The effect of copper deficiency on heart microsomal phosphatidylcholine biosynthesis and concentration.

The effect of dietary copper deficiency on phosphatidylcholine biosynthetic enzymes, phosphatidylethanolamine methyltransferase, phosphatidyldimethyltransferase and choline phosphotransferase of heart microsomes was measured in rats. The data indicated that dietary copper deficiency can alter phosphatidylcholine biosynthesis and concentration in microsomal membranes of the heart. There was a significant decrease in the specific activity of choline phosphotransferase. There was a significant decrease in the concentration of total phospholipid-P, phosphatidylcholine-P, phosphatidylethanolamine-P, phosphatidylinositol-P, sphingomyelin-P and cardiolipin-P in the microsomes of the copper deficient animals. There was a significant decrease in the concentration of copper in microsomes of heart and liver in the copper deficient animals.

Effect of Friend virus infection on the biosynthetic enzymes of phosphatidylcholine biosynthesis in spleen microsomes.

The effects of Friend virus infection on phosphatidylethanolamine methyltransferase, phosphatidylmonomethylethanolamine methyltransferase, phosphatidyldimethylethanolamine methyltransferase and choline phosphotransferase in mouse spleen microsomes were investigated 14 days following viral inoculation. There was a significant 9.2 fold increase above control values in the specific activity (pmol phosphatidylcholine formed/min/mg microsomal proteins) of choline phosphotransferase. No stimulation occurred in the activities of phosphatidylethanolamine methyltransferase, phosphatidylmonomethylethanolamine methyltransferase or phosphatidyldimethylethanolamine methyltransferase, the three enzymes involved in the methylation phosphatidylethanolamine (PE) to phosphatidylcholine (PC). The concentration of S-adenosylmethionine (AdoMet) and S-adenosylhomocysteine (AdoHcy), the substrate and physiological inhibitor of transmethylation reactions, respectively, were measured in the spleens of control and viral infected mice. AdoHcy is a competitive inhibitor of the phosphatidylethanolamine methyltransferase. A significant increase in the AdoHcy concentration and the resultant decrease in the AdoMet/AdoHcy ratio are sufficient to prevent stimulation of the transmethylation of PE to PC.

The effect of hyper and hypothyroidism, hypophysectomy and adrenalectomy on phosphatidylethanolamine methyltransferase, phosphatidyldimethyl-ethanolamine methyltransferase and choline phosphotransferase of rat liver microsomes.

The effect of hyper- and hypothyroid, hypophysectomy and adrenalectomy on phosphatidylcholine biosynthetic enzymes, phosphatidylethanolamine methyltransferase, phosphatidyldimethylethanolamine methyltransferase and choline phosphotransferase of liver microsomes was measured in rats. There was a significant increase in the specific activity of phosphatidylethanolamine methyltransferase in the hyperthyroid rats. There was a significant reduction in the specific activity of phosphatidylethanolamine methyltransferase and phosphatidyldimethylethanolamine methyltransferase in the hypothyroid states. The choline phosphotransferase increased significantly in the hyperthyroid state and decreased in the hypothyroid animals. Hypophysectomy resulted in a significant increase in specific activity of choline phosphotransferase. A reduction in the specific activity of the phosphatidylethanolamine methyltransferase occurred after 28 days of hypophysectomy. Adrenalectomy resulted in a significant stimulation of the specific activity of phosphatidylethanolamine methyltransferase and choline phosphotransferase in liver microsomes.

The effect of embryological development on phosphatidylethanolamine methyltransferase, phosphatidyldimethylethanolamine methyltransferase and choline phosphotransferase of rabbit liver microsomes.

