John T. Smith

Effect of pyridoxine deficiency on phospholipid methylation in rat liver microsomes.

The effect of altered methionine metabolism during pyridoxine deficiency on the activity of phosphatidyl-ethanolamine methyltransferase (EC 2.1.1.17) and the levels of phosphatidylethanolamine (PE) and phosphatidylcholine (PC) has been evaluated in rat liver microsomes. Animals fed a pyridoxine-deficient diet for 7 wk displayed a fivefold increase in the hepatic tissue level of S-adenosylhomocysteine when compared to either control or pair-fed animal counterparts. When PE methyltransferase was assayed in vitro, a significant increase in specific activity was observed using enzyme preparations from either pair-fed or pyridoxine-deficient rats. On the other hand, phospholipid levels did not conform to the measured enzyme activity. The level of PC in microsomes from either pyridoxine-deficient or pair-fed animal groups was significantly lower than that determined for the control group of rodents. However, the level of PC was noticeably lower in microsomes from pyridoxine-deficient animals than that from pair-fed animals, which received 45% of the feed intake of the control animals. In addition, the level of PE in microsomes from pair-fed and pyridoxine-deficient animals was significantly higher than that analyzed from the control animals, further confirming decreased methylation of substrate to product. It is concluded that pyridoxine deficiency may alter the methylation of phospholipid in the endoplasmic reticulum above and beyond that produced by feed restriction alone.

A relationship between vitamin B12, folic acid, ascorbic acid, and mercury uptake and methylation

Ingestion of megadoses of certain vitamins appears to influence the in vivo methylation of mercuric chloride in guinea pigs. The addition of megadoses of vitamin B12 fed either singularly or in combination with folic acid resulted in increased methylmercury concentrations in the liver. Moreover, percent methylmercury levels were significantly increased with B12 treatment in the liver (B12 only and B12/folic acid) and brain (B12/vitamin C). Incorporation of high levels of folic acid into the dietary regime also increased the methylmercury concentration particularly in the liver and hair tissues. The addition of vitamin C in the diet, particularly in combination with B12 (brain) or folic acid (muscle) resulted in increased methylmercury levels in these tissues and percent methylmercury values with B12 in the muscle and brain tissue.

An Effect of Dietary Sulfur on Liver Inorganic Sulfate in the Rat

The effect of dietary sulfur on liver inorganic sulfate concentration was determined by feeding rats diets containing 0.0002, 0.02, 0.1 and 0.42% of inorganic sulfate for a period of 17 days. Each diet contained 15% of casein supplemented with decreasing levels of methionine as the dietary inorganic sulfate was increased, to keep the sulfur as sulfate concentration constant at 0.67%. The molarity of the liver sulfate calculated on the basis of moles of sulfate per 1,000 g of wet tissue was 0.98, 1.3, 2.2 and 1.5 mM for rats fed the diets containing 0.0002, 0.02, 0.1 and 0.42% of inorganic sulfate, respectively. Thus it appears that the liver sulfate pool is limited by a combination of the rate of oxidation of the sulfur-containing amino acids and the extraction of sulfate from the portal system.

A method for determining tissue sulfate.

A method for the determination of the inorganic sulfate present in rat liver homogenates has been developed. In order to determine sulfate, a protein-free extract is required. The classical protein precipitation methods of preparing protein-free extracts gave 2.5-40% recovery of added /sup 35/SO/sub 4//sup 2 -/. Separation of the protein by ultrafiltration gave only 29% recovery when 0.15 M KCl was the homogenizing medium. A homogenization medium containing 0.154 M NH/sub 4/OH and 20 g EDTA per liter gave 102 +/- 11% recovery of added /sup 35/SO/sub 4//sup 2 -/ when the protein was separated by ultrafiltration.

Effect of S-adenosylhomocysteine on sulfhydryl xenobiotic transmethylases in rat liver

Rat liver cytosolic thiopurine methyltransferase and microsomal thiol methyltransferase were each found to be subject to control by the absolute molar ratio of S-adenosylmethionine to S-adenosylhomocysteine using cell-free enzyme preparations. As this ratio was lowered, inhibition of both sulfhydryl xenobiotic transmethylases occurred. On the other hand, when the ratio was decreased in vivo by the administration of D,L-homocysteine thiolactone to animals, this alteration was accompanied by an inhibition of only thiopurine methyltransferase activity. Thiol methyltransferase activity was not significantly affected after drug treatment, which would suggest that there is a compartmentalization of S-adenosylhomocysteine in the intact hepatocyte.

