Purification and characterization of a proteinase inhibitor from white croaker skeletal muscle (Micropogon opercularis).
A trypsin proteinase inhibitor has been purified to homogeneity from the skeletal muscle of white croaker (Micropogon opercularis). Previously, we had described the occurrence in fish muscle of a serine protease (proteinase I) which showed a great capacity to degrade whole myofibrils in vitro and an endogenous inhibitor that prevented the action of the protease, both on natural and artificial substrates. In this paper, we report the purification and further biochemical characterization of the endogenous trypsin inhibitor. The purification was carried out by DEAE-Sephacel, Con A-Sepharose, Sephacryl S-300 and Mono Q. Throughout the purification procedure, trypsin inhibitory activity was assayed using azocasein as substrate. The molecular mass of the inhibitor was 65 kDa, as estimated by SDS-PAGE and gel filtration. The trypsin inhibitor is a glycoprotein, as deduced by the fact that it binds to Con A-Sepharose and stains with PAS and showed a wide range of pH stability (from 5 to 11). The thermal stability of the inhibitor considerably decreased at temperatures >60 degrees C. Assays of the inhibitor against various proteases indicated that it is highly specific for serine proteases, since it did not inhibit proteases belonging to any other groups. The inhibitor was able to inhibit the endogenous target enzyme (proteinase I) in a dose-dependent manner, with a 50% inhibition at a molar ratio close to 1. The present work contributes to improving our understanding of the physiological role of the proteinase I-inhibitor system in muscle protein breakdown, as well as its influence on post mortem proteolysis.
Sangorrín
M P
MP
Laboratorio de Microbiología, Facultad de Ingeniería, UNCOMA, Casilla de Correo 790, 8300, Neuquén, Argentina.
Folco
E J
EJ
Martone
C M
CM
Sánchez
J J
JJ
D000818:Animals; D002364:Caseins / Q000378:metabolism; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D008970:Molecular Weight; D018482:Muscle, Skeletal / Q000737:chemistry* / Q000201:enzymology; D010473:Perciformes / Q000378:metabolism*; D012697:Serine Endopeptidases / Q000378:metabolism; D015842:Serine Proteinase Inhibitors / Q000737:chemistry / Q000302:isolation & purification* / Q000494:pharmacology; D013696:Temperature; D014361:Trypsin Inhibitors / Q000737:chemistry / Q000302:isolation & purification* / Q000494:pharmacology
D002364:Caseins; D015842:Serine Proteinase Inhibitors; D014361:Trypsin Inhibitors; C026018:azocasein; D012697:Serine Endopeptidases