The effect of embryological development on the two biosynthetic enzymes involved in phosphatidylcholine biosynthesis in liver microsomes of -12, -9, 0, +4, +14, +36 day old rabbits has been determined. The specific activity (pmol phosphatidylcholine formed/min/mg microsomal protein) of the phosphatidylethanolamine methyltransferase in the liver microsomes is very low before birth and a 33% increase at birth occurs when compared to the -12 day old fetal livers. The pmol of phosphatidylcholine formed/min/mg protein by the choline phosphotransferase pathway in fetal liver microsomes is 5, 10, 73, 199, 107 and 307 times greater than by the phosphatidylethanolamine methyltransferase pathway for -12, -9, 0, +4, +14, +36 day old rabbits, respectively. The specific activities of the choline phosphotransferase in the liver microsomes increased from the -12 day old fetal livers to 1.6, 19, 73, 39, 27 times for the -9, 0, +4, +14 and +36 day old animals, respectively. The choline phosphotransferase pathway in comparison to the phosphatidylethanolamine methyltransferase pathway is providing the major phosphatidylcholines in the membranes of the endoplasmic reticulum before birth and early fetal development of the rabbit.

Effect of zinc deficiency on the biosynthesis of phosphatidylcholine in rat microsomes.

Phosphatidylcholine is the major lipid of all cellular membranes. Phosphatidylcholine biosynthesis in microsomes involves two enzyme pathways, choline phosphotransferase and phosphatidyl-ethanolamine methyltransferase. The present study was designed to examine the effect of zinc deficiency on these two enzymes. Male, weanling Long-Evans rats were fed a biotin-enriched 20% egg white diet deficient in zinc for 15-45 d. The specific activity (pmol phosphatidylcholine formed/min/mg microsomal protein) of choline phosphotransferase, phsophatidylethanolamine methyltransferase, and phos-phatidyldimethylethanolamine methyltransferase was determined. The latter assay measures the third methylation of phosphatidyl-ethanolamine to phosphatidylcholine. Zinc deficiency resulted in a significant increase over controls in the specific activity of phospha-tidylethanolamine methyltransferase and phosphatidyldimethyl-ethanolamine methyltransferase in liver and spleen microsomes. A significant increase in the picomoles of phosphatidylcholine formed by the choline phosphotransferase pathway occurred in liver microsomes of zinc-deficient animals. In the brain microsomes a significant decrease in specific activity of phosphatidylethanolamine methyltransferase, phosphatidyldimethylethanolamine methyltransferase, and choline phosphotransferase occurred among zinc-deficient ani-mals. These data suggest that zinc deficiency alters the biosynthesis of phosphatidylcholine, the major lipid of cellular membranes.

Arsenic possibly influences carcinogenesis by affecting arginine and zinc metabolism.

The role of arsenic in carcinogenesis is controversial. There is no doubt arsenic can influence carcinogenesis under certain conditions. However, a review of the findings relating arsenic to cancer indicates that arsenic mainly affects carcinogenesis indirectly by influencing other metabolic systems (i.e., immune system) or nutrients (i.e., arginine, zinc) that may have a more direct role in the carcinogenic process. Depending upon the level of exposure, arsenic can either inhibit or activate interferon, an inhibitor of virus replication. Furthermore, arsenic can apparently inhibit some virusinduced tumorigenesis. However, once a tumor is initiated, arsenic enhances tumor growth, possibly by affecting the immune response. Recent experiments in our laboratory demonstrated that arsenic metabolically interacts with arginine and zinc, both of which apparently influence the immune response. Arsenic evidently has a role that strongly influences the metabolism of arginine, which is an immunostimulatory amino acid. Furthermore, the effect of arsenic on arginine metabolism is apparently modified by the zinc status of the animal. Because arsenic can apparently affect cancer development through several indirect or direct mechanisms, probably the only general conclusion that can be made about arsenic and cancer is that arsenic, depending upon dosage, route of administration, and chemical form, modifies the induction or development of some tumors.

Microsomal phosphatidylethanolamine methyltransferase: inhibition by S-adenosylhomocysteine.

