Purification and properties of beta-citryl-L-glutamate-hydrolysing enzyme from rat testis particulate.
beta-Citryl-L-glutamate-hydrolysing enzyme (beta-CGHE) was purified from rat testis particulate fraction 13,000-fold, at a yield of 7%. The enzyme was purified by ammonium sulfate fractionation, hydroxyapatite, chelating Sepharose, beta-CG-Sepharose affinity chromatography and Sephacryl S-300 gel filtration. The purified enzyme usually migrated as two periodic acid Schiff's-stained bands on native polyacrylamide gel-electrophoresis (PAGE) with molecular weights of 350 and 420 kDa. Both bands hydrolyzed beta-citryl-L-glutamate (beta-CG) to citrate and glutamate. The 420 kDa band was changed by digestion with N-glycosidase F, into 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 conditions. The purified enzyme was pharmacologically similar to the beta-CGHE activity partially purified from rat testis. This enzyme required manganese ions for full activity and it was strongly inhibited by nucleotides such as ATP or GTP and phosphate ions. beta-CGHE was also potently 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 purified enzyme reacted mainly to the 115 kDa band on the SDS-PAGE and precipitated the enzyme activity from the crude and purified enzyme solution.
Asakura
M
M
Faculty of Pharmaceutical Sciences, Kobe-Gakuin University, Japan.
Nagahashi
Y
Y
Hamada
M
M
Kawai
M
M
Kadobayashi
K
K
Narahara
M
M
Nakagawa
S
S
Kawai
Y
Y
Hama
T
T
Miyake
M
M
D000581:Amidohydrolases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000302:isolation & purification*; D000818:Animals; D002413:Cations, Divalent; D007106:Immune Sera / Q000737:chemistry; D008297:Male; D008970:Molecular Weight; D009711:Nucleotides / Q000494:pharmacology; D011233:Precipitin Tests; D016318:Quisqualic Acid / Q000494:pharmacology; D051381:Rats; D017208:Rats, Wistar; D013379:Substrate Specificity; D013737:Testis / Q000201:enzymology*
D002413:Cations, Divalent; D007106:Immune Sera; D009711:Nucleotides; D016318:Quisqualic Acid; D000581:Amidohydrolases; C039451:N-formylglutamate deformylase