Cholesteryl ester hydrolytic acitivity of rat liver plasma membrane.

Cholesteryl ester hydrolyzing activity of rat liver plasma membranes was studied using acetone-dispersed [4-14-C] cholesteryl oleate as substrate. In contrast to whole liver homogenates which displayed ample activity at both acid (4.5) and neutral (6.2-7.4) pH, purified plasma membrane fractions contained little activity at neutral pH as compared to acid pH. Moreover, rate-zonal sucrose density-gradient centrifugation patterns of plasma membrane rich fractions suggested a specific association with plasma membrane only in the case of the acid activity. These findings suggest that in vivo hepatic cell surface membranes contain little or no cholesteryl ester hydrolytic activity at extracellular pH. They support the possibility that plasma lipoprotein cholesteryl esters enter hepatic parenchymal cells prior to hydrolysis.     

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.     

Induction of liver lysosomal enzymes during the autophagic phase following phenobarbital treatment of rat

Phenobarbital was given to male rats as a single injection and as repetitive injections for 7 days. The effects of treatment on the lysosomal hydrolases acid phosphatase, cathepsin D, and aryl sulfatase were analyzed at different intervals ranging from 1 to 15 days after seven injections, and from 1 to 48 h after a single injection. In both cases, microsomal protein and NADPH-cytochrome c reductase were measured to ensure proper induction. After a single injection, a slight decrease in hydrolytic activities was observed. Repetitive administration of phenobarbital gave rise to a marked decrease of lysosomal enzyme activities 1 day after cessation of treatment. This decrease was followed by a continuous increase in activity up to day 3 and 4. One or 2 weeks after treatment, enzyme activities declined to control values. The increase in activity of lysosomal hydrolytic enzymes was correlated with the onset of induced autophagy of endoplasmic reticulum membranes described as occurring in liver upon cessation of phenobarbital exposure. It is concluded that phenobarbital treatment per se decreases lysosomal enzyme activities, whereas the induced autophagy following cessation of exposure is associated with enhanced levels of lysosomal hydrolases in rat liver.     

Expression and characterization of a PNPLA3 protein isoform (I148M) associated with nonalcoholic fatty liver disease

A genetic variant of PNPLA3 (patatin-like phospholipase domain-containing 3; PNPLA3-I148M), a serine protease of unknown function, is associated with accumulation of triacylglycerol (TAG) in the liver. To determine the biological substrates of PNPLA3 and the effect of the I148M substitution on enzymatic activity and substrate specificity, we purified and characterized recombinant human PNPLA3 and PNPLA3-I148M. Maximal hydrolytic activity of PNPLA3 was observed against the three major glycerolipids, TAG, diacylglycerol, and monoacylglycerol, with a strong preference for oleic acid as the acyl moiety. Substitution of methionine for isoleucine at position 148 markedly decreased the V(max) of the enzyme for glycerolipids but had only a modest effect on the K(m). Purified PNPLA3 also catalyzed the hydrolysis of oleoyl-CoA, but the V(max) was 100-fold lower for oleoyl-CoA than for triolein. The thioesterase activity required the catalytic serine but was only modestly decreased by the I148M substitution. The enzyme had little or no hydrolytic activity against the other lipid substrates tested, including phospholipids, cholesteryl ester, and retinyl esters. Neither the wild-type nor mutant enzyme catalyzed transfer of oleic acid from oleoyl-CoA to glycerophosphate, lysophosphatidic acid, or diacylglycerol, suggesting that the enzyme does not promote de novo TAG synthesis. Taken together, our results are consistent with the notion that PNPLA3 plays a role in the hydrolysis of glycerolipids and that the I148M substitution causes a loss of function, although we cannot exclude the possibility that the enzyme has additional substrates or activities.     

