Recenity, we, developed the new synthetic substrates for various proteases, α-naphthylester derivatives. Principle of the assay method with these substrates was based upon diazo-coupling reaction between α-naphthol released by the enzyme reaction and diazonium salt. Among the various diazonium salts, Fast Violet B salt has been used in this assay system. However, Fast Violet B salt was possible to use only in neutral pH region, and in the absence of reducing or oxidizing reagent.
In this work, to establish the methods of microassay and zymogram on the proteases which have an optimum in acidic pH region, we tested the reactivities between a-naphthol and two diazonium salts, Fast Red ITR salt and Fast Blue BB salt. Consequently, only Fast Red ITR salt was found to react in acidic pH region (pH 4.0). λmax and ε values of the diazo-color with Fast Red ITR salt in acidic pH region were 475nm and 26, 000, respectively. Further, although the reaction between Fast Red ITR salt and α-naphthol was inhibited by the reducing reagent such as dithiothreitol, its inhibition was found to be removed in the coexistence of oxidizing reagent. These results indicate that, if Fast Red ITR salt is used as coloring reagent, α-naphthylester substrate is very effective for the investigation of lysosomal protease such as cathepsin B like enzymes.
Zymogram on lysosomal protease of rat liver was prepared in acidic pH region using Pro-Phe-Arg-α-naphthylester as the substrate and Fast Red ITR salt. Two bands were visualized, and the appearance of these bands was promoted in the presence of dithiothreitol, and completely inhibited by the addition of leupeptin. Therefore, these bands may be due to cathepsin B like enzyme.

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FK-448, 4-(4-isopropylpiperadinocarbonyl) phenyl 1, 2, 3, 4-tetrahydro-l-naphthoate methanesulfonate was prepared and its inhibitory effect on chymotrypsin, trypsin, thrombin, plasmin, pancreatic kallikrein, plasma kallikrein and cathepsins A, B, C, G and H were examined. Among the various enzymes tested, chymotrypsin was strongly inhibited by FK-448 (IC₅₀: 7.1 X 10^-7M).
We also examined the effect of FK-448 on the intestinal absorption of insulin in the rat. When insulin and FK-448 were administered into the lumen of intestine, the decrease of blood glucose level was observed. In vitro, insulin was degradated by pancreatic enzyme or homogenates of intestine or liver. FK-448 supressed the decrease of insulin activity by pancreatin and its enhancement of intestinal absorption of insulin was found to be related to its inhibition of digestive enzymes, especially, chymotrypsin. Its effect in increasing intestinal absorption of insulin was enhanced by citric acid: the percentage absorptions of insulin from the intestine were 0.72% with 2.5 mg (63u) /kg of insulin with 20 mg/kg of FK-448, and and 1.47% with those doses of insulin and FK-448 in combination with 15 mg/kg of citric acid.

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A sensitive and rapid nonradiometric determination of human erythrocyte adenine phosphoribosyltransferase (APRT, EC 2.4.2.7) and hypoxanthine-guanine phosphoribosyltransferase (HGPRT, EC 2.4.2.8) activities is described. The procedure was based on a reversedphase high performance liquid chromatographic (HPLC) separation of purine bases, nucleosides, and nucleotides, after the enzyme reaction. APRT activity was assayed with adenine as a substrate and the AMP and AMP metabolites which were produced were converted to inosine by the enzymes alkaline phosphatase (EC, 3.1.3.1) and adenosine deaminase (EC, 3.5.4.4). The inosine was determined by the HPLC. HGPRT activity was assayed with hypoxanthine or guanine as a substrate and the IMP or GMP was determined by the HPLC. The activities of APRT, HPRT, and GPRT assayed by this method in erythrocytes from thirty normal adults were 0.35±0.14, 2.02±0.42, and 3.22±0.56μmol/min/gHb (mean±2SD), respectively.

