Yasuo Nakazawa

Induction of choline kinase by polycyclic aromatic hydrocarbon carcinogens in rat liver

Abstract The administration to rats of polycyclic aromatic hydrocarbons such as 3-methylcholanthrene, 3,4-benzo(a) pyrene and β-naphthoflavone caused a significant elevation of hepatic choline kinase activity. On the other hand, phenobarbital-type inducers (phenobarbital, 1,1,1-trichloro 2,2-bis (ρ-chlorophenyl) ethane (DDT) and hexachlorobenzene) did not stimulate the activity at all. The administration of either cycloheximide or actinomycin D completely depressed the elevation of choline kinase activity induced by polycyclic aromatic hydrocarbons, indicating that the elevated activity by these chemicals could be due to the change in the enzyme level. These results strongly suggest that induction of choline kinase are involved in the sequence of events leading to the induction of hepatic drug metabolism by polycyclic aromatic hydrocarbons.

Lysozyme substrates. Chemical synthesis of p-nitrophenyl O-(2-acetamido-2-deoxy-β-D-glucopyranosyl)-(1 → 4)- 2-acetamido-2-deoxy-β-D-glucopyranoside and its reaction with lysozyme☆

Abstract In order to obtain a lysozyme substrate which can be assayed colorimetrically, p- nitrophenyl -O-(2- acetamido -2- deoxy -β- D - glucopyranosyl )-(1 → 4)-O-(2- acetamido -2- deoxy -β- D - glucopyranosyl )-(1 → 4)-2- acetamido -2- deoxy -β- D - glucopyranoside was synthesized. The action of egg-white lysozyme (N-acetylmuramide glycanohydrolase, EC 3.2.1.17) on this compound was found to be random, hydrolysis occurring by two pathways, namely one which liberates p-nitrophenol and another which liberates p-nitrophenyl-2-acetamido-2-deoxy-β- D -glucopyranoside. The reaction of the substrate with lysozyme was inhibited non-competitively by N,N-diacetylchitobiose or N-acetyllactosamine, whereas N,N,N-triacetylchitotriose inhibited the reaction competitively. These results suggest that at least three monosaccharide units are necessary for binding to the active site of lysozyme.

Choline/ethanolamine kinase from rat kidney.

Publisher Summary Choline kinase (ATP:choline phosphotransferase) and ethanolamine kinase (ATP:ethanolamine O-phosphotransferase) catalyze the phosphorylation of choline/ethanolamine by ATP in the presence of Mg 2+ , yielding phosphocholine/phosphoethanolamine and ADP. This enzyme step commits choline (ethanolamine) to the CDPcholine (CDPethanolamine) pathway for the biosynthesis of phosphatidylcholine (phosphatidylethanolamine) in all animal cells. The chapter discusses different methods to assay choline kinase activity. The purification of choline/ethanolamine kinase from rat kidney is also discussed. Purification is started with the high-speed supernatant fraction from fresh kidneys (∼200 g wet weight). All procedures are carried out below 5 ° C. One of the most important features of choline/ethanolamine kinase is its inducibility in various experimental systems. Although the physiological significance of the inducibility has not yet been fully understood, the induction of choline/ethanolamine kinase by several means suggests that more than one mechanism could be involved in this process. Distinct mechanisms of choline kinase induction have been demonstrated between rooster liver by estrogens and rat liver by certain hepatotoxins. A new isozyme of choline/ethanolamine kinase appears to be preferentially induced in the latter case.

Actinomycin D-sensitive induction of choline kinase by carbon tetrachloride intoxication in rat liver

A single intraperitoneal dose(1 ml/kg body weight) of carbon tetrachloride (CCl4) caused a rapid and drastic induction of choline kinase activity in rat liver cytosol. The administration of either cycloheximide or actinomycin D completely blocked the CCl4-mediated induction of choline kinase activity, indicating that the elevated activity could be due to the change in the enzyme level. The pretreatment of rats with phenobarbital did not cause any significant effect on hepatic choline kinase induction by CCl4, suggesting that the induction may not be directly related to the metabolic rate of CCl4. A considerable part of induced form(s) of choline kinase appeared not to be a form present in the liver of untreated rats. The contribution of adrenals to the CCl4-mediated hepatic choline kinase induction could be ruled out.

