Isolation and characterization of ATP-dependent proteolytically active ubiquitin in cock testis.
1. We have successfully isolated and purified ubiquitin from cock testis by using an inhibitor, p-CMB (p-chloromercuribenzoate), which is one of the inhibitors specific for thiol-proteases and with the following procedures: heating up to 85 degrees C, ammonium sulfate fractionation, gel filtration on Sephadex G-75, chromatography on DE-52 and CM-11 and lyophilization. 2. Amino-acid analysis showed that Ub isolated from cock testis has 76 residues including 6 glycines. 3. Hydrazinolysis and carboxypeptidase digestion were also performed: the C-terminal residue is glycine. 4. The purity was checked by analytical SDS-PAGE and the isolated Ub exhibited only one band. 5. The Ub-dependent proteolysis experiment showed that this Ub was ATP-dependently proteolytically active. 6. In this paper we present evidence that a thiol enzyme is present during the purification procedure.
Sun
N E
NE
Department of Biochemistry, University of Nanjing, People's Republic of China.
Zhu
D X
DX
Han
K K
KK
Hemon
B
B
Belaiche
D
D
Sautiere
P
P
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002645:Chickens; D002729:Chloromercuribenzoates; D005260:Female; D008297:Male; D010447:Peptide Hydrolases / Q000032:analysis; D013737:Testis / Q000032:analysis*; D014452:Ubiquitins / Q000302:isolation & purification*; D020245:p-Chloromercuribenzoic Acid
D000596:Amino Acids; D002729:Chloromercuribenzoates; D014452:Ubiquitins; D020245:p-Chloromercuribenzoic Acid; D000255:Adenosine Triphosphate; D010447:Peptide Hydrolases