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Rat linver microsomes were solubilized by treatments of detergents, hydrolyzing enzymes, sonication and freezing-thawing, and the effects of these solubilizing treatments On the release of four enzymes, NADH-cytochrome b5 reductase (Fp₁), NADPH-cytochrome c reductase (Fp₂), γ-glutamyltranspeptidase (γ-GTP) and glucose-6-phosphatase (G-6-Pase) from the microsomes were investigated.
Fp₁, Fp₂ and γ-GTP were releared easily by Triton X-100, a nonionic detergent.Fp₁ and Fp₂ were releared much more than γ-GTP by cholic acid, an anionic detergent.Trypsin or lipase digestion caused only the release of Fp₂ from the microsomes.ln the case of sonication treatment, the release of each enzyme with an increase in treatment time was not observed.These enzymes were released only a little by twenty times of freezing-thawing.
The present results suggest that Fp₂ might be most released from the membrane of damaged linver microsomes.

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It is well known that serum apolipoprotein A-I (apo A-I) is an important component of high density lipoproteins (HDL) and also plays some important physiologic roles. Although several technics for quantitating apo A-I have been reported, they are complicated technically and are not feasible for automation.
In this paper, we describe a new immunoturbidimetric assay for the determination of apo A-I by automated analysers.High precision and good correlation with the single radial immunodiffusion (SRID) method were obtained. The normal value of Apo A-I levels in male (n=82) and in female (n=82) were 122±24 mg/di (mean±S.D.) and 124±24 mg/di respectively. Apo A-I levels in patients with hepatitis, nephritisand old myocardial infarction were significantly decreased as compared with those of normals.
This method is useful and widely acceptable for the prediction of the risk of coronary heart disease in addition to HDL-cholesterol determination.

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Reaction rate in enzyme catalysis, as the general chemical reactions, is a function of temperature, that is, increase of teperature causes increase of reaction rate (enzyme activity).in the enzyme reaction, generally, change of temperature of 1° Ccauses change of 7 to 10% of reaction rate. in this paper, we describe the relation of incubation temperature and enzyme activity using UV rate method which employs α-ketobutyric acid and NADH as substrate, and using Purified LDH isoenzymes, especially LDH₁ and LDH₂ which contain α-HBDH activity. As the results, 12% increase, only a little increase, of α-HBDH activity was found by the change of incubation temperature from 30° to 37°. We also found that the α-ketobutyric acid, as substrate, was used in low concentration to determine the α-HBDH activity in LDH₁ and LDH₂.

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A simple and rapid method for the determination of glycerol-3-phosphate dehydrogenase (EC, 1. 1. 1. 8, GPD) isoenzymes in human tissues was examined using Cellogel membrane electrophoresis. An LL isoenzyme of glycerol-3-phosphate dehydrogenase was partially isolated from human heart muscle and determined apparent Km value for NADH.Then actMties and isoenzymes of glycerol-3-phosphate dehydrogenase and lactate dehydrogenase in 100000xG supernatant were measured in human tissues. The glycerol-3-phosphate dehydrogenase apPeared to form dimeric combinations of two different subunits, H and L, and formed three isoenzymes HH, HL and LL. The HH form represents the major heart isoenzy me and LL represents the liver. These results are in agreement with the report of McGinnis and Vellis. in skeletal muscle such as femoral and rectal abdominal muscle and kidney, the LL isoenzyme was more than 80% of total activity and a hybrid form HL was about 20%.
The apparent Km value for NADH of LL form was measured to be 7.8μM which value was lower than that of lactate dehydrogenase (LD) isoenzymes.The calculated activity ratios, LD (Total) /GPD (LL), LD (H-subunit) /GPD (LL) and LD (M-subunit) /GPD (LL), of rectal abdominal muscles and livers were lower than LD (H-subunit) /GPD (LL) of femoral muscies.These results indicate the abnormal participation of the glyCerol-3-phosphate dehydrogenase to the reoxidation of NADH in the muscle and the liver under anaerobic conditions.

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Studies are reported on a colorimetric microdetermination of citric acid using pyruvate oxidase. The method is based on formation of a red quinone compound in a reaction of citric acid together with citrate lyase, oxalacetate decarboxylase, pyruvate oxidase and peroxidase.
The optimum pH (0.2 M Tris-HCI buffer) was 8.8, and the color product was measured at 505nm. in this method the calibration graph was rectilinear in the range of 50 to 200 nmol of citric acid and the detection limit was 13nmol.
This enzymic method wouid be more advantageous in some applied sciences than that with citrate lyase and malate dehydrogenase because of the visible colordevelopment.

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