Tissue distribution and characterization of a 30-kDa cysteine proteinase inhibitor from bovine skeletal muscle.
Gel chromatography on a Sephadex G100 column of a crude extract obtained from bovine diaphragma muscle separated four fractions (F-I, F-II, F-III and F-IV) in the range of 12-70 kDa that were active against either papain, trypsin or both. From the F-III fraction, a cysteine proteinase inhibitor was purified by two successive anionic exchange chromatographies on Q-Sepharose and Mono Q columns. The pooled active fraction had a Mw of approximately 30 kDa, and isoelectrofocusing revealed one band with a pI of 6.7. The papain-inhibiting activity was unaffected by dithiothreital or 2-mercaptoethanol treatment, and only one band was obtained after SDS-poly acrylamide gel electrophoresis under both reducing and non-reducing conditions. These results suggest that the 30-kDa muscle cysteine proteinase inhibitor did not contain disulphide bonds essential for activity and the protein was a monomer. This proteinase inhibitor is stable between 40 and 80 degrees C and pH 5-12. Furthermore, the 30-kDa inhibitor is stable to papain proteolysis. The tissue distribution of this inhibitor was investigated using double immunodiffusion and Western blot techniques that provided evidence for its presence in bovine heart, spleen, liver and lung and its absence in bovine plasma.
Berri
M
M
Muscle Biochemistry Laboratory, SRV, INRA de Theix, St. Genès Champanelle, France.
Venien
A
A
Levieux
D
D
Ouali
A
A
D000818:Animals; D002417:Cattle; D002852:Chromatography, Ion Exchange; D003546:Cysteine Endopeptidases / Q000378:metabolism; D015853:Cysteine Proteinase Inhibitors / Q000737:chemistry / Q000302:isolation & purification* / Q000494:pharmacology; D004355:Drug Stability; D004591:Electrophoresis, Polyacrylamide Gel; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D005779:Immunodiffusion; D008970:Molecular Weight; D018482:Muscle, Skeletal / Q000378:metabolism*; D009928:Organ Specificity; D010084:Oxidation-Reduction
D015853:Cysteine Proteinase Inhibitors; D003546:Cysteine Endopeptidases