Phospholipase D Activity of Rat Brain Neuronal Nuclei

Abstract: Phospholipase D activity of rat brain neuronal nuclei, measured with exogenous phosphatidylcholine as substrate, was characterized. The measured activity of neuronal nuclei was at least 36-fold greater than the activity in glia nuclei. The pH optimum was 6.5, and unsaturated but not saturated fatty acids stimulated the enzyme. The optimal concentration of sodium oleate for stimulation of the enzyme activity was 1.2 m M in the presence of 0.75 m M phosphatidylcholine. This phospholipase D activity was cation independent. In the absence of NaF, used as a phosphatidic acid phosphatase inhibitor, the principal product was diglyceride; whereas in the presence of NaF, the principal product was phosphatidic acid. The phospholipase D, in addition to having hydrolytic activity, was able to catalyze a transphosphatidylation reaction. Maximum phosphatidylethanol formation was seen with 0.2–0.3 M ethanol. GTPγS, ATPγS, BeF₂, AIF₃, phosphatidic acid, and phosphatidylethanol inhibited the neuronal nuclei phospholipase D activity. The addition of the cytosolic fraction of brain, liver, kidney, spleen, and heart to the incubation mixtures resulted in inhibition of the phospholipase D activity. Phospholipase D activity was detectable in nuclei prepared from rat kidney, spleen, heart, and liver.     

Biocatalytic properties of a novel crude glycyrrhizin hydrolase from the liver of the domestic duck

A novel crude glycyrrhizin (GL) hydrolase preparation from the liver of domestic duck was used to produce glycyrrhetic acid monoglucuronide. To characterize the biocatalytic profiles of the crude enzyme, some effect factors were investigated. It had an apparent optimal pH of 6.0 and an optimal temperature at 55 °C. Most of the metal ions tested and ethylene diamine tetra acetic acid showed little effect on the crude enzyme activity except Cu²⁺. The enzyme was stable only at pH 6. It was more prone to inactivity at high pH conditions than at low pH conditions. It was stable at temperatures below 55 °C and it will lost 90% GL hydrolytic activity exposed at 70 °C. GL hydrolytic activity declined by 30% compared with the control in aqueous solution (buffer pH 6.0) when pre-equilibrated at 55 °C for 5 days. It indicated that the novel crude GL hydrolase preparation had good biocatalytic ability for selective hydrolysis of one glucuronic acid from GL.     

Species differences for stereoselective hydrolysis of propranolol prodrugs in plasma and liver

Species differences and substrate specificities for the stereoselective hydrolysis of fifteen O-acyl propranolol (PL) prodrugs were investigated in pH 7.4 Tris-HCl buffer and rat and dog plasma and liver subfractions. The (R)-isomers were preferentially converted to propranolol (PL) in both rat and dog plasma with the exception of isovaleryl-PL in rat plasma, although the hydrolytic activities of prodrugs in rat plasma were 5–119-fold greater than those in dog plasma. The prodrugs with promoieties (C(=O)CH(R)CH₃) based on propionic acid showed marked preference for hydrolysis of the (R)-enantiomers in plasma from both species (R/S ratio 2.5–18.2). On the other hand, the hepatic hydrolytic activities of prodrugs were greater in dog than rat, especially in cytosolic fractions. The hydrolytic activity was predominantly located in microsomes of the liver in rat, while the cytosol also contributed to hepatic hydrolysis in dog. Hepatic microsomal hydrolysis in dog showed a preference for the (R)-isomers except acetyl- and propionyl-PL. Interestingly, in rat liver all types of prodrugs with substituents of small carbon number showed (S)-preference for hydrolysis. The hydrolyses of (R)- and (S)-isomers of straight chain acyl esters in rat liver microsomes were linearly and parabolically related with the carbon number of substituents, respectively, while these relationships were linear for both isomers in dogs. Chirality 9:661–666, 1997. © 1997 Wiley-Liss, Inc.     

Differences in phosphatidate hydrolytic activity of human alkaline phosphatase isozymes

Hydrolytic activities of human alkaline phosphatase isozymes were investigated using phosphatidases with various fatty acyl chains (egg phosphatidate and dioleoyl, distearoyl, dipalmitoyl, dimyristoyl and dilauroyl phosphatidates). In the presence of sodium deoxycholate, purified human placental and intestinal alkaline phosphatases hydrolyzed all the phosphatidates examined. The hydrolytic activity was maximal in the presence of 10 g/l sodium deoxycholate. Of the phosphatidates, dilauroyl phosphatidate was the best substrate. Using the same unit of the enzyme, the phosphatidate hydrolytic activity of placental alkaline phosphatase was 2- to 3-times higher than that of the intestinal enzyme. In contrast, liver alkaline phosphatase did not hydrolyze phosphatidates with long fatty acyl chains (C16-18) even in the presence of sodium deoxycholate. The liver enzyme hydrolyzed dimyristoyl and dilauroyl phosphatidates very slowly. These results show that the phosphatidates with long fatty acyl chains were useful to differentiate placental and intestinal alkaline phosphatases from the liver enzyme, and suggest that the former enzymes play a different physiological role from the liver enzyme.     

