Partial Purification of Cysteine Synthase (O-Acetylserine Sulfhydrylase) from Onion (Allium cepa).

Purification of 0-acetylserine sulfuydrylase (OASS) from seedlings of two species of Phaseolus reveals the presence in both species of two forms of this enzyme. The isolation and purification procedure gives purification of 7-to 160-fold for individal isoenzymes with specific activities ranging from 33 IU mg-' to 775 IU mg-' protein.Detailed study of the basic kinetic parameters of the OASS isoenzymes indicates that both forms from Phaseolus vulgaris (which are of about equal specific activity) display substrate inhibition by S2-above 1 mm and positive cooperativity at lower concentrations of S2-. With respect to 0-acetylserine (OAS), the second substrate of the reaction, one P. vulgaris isoenzyme shows substrate inhibition by OAS concentrations above 10 mM, while the second is unaffected by OAS concentrations up to 50 mm. The isoenzymes from Phaseolus polyanthus (one of which has a specific activity 24 times higber than the other) are sligtly and approximately equally inhibited by both S2-and OAS.There is increasing evidence that a variety of plants including spinach (6), kidney bean (20,22), and species of rape, snapdragon, sunflower, wheat, barley, carrot, aster, flax, and pumpkin (18) synthesize cysteine by a two-step process similar to that known to occur in bacteria (12). First, OAS' is formed in a reaction catalyzed by serine transacetylase. Then, cysteine is synthesized from OAS and sulfide by OASS (15,(20)(21)(22)(23). Unlike bacteria, there is no evidence for the existence of a multifunctional protein complex of serine transacetylase-OASS in higher plants (20,22).This report is concerned with comparison of some of the properties of OASS from 5-day etiolated seedlings of Phaseolus vulgaris and Phaseolus polyanthus. These species were chosen because the amount of cysteine in seed proteins of P. polyanthus was approximately three times that of P. vulgaris (2). A relatively simple method of isolation which gives a good yield of the enzymes and which leads to purification to homogeneity as judged by disc gel electrophoresis has been developed. These studies indicate the existence of two isoenzymes of OASS from each species of legume investigated.

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“…Corp.) were added according to the procedures of Granroth (8). Finely ground ammonium sulfate (Schwarz/Mann) was added slowly to 40% saturation.…”

Section: Methodsmentioning

confidence: 99%