19. Presence O F Creatinase and Sarcosine Dehydrogenase In
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No. 2] Proc. Japan Acad., 56, Ser. B (1980) 95 19. Presence of Creatinase and Sarcosine Dehydrogenase in Human Skeletal Muscle Proposal f or Creatine-Urea Pathway By Kazuo MIYOSHI,Akira TAIRA,Kenzo Y0sHIDA, Katsuya TAMURA,and Shigetoshi UGA The First Department of Internal Medicine,School of Medicine, TokushimaUniversity, Tokushima (Communicated by Setsuro EBASHI,M. J. A., Feb. 12, 1980) The presence of creatinase (creatine amidinohydrolase, EC 3.5.3.3) which catalyzes the reaction, creatine-sarcosine + urea, has been reported only in pseudomonas.lDuring a course of study on creatine metabolism in human skeletal muscle,4~ we found the presence of creatinase activity. We also found the activity of Sarcosine de- hydrogenase (sarcosine : oxygen oxydoreductase, EC 1.5.3.1) which catalyzes the reaction, sarcosine- glycine + formaldehyde, in human skeletal muscle. This paper describes evidence for the presence of these two enzymes and proposal for the existence of creatine-urea pathway in human skeletal muscle. Materials and methods. Human skeletal muscle was obtained from 6 non-myopathy patients, aged 22-69 (male and female), at autopsy and stored at -60°C. The muscles were homogenized in 5 volumes of physiological saline using a Potter-Elvehj em homogenizer. The homogenate was centrifuged at 18,000 x g for 30 min and the supernatant was used as the enzyme source. The supernatant was applied onto a Sephadex G-150 column (2.8x40 cm), eluted with 0.1 M sodium phosphate buffer (pH 6.2), and every 8 ml fraction was collected. Active fractions were combined and used as the enzyme source in some experiments. The protein content of the sample was measured by the biuret methods. Creatinase activity was assayed either by urea production or by creatine consumption. For urea production, enzyme reaction was carried out in 0.1 M sodium phosphate buffer (pH 7.8) at 30°C for 1 hr and urea produced was measured by the diacetylmonoxime- thiosemicarbazide method. For creatine consumption, enzyme reac- tion was carried out in 0.1 M sodium phosphate buffer (pH 6.2) at 37°C for 30 min and the remaining creatine was determined by the diacetyl a-naphthol method. Sarcosine dehydrogenase activity was assayed in 0.05 M sodium 96 K. Miyosrn et al. [Vol. 56(B), phosphate buffer (pH 7.5) by the method of Hoskins and Mackenzie~~ using phenazine methosulfate and 2,6-dichlorophenolindophenol (DCIP). Results. The presence of creatinase activity in human skeletal muscle was demonstrated by formation of urea from creatine in the presence of the muscle extract (Fig. 1). The creatinase activity can Figs. 1-4. 1: Creatinase activity in 18,000xg supernatant of human skeletal muscle homogenate. 16 mM of creatine was initially present in total reaction mixture of 3 ml. 2: Creatinase activity in combined active fractions of Sephadex G-150 eluate from skeletal muscle. Initial concentration of creatine was 1.7 mM in total reaction mixture of 1.2 ml. 3 : Elution pattern of creatinase activity from Sephadex G-150 column (2.8X40 cm). 8 ml fractions were collected. 4: Effect of creatine concentration on creatinase activity. 0.8 mg protein of combined active fractions of gel filtrate was incubated in tatal reaction mixture of 1.2 ml. No. 2] Creatinase and Creatine-Urea Pathway in Human Muscle 97 also be assayed by measuring the consumption of creatine in the reaction mixture (Fig. 2). On Sephadex G-150 gel filtration, the peak of creatinase activity was eluted at 144 ml of the effluent (Fig. 3), corresponding to the molecular weight of approximately 50,000. Varying the pH of 0.1 M sodium phosphate buffer in the reaction mixture from 4.3 to 9.0, the optimum pH of muscle creatinase was found at pH 6.2. Creatinase activity with respect to increasing creatine concen- trations gave a hyperbolic curve (Fig. 4) and the Km value for creatine was calculated to be 8.0 x 10-5 M. The presence of sarcosine dehydrogenase activity in human skeletal muscle was also demonstrated by the time-dependent reduc- tion of DCIP in the presence of the supernatant fraction of the muscle homogenate (Fig. 5). Fig. 5. Sarcosine dehydrogenase activity in 18,OOO><g supernatant of human skeletal muscle homogenate. 10 mM of sarcosine and 16 mg of supernatant protein were initially present in total reac- tion mixture of 3 ml. Discussion. Although creatinase has been found in a few species of pseudomonas,1>~3> no report of its presence in mammals has yet appeared in the literature. Thus, the only product of creatine and phosphocreatine in muscle metabolism has long been assumed to be creatinane. The present demonstration of creatinase activity in human skeletal muscle prompts us to propose a new pathway of urea synthesis in man, i.e. creatine-urea pathway. The concentration of urea in human skeletal muscle was found to be 0.4-1.1 mg/g wet weight as compared with the value of 0.8-0.9 mg/g wet weight of liver (our unpublished results). The relative contribution of skeletal muscle and liver to the whole-body urea output is the subject of future studies. 98 K. MIY0sHI et al. [Vol. 56(B), Sarcosine, another product of creatinase reaction, will be con- verted to glycine and formaldehyde by the presently found sarcosine dehydrogenase in skeletal muscle. Sarcosine dehydrogenase has been found in liver mitochondria of many mammalian species, but the present report is the first of its occurrence in skeletal muscle. Abnormal properties of creatinase in the skeletal muscle of Duchenne muscular dystrophy will be reported in a separate paper.4) A preliminary report of this work was presented elsewhere.6> References 1) Akamatsu, S., and Kanai, Y.: Bacterial decomposition of creatine. I. Creati- nomutase. Enzymologia, 15, 122 (1951). 2) Appleyard, G., and Woods, D. D.: The pathway of creatine catabolism by pseudomonas ovalis. J. Gen. Microbiol., 14, 351 (1956). 3) Yoshimoto, T., Oka, I., and Tsuru, D.: Purification, crystalization, and some properties of creatine amidinohydrolase from pseudomonas putida. J. Bio- chem., 79, 1381 (1976). 4) Miyoshi, K., Taira, A., Yoshida, K., Tamura, K., and Uga, S.: Abnormalities of creatinase in skeletal muscle of patients with Duchenne muscular dys- trophy. Proc. Japan Acad., 56B, 99 (1980). 5) Hoskins, D. D., and Mackenzie, C. G.: Solubilization and electron transfer flavoprotein requirement of mitochondrial sarcosine dehydrogenase and dimethylglycine dehydrogenase. J. Biol. Chem., 236, 177 (1961). 6) Miyoshi, K.: Creatine metabolism in muscular dystrophy. Report to Research Group of Etiology of Muscular Dystrophy, Ministry of Health and Welfare, Japan, March 1979, p. 175..