of testosterone, p-nitrophenol or oestrone. The 3 alpha-hydroxysteroid UDP-glucuronosyltransferases from livers of Wistar and Sprague-Dawley rats were isolated and highly purified by using Chromatofocusing and affinity chromatography. The amount of 3 alpha-hydroxysteroid UDP-glucuronosyltransferase in the liver of Wistar rats exhibiting low rates for androsterone glucuronidation is about 10% or less than that found in hepatic microsomal fractions obtained from Wistar rats having high rates for androsterone glucuronidation. The apparent Km for androsterone with purified 3 alpha-hydroxysteroid UDP-glucuronosyltransferase from Wistar rats with high glucuronidation activity (6 microM) was not different from that observed for the enzyme purified from Sprague-Dawley animals, whereas that for the enzyme purified from Wistar rats with low glucuronidation activity was substantially higher (120 microM). Despite the differences in apparent Km values for androsterone, the apparent Km for UDP-glucuronic acid (0.3 mM) was not different in the different populations of rats.     

Relationship of sulfation to ongoing chondroitin polymerization during biosynthesis of chondroitin 4-sulfate by microsomal preparations from cultured mouse mastocytoma cells

A microsomal preparation from chondroitin 4-sulfate-synthesizing cultured mouse mastocytoma cells was incubated with UDP-[3H]GalNAc, UDP-GlcA, and 3'-phosphoadenylylphosphosulfate (PAPS) for 30 s at 10 degrees C and with UDP-[14C]GlcA, UDP-GalNAc, and PAPS for 4 h at 37 degrees C for synthesis of 3H- and 14C-labeled chondroitin/chondroitin sulfate. The latter incubation provided more than 100 times as much product as did the short incubation at 10 degrees C. Upon chromatography of the isolated labeled glycosaminoglycans on a Sepharose CL-6B column, most of the [14C]glycosaminoglycan from the 4 h, 37 degrees C incubation was excluded from the column, indicating that this nascent glycosaminoglycan had been polymerized fully. In contrast, most of the [3H]glycosaminoglycan from the 30 s, 10 degrees C incubation was mostly retarded upon cochromatography on this same column, indicating that the nascent glycosaminoglycan was still growing in size. The labeled fractions representing chondroitin/chondroitin sulfate of varying sizes were analyzed for degree of sulfation by degradation with chondroitin ABC lyase followed by paper electrophoresis of the products. Results indicated that the [14C]chondroitin/chondroitin sulfate formed in the 4-h incubation was 60-70% sulfated. Incomplete chains of [3H]chondroitin/chondroitin sulfate formed in the 30-s incubation were also sulfated as much as 20-25%. As the size of the [3H]chondroitin/chondroitin sulfate increased, there was a concomitant increase in sulfation. These results demonstrate that in this microsomal system sulfation takes place while the nascent chondroitin glycosaminoglycan chains are still actively growing in length, although the sulfation lags somewhat behind the polymerization. This not only indicates a common membrane location for both polymerization and sulfation of chondroitin but also demonstrates that the sulfation of chondroitin by these mastocytoma cells may occur during the process of glycosaminoglycan polymerization rather than subsequent to completion of the glycosaminoglycan chains.     

Diurnal variations of rat liver enzymes catalyzing cholesterol ester hydrolysis

Cholesterol ester hydrolase activity was determined at 3 h time intervals over 24 h in lysosomes, cytosol and microsomes from ad libitum-fed and 24 h food-deprived female rat liver. Diurnal rhythms were identified for the acid and neutral esterases, which were strikingly changed by fasting. In fed animals, lysosomal esterase specific activity exhibited a peak at noon and a sustained medium rate at early darkness, whereas total esterase was maximal at midnight. The circadian patterns of the cytosolic and the microsomal esterases paralleled each other, though the amplitude of rhythms differed, showing higher activities around midnight. After fasting, cholesterol esterase activity from all cell fractions reached a maximum near dark onset. These results are the first to indicate that cholesteryl ester hydrolysis may play a role in generating the diurnal rhythm of hepatic cholesterol.     

Product inhibition of o-aminophenol glucuronidation by p-nitrophenyl glucuronide in detergent-treated microsomal fractions.

