Regulation of ascorbic acid and of xylulose synthesis in liver extracts. The effect of starvation in various animals

1. The effect of starvation on the synthesis of ascorbic acid and of xylulose from glucuronolactone and from gulonate has been studied with liver extracts from cats, rabbits, hamsters, mice and guinea pigs. 2. The synthesis of ascorbic acid from glucuronolactone is decreased in all species except cats, and that from gulonate is decreased in hamsters and mice only. 3. The synthesis of xylulose from glucuronolactone was decreased in all species except cats and mice, whereas from gulonate it was enhanced in all the species examined.     

Biosynthesis of ascorbic acid by extant actinopterygians

Polypterus senegalus , the longnose gar Lepisosteus osseus and the bowfin Amia calva had gulonolactone oxidase activity in the kidney and thus can synthesize ascorbic acid de novo . The enzyme activity was associated with the microsomal fraction. The common carp Cyprinus carpio and the goldfish Carassius auratus had no gulonolactone oxidase activity. Antibodies directed against white sturgeon gulonolactone oxidase showed cross-reactivity with lake sturgeon, bowfin and longnose gar kidney enzymes, but not with enzymes from Polypterus , sea lamprey, and tadpole kidney or pig liver. Given cross-reactivity, gulonolactone oxidase relatedness matched actinopterygian phylogeny, and suggested homology of the character throughout fishes. Modern teleosts may have lost the ability to synthesize ascorbic acid since the late Triassic as a result of a single reversal in the founding population. Wild bowfin and longnose gar exhibited high ascorbate concentrations in liver and spleen when compared with the teleosts rainbow trout Oncorhynchus mykiss and common carp fed vitamin C-supplemented diets.     

Scurvy-prone animals, including man, monkey, and guinea pig, do not express the gene for gulonolactone oxidase

Man, monkeys, and guinea pigs cannot synthesize ascorbic acid due to a lack of gulonolactone oxidase activity. Recently, using two immunological methods, immunoprecipitation and microcomplement fixation, we reported that guinea pigs do not contain antigenic material related to gulonolactone oxidase. Now, using such immunologie techniques as double immunodiffusion, microcomplement fixation, antibody affinity chromatography, and a more sensitive radioimmunoassay, we have found that all three of these species do not contain immunologically cross-reacting material to gulonolactone oxidase. On the other hand, comparable extracts from tissues of all other species that were investigated and that do possess gulonolactone oxidase did cross-react with antiserum to enzyme from two widely differing species, rat and goat. We conclude that the gene for gulonolactone oxidase is not expressed in these scurvy-prone animals.     

Induction and peroxisomal appearance of gulonolactone oxidase upon clofibrate treatment in mouse liver

Various antihyperlipemic peroxisome proliferators are known to be carcinogenic in rodents but not in human, other primates and guinea pig, which species lost their ability to synthesize ascorbate due to mutations in the gulonolactone oxidase gene. Ascorbate synthesis is accompanied by H2O2 production, consequently its induction can be potentially harmful; therefore, the in vivo effect of the peroxisome proliferator clofibrate was investigated on gulonolactone oxidase expression in mouse liver. Liver weights and peroxisomal protein contents were increased upon clofibrate treatment. Elevated plasma ascorbate concentrations were found in clofibrate-treated mice due to the higher microsomal gulonolactone oxidase activities. Remarkable gulonolactone oxidase activity appeared in the peroxisomal fraction upon the treatment. Increased activity of the enzyme was associated with an elevation of its mRNA level. According to the present results the evolutionary loss of gulonolactone oxidase may contribute to the explanation of the missing carcinogenic effect of peroxisome proliferators in humans.     

维生素C发酵中2—酮—L—古龙酸还原酶的研究

由于２酮Ｌ古龙酸还原酶（简称ＫＧＲ）的存在,理论上造成了Ｖｃ合成前体２酮Ｌ古龙酸（简称２ＫＬＧ）部分被还原成Ｌ艾杜糖酸［１,２］,影响了Ｖｃ二步发酵的产率。而从Ｌ山梨糖到２酮Ｌ古龙酸的转化中,除被ＫＧＲ催化的还原负反应外,均属...     

Regulation of amino acid transport in Thiobacillus thioparus.

