Previously N.KATUNUMA et al. discovered that GOT from pig liver consist of two types of isozyme, the one being localized in cell supernatant fraction (GOTs) and the other in mitochondria (GOTM). Subsequently they succeeded in purifying the isozyme to almost single protein. Using the technique of column chromatographic separation, the author could elucidate the mechanism of the leakage of the GOT-isozvme from the heart muscles into serum in the case of experimental myocardial infarction. Method s In dogs, experimental myocardial infarction was produced by ligation of the circumflex branch of the left coronarv arterv, and a few to several dogs were killed at a certain period of time after the occlusion (24 hr, 2 day, 3 day, 4 day, 5 day, 7 day, 10 day, 14 day after operation) and the direct ECG was recorded respectively, before slaughter. Then, the removed heart was separated into the infarcted and non-infarcted portions, and the GOT ac-tivity in the heart muscles obtained from each portion was measured. In order to separate the GOT-isozyrne quantitatively, HUZlNO-KATUNUMA'S method was used, and the GOT activity was expressed by a modified method of FRIEDMANN-HAUGEN. Results 1. GOT-isozyme in heart muscles. 1) The ratio of the total activity of GOTS to that of GOTM in normal heart muscles was about 4 to 1 on the average of 2 dogs. 2) At 24 hours after the occlusion, the ratio of the total activitv of GOTS to that of GOTM in the non-infarcted portion was about 4 to 1 on the average of 5 dogs, while in the infarcted portion, it was about 1.2 to 1. Namely, the total activity of GOTS in the infarcted portion was decreased to about 2S per cent of that in the non-infarcted portion. On the contrary, any significant change of the total activitv of GOT_M in the infarcted portion could not be observed compared with that of the non-infarcted portion. 3) At 2 days, 3 clays and 4 clays after the occlusion in 2 dogs respectively, no change of the total activity of GOT_M could be observed between the infarcted portion and non-infarctecl portlon, just like at 24 hours after the occlusion. As for the total activity of GOTs, the rates of decrease were 71.8 and 72.1 per cent after 2 days, 74.8 and 77.7 per cent after 3 days, 73.9 and 77.5 per cent after 4 days respectively in the infarcted portion compared with that in the non-infarcted portion.

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Previously KATUNUMA has shown that GOT from pig liver consists of two types of isozyme, the one being localized in cell supernatant (GOTs) and the other in mitocondria(GOTM), and subsequently purified them to almost single protein. Using the technique of colum chromatographic separation, the author elucidated the changes of GOT-isozyme and effects of some medicines on the enzymatic activity in infarcted heart muscles togather with subsequent changes of pattern of ECG in experimental infarction in order to develop the way to their clinical application. Method of the Experiment Experimental myocardial infarction was made by ligating the coronary artery of dogs at the circumflex branch of the left coronary artery. The direct ECG were recorded before or twenty-four hours after occlusion respectively. The dogs were sacrificed for the enzymatic assay. Furthermore, in order to determine the effect of some medicines, medicines were injected intravenously before or immediately after the ligation of coronary artery. Dogs were killed and the removed heart muscle was divided into the infarcted portion and non-infarcted portion. The isozymic activity were measured on the materials obtained from each portion. HUZlNO-KATUNUMA'S method was employed for the separation and the measurement of the isozymic activity. Results 1. GOT-isozyme in Normal Heart Muscles: The ratio of the activity of GOTS to GOTM in normal heart muscles was about 4 to I on the average. 2. Twenty-four Hours after Occlusion: 1) Control groups without medicines The average ratio of the activity of GOTS to GOTM in the non-infarcted portion were almost the same value as that in the normal heart muscles. The average activity of GOTS in the infarcted portion decreased about 70 per cent compared with that in the non-infarcted portion. 2) The effect of some medicines on the GOT-isozymes in the infarcted portion a) Control group (physiological saline solution) The rates of decrease of GOTS were 62.8 per cent and 73.6 per cent respectively, and, in comparison with the control group to which no medicines were administered, any difference could not be observed.