Acid hydrolases of the rat uterus in relation to pregnancy, post-partum involution and collagen breakdown

1. The rat uterus contains acid cathepsin, beta-glucuronidase, beta-galactosidase, acid phosphatase and deoxyribonuclease II at concentrations comparable with those found in liver. Two non-hydrolytic uterine enzymes, cytochrome c oxidase and aspartate aminotransferase, display only 2-6% of the activity found in liver. 2. The concentrations of acid cathepsin and beta-glucuronidase are significantly decreased in pregnancy and increase 3-4-fold during post-partum involution. 3. The concentrations of beta-galactosidase and acid phosphatase are not decreased in pregnancy and increase only 2-3-fold during involution. 4. The concentrations of these four acid hydrolases increase linearly during the first 4 days post partum and reach their peak values at the same time that wet weight and collagen content fall to their lowest point. 5. The concentration of deoxyribonuclease is depressed in pregnancy but does not rise above normal in the post-partum period. 6. Only a small proportion of each hydrolytic activity can be isolated in the mitochondrial-lysosomal fraction of sucrose homogenates of the rat uterus. This proportion increases during involution. However, the extensive mitochondrial rupture occurring during homogenization indicates that the technique is probably too harsh to obtain a true measure of the proportion of lysosomes present in the intact tissue. 7. There are no significant changes in either the concentration or subcellular distribution of the five acid hydrolases in the livers of the experimental rats during pregnancy or involution. In each case the largest proportion of the activity is found in the mitochondrial-lysosomal fraction of liver homogenates. 8. The results are interpreted in terms of the lysosomal theory of intracellular digestion.     

Ethanediol monoester hydrolysis by monoacylglycerol lipase of rat liver microsomes.

The hydrolysis of long-chain monoester of ethanediol by rat,liver subcellular fractions was investigated in order to define the carboxylic acid ester hydrolase involved and to localize the enzymic activity. We found that with 1-O-hexadecanoyl [U-14C]ethanediol as substrate, hydrolytic activity was foremost associated with the rough microsomal fraction. The pH optimum occurred at 8.5. The apparent Km and V values were 6.5 . 10(-4) M and 13 mumol/h per mg microsomal protein, respectively. Enzymic activity was inhibited by p-chloromercuribenzoate and by diisopropylfluorophosphate, whereas NaF was less effective and CaCl2 did not affect apparent activity. Amongst a number of carboxylic acid esters tested as substrate, only long-chain 1-acyl and 2-acyl glycerols inhibited acyl diol hydrolysis competitively (Ki approximately 0.9 mM). It was concluded that long-chain monoesters of ethanediol are hydrolyzed by the monoacyl glycerol lipase system associated with the rat liver microsomal fraction. Because diol monoester is also utilized by the cholinephosphotransferase system of liver to form highly lytic acyl diol phosphocholines, efficient diol monoester hydrolysis by monoglyceride lipase may be a significant step in regulating acyl diol phosphocholine levels in biological systems.     

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.     

Subcellular localization and isolation of gamma-glutamyltransferase from rat hepatoma cells

Cultured rat hepatoma cells were homogenized and subjected to subcellular fractionation by analytical sucrose density centrifugation to determine the localization of gamma-glutamyltransferase ((5-glutamyl-)-peptide: amino acid 5-glutamyltransferase, EC 2.3.2.2). The activity was exclusively localized to the plasma membrane. Diazotized sulphanilic acid was used as a non-penetrant membrane reagent which inactivates ectoenzymes. With both intact and sonicated cells, only 70-75% inhibition of gamma-glutamyltransferase activity was observed. At least 12% of the total cell complement of gamma-glutamyltransferase activity is highly resistant to inactivation by diazotized sulphanilic acid even after Triton X-100 solubilization. The enzyme was purified from hepatoma cells and its properties compared with enzyme from normal liver. Apart from the striking increase in Vapp there were only minor differences between the enzymes from the two sources. In contrast to the complete abolition of transpeptidase activity of the purified hepatoma enzyme by diazotized sulphanilic acid, the hydrolytic activity of this preparation was only slightly inhibited.     

