Dennis R. Hoffman

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 phosphatidyl-ethanolamine 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 μM and 68 μM 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 methyl-transferase 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 phosphatidyl-ethanolamine 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 p mol/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 μM) than in guinea pig liver (Km=302 μM).

Effect of diet on choline phosphotransferase, phosphatidylethanolamine methyltransferase and phosphatidyldimethylethanolamine methyltransferase in liver microsomes

Phosphatidylcholine (PC) biosynthesis has been investigated in female rats fed a liquid amino acid, choline-methionine-free diet by assaying in liver microsomes the specific and total activities of choline phosphotransferase, phosphatidyldimethylethanolamine methyltransferase and phosphatidylethanolamine methyltransferase. There was a significant decrease in the specific activity (sp act) of choline phosphotransferase in the liver of rats fed a choline-methionine-free diet. The dietary omission of methionine for 2 wk resulted in a significant decrease in the sp act of choline phosphotransferase. The dietary omission of choline, methionine, B12, folic acid and the addition of a methyl group acceptor, guanidoacetic acid, decreased further the sp act of choline phosphotransferase. The phosphatidyl-ethanolamine methyltransferase sp act increased with the dietary omission of choline and methionine. The dietary omission of choline, methionine, B12, folic acid and the addition of a methyl group acceptor, guanidoacetic acid, resulted in a decrease in the sp act of phosphatidyldimethylethanolamine methyltransferase and an increase in phosphatidylethanolamine methyltransferase. The dietary omission of choline, methionine, B12, folic acid and the addition of a methylation inhibitor, 2-amino-2-methyl-1-propanol, did not result in a significant decrease in the sp act of choline phosphotransferase; however, it did significantly decrease the sp act of phosphatidylethanolamine methyltransferase. The addition of dietary methionine with the inhibitor resulted in a significant decrease in the sp act of the choline phosphotransferase and phosphatidylethanolamine methyltransferase when compared to control and/or when compared to deficient with or without inhibitor. The dietary supply of methionine, as a source of choline, did affect the activity of the enzymes that synthesize PC. The ratio of the substrate, S-adenosylmethionine, and the inhibitory product, S-adenosylhomocysteine, affected the enzymatic activity of phosphatidylethanolamine methyltransferase. It is suggested that the concentrations of these 2 compounds may be important in regulating the methylation of phosphatidylethanolamine in the liver cell.

Effect of Alloxan Diabetes on Phosphatidylcholine Biosynthetic Enzymes

Abstract Phosphatidylethanolamine methyltransferase, phosphatidyldimethylethanolamine methyltransferase, and choline phosphotransferase enzymatic activities (nmole PC formed min/mg protein) have been determined in liver microsomes of alloxan diabetic rats. There was a significant reduction in the methylation pathway in the conversion of phosphatidylethanolamine to phosphatidyl choline as demonstrated in the low value of the phosphatidylethanolamine methyltransferase and phosphatidyldimethylethanolamine methyltransferase in those diabetic rats with blood glucose levels greater than 600 mg%. The reduction was 49 and 48% decrease over controls, respectively. There was a significant increase in the choline phosphotransferase in the diabetic rats. The increase was 92% over controls for the rats with blood glucose of 461 mg% and 55% over controls for the rats with blood glucose 946 mg%.

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<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 or 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.

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 phosphatidyletanolamine 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.

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.

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.

Effects of diethanolamine on phosphatidylcholine biosynthetic enzymes of rat liver microsomes

Abstract 1. 1. The effect of administration of diethanolamine on the phosphatidylcholine biosynthetic enzymes. phosphatidylethanolamine methyltransferase, phosphatidyldimethylethanolamine methyltransferase and choline phosphotransferase of liver microsomes was measured in rats. 2. 2. In animals fed a stock diet for 3 days plus 0.5 mmol of diethanolamine by intraperitoneal injection. a significant stimulation of the activity of phosphatidylethanolamine methyltransferase was observed. There was a significant inhibition of choline phosphotransferase. 3. 3. Rats stomach tubed with 1 mmol diethanolamine for 3 days showed a significant stimulation of phosphatidylethanolamine methyltransferase and phosphatidyldimethylethanolamine methyltransferase activities. There was a significant inhibition of choline phosphotransferase. 4. 4. A cholinemethionine deficient diet was used to facilitate the incorporation of diethanolamine into an atypical phospholipid fraction. Feeding this diet containing 1% diethanolamine for 7 days resulted in a significant decrease in the in vitro activity of the methylating enzymes phosphatidylethanolamine methyltransferase, phosphatidylmonomethylethanolamine methyltransferase and phosphatidyldimethylethanolamine methyltransferase.

METHYLATION IN CELLULAR MEMBRANES OF FRIEND VIRUS TUMORS OF MICE

Publisher Summary This chapter discusses the principles of methylation in the cellular membranes of friend virus tumors of mice. Phosphatidylcholine (PC) is the major lipid in all cellular membranes. PC biosynthesis occurs by two major different pathways. One pathway involves the methylation of phosphatidylethanolamine (PE) to phosphatidylcholine. This pathway is catalyzed by endoplasmic reticulum membrane enzyme—phosphatidylethanolamine methyl transferase (PEMT)—and requires three S-adenosylmethionine (AdoMet) for methyl groups to produce three S-adenosylhomocysteine (AdoHcy). It is known that AdoHcy is a competitive inhibitor of PEMT. The second pathway of PC biosynthesis is catalyzed by choline phosphotransferase. The phospholipid methylation of PE to PC can alter the structure and function of biomembranes. The phosphatidylcholines produced by the methylation pathway have more polyunsaturated fatty acids than PC from the CDP-choline pathway.