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The changes of hepatic lysosomal cathepsins, alanine aminotransferase (AAT) activities and serum amino acids were studied on rat fed on high and low protein diets with or without B_6. The group fed on high protein diet without B_6 caused the marked suppression of growth and also in the pathological bad lie of hair with dermatosis, while these pathological syndromes were not observed in rats fed on low protein diet without B_6. The activities of cathepsins B, L, H and J in rats fed on high protein diet these cathepsin activities were not affected by B_6 deficiency. Therefore, the biosynthesis and also degradation of these cathepsins are not influenced by B_6 deficiency. Some amino acid levels in serum were increased by high protein diet intake and B_6 deficiency. The levels of glycine were significantly decreased in rat fed on high protein diet. The levels of aspartate and glycine were markedly elevated in the cases of B_6 deficiency and those of alanine and hydroxyproline were decreased in the cases of B_6 deficiency.

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Actomyosin prepared from the ordinary muscle of pacific mackerel Scomber japonicus was denatured by the crude enzymes obtained from the above mentioned muscle of mackerel at pH 5.0 in the presence and the absence of pepstatin, the typical inhibitor of cathepsin D. Thus, it has been presumed that other than cathepsin D there were some proteases which denatured the actomyosin of the ordinary muscle of pacific mackerel at about pH 5.
Optimum pH's of hemoglobin substrate were 5.4 being measured by the Cu-Folin method and at 5-6 by the ninhydrin one.
The enzymes fractionated by Sephadex G-75 column chromatography, were inhibited by leupeptin and iodoacetic acid and slightly activated by L-cysteine. From this result, it has been assumed that the enzymes would be acid thiol proteases.
The enzymes hydrolyzed the synthetic substrate Z-Phe-Arg-MCA which is sensitive to both cathepsins B and L, but inactive on Arg-MCA which is known to be susceptible to cathepsin H.
From these results, it has been confirmed that there were acid thiol proteases which denatured or autolyzed the actomyosin of the ordinary muscle of pacific mackerel, and that the proteases might be cathepsins B or L, or the like.

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わたくしは癌組織においては,変性組織と明らかに異なる電子構造の異常が存在し,少なくともその一部が細胞顆粒に存在する燐脂質を含む機能リポ蛋白(または活性錯化合物複合体)におけるradicalのtrap,またはspin relaxationが正常のそれと異なることに依存するという実験成績をえた.そして量子生物学的にみて,これが癌組織内過酸化物に生ずるcis-trans共軛methylene dieneにその源を求め,この常磁性の変化が癌特有の酵素活性の変動を招くものと推論した. B₂-ButはDAB変性肝にけるESRスペクトルの強度を減ずるが,発癌肝にける特異なESRのシグナル(g=2.035)には影響はおよぼさなかつた.同様のことは, mitomycin C投与白鼡肝においても見られ,この種の特異的変化に対する薬物の開発は,今後の研究に俟たれることとなつた,しかしいずれにせよこれらの変化は未だ推測の領域をでず,その本質的解明は今後行なわれる幾多のモデル実験を総合した上でえられることはいうまでもない.

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The existence of two forms of GOT was first shown enzymatically by Katunuma et al. Partially purification and dissimilar properties of mitochondrial GOT (GOT_M) and soluble fraction GOT (GOT_S) have been reported by the previous paper from our laboratory. The present paper describes the procedure of further purification and distinct characteristics of the enzymes. The use of (NH_4)_2SO_4 fractionation, heat treatment and chromatography on DEAE-cellulose effected both about 100-fold purification of GOT_M and GOT_S. Purified preparations obtained were homogeneous column chromatographycally, electrophoretically and ultracentrifugal analytically. Compared to GOT_S, 5.8S, GOT_M had a smaller S_<20>W 3.8S. The purified enzymes also differd in other physicochemical natures. GOT was retained and GOT unretained by DEAE-cellulose, whereas the reverse seemed to be true of the IRC-50. On zone electrophoresis, GOT_M moved to anode (-0.4 cm/hr) and GOT_S to cathode (+0.05 cm/hr) at pH 8 in veronal buffer. GOT_M (GOT_S) gave the following Michaelis constant : for aspartic acid, 1.8×10^<-3> (8.0×10^<-3>), for α-ketoglutaric acid, 5.8×10^<-3> (4.3×10^<-3>). Absorption spectra of the enzyme were recorded and showed typical spectra of vitamin B_6-enzyme without a trace of soret band. Spectrophotometric studies of the enzymes, especially of GOT_M, provided the information that the addition of L-aspartic acid, to pyridoxal phosphate or GOT at pH 5.4 decreased the absorbancy at 430mμ and increased at 340 mμ, showing the possibility of interconversion of the enzyme between pyridoxal phosphate form and pyridoxamine phosphate form even at low pH. Upon addition of L-alanine or L-ornithine, such a shift was not observed.

