STUDIES ON THE ENZYMATIC DEGRADATION OF COCARBOXYLASE

概要

論文の詳細を見る
Thiamine diphosphate-decomposing activities in the homogenates of rat brain, dog kidney' and bovine kidney were studied. Those in the autolysate of dog kidney, bovine kidney and yeast were fractionated, some of which were partially purified, and the enzymological properties were also studied with the following results.1. When the liberation of inorganic phosphate was measured for determining thiamine diphosphate-decomposing activities, a turbidity caused by the reaction between thiamine and phosphomolybdate appeared. However, under suitable conditions, and in a settled concentration range, the extinction could be used as an indicator of degradation. Furthermore, the method of using ammonium chloride to extinguish the turbidity was retested and proved to be excellent and useful. It was applied to the present experiments.2. β-Glycerophosphate-dephosphorylating activity in rat brain homogenates showed, under conditions used, two maxima at pH 9 and 5. On the contrary, thiamine diphosphate-decomposing activity showed the highest value at approximately pH 9, and, in addition, an accessary peak between pH 6 and 7. The degree of the inhibition for thiamine diphospate-decomposing activity by fluoride was much less than that for β-glycerophosphate-dephosphorylating activity, especially at pH 6.9. The activity for thiamine diphosphate was influenced to some extent by the change in osmotic pressure. The enzyme was presumed to exist in both soluble and insoluble fractions.3. Thiamine diphosphate-degrading activity in dog kidney homogenates showed a maximum between pH 8 and 9, but the activity was also observed below pH 6. The accessory activity peak was observed adjacent to pH 4.9.An enzyme preparation was obtained from the supernatant of the kidney homogenate by fractionating at various pH's. It decomposed thiamine diphosphate at acidic pH range, but its activity was not so high. Another enzyme preparation was obtained from the extract prepared by autolyzing kidneys in the mixed solvent containing water, acetone, toluene and ethyl acetate. This preparation degraded thiamine diphosphate more markedly at pH 9.2.4. Bovine kidney homogenates showed strong decomposing activities for inorganic pyrophosphate over a wide pH range. Besides these, they showed relatively high decomposing activities for thiamine diphosphate at pH 9 and 6, and also for β-glycerophosphate at pH 9 and 5 (so-called alkaline and acid phosphatases).5. Using the autolysate of bovine kidneys as the starting material, attempts for purification of the enzyme were made. In the partially purified enzyme preparations, no inorganic pyrophosphatase activity at pH 9.2 was found. However, decomposing activities for both thiamine diphosphate and β-glycerophosphate were found, and it was difficult to separate them. These two activities showed the same behaviour when tested with ordinary zone electrophoresis and column chromatography. With ammonium sulfate fractionation, trypsin treatment, and acetone fractionation, especially with repeated acetone fractionations, the activity ratio (TDPase/GPase) somewhat shifted to the larger. But it was not clarified whether ther could be separated or not. It was presumed that the thiamine diphosphate-decomposing enzyme and the β-glycerophosphate-decomposing enzyme would be of a very similar nature.6. Thiamine diphosphate-decomposing enzyme preparations obtained from bovine kidneys could also decompose ATP, adenosine-5′-phosphate, thiamine monophosphate, and β-glycerophosphate.Presumptive mechanism for degradation is as follows.TDP→TMP+iP, TMP→T+iPThe activities for both thiamine diphosphate and thiamine monophosphate was inhibited by the increase in substrate concentration, but that for β-glycerophosphate was not. Thiamine showed no inhibitory action
日本ビタミン学会の論文

著者

Horie Shigeo
Department Of Biochemistry Kitasato University School Of Medicine

STUDIES ON THE ENZYMATIC DEGRADATION OF COCARBOXYLASE

スポンサーリンク

概要

著者

関連論文

スポンサーリンク