Gumpei Urata

Measurement of δ-Aminolevulinic Acid Synthetase Activity in Human Erythroblasts

Abstract A new, specific, and simple method for the determination of δ-aminolevulinic acid (ALA) synthetase activity in human bone marrow cells has been developed. ALA synthetase of erythroblasts was partially purified so as to permit the use of [14C]succinyl-CoA as a substrate for this enzyme. In this enzyme preparation there were negligible activities of succinyl-CoA hydrolase, α-ketoglutarate dehydrogenase, and succinyl-CoA synthetase and there was no activity of ALA dehydrase. The ALA formed from [14C]succinyl-CoA has been isolated by column chromatography. Radioactivity in the eluate from the column has been proved by paper chromatography to be exclusively that of [14C]ALA. The entire assay can be completed within 4 h, and [14C]succinyl-CoA was incorporated into [14C]ALA on the order of several percent. Moderate to marked decreases of ALA synthetase activity have been demonstrated in the erythroblasts of all cases of sideroblastic anemia. In the cases of iron deficiency anemia, on the other hand, normal or slightly elevated activity has been obtained.

A simple method for the quantitative analysis of urinary delta-aminol evulinic acid to evaluate lead absorption

Wada, O., Toyokawa, K., Urata, G., Yano, Y., and Nakao, K. (1969).Brit. J. industr. Med.,26, 240-243. A simple method for the quantitative analysis of urinary delta-aminolevulinic acid to evaluate lead absorption. A procedure is given for the rapid, quantitative determination of urinary delta-aminolevulinic acid (ALA). Interfering substances are removed by n-butanol extraction. After pyrrole formation with ethyl acetoacetate, Ehrlichs reagent is added to produce the chromophore, which is then extracted with chloroform and measured spectrophotometrically or by comparison of the depth of colour with standard colour solutions. The recoveries were about 91% and the results agreed well with those obtained using ion-exchange column chromatography (r=0·985). This assay is simple, dependable, and suitable for large-scale screening of industrial workers exposed to lead poisoning, because the critical level of urinary ALA (20 mg./l. urine), which indicates dangerous lead absorption, gives a convenient absorbance.

Purification and some properties of δ-aminolevulinate(ALA) synthetase in rabbit reticulocytes

Abstract ALA synthetase has been purified approximately 4,400 fold from rabbit reticulocytes. The purified enzyme was demonstrated to be homogeneous by disc gel electrophoresis. The enzyme had a pH optimum of 7.6. Isoelectric point was 5.9. The enzyme required pyridoxal phosphate as a cofactor. Iron inhibited, but α,α′-dipyridyl neither inhibited nor activated the activity. Hemin inhibited ALA synthetase activity about 40% at the concentration of 10−5M. Km for succinyl-CoA was 6.0 × 10−5M and Km for glycine was 1.0 × 10−2M.

A new protease inactivating δ-aminolevulinic acid synthetase in mitochondria of human bone marrow cells

Summary A new protease which inactivates specifically apo-forms of certain pyridoxal phosphate enzymes was found in the human bone marrow cells. The enzyme, which is located in the mitochondrial fraction, was purified so as to be nearly homogeneous as judged by polyacrylamide disc gel electrophoresis. It is considered to be a seryl protease, because diisopropylfluorophosphate (10−5 M) inhibited the protease activity completely, while p-chloromercuribenzoate (10−3 M) had no effect. It had some resemblance to elastase, but had no elastinolytic activity.

Concept on mechanism and regulation of intracelluar enzyme degradation in mammalian tissues

In this review, we focus on the following points regarding intracellular enzyme degradation. We have proposed, in fact, that at least two conformations of each enzyme protein exist in the cell and the unfolding form in the tertiary structure of the substrate protein is in physiological conditions a prerequisite to proteolysis. Conversion from a non-susceptible holoenzyme to a susceptible form, apoenzyme, via interaction with coenzymes and/or substrates might be the starting point in the catabolism of pyridoxal enzymes. It was pointed out that intracellular enzyme degradation may have to occur in a step-wise manner and the initiating protease for intracellular enzyme degradation may have to catalyze a strictly limited proteolysis. The mode of proteolysis by group specific proteolysis on two representative pyridoxal enzymes, ornithine aminotransferase and cystathionase, was demonstrated. We have shown in this review that the group specific protease activities are altered under various physiological and pathological conditions and in the alteration the specific inhibitor may be involved. The possibility of participation of group specific proteases in degradation of pyridoxal enzymes in vivo in normal and B6 deficient rats was investigated using the trapping method of degradative intermediate products by antibody.

Aminoacetone formation and decomposition in liver.

Abstract The formation of aminoacetone by guinea pig liver mitochondria from pyruvate and glycine has been shown. Aminoacetone is decomposed by liver mitochondria and nuclei. A cycle for glycine oxidation via aminoacetone is suggested.

Porphyrias in Japan: Compilation of all cases reported through 2002

The first case of porphyria on record in Japan was a patient with congenital erythropoietic porphyria (CEP) reported by Sato and Takahashi in 1920. Since then until the end of December 2002, 827 cases of porphyrias have been diagnosed from characteristic clinical and/or laboratory findings (463 males, 358 females, and 6 of unknown sex). Essentially all inherited porphyrias have been found in Japan, with the incidences and clinical symptoms generally being similar to those reported for other countries. The male-female ratio was approximately 1:1 for CEP, whereas it was higher for erythropoietic protoporphyria. In contrast, preponderances of female patients exist with acute hepatic porphyrias, such as acute intermittent porphyria (AIP), variegate porphyria (VP), and hereditary coproporphyria (HCP), and with undefined acute porphyria. Although porphyria cutanea tarda (PCT) is believed to be increasing recently in women in other countries because of smoking and the use of contraceptives, it is still by far more prominent in males in Japan than in females. The recent increasing contribution of hepatitis C virus infection to PCT in Japan has also been recognized, but there have been no PCT cases in Japan with HFE gene mutations. Familial occurrence and consanguinity were high for CEP, as expected; however, significant consanguinity was also noted in families where CEP, AIP, HCP, VP, or PCT occurred as a single isolated case without a family history of disease. This survey also revealed that as many as 71% of acute hepatic porphyria cases were initially diagnosed as nonporphyria and later revised or corrected to porphyria, indicating the difficulty of diagnosing porphyria in the absence of specific laboratory testing for por-phyrins and their precursors in urine, stool, plasma, and erythrocyte samples.