Isolation and Properties of a Homogeneous Preparation of Cystathionine Synthetase-l-Serine and l-Threonine Dehydratase

Abstract

Selim and Greenberg (1, 2) achieved a considerable degree of purification of L-serine dehydratase (L-serine hydro-lyase (deaminating), EC 4.2.1.13)from rat liver and demonstrated that this protein preparation contained the cystathionine-synthesizing activity of the liver (L-serine hydro-lyase (adding L-homocysteine), EC 4.2.1.21).These workers (2) also observed activity of their enzyme preparation on L-threonine.Subsequently, the work of Goldstein, Knox, and Behrman (3) indicated that the L-threonine dehydratase activity of rat liver was a function of the same enzyme protein.Pitot, Potter, and Morris (4) demonstrated a large increase in the threonine and serine dehydratase activities of the livers of rats fed a high protein diet.This observation was confirmed by Goldstein et al. (3).This ability to increase the content of the enzyme in the liver offers a decided advantage in its purification.From livers of rats fed a high protein diet, we have succeeded in obtaining enzyme preparations apparently consisting of a single homogeneous protein.Various properties of the purified enzyme have been studied.lAn observation of considerable interest is that the /3-hydroxyl group is not an essential characteristic for substrate activity.Chloride, for example, can be substituted for the hydroxyl group. EXPERIMENTAL PROCEDUREJ~aterials-L-Serine, DL-+ nn-allocystathionine, nn-homocysteine thiolactone hydrochloride, and pyridoxal phosphate were purchased from Calbiochem.The thiolactone hydrochloride was converted to homocysteine by treatment with alkali immediately before incubation with the enzyme preparation.DEAE-cellulose was obtained from Schleicher and Schuell.The material was washed successively with 1 N NaOH, 1 N HCl, ethanol, 1 N NaOH, and finally with water.Analytical Jdethods-The or-keto acids derived from the enzymatic decomposition of serine, threonine, and the 6.chloroamino acids were determined by the direct method of Friedemann and Haugen (7), with the mod.ification described by Sayre and Greenberg (8).Standard curves were prepared from salts of a-ketobutyrate and pyruvate.