The osmotic stability of lysosomes from adult and foetal guinea-pig liver tissue

1. Lysosome-rich fractions were obtained from foetal liver tissues as early as 35 days uterine age. Foetal lysosomes showed the same ;structure-linked latency' and acid hydrolytic potentiality characteristic of their adult counterparts. 2. The osmotic stability of lysosome-rich fraction from foetal guinea-pig liver tissue was greater than that of the corresponding adult lysosome fractions, p-nitrophenyl-phosphatase being used as marker enzyme. 3. The observation was confirmed by using beta-glycerophosphatase and phenolphthalein beta-glucuronidase as alternative marker enzymes. p-Nitrophenyl phosphate and beta-glycerophosphate appear to act as substrates for the same enzyme. 4. By using p-nitrophenylphosphatase activity measurements it was shown that the osmotic stability of foetal lysosomal fractions decreased with increasing foetal age, but at no time achieved the degree of osmotic instability characteristic of adult lysosomal fractions. 5. The correlation of these findings with the intracellular environment of lysosomes is discussed.     

Pyrophosphatase and glucuronosyltransferase in microsomal UDPglucuronic-acid metabolism in the rat liver.

1. A radiochemical method for the studies on the microsomal UDPglucuronic acid metabolism has been developed. 2. The rat liver microsomes caused a rapid hydrolysis of UDPglucuronic acid to D-glucuronic acid 1-phosphate and further although much slower to free D-glucuronic acid. In Tris-HCl buffer (pH 7.4) they were produced in ratio 72 : 1. No other metabolites were found in measurable amounts. The pyrophosphatase splitting UDPglucuronic acid showed a pH optimum at 8.9, but the liberation of D-glucuronic acid from UDPglucuronic acid had two pH maxima (pH 3.5 and 8.5). EDTA appeared to be less powerful inhibitor of pyrophosphatase than previously suggested. About 25 per cent of the UDPglucuronic acid hydrolyzing activity was still remaining in the presence of 10 mM EDTA. D-Glucaro-1,4-lactone was found to have a slight inhibitory action on the pyrophosphatase activity. Citrate inhibited powerfully the hydrolysis of UDPglucuronic acid and the liberation of free D-glucuronic acid. Phosphate was also inhibitory. 3. In the presence of an exogenous UDPglucuronosyltransferase substrate, 4-nitrophenol, the formation of D-glucuronic acid 1-phosphate and free D-glucuronic acid were slightly reduced, and D-glucuronic acid 1-phosphate, 4-nitrophenylglucuronide and free D-glucuronic acid were produced in ratio 78 : 23 : 1. When 10 mM EDTA was added to diminish the hydrolytic consumption of the glucuronyl donor substrate, the corresponding ratio was still as unfavorable as 19 : 2.6 : 1. The measurable activity of UDPglucuronosyltransferase was lower in the presence of phosphate or citrate than in Tris-HCl buffer, although they protected the glucuronyl donor substrate against hydrolysis. 4. The results indicate that even in the presence of added glucuronyl acceptor substrate the hydrolysis of UDPglucuronic acid predominates the conjugation in rat liver microsomes. The rate of the hydrolysis of UDPglucuronic acid is quite considerable even in the presence of EDTA, and it is recommended to control the UDPglucuronic acid pyrophosphatase activity when UDPglucuronosyltransferase and glucuronidation reactions are studied. Free D-glucuronic acid appears to be produced from UDPglucuronic acid for further use via D-glucuronic acid 1-phosphate, the rate-limiting step being the hydrolysis of this intermediate. UDP-glucuronosyltransferase, glucuronides of either endogenous or exogenous aglycones and beta-glucuronidase have only a minor role in this respect in rat liver microsomes.     

The use of phospholipid vesicles for in vitro studies on cholesteryl ester hydrolysis.

