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l- kang rang xue suan zhi sheng wu he cheng l- gu luo tang suan - - nei zuo ji l- ban ru tang suan - - nei zuo zhi yang hua
Pages: 190-204
Year: Issue:  2
Journal: Acta Physiologica Sinica

Keyword:  生物合成半乳糖酸血酸酶活力酶作用氧化大白鼠乙二胺四乙酸磷酸古罗糖;
Abstract: The last step in the biosynthesis of vitamin C has been shown to be carried out enzymatically by a particulate preparation from rat liver.In the presence of oxygen and glutathione,L-gulono-or L-galactono-γ-lactone is oxidized to L-ascorbic acid. When the mitochondria and the microsomes are separated by means of differential centri- fugation,the enzyme activity is found in both fractions but is much higher in the microsomes. In the particulate form,the enzyme is insensitive to inhibitors such as cyanide,azide,iodoacetate, arsenite and atebrin but is stimulated by 2,4-dinitrophenol. Upon treatment with sodium desoxycholate,the microsomes can be partially solubilized.The solubilized enzyme can be purified 5—6 fold by adsorption on alumina C_γ gel and precipitation by ammonium sulfate at 0.2 saturation.The partially purified enzyme still requires oxygen for its action and is incapable of utilizing ferricytochrome c as acceptor or artificial hydrogen acceptors such as ferricyanide,methylcne blue and 2:6-dichlorophenol indophenol.It still acts only on the lactones and cannot utilize the acid form of the substrates.With the iemoval of most of the substances capable of oxidizing ascorbic acid, glutathione is no longer a necessary factor in the reaction medium.Its addition,however, slightly enhances the reaction.This and the facts that p-chloromercuribenzoate strongly inhibits it while metal-chelating agents such as 8-hydroxyquinoline and thiocyanate enhance its activity show that the enzyme probably contains essentiai-SH-groups.The enzyme action is still in- sensitive to cyanide,being only slightly depressed by very high concentration of KCN(10~1M). The inhibitory effect,however,is more marked upon incubation in the absence of the substrate. The ()nzyme is also inhibited by BAL but not by atebrin,arsenite or fluoride,while 2,4-dinitro- phenol has a markedly stimulating effect.Inhibitors that are known to affect catalase activity, such as hydroxylamine and azide have an inhibitory effect on our enzyme only when gluta- thione is absent.It is suggested that in the oxidation of the substrate,H_2O_2 is produced and normally is immediately decomposed by catalase which accompanies our enzyme and proves to be very difficult to remove completely.In the presence of inhibitors which act on catalase, however,H_2O_2 accumulates resulting in either the oxidation of ascorbic acid or inactivation of the enzyme or both.Glutathione,if present,protects both the product and the enzyme from destruction.The action of KCN which,in addition to its strong effect on catalase,also protects L-ascorbic acid,is not influenced by glutathione in the same way.The enzyme shows a decided requirement for inorganic phosphate which can only be partially replaced by metal chelating agents such as versene(ethylene diamine tetraacetic acid). Work on the further purification of the enzyme and detailed investigation on its nature and action is still in progress.
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