W. E. Cornatzer

Rat kidney glycerylphosphorylcholine diesterase

Abstract The characterization of rat kidney glycerylphosphorylcholine diesterase ( l -3-glycerylphosphorylcholine glycerophosphohydrolase, EC 3.1.4.2) is described. Of eight tissues tested, rat kidney shows the greatest activity and substantial amounts are found in spleen and lungs. Considerably lower levels of activity are present in heart, skeletal muscle, liver, brain and intestine. Among subcellular components the microsomal fraction contained the greatest amount of the diesterase. Optimal activity occurs at pH 9.2. The enzyme preparation shows a narrow substrate specificity. Of six phosphodiesters tested, only glycerylphosphorylcholine and glycerylphosphorylethanolamine showed any activity. Michaelis constants for these compounds are 2.2 · 10 −3 M and 11.5 · 10 −3 M, respectively. Identical resistance toward heat inactivation substantiates the common identity of the two activities. The enzyme is inhibited by ethanolamine, N -methylethanolamine, N ′, N -dimethylethanolamine and choline. The selective inhibitors of the cholinesterases, prostigmine and physostigmine, do not significantly affect the activity of the diesterase. EDTA inhibits the enzyme. However, it has not been possible to demonstrate the participation of a metal cofactor. Sulfhydryl groups do not appear to be required for activity. The diesterase is remarkably stable to organic solvents, losing only 24% of its original activity when dissolved in 90% isopropanol. It is however, less stable in methanol, acetone and ethanol.

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).

Choline phosphotransferase and phosphatidyl ethanolamine methyltransferase activities

Abstract 1. 1. The two enzymes, choline phosphotransferase and phosphatidyl ethanolamine methyl-transferase involved in phosphatidyl choline biosynthesis has been assayed in liver homogenates, nuclear, mitochondria and microsome of female rats. 2. 2. These two enzymes are microsomal and a tissue distribution study of liver, kidney, spleen, lung, heart, and brain showed liver having the greatest activity. 3. 3. A species study showed the greatest activity of phosphatidyl ethanolainine methyltransferase in liver microsomes in mouse, chicken, rat, dog, pigeon, human, beef, rabbit, turtle, guinea pig respectively. Choline phosphotransferase activity in liver microsomes decreased in chicken, mouse, human, rat, rabbit, turtle, pigeon, guinea pig, beef and dog, respectively. 4. 4. Female rats fed a purified 25 % casein diet decreased significantly the activity of choline phospho-transferase when compared to Purina lab chow. Intraperitoneal injection daily of 30 mg choline chloride decreases significantly the activity of phosphatidyl ethanolamine methyltransferase.

Phospholipid metabolism in mitochondria and microsomes of rabbit liver during development

Abstract The protein, total phospholipid, phosphatidyl inositol, sphingomyelin, phosphatidyl choline, phosphatidyl serine, and phosphatidyl ethanolamine concentrations of mitochondria and microsomes have been determined m the liver of −12, −9, 0, +2, +9, and +14-day-old rabbits. The pattern of growth and development of the liver and liver mitochondria and microsomes was demonstrated by increased wet weight of the liver, by increased mitochondrial and microsomal protein concentration per whole tissue, and by increased mitochondrial and microsomal phospholipid phosphorus per whole tissue. The protein concentration per g of wet tissue was observed to increase in both mitochondria and microsomes after birth. The concentration of total and individual phospholipid phosphorus per mg of mitochondrial protein does not change during development and would suggest that the pattern of mitochondrial phospholipids has been established prior to the development interval studied (−12 to +14 days). This observation would suggest that the duplicating machinery for the phospholipids of membranes of rabbit-liver mitochondria has already been developed 12 days before birth. However, this is not true for the microsomes. The concentration of phospholipid phosphorus per mg of microsomal protein showed an increase in the amount of total phospholipid and phosphatidyl choline before birth and phosphatidyl ethanolamine and phosphatidyl inositol after birth. The incorporation of radioactive phosphorus into the phospholipid fractions of mitochondria and microsomes seemed to indicate a relatively constant rate of synthesis during development.

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 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<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 pairfed the isocaloric diet with or without choline. Ethanol administration with or without dietary choline for 2 weeks stimulated significantly (P<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<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.