Radiolabeled cholesteryl oleate was incorporated into vesicles prepared from egg yolk lecithin and utilized as a substrate for studies of sterol ester hydrolases present in rat liver homogenates. The cholesteryl oleate was shown to be associated with vesicles (unilamellar liposomes) using Sepharose 4B chromatography. With this substrate, two different cholesteryl ester hydrolytic enzymes were demonstrated in subcellular fractions from the liver homogenates. In the lysosome-rich fraction an acid hydrolase was present, while in the cytosol fraction (150,000 g supernatant), hydrolytic activity was shown to occur with an optimum pH between 8 and 8.5. The substrate was characterized by Sepharose chromatography both before and after incubation with the liver fraction and was not dramatically altered even by rigorous incubation conditions. The lysosomal enzyme preparation was capable of hydrolyzing almost all the cholesteryl oleate in the vesicles. Hydrolysis of the phospholipid was proportionately much less than that of the cholesteryl oleate. Comparisons were performed between the vesicle preparation and an alternate substrate preparation involving the direct addition of cholesteryl oleate in acetone solution. The vesicles appeared to be a better substrate for the lysosomal enzyme whereas the activity in the cytosol fraction did not distinguish between the two substrate preparations. Unsonicated suspensions of cholesteryl oleate and lecithin did not serve as suitable substrates for the enzymes. These studies demonstrate the applicability of cholesteryl ester-containing vesicles as a useful substrate for studying cholesteryl ester hydrolysis in vitro.     

The relation of the soluble thiamine triphosphatase activity of various rat tissues to nonspecific phosphatases

Polyacrylamide gel electrophoresis was used to investigate the relation of the soluble thiamine triphosphatase activity of various rat tissues to other phosphatases. This technique separated the thiamine triphosphatase of rat brain, heart, kidney, liver, lung, muscle and spleen from alkaline phosphatase (EC 3.1.3.1), acid phosphatase (EC 3.1.3.2) and other nonspecific phosphatase activities. In contrast, the hydrolytic activity for thiamine triphosphate in rat intestine moved identically with alkaline phosphatase in gel electrophoresis. Thiamine triphosphatase from rat liver and brain was also separated from alkaline phosphatase and acid phosphatase by gel chromatography on Sephadex G-100. This gave an apparent molecular weight of about 30,000 and a Stokes radius of 2.5 nanometers for brain and liver thiamine triphosphatase. The intestinal thiamine triphosphatase activity of the rat was eluted from the Sephadex G-100 column as two separate peaks (with apparent molecular weights of over 200,000 and 123,000) which exactly corresponded to the peaks of alkaline phosphatase. The isoelectric point (pI) of the brain thiamine triphosphatase was 4.6 (4 degrees C). The partially purified thiamine triphosphatase from brain and liver was highly specific for thiamine triphosphate. The results suggest that, apart from the intestine, the rat tissues studied contain a specific enzyme, thiamine triphosphatase (EC 3.6.1.28). The specific enzyme is responsible for most of the thiamine triphosphatase activity in these tissues. Rat intestine contains a high thiamine triphosphatase activity but all of it appears to be due to alkaline phosphatase.     

Histochemical mapping of certain enzymes in few fresh water teleosts. I. Acid phosphatase.

A detailed investigation of the distribution pattern of acid phosphatase in the different parts of alimentary canal and associated glands of Colisa fasciatus, Macrognathus aculeatus, Notopterus notopterus and Nandus nandus has been made. Though this enzyme shows its hydrolytic activity in all the parts of the digestive system yet its intense activity has been noted in the intestine, pyloric caeca, liver and pancreas of all the 4 fishes. Mucosal and submucosal layers of all the parts of the alimentary canal are the main seat of localization of this enzyme.     

