Acta Physiologica 25. (1964)

2. szám - Telegdi Marianna-Keleti T.: The Role of Sulphydryl Groups in A-Glycerophosphate Dehydrogenase (L-Glycerol-3-Phosphate: NÁD Oxideroductase 1.1.1.8) Activity

THE ROLE OF SULPHYDRYL GROUPS l \ «-GLYCERO­PHOSPHATE DEHYDROGENASE (L-GLYCEROL-.L PHOSPHATE: NAD OXIDOREDUCTASE 1.1.1.8) ACTIVITY By Marianna Telegdi and T. Keleti INSTITUTE OF BIOCHEMISTRY, HUNGARIAN ACADEMY OF SCIENCES, BUDAPEST (Received November 9, 1963) The a-glycerophosphate dehydrogenase isolated from rabbit muscle contains 9 to 11 SH-groups per protein molecule. Blocking the SH-groups with PCMB the reaction catalyzed by the enzyme is inhibited in both directions. Blocking of 6 to 7 S11-groups is required for complete inactivation. The inhibition of enzymatic activity is due to the blocking of SH-groups, it is instantaneous and can be reverted by cysteine. This process is followed by a time-consuming reaction, the alteration of the steric structure, revertible by cysteine only partly and resulting in a decreased ability to be reactivated. Under certain circumstances the enzyme is undergoing spontaneous inactivation, accompanied by the disappearance of some of its free, titrât able SH- groups. This inactivation cannot be reverted by cysteine. The role of the SH-groups of a-glycerophosphate dehydrogenase in enzymatic catalysis has been studied by several authors |1, 2, 3, 4]. By the use of our new method [5] for the isolation of the enzyme in crystalline form, an enzyme preparation with a specific activity about 8 times higher than that of the best ones described in the literature [1, 6] can be pro­duced. It was therefore reasonable to examine the role of SH-groups in the activity in this highly active enzyme preparation. Methods The enzyme was prepared by our method [5] from rabbit skeletal muscle. Enzyme preparations of maximum activity were used. The number of SH-groups was determined by the spectrophotometric method of Boyer [7]. The PCMB preparation used was of 94 per cent purity, as determined from the molar extinctioh coefficient of the solution [71. Enzymatic activity was assayed by the optical test of Warburg, using a reaction mixture already described [3]. As ingredients, a-glycerophosphate (Rhône-Poulenc), dihydroxyacetone phosphate (Sigma), NAD andNADII (Boehringer) were used. The measure­ments were carried out in a Hilger (Uvispek) spectrophotometer. The tryptic digestibility of the protein was controlled by the method of Szabolcsi and Szörényi [8], in 0.1 M phosphate buffer, pH 7.0, at room temperature, using IXlO-4 ITU]?*? trypsin concentration in a 2 mg per ml a-glycerophosphale dehydrogenase solution. The trypsin was a three times recrystallized Tripure-Novo preparation. Proteolytic activity was checked by Anson's test. 5 Acta Physiologica XXV/2.

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