Amino acid transport in amino acid auxotrophs of Thiobacillus thioparus was enhanced during growth on rate-limiting amino acid concentration. A pleiotropic mutation enhanced general amino acid transport as manifested by higher values of Vmax of amino acid transport. Affinity constants remained unaltered. Mutants with enhanced transport properties did not show changes in oxidation of thiosulfate, did not oxidize various organic compounds, and did not increase the heterotrophic potential of T. thioparus. The mutations for enhanced transport caused increased synthesis of amino acid transport system components. A method for genetic transformation of T. thioparus is described.     

The mechanism of enhancement by fatty acid hydroperoxides of anaphylactic mediator release.

Indomethacin augmented the release of histamine and SRS-A but abolished synthesis of TxB2. Compound CLI that inhibited both cyclo-oxygenase and lipoxygenase pathways of arachidonic acid metabolism did not augment release of anaphylactic mediators. 13-HPLA enhanced mediator release from lungs in which arachidonic acid metabolism was blocked by compount CLI. Thus, it is concluded that 13-HPLA enhances mediator release not by altering the balance of arachidonic acid metabolites, e.g. by inhibiting synthesis of prostacyclin, but by a direct effect on lung mast cells. A corollary to this conclusion is that the fatty acid hydroperoxide (HPETE) formed by lipoxygenase from arachidonic acid may also augment the release of anaphylactic mediators. Thus, the enhancement of mediator release by indomethacin may be attributed to increased synthesis of HPETE following inhibition of cyclo-oxygenase.     

The mechanism underlying the hypolipemic effect of perfluorooctanoic acid (PFOA), perfluorooctane sulphonic acid (PFOSA) and clofibric acid.

The influence of the peroxisomal proliferators perfluorooctanoic acid (PFOA), perfluorooctane sulphonic acid (PFOSA) and clofibric acid on lipid metabolism in rats was studied. Dietary treatment of male Wistar rats with these three compounds resulted in rapid and pronounced reduction in both cholesterol and triacylglycerols in serum. The concentration of liver triacylglycerols was increased by about 300% by PFOSA. Free cholesterol was increased by both perfluoro compounds. Cholesteryl ester was reduced to 50% by PFOSA as well by clofibrate. In hepatocytes from fed rats, all the compounds resulted in reduced cholesterol synthesis from acetate, pyruvate and hydroxymethyl glutarate, but there was no reduction of synthesis from mevalonic acid. The oxidation of palmitate was also increased in all groups. The perfluoro compounds, but not clofibrate, caused some reduction in fatty acid synthesis. The activity of liver HMG-CoA reductase was reduced to 50% or less in all treatment groups and all three compounds led to lower activity of acyl-CoA:cholesterol acyltransferase (ACAT). Changes in other enzymes related to lipid metabolism were inconsistent. The present data suggest that the hypolipemic effect of these compounds may, at least partly, be mediated via a common mechanism; impaired production of lipoprotein particles due to reduced synthesis and esterification of cholesterol together with enhanced oxidation of fatty acids in the liver.     

Orotic acid biosynthesis in arginine-deficient rats.

Arginine deficiency is associated with a mild orotic aciduria. Liver slices from rats fed a purified l-amino acid diet with (control) and without arginine supplementation were used for studies of [¹⁴C]bicarbonate incorporation into orotic acid. The nanomoles of orotic acid synthesized in isolated liver slices from both control and arginine-deficient animals increased linearly with time. Orotic acid biosynthesis was significantly greater in liver slices than slices of heart, muscle, kidney, and minced spleen. The order of orotate biosynthesis from [¹⁴C]bicarbonate was liver > spleen = kidney > muscle > heart. Arginine deficiency resulted in a significant stimulation of liver orotic acid biosynthesis. This stimulation in pyrimidine biosynthesis can account for a major portion of the orotic aciduria. Orotic acid synthesis from spleens isolated from arginine-deficient rats was also enhanced compared with controls. Although the rate of orotic acid biosynthesis is small relative to liver production, the spleen may contribute slightly to increased orotic aciduria in the arginine-deficient rat. Arginine supplementation in vitro to livers from rats fed either the control of arginine-deficient diet resulted in a significant reduction in synthesis of orotic acid. Dietary arginine may play a key role in regulating mitochondrial carbamoyl phosphate utilization into both pyrimidine and urea biosynthesis.     

Effect of mercaptopicolinic acid and of transaminase inhibitors on glycogen synthesis by rat hepatocytes.