Human carboxylesterases and their role in xenobiotic and endobiotic metabolism

Carboxylesterases (CEs) are traditionally regarded as xenobiotic metabolizing enzymes that hydrolyze esterified xenobiotics to alcohol and carboxylic acid products. However, there is a growing appreciation for the role of CEs in the processing of endobiotics, including cholesteryl esters and triacylglycerols. Human liver microsomes (HLMs) are often used in reaction phenotyping studies to discern interindividual variability in xenobiotic metabolism. The two major CE isoforms expressed in human liver are hCE1 and hCE2. These two isoforms are different gene products. We have begun studies to evaluate the CE phenotype' of human liver samples, i.e. to determine both the levels of hCE1 and hCE2 protein and the hydrolytic activity of each. We have previously shown that there is little variation in hCE1 protein expression in HLM samples from 11 individuals [a 1.3-fold difference between the highest and lowest individuals; coefficient of variation (CV), 9%]. hCE2 protein expression in individual HLMs is only slightly more variable than hCE1 (2.3-fold difference between the highest and lowest individuals; CV, 36%). However, hCE1 protein is found in 46-fold higher amounts in HLMs than hCE2 protein (64.4 +/- 16.5 microg hCE1/mg microsomal protein compared to 1.4 +/- 0.2 microg hCE2/mg microsomal protein). The hydrolytic activity specifically attributable to hCE1 and hCE2 in individual HLMs was measured using bioresmethrin (a pyrethroid insecticide hydrolyzed specifically by hCE1, but not by hCE2) and procaine (an analgesic drug hydrolyzed by hCE2, but not by hCE1). The hydrolytic activity of individual HLMs toward bioresmethrin and procaine did not correlate with the protein content of hCE1 and hCE2. Thus, the mere abundance of CE proteins is not a good predictor of CE activity in HLMs. Identification of the factors that lead to altered CE activities in HLMs will be important to characterize since several pharmaceutical agents, environmental toxicants, and endobiotics are metabolized by these enzymes.     

Cytochemical response of kidney, liver and nervous system of fluoride ions in drinking water.

Morphological and cytochemical studies on the squirrel monkey have been made after maintaining the sujects on pure distilled water and fluoridated distilled water for 18 months with the objective of determining the effect of fluoride on the activity of some hydrolytic and oxidative enzymes in the kidney, liver and nervous system. Daily water intake by individual animals was measured over the final 10 months of the animal's exposure to 0,1 and 5 ppm fluoride. Water consumption was considerably higher in the animals on higher fluoride intake. Whereas the nervous system remained totally unaffected by this experimental procedure, the liver showed a slightly enhanced acivity of Krebs citric acid cycle enzymes. The kidneys, however, showed significant cytochemical changes, especially in the animals on 5 ppm fluoride intake in their drinking water. In these animals, the glomeruli showed an increase in the activity of acid phosphatase and the enzymes belonging to the citric acid cycle and the pentose shunt, whereas lactate dehydrogenase, a resentative of the anaerobic glyoclytic pathway, remained unchanged or only slightly changed. These observations suggest that fluoride in concentrations as low as 5 ppm interferes to some extent with the intracellular metabolism of the excretory system.     

Muscarinic acetylcholine receptor regulates phosphatidylcholine phospholipase D in canine brain

The hydrolytic activity of phosphatidylcholine phospholipase D in the synaptosomes from canine brain was examined using a radiochemical assay with 1,2-dipalmitoyl-sn-glycerol-3-phosphoryl[3H]choline as the exogenous substrate. The involvement of G protein(s) in regulation of this enzyme was demonstrated by a 2- to 3-fold stimulation of the basal activity (4.81 +/- 0.44 nmol choline released/mg protein/h) with guanosine 5'-(3-O-thiol)triphosphate (GTP gamma S), guanyl-5'-yl-(beta, gamma-methylene)diphosphonate, aluminum fluoride, or cholera toxin. The stimulation of phospholipase D hydrolytic activity by GTP gamma S was inhibited by 2 mM guanosine 5'-(2-O-thiol)diphosphate. GTP gamma S at the maximum stimulatory concentration (10 microM) had an additive effect on the maximum cholera toxin stimulation of phospholipase D activity. However, the reverse was not true, thus indicating the possibility that more than one G protein may be involved. Furthermore, cholinergic agonists, including acetylcholine, carbachol, and muscarine, were able to increase the phospholipase D hydrolytic activity at low but not maximally stimulatory concentrations of guanine nucleotide. These cholinergic stimulations were antagonized by atropine, a muscarinic blocker. In addition, O-tetradecanoylphorbol 13-acetate, a protein kinase C activator, was able to stimulate the hydrolytic activity of phospholipase D more than 300% in the presence of 0.2 microM GTP gamma S. However, in the absence of GTP gamma S, stimulation was less than 60%. Our results not only indicate that the receptor-G protein-regulated phospholipase D may be directly responsible for the rapid accumulation of choline and phosphatidic acid in the central nervous system but also reveal that muscarinic acetylcholine receptor-G protein-regulated phospholipase D is a novel signal transduction process coupling the neuronal muscarinic receptor to cellular responses.