Inhibition by S-adenosylhomocysteine (AdoHcy) of the three reactions of phosphatidylethanolamine methyltransferase which catalyzes the production of phosphatidylcholine from phosphatidylethanolamine in guinea pig and rat liver microsomes has been evaluated. Five of the six methylation reactions in these two species exhibit greater affinity for inhibitor, AdoHcy, than for substrate, S-adenosylmethionine (AdoMet). The Ki values for the rate-limiting reactions were 3.8 microM and 68 microM in rat and guinea pig livers, respectively. An AdoMet : AdoHcy ratio of 12 : 1 in developing liver was found to decline to a constant value in the adult of 5 : 1. The concentration of AdoHcy in rat and guinea pig liver increases markedly following death of the animal. A concomitant decrease in the AdoMet level was observed in guinea pig liver. A comparison of phosphatidylethanolamine methyltransferase activity with the hepatic concentrations of AdoMet and AdoHcy in mouse, rat, rabbit and guinea pig is presented. Regulation of the methylation pathway is discussed.

Microsomal phosphatidylethanolamine methyltransferase: some physical and kinetic properties.

Some physical and kinetic properties of the microsomal enzyme(s) that convert phosphatidylethanolamine to phosphatidylcholine in rat and guinea pig livers have been investigated. The pH optima of the reactions were 9.8, 9.3 and 9.5 for the first, second and third methylation reactions, respectively. Incomplete heat denaturation of the protein catalyzing the first reaction contrasts with inactivation at 60 C of the enzymes catalyzing the second and third methylations. The maximal velocity of the first reaction of the guinea pig liver enzyme is 48 pmol/min/mg protein, substantially less than exhibited rate-limiting reaction of the three step methylation sequence in rat liver, 114 pmol/min/mg. The affinity of the microsomal enzyme for S-adenosylmethionine is greater in rat liver (Km = 18.2 microM) than in guinea pig liver (Km = 302 microM).

Effects of a methyl-deficient diet on rat liver phosphatidylcholine biosynthesis.

To produce a severe choline-methionine deficiency, a synthetic L-amino acid diet, free of choline, methionine, vitamin B12, and folic acid and supplemented with guanidoacetic acid, a methyl group acceptor, was fed to female rats for 2 weeks. The in vitro activity of liver microsomal phosphatidylethanolamine methyltransferase was stimulated twofold when compared with basal diet controls. The activity of choline phosphotransferase was depressed by 86%; thus, the contribution of the methyltransferase in the overall synthesis of phosphatidylcholine apparently increased. However, measurement of the in vivo methylation of phosphatidylethanolamine by incorporation of [1,2-14C]ethanolamine into phosphatidylcholine indicates that the methylation pathway is markedly depressed in methyl deficiency. Hepatic concentrations of the methyltransferase substrate, S-adenosylmethionine, and the inhibitory metabolite, S-adenosylhomocysteine, were significantly altered such that an unfavorable environment for methylation was present in the deficient animal. The ratio of substrate to inhibitor was depressed from 5.2:1 in the controls to 1.7:1 in the livers of methyl-depleted rats. Control of transmethylation in accordance with the availability of substrates, phosphatidylethanolamine, or S-adenosylmethionine, and the level of S-adenosylhomocysteine is discussed.

S-Adenosylmethionine and S-adenosylhomocystein metabolism in isolated rat liver. Effects of L-methionine, L-homocystein, and adenosine.