Inhibition of mitochondrial palmitate oxidation by calmodulin antagonists

1. The effect of calmodulin antagonists on the rate of palmitate oxidation by isolated rat liver mitochondria was studied. 2. In the presence of 100 microM amitriptyline, chlorpromazine, prenylamine, N-(4-aminobutyl)-5-chloro-2-naphthalenesulfonamide or N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide, palmitate oxidation was inhibited by 17, 34, 49, 31 and 37%, respectively. 3. The degree of inhibition of palmitate oxidation exerted by these chemical compounds did not appear to correlate appreciably with changes in mitochondrial membrane fluidity.

Glutathione: an endogenous substrate for thiopurine methyltransferase?

An investigation was initiated to determine if glutathione is an endogenous substrate for thiopurine methyltransferase. Glutathione, as well as S-methylglutathione, were each capable of inhibiting the enzyme in a concentration-dependent manner, which suggested competitive and product inhibition, respectively. However, radiochromatography revealed that S-methylation of glutathione is not a catalytic activity of this sulfhydryl transmethylase. Subsequent experiments indicated that the inhibitory effects of both glutathione and S-methylglutathione on thiopurine methyltransferase may be due to their acidic natures, changing the reaction mixture pH away from the optimal range for the enzyme.

Regulation of rat liver protein methylase III by S-adenosylhomocysteine after D,L-homocysteine thiolactone administration and during pyridoxine deficiency

Abstract Since S-adenosylhomocysteine is a potent inhibitor of rat liver protein methylase III in vitro , this enzyme appears to be regulated by modulation of the intracellular molar ratio of S-adenosylmethionine to S-adenosylhomo-cysteine (SAM:SAH ratio). The intraperitoneal administration of D,L-homocysteine thiolactone to animals (500 or 1000 mg/kg), followed by sacrifice 20 minutes later, decreased the SAM:SAH ratio in vivo from 4.35 in the control rats to 1.33 and 0.56 and caused a 62 and 82% inhibition of protein methylase III activity, respectively. Similarily, rats maintained on a pyridoxine-deficient diet for 7 weeks displayed a 5-fold reduction in the hepatic SAM:SAH ratio, when compared either to control or pair-fed counterparts, and a significant 26% reduction in the activity of protein methylase III. Thus, it would appear that a pyridoxine deficiency affects protein methylase III activity via altered SAM:SAH ratios.

Down-regulation of rat liver β-adrenergic receptors by cysteine

Abstract Down-regulation of hepatic β-adrenergic receptors was indicated by a 56% decrease in the specific activity of 125 I-iodocyanopindolol bound to rat liver membrane preparations from rats fed diets containing 15% of casein supplemented with cysteine, instead of methionine or unsupplemented. Down-regulation of hepatic β-adrenergic receptors by cysteine appears to be mediated through an effect of cysteine on the tissue concentration of S-adenosyl methionine (SAM). The liver tissue concentration of SAM in rats fed cysteine-supplemented diets decreased 53% compared to those fed diets supplemented with methionine. The decrease in liver SAM in rats fed the diet supplemented with cysteine appears to reflect a non-competitive inhibition of methionine adenosyl-transferase by cysteine. Lineweaver-Burk plots demonstrated a dose-related V max response to cysteine but did not change the apparent K m at any concentration tested.

Phenylethanolamine-N-Methyl transferase may control methionine demethylation

Abstract Rats fed diets which contained 15% of casein and 0.620% of methionine with 0.0002, 0.02 and 0.42% of dietary inorganic sulfate had a dietary sulfate-related change in methionine metabolism. Rats fed the diets low in sulfate (0.0002%) had a 35% increase in methionine metabolism compared to rats fed the diets high in sulfate (0.42%). In contrast, rats fed the diet low in sulfate (0.0002%) had the lowest level of tissue S-adenosyl-methionine and the highest activity of phenylethanolamine-N-methyltransferase activity. Those animals fed the diet normal with respect to sulfate (0.02%) had intermediate levels of S-adenosylmethionine and phenylethanolamine-N-methyl transferase activity. Rats fed the diet high in sulfate (0.42%) had the highest level of tissue S-adenosyl-methionine and the lowest phenylethanol-amine-N-methyl transferase activity. Due to the inverse relationship between S-adenosylmethionine and phenylethanolamine-N-methyl transferase activity, it appears that the catecholamines may function as a methyl sink for the increase in the metabolism of methionine required to provide sulfate for rats fed diets low in sulfate.