Liver lipids during development

The fatty acid composition of the major liver microsomal phospholipids has been studied during pre- and postnatal development of the rabbit. The fatty acid composition of the total lipids, phosphatidyl choline, and phosphatidyl ethanolamine from animals −6, −3, 0, +3, +6, +9, +16, and +112 days of age was determined. Fatty acid composition is similar in phosphatidyl choline and phosphatidyl ethanolamine for oleic acid at +3, +6, +9, and +16 day old animals; palmitoleic acid at +9 day old animals and linoleic acid at −6, −3, and 0 day old animals.Palmitoleic acid demonstrated a uniform decrease during early development in the total lipids and in both phosphatidyl choline and phosphatidyl ethanolamine; however, in the 112 day animal, the amount was just slightly lower than that observed for the earliest prenatal animal studied. Oleic acid decreased considerably during early postnatal development in the total lipids, phosphatidyl choline and phosphatidyl ethanolamine, but an increase in the 112 day animal was observed. Linoleic acid fluctuated considerably throughout postnatal development in the total lipids as well as in the two major phosphatides.Lecithin biosynthesis has been studied by two pathways during development of rabbit liver from −6 days to +110 days. The two pathways of lecithin biosynthesis were evaluated by assaying the activities of the liver enzymes choline phosphotransferase and phosphatidylmethyltransferase at different time intervals during development. The greater enzymatic activity was observed in the cholinephosphotransferase during development.

Biliary copper excretion in the rat.

Summary Gel filtration chromatography and spectral analysis of copper-containing fractions from rat bile suggest that the major fraction of biliary copper is associated with amino acids and small peptides. Following adrenalectomy or hypophysectomy? bile flow was significantly decreased suggesting that adrenal steroids regulate biliary copper excretion by choleretic action.

Studies on liver phosphatidyl cholines: I. Effects of fatty liver induction on phosphatidyl cholines from liver mitochondria and microsomes

Protein, total phospholipid, phosphatidyl cholines and phosphatidyl choline fractions from liver mitochondria and microsomes of female rats were analyzed after treatment with CCl4 (0.3 ml of CCl4 suspended in corn oil) or ethionine (50 mg in 0.9% saline) or after feeding a choline deficient, low protein diet for seven days. Phosphatidyl cholines were separated into four fractions differing in the degree of fatty acid unsaturation. Over 50% of total phosphatidyl choline phosphorus was present in fraction 3 of liver mitochondria and microsomes. The major fatty acid in fraction 1 was docosahexaenoic acid. Fraction 4 contains oleic and linoleic acids. Arachidonic acid occurs in fraction 2 and 3. Ethionine decreased the amount of microsomal protein and phosphatidyl choline fraction 1 of mitochondria. Microsomal protein was decreased by CCl4. The choline deficient, low protein diet caused a decrease in mitochondrial and microsomal phospholipids. The amount of the mitochondrial phosphatidyl choline decreased. Corn oil increased the level of phosphatidyl choline fraction 3. Choline deficiency decreased the amount of phosphatidyl choline fraction 3, increased fraction 4 of mitochondria and microsomes and increased fraction 1 of microsomes.Protein, total phospholipid, phosphatidyl cholines and phosphatidyl choline fractions from liver mitochondria and microsomes of female rats were analyzed after treatment with CCl4 (0.3 ml of CCl4 suspended in corn oil) or ethionine (50 mg in 0.9% saline) or after feeding a choline deficient, low protein diet for seven days. Phosphatidyl cholines were separated into four fractions differing in the degree of fatty acid unsaturation. Over 50% of total phosphatidyl choline phosphorus was present in fraction 3 of liver mitochondria and microsomes. The major fatty acid in fraction 1 was docosahexaenoic acid. Fraction 4 contains oleic and linoleic acids. Arachidonic acid occurs in fraction 2 and 3. Ethionine decreased the amount of microsomal protein and phosphatidyl choline fraction 1 of mitochondria. Microsomal protein was decreased by CCl4. The choline deficient, low protein diet caused a decrease in mitochondrial and microsomal phospholipids. The amount of the mitochondrial phosphatidyl choline decreased. Corn oil increased the level of phosphatidyl choline fraction 3. Choline deficiency decreased the amount of phosphatidyl choline fraction 3, increased fraction 4 of mitochondria and microsomes and increased fraction 1 of microsomes.