The effects of varying concentrations of L-methionine, L-homocysteine, and adenosine on the tissue levels of S-adenosylmethionine (AdoMet) and S-adenosyl-homocystein (AdoHcy) were investigated in perfused liver. In the normal liver, the intracellular concentration of AdoMet was dependent upon the availability of methionine. In the presence of high concentrations of methionine the maximum level of AdoMet attainable was 300 nmol/g of liver. The exogenous concentration of methionine did not alter the hepatic concentration of AdoHcy (8 to 20 nmol/g) while adenosine or homocysteine blocked hydrolysis of AdoHcy resulting in elevated levels of AdoHcy (400 to 600 nmol/g) and AdoMet (300 to 600 nmol/g). The addition of both adenosine (4mM) and homocysteine (3.4 mM) to the perfusate further increased the levels of AdoHcy (4 mumol/g) and AdoMet (1.2 mumol/g). As the concentration of AdoHcy increased, significant amounts of this compound were released into the perfusate, while AdoMet was not detected. Under all conditions where AdoHcy accumulated in the cell, a concomitant increase in the AdoMet level occurred. Apparently AdoHcy acts as a positive effector of the S-adenosylmethionine synthase. The hepatocytes did not take up significant amounts of [methyl-14C]AdoMet from the perfusate nor were any [14C]methyl groups from this compound incorporated into histones, DNA, or phospholipids. In contrast, [14C]methyl groups were readily incorporated into these macromolecules from exogenous [methyl-14C]methionine. The addition of adenosine (4 mM) and homocystein (3.4 mM) shifted the AdoMet:AdoHcy ratio from 8.2 to 0.3. Under these conditions, transmethylation was inhibited markedly.

Stimulation by polyamines of carbamylphosphate:glucose phosphotransferase and glucose-6-phosphate phosphohydrolase activities of multifunctional glucose-6-phosphatase.

The effects of added polyamines on carbamylphosphate (carbamyl-P):glucose phosphotransferase and glucose-6-phosphate (Glc-6-P) phosphohydrolase activities of rat hepatic D-Glc-6-P phosphohydrolase (EC 3.1.3.9) of intact and detergent-treated microsomes have been investigated. With the former preparation, in the presence of 1.4 mM phosphate substrate and 90 mM D-glucose (phosphotransferase), 1 mM spermine, spermidine, and putrescine activated Glc-6-P phosphohydrolase 67%, 57%, and 35%, respectively. Carbamyl-P:glucose phosphotransferase, under comparable conditions, was activated 57%, 34%, and 18%. NH+4 (0.25--5.0 mM) produced at best but a minor activation (0--14%), while poly(L-lysine) (Mr = 3400; degree of polymerization 16) equimolar relative to other polyamines with respect to ionized free amino groups activated the hydrolase 358% and the transferase 222%. Treatment of microsomes with the detergent deoxycholate reduced, but did not abolish, polyamine-induced activation. The stimulatory effects of polyamines persisted in the presence of excess catalase, indicating their independence from H2O2 formation; and were eliminated in the presence of Ca2+. Kinetic analysis revealed that all tested polyamines decreased the apparent Michaelis constant values for carbamyl-P and Glc-6-P, but had no effect on the Km for glucose. Poly(L-lysine) increased the V value for both Glc-6-P phosphohydrolase and apparent V values for phosphotransferase extrapolated to infinite concentrations of either carbamyl-P or glucose. The other tested polyamines elevated only this last velocity parameter. It is proposed that a major mechanism by which polyamines activate glucose-6-phosphatase-phosphotransferase is through their electrostatic interactions with phospholipids of the membrane of the endoplasmic reticulum of which this enzyme is a part. Conformational alterations thus induced may in turn affect catalytic behavior. It is suggested that polyamines, or similar positively charged peptides, might participate in the cellular regulation of synthetic and hydrolytic activities of glucose-6-phosphatase.

Relationship between tissue levels of S-adenosylmethionine, S-adenylhomocysteine, and transmethylation reactions.

