PURIFICATION AND PROPERTIES OF BETA-CITRYL-L-GLUTAMATE-HYDROLYZING ENZYME FROM RAT TESTIS PARTICULATE

beta-Citryl-L-glutamate-hydrolysing enzyme (beta-CGHE) was purified fr om rat testis particulate fraction 13 000-fold, at a yield of 7%. The enzyme was purified by ammonium sulfate fractionation, hydroxyapatite, chelating Sepharose, P-CG-Sepharose affinity chromatography and Sepha cryl S-300 gel filtration. The purified enzyme usually migrated as two periodic acid Schiff's-stained bands on native polyacrylamide gel-ele ctrophoresis (PAGE) with molecular weights of 350 and 420 kDa. Both ba nds hydrolyzed beta-citryl-L-glutamate (beta-CG) to citrate and glutam ate. The 420 kDa band was changed by digestion with N-glycosidase F, i nto a 350 kDa band on native PAGE. The purified enzyme was composed of 90, 100, 115 and 130 kDa subunits on SDS-PAGE under non-reduced condi tions. The purified enzyme was pharmacologically similar to the beta-C GHE activity partially purified from rat testis. This enzyme required manganese ions for full activity and it was strongly inhibited by nucl eotides such as ATP or GTP and phosphate ions. beta-CGHE was also pote ntly inhibited by an excitatory amino acid agonist, L-quisqualate, but not by another agonists, N-methyl-D-aspartate and kinate. It had high substrate specificity for beta-CG. The antibodies against the purifie d enzyme reacted mainly to the 115 kDa band on the SDS-PAGE and precip itated the enzyme activity from the crude and purified enzyme solution .