Effect of Cystathionase on Isovalthine.

In the course of studies on the cleavage reaction of S-(isopropylcarboxymethyl) glutathione (GSIV) into isovalthine in kidney homogenate or glutathionase preparation, it has sometimes been observed that the amount of isovalthine formed is far le than that of GSIV decomposed. Furthermore, when such reaction mixture is analyzed on an automatic amino acid analyzer, prominent peak corresponding to the reasonable amount of S-(isopropy1carboxymethyl)cysteinylglycine which is an expected intermediate of the GSIV cleavage reaction cannot be found up to 400 effluent ml. Though several reasons may be considered for the explanation of the above curious phenomenon, the effect of cystathionase on isovalthine is at first examined here. But the result was negative. Land L-Alloisovalthineused as substrate were prepared by the method of OHMORI. Homoserine and purified cystathionase in ammonium sulfate solution prepared according to the method of GREENBERGB were kindly furnished by Prof. M. Suda of Osaka University. Incubation mixture contains 0.1 ml of enzyme solution, 1.0 ml of 0.2 M borate buffer (pH 8.0) containing 2×10-M cysteine, 0.lml of 0.1 M substrate, and 0.8ml of deionized water containing 5×10-4M EDTA. The mixture was shaken at 37◦C for 30 minutes in the air. The reaction was terminated by adding 2ml of 10% trichloroacetic acid and the α-keto acids formed were determined by the method of FRIEDEMANN and HAUGEN4 with a following modification: toluene extract was washed once with 8 ml of 10% sodium sulfate. The results obtained are summarized in Table l. When the reaction mixtures are analyzed before or after incubation on an automatic amino acid analyzer, the amount of Lor L-Alloisovalthine is found to be unchanged. Furthermore, as indicated in Table 1, L-isovalthine showed no inhibitory effect on the homoserine cleavage by cystathionase. Since amino acid oxidases have already been reported to have no effect on isovalthine, the curious phenomenon above cited may have to be explained by other reaction mechanism such as transpeptipation reaction. ∗PMID: 14245866 [PubMed indexed for MEDLINE] Copyright c ©OKAYAMA UNIVERSITY MEDICAL SCHOOL Acta Med. Okayama 18, 239-240 (1964) BRIEF NOTES EFFECT OF CYSTATHIONASE ON ISOVALTHINE* Toshihiko UBUKA, Katsumi HORIUeHI, Takehira SHIMOMURA and Tsukasa AZUMI Department of Biochemistry, Okayama University Medical School, Okayama (Director: Prof. S. Mizuhara) Received for publication, May 7,1964 In the course of studies on the cleavage reaction of S-(isopropylcarboxymethyl)glutathione (GSIV) into isovalthine in kidney homogenate or glutathionase preparation, it has sometimes been observed that the amount of isovalthine formed is far less than that of GSIV decomposed • Furthermore, when such reaction mixture is analyzed on an automatic amino acid analyzer, prominent peak corresponding to the reasonable amount of S-(isopropy1carboxymethyl)cysteinylglycine which is an expected intermediate of the GSIV cleavage reaction cannot be found up to 400 effluent ml. Though several reasons may be considered for the explanation of the above curious phenomenon, the effect of cystathionase on isovalthine is at first examined here. But the result was negative. Land L-Alloisovalthineused as substrate were prepared by the method of OHMORI• Homoserine and purified cystathionase in ammonium sulfate solution prepared according to the method of GREENBERGBwere kindly furnished by Prof. M. Suda of Osaka University. Incubation mixture contains 0.1 ml of enzyme solution, 1.0 ml of 0.2 M borate buffer (pH 8.0) containing 2 X la-BM cysteine, O.lml of 0.1 M substrate, and 0.8ml of deionized water containing 5 X 10-M EDTA. The mixture was shaken at 37°C for 30 minutes in the air. The reaction was terminated by adding 2ml of 10% trichloroacetic acid and the a-keto acids formed were determined by the method of FRIEDEMANN and HAUGEN with a following modification: toluene extract was washed once with 8 ml of 10% sodium sulfate. The results obtained are summarized in Table l. When the reaction mixtures are analyzed before or after incubation on an automatic amino acid analyzer, the amount of Lor L-Alloisovalthine is found *This work was supported by research grants from the U. S. National Institutes of Health (HE07419) and the Ministry of Education of Japan. 239 1 Ubuka et al.: Effect of cystathionase on isovalthine Produced by The Berkeley Electronic Press, 1964 240 T. UBUKA, K. HORIUCHI, T. SHIMOMURA and Table 1 Effect of Cystathionase on Isovalthine T. AzuMI Ilmoles of a-keto acid formed Substrates I Experiment I Experiment II Homoserine I 0.72 0.64 Homoserine + L-Isovalthine I 0.64 L-Isovalthine , 0.00 0.00 L-Alloisovalthine 0.00 Reactian mixture contains 0.1 ml of cystathionase solution, 1. 0 ml of 0.2 M borate buffer (pH 8.0) containing 2X10-3 M cysteine, O.lml of O.lM substrate, and 0.8ml of water containing 5 X10-4M EDTA. Shaken at 37°C, 30 min. in air. to be unchanged. Furthermore, as indicated in Table 1, L-isovalthine showedno inhibitory effect on the homoserine cleavage by cystathionase.Since amino acid oxidases have already been reported to have no effect onisovalthine, the curious phenomenon above cited may have to be explained byother reaction mechanism such as transpeptipation reaction. REFERENCES1. KUWAKI, T.: in preparation2. OHMORI, S.: Arch. Biocllem. BioPhys. 104, 509, 19643. GREENBERG, D. M.: Methods in Enzymology, by Colowick, S. P., and Kaplan, O. AcademicPress Inc., New York, Vol. V, p. 936, 19624. FRIEDEMANN, T. E., and HAUGEN, G. E.: ]. Bioi. Chem. 147, 415, 19435. MIZUHARA, S., KUWAKI, T., UBUKA, T., and FRIMPTER, G. W.: ]. Biochem. 52, 59,1962 2Acta Medica Okayama, Vol. 18 [1964], Iss. 4, Art. 6 http://escholarship.lib.okayama-u.ac.jp/amo/vol18/iss4/6