The glucuronidation of o-aminophenol is unaffected by p-nitrophenyl gluronide when native microsomal fractions are the source of UDP-glucuronyltransferase. When microsomal fractions treated with Lubrol detergent are the source of the enzyme, however, p-nitrophenyl glucuronide exhibits competitive inhibition of o-aminophenol glucuronidation. In addition, the apparent K1 for p-nitrophenyl glucuronide is the same whether o-aminophenol or p-nitrophenol is the acceptor substrate. The data suggest that UDP-glucuronyltransferase has one binding site for the two phenols and that the absence of inhibition observed in native microsomal fractions is dependent on an intact microsomal membrane.     

Optimal assay conditions for liver UDP-glucuronosyltransferase from the rainbow trout, Salmo gairdneri

Several kinetic characteristics and assay dependence of UDP-glucuronosyltransferase were studied with microsomal preparations made from liver of rainbow trout. The optimal enzyme assay, performed by incubating less than 5 mg microsomal protein/ml assay buffer for 20 min at 25 degrees C and in pH 7.0, contains 2.5 X 10(-5) M p-nitrophenol (p-NP) and 2.5 X 10(-3) M UDP-glucuronic acid (UDPGA). Apparent Km values revealed that the affinity of trout enzyme for p-NP and UDPGA is, respectively, about 70 and 10 times higher than that of rat enzyme. The optimized method will be used for aquatic bioassays, e.g. when assessing the influencing of toxic effluents from the pulp and paper industry.     

Characteristics of renal UDP-glucuronyltransferase

Rat renal microsomes catalyzed the glucuronidation of l-naphthol, 4-methylumbelliferone and p-nitrophenol, whereas morphine and testosterone conjugation were not detected. In contrast, all five substrates were conjugated by hepatic microsomes; the activity was typically 5-10 times greater than with renal microsomes. Renal microsomal UDP-glucuronyltransferase toward l-naphthol was fully activated (six-fold) by 0.03% deoxycholate while the hepatic enzyme was fully activated (eight-fold) by 0.05% deoxycholate. Full activation of hepatic UDP-glucuronyltransferase occurred when microsomes had been preincubated at 0 C with deoxycholate for 20 min. This effect of preincubation was not observed with renal microsomes. The presence of 0.25M sucrose in the buffers during renal microsomal preparation resulted in a two-fold greater rate of l-naphthol conjugation in both unactivated and activated microsomes than renal microsomes prepared in phosphate buffers alone. Preparation of hepatic microsomes with or without 0.25M sucrose had no effect on UDP-glucuronyltransferase activity. Unactivated (-deoxycholate) renal enzyme was activated when incubations were done at a low pH (5.7), whereas fully activated (0.03% deoxycholate) renal microsomal UDP-glucuronyltransferase displayed a pH optimum at 6.5. Renal microsomal UDP-glucuronyltransferase activity toward l-naphthol, p-nitrophenol and 4-methylumbelliferone was induced by pretreatment of rats with beta-naphthoflavone and trans-stilbene oxide but not by phenobarbital or 3-methylcholanthrene. These data demonstrate that renal UDP-glucuronyltransferases are different from the hepatic enzymes with regard to biochemical properties, substrate specificity and in response to chemical inducers of xenobiotic metabolism.     

Enzymatic reaction of chlorotrifluoroethene with glutathione: 19F NMR evidence for stereochemical control of the reaction

Chlorotrifluoroethene, a potent nephrotoxin, is a substrate for the glutathione S-transferases present in the cytosolic and microsomal fractions of rat liver. The glutathione conjugate formed by both subcellular fractions has been identified as S-(2-chloro-1,1,2-trifluoroethyl)glutathione by 1H and 19F NMR and by secondary ion mass spectrometry. The conjugate formed by the cytosolic fraction is an equimolar mixture of two diastereomers, whereas the conjugate formed by the microsomal fraction is predominantly one diastereomer, as judged by the 19F NMR spectra. No evidence for the formation of S-(trihalovinyl)glutathione derivatives by an addition/elimination reaction was found. High-performance liquid chromatography was employed to measure the rates of glutathione conjugate formation in vitro. The rates of S-(2-chloro-1,1,2-trifluoroethyl)glutathione formation were 75-107 nmol min-1 (mg of protein)-1 and 151-200 nmol min-1 (mg of protein)-1 catalyzed by the cytosolic and microsomal fractions, respectively (measured at pH 7.4, 37 degrees C, with 5 mM glutathione). These results suggest that glutathione conjugation occurs at high rates in vivo to produce the highly nephrotoxic S-(2-chloro-1,1,2-trifluoroethyl)glutathione.     

