Purification and characterization of bile salt hydrolase from Clostridium perfringens.
1988; Elsevier BV; Volume: 29; Issue: 8 Linguagem: Inglês
10.1016/s0022-2275(20)38464-9
ISSN1539-7262
AutoresRashmi Gopal-Srivastava, P B Hylemon,
Tópico(s)Biochemical effects in animals
ResumoBile salt hydrolase (cholylglycine hydrolase, EC 3.5.1.24)has been purified to homogeneity (792-fold) from Clostridium pcrfrrngens using high performance DEAE-chromatography.The purified enzyme showed a single detectable protein band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with a relative molecular weight ca.56,000.The intact enzyme had a relative molecular weight (M,) of ca.250,000 as determined by nondenaturing PAGE.The NH2-terminal sequence of bile salt hydrolase was determined to be Met-(Ser/C ys)-Arg-Thr-Lys-Leu-Val-Ileu-Thr-Ileu-Gly-Ala-Ser.The purified enzyme was active towards both glycine and taurine conjugates of cholate.The apparent K , and V , , of the enzyme for glycocholate was estimated to be 0.5 mM and 107 nmol/min mg protein, respectively.The pH optimum was in the range of 5.8 to 6.4.The enzyme was inhibited 85%, 81%, and 83% by 2 mM iodoacetate, p-chloromercuribenzoate, and phenylmethanesulfonylfluoride, respectively.Rabbit polyclonal antibody was prepared and used to demonstrate a single form of the enzyme in crude cell extracts.-Gopal-Srivastava,R., and P. B. Hylemon.Purification and characterization of bile salt hydrolase from Clostridium per-
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