Effect of polychlorinated biphenyls (PCBs) administration on phospholipid biosynthesis in rat liver

Abstract The effect of PCBs or phenobarbital on the biosynthesis of phospholipids in hepatic endoplasmic reticulum of rats was studied by the intraperitoneal injection of [ 32 P]orthophosphate, [Me− 14 C]choline or [2− 3 H]glycerol. Significant increases in liver microsomal phospholipid content after the administration of either PCBs or phenobarbital indicated the actual proliferation of endoplasmic reticulum membranes. The rate of both [ 32 P] and [ 14 C] incorporations into microsomal choline-containing phospholipids, such as phosphatidylcholine, sphingomyelin and lysophosphatidylcholine, was reduced to one fifth by PCBs administration compared with control animals. The incorporation of [ 32 P]orthophosphate into phosphatidylethanolamine or other phospholipid classes was less or not affected, respectively, by PCBs administration. The specific inhibitory effect of PCBs on the incorporation into cholinecontaining phospholipids was not observed when [2− 3 -H]glycerol was used as a precursor. Phenobarbital administration, however, increased significantly the rate of [ 32 P] incorporation into liver phospholipids, especially phosphatidylcholine. It is suggested that the increase in microsomal phospholipid content by PCBs administration is not due to the stimulation of synthesis but to the inhibition of the catabolism of membrane phospholipids and that the increase in content caused by phenobarbital is due at least in part, to the stimulation of synthesis. The possible site(s) of PCBs-induced inhibition of phospholipid biosynthesis in rat liver is discussed.

Induction of choline kinase by polycyclic aromatic hydrocarbons in rat liver: II. Its relation to net phosphatidylcholine biosynthesis

The effect of a single dose (50 mg/kg body weight) of 3-methylcholanthrene on de novo phosphatidylcholine biosynthetic activities in rat liver was studied both in a cell-free system and with slice experiments. 3-Methylcholanthrene caused a significant depression of either [methyl-14C]choline or [2-(3)H]glycerol incorporation into phosphatidylcholine when the precursor was incubated with liver slices. At the same time, there occurred a significant accumulation of radioactivity in either cholinephosphate or diacylglycerol molecule from [14C]choline or [3H]glycerol, respectively, suggesting that 3-methylcholanthrene could cause an inhibitory effect on hepatic phosphatidylcholine synthesis at the cholinephosphotransferase or/and cholinephosphate cytidylyltransferase step. Subsequent studies, where the activities of the three enzymes involved in de novo phosphatidylcholine synthesis were compared between control and 3-methylcholanthrene-pretreated rat liver subcellular fractions, demonstrated that the cholinephosphotransferase step could be the site of inhibition by 3-methylcholanthrene. On the other hand, 3-methylcholanthrene caused a significant induction of choline kinase activity in a time-dependent manner and, at the same time, the cholinephosphate pool size in liver cytosol was enlarged 2-3-fold when compared to the respective control. The overall results suggested strongly that 3-methylcholanthrene causes the counteractive effects on the de novo phosphatidylcholine biosynthesis, induction of choline kinase activity and inhibition of cholinephosphotransferase activity, both of which could participate in a concomitant increase in cholinephosphate pool size in rat liver.

Diacylglycerol generated in the phospholipid vesicles by phospholipase C is effectively utilized by diacylglycerol lipase in rat liver cytosol

Diacylglycerol was generated in phosphatidylcholine vesicles by incubation with Clostridium welchii phospholipase C. Newly formed diacylglycerol was rapidly converted to monoacylglycerol and glycerol when rat liver cytosol fraction was present in the incubation mixture, suggesting the presence of di- and monoacylglycerol lipase activities in this subcellular fraction. On the other hand, 3H-labeled diacylglycerol co-emulsified with non-radioactive phosphatidylcholine was found to be a poor substrate for the diacylglycerol lipase. These results indicate that enzymatic generation of diacylglycerol provide a substrate having a suitable physical state for the expression of diacylglycerol lipase activity. It was also found that the rate of diacylglycerol hydrolysis was dependent upon the rate of diacylglycerol generation, but not upon the absolute concentration in the incubation mixture. When the rate of diacylglycerol hydrolysis was plotted against the rate of diacylglycerol generation, a saturation curve was obtained and the double-reciprocal plot gave a straight line. It is not known why a relationship similar to Michaelis-Menten type kinetics was obtained between the rate of diacylglycerol hydrolysis and diacylglycerol generation instead of diacylglycerol concentration, but it may be best explained by the following assumptions: (1) diacylglycerol molecules are generated at the surface of the lipid vesicles where they are readily accessible to diacylglycerol lipase; (2) soon after the generation, diacylglycerol molecules migrate into inside the vesicles where they are inaccessible to the enzyme; (3) the effective concentration of diacylglycerol, i.e., the concentration of diacylglycerol located in the surface layer of the vesicles is proportional to the rate of diacylglycerol generation.