Purification and immunological characterization of catfish (Heteropneustes fossilis) metallothionein

Catfish hepatic metallothionein was purified to homogeneity by Sephadex G-75 gel filtration, DEAE-Sephadex A-25 column chromatography and preparative polyacrylamide gel electrophoresis. Induction by cadmium and zinc, characteristic UV spectrum, cadmium binding property and its low MW established that it was a metallothionein. Antibody was raised in rabbit against catfish metallothionein. Catfish antimetallothionein cross-reacted with other fish metallothioneins but not with chicken or rodent metallothionein. Catfish metallothionein is more electronegative as compared to mouse, rat, chicken or hamster metallothionein. Catfish MT appeared to aggregate readily on storage and to be less electronegative.     

The biosynthesis of tyramine glucuronide by liver microsomal fractions.

Labelled tyramine glucuronide was synthesized in vitro from UDP-[14C]glucuronic acid, [14C]tyramine or [3H]tyramine. The glucuronidation was carried out at pH9.2 in the presence of a monoamine oxidase inhibitor, trans-2-phenylcyclopropylamine. The Km values for tyramine were 69 and 125 micrometer and those for UDP-glucuronic acid were 260 and 290 micrometer respectively for guinea-pig and rat liver microsomal preparatons. The specific activities of microsomal glucuronyltransferase measured in fresh hepatic preparations of guinea pig, mouse and rat were respectively 601, 251 and 235 pmol of [14C]tyramine glucuronide/min per mg of protein. Increase in activity ranged from 2- to 6-fold in preparations which were frozen and thawed once and 5.4- to 10-fold when the freezing and thawing was repeated. Rabbit liver has very low activity, and monkey liver and intestine were completely devoid of this conjugating capacity.     

Diacylglycerol metabolism in neonatal rat liver: characterization of cytosolic diacylglycerol lipase activity and its activation by monoalkylglycerols.

Diacylglycerol lipase (glycerol ester hydrolase, EC 3.1.1.3) activities were investigated in subcellular fractions from neonatal and adult rat liver in order to determine whether one or more different lipases might provide the substrate for the developmentally expressed, activity monoacylglycerol acyltransferase. The assay for diacylglycerol lipase examined the hydrolysis of sn-1-stearoyl,2- [14C]oleoylglycerol to labeled monoacylglycerol and fatty acid. Highest specific activities were found in lysosomes (pH 4.8) and cytosol and microsomes (pH 8). The specific activity from plasma membrane from adult liver was 5.8-fold higher than the corresponding activity in the neonate. In other fractions, however, no developmental differences were observed in activity or distribution. In both lysosomes and cytosol, 75 to 90% of the labeled product was monoacylglycerol, suggesting that these fractions contained relatively little monoacylglycerol lipase activity. In contrast, 80% of the labeled product from microsomes was fatty acid, suggesting the presence of monoacylglycerol lipase in this fraction. Analysis of the reaction products strongly suggested that the lysosomal and cytosolic diacylglycerol lipase activities hydrolyzed the acyl-group at the sn-1 position. The effects of serum and NaCl on diacylglycerol lipase from each of the subcellular fractions differed from those effects routinely observed on lipoprotein lipase and hepatic lipase, suggesting that the hepatic diacylglycerol lipase activities were not second functions of these triacylglycerol lipases. Cytosolic diacylglycerol lipase activity from neonatal liver and adult liver was characterized. The apparent Km for 1-stearoyl,2-oleoylglycerol was 115 microM. There was no preference for a diacylglycerol with arachidonate in the sn-2 position. Bovine serum albumin stimulated the activity, whereas dithiothreitol, N-ethylmaleimide, and ATP inhibited the activity. Both sn-1(3)- and 2-monooleylglycerol ethers stimulated cytosolic diacylglycerol lipase activity 2-3-fold. The corresponding amide analogs stimulated 28 to 85%, monooleoylglycerol itself had little effect, and 1-alkyl- or 1-acyl-lysophosphatidylcholine inhibited the activity. These data provide the first characterization of hepatic subcellular lipase activities from neonatal and adult rat liver and suggest that independent diacylglycerol and monoacylglycerol lipase activities are present in microsomal membranes and that the microsomal and cytosolic diacylglycerol lipase activities may describe an ambipathic enzyme. The data also suggest possible cellular regulation by monoalkylglycerols.     

