Tsunehiko Katsunuma

Studies on New Intracellular Proteases in Various Organs of Rat

1. Specific proteases which inactivate the apo-proteins of many pyridoxal enzymes were found in skeletal muscle, liver and small intestine of rats. The protease from these three organs were purified and their properties were compared. 2. The purified proteases from liver and skeletal muscle appeared homogeneous on acrylamide gel electrophoresis. Two different proteases were separated from small intestine. A homogeneous, crystalline enzyme was obtained from the muscle layer while enzyme from the mucosa was partially purified. 3. They showed substrate specificity for pyridoxal enzymes. Their pH optima were in an alkaline region. They showed activity with the substrate of chymotrypsin, N-acetyl-L-tyrosine ethyl ester, but not with that of trypsin, p-toluenesulfonyl-L-arginine ethyl ester. They were inhibited by pyridoxal phosphate or pyridoxamine phosphate and seryl residues were involved in their active center. 4. The four enzymes differed in the following characters: (a) molecular weights; (b) patterns of elution from a CM-Sephadex column; (c) rates of inactivation of substrate enzymes; (d) rates of cleavage of N-acetyl-L-tyrosine ethyl ester; (e) reactivities with antiserum against the enzyme from the muscle layer of small intestine; (f) specific activities. 5. The amino acid composition and effect of chemical modifications of the crystalline enzyme from the muscle layer of small intestine were examined to elucidate its active sites and mode of action. Serine and histidine residues were found to be essential for protease activity. A tyrosine residue was also necessary for activity. Modifications of its sulfhydryl group, amino residues and carboxyl group had no effect on its activity.

Studies on a tryptophan synthase inactivating system from yeast

Abstract Manney demonstrated in 1968 (4) that under certain growth conditions the tryptophan synthase of yeast extracts is rapidly inactivated upon standing. We have purified two different tryptophan synthase inactivating enzymes about 1000-fold. Among different yeast enzymes studied as substrates, the inactivating enzymes show a certain specificity for tryptophan synthase. Evidence is presented which strongly suggests that the mechanism of inactivation is proteolytic. A macromolecular inhibitor of the inactivating enzymes, which prevents inactivation of tryptophan synthase, has been purified about 30-fold from yeast. The inhibitor is probably a protein. It is resistant to boiling and can be treated with trichloroacetic acid without loss of activity. In batch cultures of yeast, tryptophan synthase inactivating activity is not found in the exponential phase of growth, but appears at high levels in the stationary phase. The biological significance of the tryptophan synthase inactivating system is discussed.

Comparative studies on the inactivating enzymes for pyridoxal enzymes from yeast and rat

Summary With protein fractionating methods an apo-ornithine transaminase inactivating activity from yeast has been partially purified. The kinetics of the inactivating reaction and the behaviour of the active principle during purification make it very probable that we are dealing with an “inactivating enzyme”. Comparison of the OTA inactivating enzyme from yeast with the previously described OTA “splitting enzyme” from rat tissues was established by cross reacting the substrate OTAs and other substrate enzymes with the inactivating enzymes from yeast and rat intestine. It has been found that the group specificity of the inactivating enzyme towards the apo-forms of pyridoxal enzymes is not only observed when the inactivating enzyme and the substrate enzymes are prepared from the same species but also when the inactivating enzymes and the substrate enzymes from yeast and rat tissues are cross exchanged.

A autopsied case of hypercitrullinemia in adult caused by partial deficiency of livcr arginosuccinate synthetase

46歳の男性で,反復する意識障害,高アンモニア血症,高シトルリン血症などの臨床所見を示しながら,明らかな肝機能障害や門脈大循環短絡を認めない1症例を経験した.剖検にて類瘢痕型肝脳疾患の病理診断を得,また剖検肝および腎にて尿素サイクル酵素系の酵素化学的検討を行い,本症例の病因が,肝Arginosuccinate Synthetase活性の低下であることを明らかにし,本症の病因と病態に関して文献的考察を行った.