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In previous report one of the authors has reported that glutamic・pyruvic transaminase located at mitochondria has quite different natures from one in soluble fraction. We have isolated glutamic-oxalacetic transaminase from pig liver mitochondria (GOTM) about 20 times active more than original mitochondria suspension using the technique of ammonium sulfate fractionation, calcium phosphate gel absorption and zone electrophoresis. On calcium phosphate gel column, GOTM is eluted with 0.1M phosphate buffer at pH 8.0 and GOT in soluble fraction (GOTS) with 0.04M phosphate buffer at pH 7.0. On starch zone electrophoresis, they showed very contrary pattern, i. e., GOTM moved to cathode and GOTS to anode. GOTM was more labile than GOTS at 70℃ heating. In detecting optimum pH and temperature, was not found any remarkable difference. Thus the presence of two different type of GOT in different cell fraction was demonstrated. We are now undergoing the efforts to elucidate the significance of their localization from the view point of respiratory regulation and protein synthesis.

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Pyridoxal (PL) or saline as a control was administered intraperitonealy to mice fed vitamin B_6 deficient diets or B_6 standard diets with normal protein level for 24 days. The mice were fasted for 16 hours and then supplied each diet for limited 30 minutes. At one and half hours after pyridoxal administration to the retake mice, the mice were sacrificed to collect their blood and livers. Body weights and serum alanine aminotransferase values of B_6 deficient groups were significantly decreased compared with that of control group. In the case of pyridoxal administration to B_6 deficient groups, the blood glucose levels as well as the insulin concentrations in serum were significantly decreased. In contrast, the amounts of coenzyme forms of B_6 (pydoxal 5'-phosphate+pyridoxamine 5'-phosphate) and glucose-6-phosphatase activities in the livers increased significantly compared with that of saline administrated group to B_6 deficient group. These results suggest that the insulin levels in serum of B_6 deficient mice decreased and the glucose-6-phosphatase activities increased by PL administration.

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In a previous publication, results on the purification, nature and mode of action of glutamic・pyruvic transaminase (GPT) from pig heart muscle were reported. In the course of the experiment, it was found that GPT was separated into two fractions on calcium phosphate gel column and starch zone electrophoresis, and they belong to the one in soluble fraction and mitochondria respectively. For solubilization of enzyme from mitochondria, several techniques, i. e., sonication (10Kc, 10min.) treatment with deoxycholate, freezing and thawing, and grinding with glass powder, gave the enzymes which have 60% of activity compared with original mitochondrial suspension. This loss is assumed to be attributed to the disruption of essential structure of mitochondria, or to the accumulation of oxalacetic acid in intact mitochondria which is measured as pyruvate. Digitonin inhibited the enzyme activity completely. GPT in soluble and in mitochondrial fractions were eluted with 0.02M phosphate buffer at pH 6.8 and 0.06M at pH 6.8 respectively on calcium phosphate gel column chromatography by stepwise and gradient eluting systems. On zone electrophoresis, they moved to cathode and separated each other. GPT preparation catalyzed the elemental reaction (C^<14>-alanine+pyruvate &rlarr;C^<14>-pyruvate+alanine, C^<14>-glutamate+α-ketoglutarate&rlarr;C^<14>-α-ketoglutarate+glutamate) more strongly than original reaction (glutamate+pyruvate&rlarr;alanine+α-ketoglutarate) with the same Km. GPT in soluble fraction has optimum pH at 9.7-9.5 and that in mitochondrial fraction at pH 8.5.

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