To explore the mechanism of the stimulation of glycogen synthesis by amino acids (1) we have studied the effects of transaminase inhibitors and of mercaptopicolinic acid, (MPA) an inhibitor of phosphoenol pyruvate carboxykinase. Mercaptopicolinic acid enhanced glycogen synthesis from fructose, dihydroxyacetone and xylitol. Stimulation of glycogen synthesis with hepatocytes from fasted rats by 0.5 mM mercaptopicolinic acid was 50–70% as effective as 10 mM glutamine. With hepatocytes from fed rats, the stimulation of glycogen synthesis by mercaptopicolinic acid was more pronounced, and stimulation by mercaptopicolinic acid and amino acids was additive. Glycogen synthesis as high as 1% in wet weight per hour was attained in hepatocytes with a high initial glycogen content. Over 80% of glycogen synthase was in the active (a) form. Amino oxyacetic acid greatly depressed or abolished the stimulatory effect of glutamine and asparagine and of mercatopicolinic acid, and induced extensive glycogen breakdown in hepatocytes of fed rats.     

Synthesis of fatty acid sterol esters using cholesterol esterase from Trichoderma sp. AS59

We recently reported the characterization of novel cholesterol esterase (EC. 3.1.1.13) from Trichoderma sp. and preliminary work on sterol ester synthesis. In the present study, we further examined the enzyme ability to synthesize cholesterol esters from cholesterol and free fatty acids of various chain lengths, and compared the fatty acid specificity in synthesis with that in hydrolysis. The enzyme catalyzed the synthesis of medium- and long-chain fatty acid cholesterol esters, but failed to synthesize short-chain fatty acid esters. The fatty acid specificities in the synthesis and hydrolysis of cholesterol esters were entirely different from each other. Unlike other lipolytic enzymes, the enzyme was largely independent of water content in the synthesis of cholesterol oleate, and it achieved near-complete esterification in the presence of an equimolar excess of oleic acid. Of additional interest is the finding that the addition of n-hexane markedly enhanced the esterification activities on all the medium- and long-chain saturated fatty acids used. Based on these findings, we attempted to synthesize stigmasterol stearate as a food additive to lower cholesterol levels in blood plasma, and found that the enzyme catalyzed effective synthesis of the ester without the need of dehydration during the reaction, indicating the potential utility of the enzyme in the food industry.     

Activation of synthesis of hexacosenoic acid by sulfhydryl reagents in swine cerebral microsomes

The elongation of icosenoyl-CoA (20:1-CoA) in swine cerebral microsomes resulted in the synthesis of docosenoic acid (22:1) and tetracosenoic acid (24:1), but the synthesis of hexacosenoic acid (26:1) was negligible. In contrast, in the presence of sulfhydryl reagents (0.6 mM N-ethylmaleimide [NEM] or 0.3 mM p-chloromercuriphenylsulfonic acid [PCMPS]) the synthesis of 26:1 was remarkably enhanced. We suggest that the synthesis of 26:1 from 20:1-CoA was more enhanced by NEM or PCMPS as a result of activation of the condensation step in the elongation of 24:1 (intermediate) to 26:1.     

Biosynthesis of xanthurenic acid 8-O-beta-D-glucoside in Drosophila. Characterization of the xanthurenic acid:UDP-glucosyltransferase activity

Xanthurenic acid 8-glucoside is a side metabolite of the tryptophan-xanthommatin pathway in Drosophila. From 3-hydroxykynurenine, two biosynthetic pathways can be envisaged, one via xanthurenic acid, and another via 3-O-glucoside of 3-hydroxykynurenine. In this report evidence is presented to show that the synthesis takes place via xanthurenic acid. (a) We have demonstrated that the Drosophila melanogaster vermilion purple mutant (unable to synthesize 3-hydroxykynurenine) synthesizes xanthurenic acid 8-glucoside when fed with xanthurenic acid; and (b) the activities required for its synthesis via xanthurenic acid have been found (3-hydroxykynurenine transaminase and xanthurenic acid:UDP-glucosyltransferase). This is the first time that a UDP-glucosyltransferase activity that utilizes xanthurenic acid has been demonstrated. The enzyme in crude extracts from Drosophila sordidula shows the following characteristics. (a) It has optimal activity at 35 degrees C at pH 7.1 (in buffer Tris-HCl), and in the presence of a divalent cation (Mg2+ or Mn2+); (b) the activity is inhibited by xanthurenic acid (above 1.5 mM), UDP, D-gluconic acid 1,5-lactone, and Triton X-100; (c) it is localized in both the microsomal and the soluble fractions; (d) the specific activity is two times higher in heads than in bodies; and (e) the activity is enhanced in flies fed with phenobarbital.     