Phospholipid dependence of UDP-glucuronyltransferase.

Very extensive hydrolysis of phospholipids with pure Bacillus cereus phospholipase C at 5 degrees C greatly inhibited the maximum demonstrable rate of glucuronidation of p-nitrophenol by UDPglucuronyltransferase in guinea pig liver microsomes. Lysophosphatidylcholine restored much of the inhibited activity but non-phospholipid surfactants or hydrolysis of diglycerides failed to reactivate. Phospholipid depletion likewise inhibited o-aminophenol glucuronidation and phospholipids restored activity. It is concluded that glucuronyltransferase specifically requires phospholipids for optimal activity. It seems unlikely that these phospholipids only serve to dissolve aglycones, or that they are direct physiological regulators of the transferase. Instead, a permissive role is ascribed to phospholipids, allowing glucuronyltransferase to be regulated by other means.     

Stimulation of microsomal uridine diphosphate glucuronyltransferase by glucuronic acid derivatives

The glucuronic acid adducts of 1-naphthol, 2-naphthol and 4-methylumbelliferone activate microsomal UDP-glucuronyltransferase (EC 2.4.1.17) when the enzyme is assayed with p-nitrophenol as aglycone. Phenyl glucuronide and oestriol 3beta-glucuronide also activate UDP-glucuronyltransferase. but to a lesser extent. Activation by glucuronides is not dependent on metal ions, but is blocked by prior treatment of microsomal fractions with p-chloromercuribenzoate. The kinetic mechanism of activation is concluded to be an increase in the affinity of the enzyme for UDP-glucuronic acid. Activation by 1-naphthyl glucuronide, at high concentrations of p-nitrophenol, is not affected by 1-naphthol. Apparently 1-naphthyl glucuronide activates the preparation by binding at a site that is separate from the site of glucuronidation of 1-naphthol. Further evidence for the existence of distinct effector sites for the glucuronides was provided by the finding that activation by glucuronides is inhibited competitively by aglycone glucosides. These glucosides do not inhibit the rate of glucuronidation of p-nitrophenol in the absence of glucuronide adducts, nor do they alter the rate of glucuronidation of 1-naphthol. When UDP-glucuronyltransferase is assayed with 1-naphthol as aglycone it is activated by p-nitrophenyl glucuronide, 4-methyl-umbelliferyl glucuronide and under appropriate conditions by its own glucuronide. These activations are similarly inhibited by aglycone glucosides. p-Nitrophenyl glucuronide also stimulates the rate of glucuronidation of o-aminophenol, o-aminobenzoate and bilirubin.     

Effects of levonorgestrel on enzymes responsible for synthesis of triacylglycerols in rat liver

The effects of levonorgestrel treatment (4 micrograms/day per kg body weight 0.75 for 18 days) on rate-limiting enzymes of hepatic triacylglycerol synthesis, namely glycerol-3-phosphate acyltransferase and phosphatidic acid phosphatase were investigated in microsomal, mitochondrial and cytosolic fractions of rat liver. Levonorgestrel treatment resulted in a significant reduction (26%) of hepatic microsomal glycerol-3-phosphate acyltransferase specific activity. Hepatic mitochondrial glycerol-3-phosphate acyltransferase specific activity was unchanged. Levonorgestrel treatment also significantly reduced (by 20%) the specific activity of hepatic microsomal magnesium-independent phosphatidic acid phosphatase. However, magnesium-dependent phosphatic acid phosphatase specific activities in microsomal and cytosolic fractions were unaffected. Cytosolic magnesium-independent phosphatidic acid phosphatase activity was also unchanged. These studies are consistent with the view that levonorgestrel lowers serum triacylglycerol levels, at least in part, by inhibition of the glycerol-3-phosphate acyltransferase (EC 2.3.1.15) step in hepatic triacylglycerol synthesis.     