Rapid hydrolysis of diacylglycerol formed during phosphatidate phosphatase assay by lipase activities in rat liver cytosol and microsomes

Side reactions which may affect the determination of phosphatidate phosphatase activity were investigated in rat liver cytosol and microsomes. Incubation of these subcellular fractions with either 14C-labeled phosphatidate bound to microsomal membranes (PAmb) or that coemulsified with microsomal lipids resulted in rapid formation of water-soluble products, most of which were identified as glycerol, in addition to diacylglycerol. Neither lysophosphatidate nor glycerol 3-phosphate accumulated under any of the conditions used and only a minute amount of activity catalyzing hydrolysis of glycerol 3-phosphate could be detected in cytosol and microsomes, suggesting that glycerol was not formed by the deacylation of phosphatidate to glycerol 3-phosphate and subsequent dephosphorylation. On the other hand, pretreatment of cytosol or microsomes with diisopropylfluorophosphate abolished the formation of water-soluble products, indicating that glycerol was formed from diacylglycerol, the product of the phosphatidate phosphatase reaction, by lipase-type activities. Rapid deacylation of diacylglycerol by these subcellular fractions was also observed with an emulsion of phosphatidate, which has been purified from the total lipid extract of PAmb as substrate. The rate of hydrolysis of diacylglycerol was maximum when the concentration of diacylglycerol was less than 20 microM with either cytosol or microsomes. The present results suggest that it is essential to characterize the reaction products before employing specific assay conditions for phosphatidate phosphatase. At least under the conditions we tested, reliable measurement of the enzyme activity in rat liver cytosol and microsomes can be achieved only by determining the release of Pi or that of water-soluble activity from 32P-labeled phosphatidate.

CDPcholine : 1,2-diacylglycerol cholinephosphotransferase from rat liver microsomes. I: Solubilization and characterization of the partially purified enzyme and the possible existence of an endogenous inhibitor

The solubilization and partial purification of cholinephosphotransferase (CDPcholine:1,2-diacylglycerol cholinephosphotransferase, EC 2.7.8.2) from rat liver microsomes were examined in the presence of ionic (sodium deoxycholate), nonionic (Triton X-100,n-octylglycoside), or zwitter ionic (CHAPS) detergents. Among the four detergents tested, only sodium deoxycholate was found to be an efficient solubilizer of cholinephosphotransferase activity from microsomal membranes, whereas the other three detergents caused irreversible inactivation of the enzyme at the solubilization step. Addition of phospholipids at the solubilization step, or after solubilization of the membrane proteins, could not preserve or reconstitute activity to any extent. The sodium deoxycholate-solubilized activity was partially purified by gel permeation chromatography (Superose 12HR). The partially purified preparation appeared to consist of a large aggregate containing phospholipids; further dissociation of the protein-phospholipid complex caused complete inactivation of the enzyme. The partially purified cholinephosphotransferase showed a specific activity of 100–130 nmol/min/mg protein, which is the highest activity reported to date from any tissue source; this amounts to a 4-fold enrichment of cholinephosphotransferase activity from the original KCl-washed rat liver microsomes. Ethanolaminephosphotransferase (CDPethanolamine:1,2-diacylglycerol ethanolaminephosphotransferase, EC 2.7.8.1) activity was copurified and 6-fold enriched with a total recovery of 60%. During the purification of cholinephosphotransferase activity, a putative endogenous inhibitor of cholinephosphotransferase was also solubilized and was isolated from the microsomal membranes. This heat-labile, nondialyzable inhibitor was shown to act specifically on cholinephosphotransferase and not on ethanolaminephosphotransferase. Further characterization of the inhibitory activity revealed that it may act at the binding step of the cholinephosphotransferase to its lipid substrate, diacylglycerol.

The effect of cadmium ions and cadmium-metallothionein on the activities of phospholipid-synthesizing enzymes of rat liver microsomes in vitro

Abstract The effects of cadmium ions or cadmium-metallothionein on the activities of acyl-CoA:1acyl- sn -glycerol 3-phosphoric acid or 1-acyl- sn -glycero 3-phosphocholine acyltransferase of rat liver microsomes have been studied, in vitro . Cadmium ions were found to cause a noncompetitive type inhibition of these two acyltransferases. The K i values were calculated, and found to be smallest (1.7 × 10 −5 m ) for palmitoyl-CoA and greatest (1.0 × 10 −4 m ) for linoleoyl-CoA, among the several fatty acyl-CoAs tested on the 1-acyl- sn -glycerol 3-phosphoric acid acyltransferases. With the 1-acyl- sn -glycero 3-phosphocholine acyltransferase, the K i values were found to be smallest for the plamitoyl-CoA acyltransferase (3.8 × 10 −5 m ) and largest for thearachidonoyl-CoA acyltransferase (1.1 × 10 −4 m ). In contrast, mouse liver cadmium-metallothionein, including 4 mol of cadmium and 2 mol of zinc in one molecule of metallothionein, was not found to be inhibitory or rather stimulative on the above two acyltransferases at the same concentration of cadmium tested in the cadmium ion inhibitor experiments. The above results demonstrate that there is a strong and irreversible inhibition by cadmium ions on acyl-CoA acyltransferases, but that when cadmium acts on the enzyme in the form of a cadmium-metallothionein complex, the inhibition effect does not occur. These findings may reflect differing degrees of toxicity of these two types of cadmium compounds in mammalian tissues.