Ascorbate-mediated electron transfer in protein thiol oxidation in the endoplasmic reticulum

Addition of, or gulonolactone oxidase-dependent in situ generation of, ascorbate provoked the oxidation of protein thiols, which was accompanied by ascorbate consumption in liver microsomal vesicles. The maximal rate of protein thiol oxidation was similar upon gulonolactone, ascorbate or dehydroascorbate addition. Cytochrome P450 inhibitors (econazole, proadifen, quercetin) decreased ascorbate consumption and the gulonolactone or ascorbate-stimulated thiol oxidation. The results demonstrate that the ascorbate/dehydroascorbate redox couple plays an important role in electron transfer from protein thiols to oxygen in the hepatic endoplasmic reticulum, even in gulonolactone oxidase deficient species.     

The Myo‐inositol pathway does not contribute to ascorbic acid synthesis

• Ascorbic acid (AsA) biosynthesis in plants predominantly occurs via a pathway with d ‐mannose and l ‐galactose as intermediates. One alternative pathway for AsA synthesis, which is similar to the biosynthesis route in mammals, is controversially discussed for plants. Here, myo‐inositol is cleaved to glucuronic acid and then converted via l ‐gulonate to AsA. In contrast to animals, plants have an effective recycling pathway for glucuronic acid, being a competitor for the metabolic rate. Recycling involves a phosphorylation at C1 by the enzyme glucuronokinase.
• Two previously described T‐DNA insertion lines in the gene coding for glucuronokinase1 show wild type‐like expression levels of the mRNA in our experiments and do not accumulate glucuronic acid in labelling experiments disproving that these lines are true knockouts. As suitable T‐DNA insertion lines were not available, we generated frameshift mutations in the major expressed isoform glucuronokinase1 (At3g01640) to potentially redirect metabolites to AsA.
• However, radiotracer experiments with ³H‐myo‐inositol revealed that the mutants in glucuronokinase1 accumulate only glucuronic acid and incorporate less metabolite into cell wall polymers. AsA was not labelled, suggesting that Arabidopsis cannot efficiently use glucuronic acid for AsA biosynthesis.
• All four mutants in glucuronokinase as well as the wild type have the same level of AsA in leaves.

     

Irreversible inactivation of chicken liver fatty acid synthetase by its substrates acetyl and malonyl CoA: Effect of temperature and NADP+ on fatty acid and triacetic acid lactone synthesis

Chicken liver fatty acid synthetase is irreversibly inactivated by malonyl CoA and by acetyl and malonyl CoA. Two active forms of the enzyme existing above and below 11.5° are inactivated at different rates. Activities for fatty acid and triacetic acid lactone synthesis are lost at about the same rate and NADP⁺ protects the enzyme against inactivation. Inactivation results from the enhanced covalent binding of malonyl groups in addition to those required for fatty acid synthesis.     

Biosynthesis of D-alanyl-lipoteichoic acid: role of diglyceride kinase in the synthesis of phosphatidylglycerol for chain elongation.

Lipophilic and hydrophilic D-alanyl-lipoteichoic acids are elongated in Lactobacillus casei by the transfer of sn-glycerol 1-phosphate units from phosphatidylglycerol to the poly(glycerophosphate) moiety of the polymer. These sn-glycerol 1-phosphate units are added to the end of the poly(glycerophosphate) which is distal to the glycolipid anchor; 1,2-diglyceride results from this addition. The presence of a diglyceride kinase was suggested by the ATP-dependent phosphorylation of 1,2-diglyceride to phosphatidic acid. Inorganic phosphate was used to initiate the synthesis of lipophilic lipoteichoic acid (LTA) and the elongation of both lipophilic and hydrophilic LTA. Three observations suggest that phosphate and other anions play a role in the in vitro synthesis of LTA and its precursors. First, the conversion of 1,2-diglyceride to phosphatidic acid by diglyceride kinase was stimulated. Second, the synthesis of phosphatidylglycerol was increased. Third, the elongation of lipophilic and hydrophilic LTA was enhanced. These observations indicated that one effect of phosphate might be to enhance the utilization of 1,2-diglyceride for the synthesis of phosphatidic acid. This phospholipid is a precursor of phosphatidylglycerol, the donor of sn-glycerol 1-phosphate for elongation of LTA.