Inhibition of rat liver UDP-glucuronosyltransferase by silymarin and the metabolite silibinin-glucuronide

The inhibitory effects of silymarin, its main constituent silibinin and the metabolite silibinin-glucuronide on UDP-glucuronosiltransferase (UGT) were evaluated in rat hepatic microsomes. Three substrates were chosen to cover both UGT1A and UGT2B family isozymes: bilirubin (substrate of UGT1A1), p-nitrophenol (UGT1A6) and ethinylestradiol (UGT2B1 and 2B3 for position C17 and UGT1A1 for position C3). The study of p-nitrophenol and bilirubin glucuronidation indicated that silymarin (SM) and silibinin glucuronide (SB-G) were enzyme inhibitors. The kinetic analysis showed that the type of inhibition was competitive in all cases and the Ki obtained were: for p-nitrophenol glucuronidation, KiSB-Gapp: 14+/-1 microg/ml and KiSMapp: 51+/-10 microg/ml and for bilirubin glucuronidation, KiSB-Gapp: 16+/-3 microg/ml. In turn, ethinylestradiol glucuronidation was not affected by any of the compounds studied suggesting that the inhibitory effect was restricted to UGT1A isozymes. Similar studies performed using human hepatic microsomes showed that SM and SB-G were also inhibitors of human UGT1A isozymes. In conclusion, administration of silymarin or its main constituent silibinin could lead to the decrease in the glucuronidation of substrates whose conjugation depends on UGT1A isozymes in a process mediated by silibinin-glucuronide, though their effect in humans needs further investigation.     

6 alpha-glucuronidation of hyodeoxycholic acid by human liver, kidney and small bowel microsomes

Kinetic constants for the glucuronidation of hyodeoxycholic acid in man were determined using microsomal preparations of liver, kidney and small bowel. The affinity of hyodeoxycholic acid for the microsomal hepatic and extrahepatic enzymes was in the same range as previously observed for the monohydroxy bile acid lithocholic acid and about 3-14-times the affinity for the dihydroxy bile acids chenodeoxycholic, deoxycholic and ursodeoxycholic acids. The Vmax values for glucuronidation of hyodeoxycholic acid with hepatic microsomes were 10-30-times higher and with kidney microsomes 50-110-times higher than for the bile acids lacking a 6 alpha-hydroxy group. The site of glucuronidation was determined by gas chromatographic-mass spectrometric analysis of derivatives of products formed after periodate and chromic acid oxidation. Hyodeoxycholic acid glucuronides synthesized with microsomal preparations from the three organs were all found to be conjugated at the 6 alpha position. This has previously been shown to be the site of glucuronidation of endogenous hyodeoxycholic acid glucuronide excreted in urine.     

Hydrolysis of WR2721 by mouse liver cell fractions

A study of the dephosphorylation of WR2721 by broken cell preparations of mouse liver revealed the presence of at least two distinctive activities. One activity was inactivated by heat treatment and was present in the nuclear and microsomal fractions. It had an optimum pH at 9 and was inhibited by sodium vanadate, EDTA, and phenylalanine. Further subcellular fractionation demonstrated the localization of this activity in plasma membrane. A second WR2721 hydrolysis activity was detected in the cytosol fraction (postmicrosomal supernatant), which changed little with pH over the range of 5 to 10; sodium vanadate did not inhibit it. The cytosolic activity in response to heat treatment was complicated since there was an initial decrease followed by an increase in catalytic activity as a function of time at 55 degrees C. Enzyme kinetic analysis of the plasma membrane-associated activity in the microsomal fraction was performed, and Km and Vmax values of 12.5 and 69.9 nmol/min/mg protein, respectively, were obtained.