The concentrations of S-adenosylmethionine (AdoMet), S-adenosylhomocysteine (AdoHcy), and various methyltransferases were determined in the cerebrum, cerebellum, and liver of rats during development and aging. The liver contained from 3 to 7 and from 10 to 15 nmol AdoHcy per gram in young and adult rats, respectively. The AdoMet concentration was 60 to 90 nmol/g liver from rats of the same age and sex. It did not vary significantly with age. In the brain the AdoMet concentration was 45 to 50 nmol/g at birth and decreased to 20 nmol/ g tissue with maturity of the organ. The level of AdoHcy in this organ was less than 1 nmol/g tissue throughout the life-span of the rat. Since the ratio of AdoMet to AdoHcy is relatively high, the rate of methylation of histones, DNA, or phosphatidylethanolamine in the liver or brain was not significantly influenced by AdoHcy. Under normal nutritional conditions, the tissue concentration of AdoMet is far above the Km values of histone and phosphatidylethanolamine methyltransferases. The levels of activity of these enzymes in liver and brain did not correlated with the cellular concentration of AdoHcy. Thi histone methyltransferase activity was elevated in rapidly proliferating tissues and declined markedly in the absence of histone biosynthesis. Phosphatidylethanolamine methyltransferase activity was elevated during development of the liver. The specific activity of the AdoHcy hydrolase remained relatively constant in the rat brain and liver. The activity of this enzyme was 10 times higher in liver than in brain, yet the concentration of AdoHcy was much lower in the latter organ. The tissue levels of this compound are evidently dependent on the rates of removal of homocysteine and adenosine. Adenosine deaminase was present in the liver and brain at relatively high concentrations, particularly during development.

Effect of ethanol ingestion on choline phosphotransferase and phosphatidyl ethanolamine methyltransferase activities in liver microsomes.

The effect of ethanol ingestion on choline phosphotransferase and phosphatidyl ethanolamine methyltransferase activities, the two enzymes involved in phosphatidyl choline biosynthesis in liver microsomes, has been investigated. Female rats were fed a 5% ethanol-liquid diet containing amino acids, minerals, vitamins, with and without choline, for 2, 6 and 10 weeks. Control animals were pair-fed the same isocaloric diet with 5% sucrose with and without choline. Ethanol administration with or without dietary choline stimulated significantly (P less than 0.001) the specific activities of phosphatidyl ethanolamine methyltransferase in liver microsomes in the animals fed 5% ethanol for 2, 6, and 10 weeks, when compared to those control animals pair-fed the isocaloric diet with or without choline. Ethanol administration with or without dietary choline for 2 weeks stimulated significantly (P less than 0.02) the specific activities of choline phosphotransferase. The specific activities of phosphatidyl ethanolamine methyltransferase continued to increase in the liver microsomes from the animals in which dietary choline was omitted for 2, 6, and 10 weeks in the sucrose controls and alcohol-fed animals. Ethanol administration stimulates significantly (P less than 0.001) the phosphatidyl ethanolamine methyltransferase specific activities in liver microsomes of animals fed the liquid diet with dietary omission of choline and methionine for 2 weeks.

Viral stimulation of choline phosphotransferase in spleen microsomes.

Choline phosphotransferase and phosphatidyl ethanolamine methyltransferase enzymatic activities (nmoles phosphatidyl choline/min/mg protein) have been determined in spleen microsomes of Rauscher virus infected balb/c male mice at 5, 10, 14, and 21 days following inoculation of the virus. There is a significant stimulation of the choline phosphotransferase activity in the virus infected spleens with the peak of activity at about 10 days of viral infection. The specific activity of choline phosphotransferase is 10 times that of the phosphatidyl ethanolamine methyltransferase at 10 days of viral infection. There is a 51-fold increase over controls for the total microsomal choline phosphotransferase at 14 days of viral infection and only an 18-fold increase over controls for the phosphatidyl ethanolamine methyltransferase activity. There is a significant (P less than 0.001) increase over controls in the concentration of total phospholipid-P, phosphatidyl choline-P, and phosphatidyl choline-P fractions as separated by argentation chromatography of microsomes from spleens of mice infected with Friend virus of Rauscher virus for 14 days. The choline phosphotransferase and phosphatidyl ethanolamine methyltransferase specific activities in liver microsomes of 14 day Friend and/or Rauscher virus are unaltered during viral infection.