Calcium-activated neutral protease from bovine ventricular muscle: Isolation and some of its properties

Abstract

A new protease has been isolated and purified from bovine ventricular muscle, by a new method which employs affinity chromatography. This protease required both millimolar concentrations of Ca 2+ and the SH-group for the activation, and it was active at neutral pH. The molecular weight of this calcium-activated neutral protease was estimated to be 92 000 and 80 000 by gel filtration and sodium dodecylsulfate polyacrylamide gel electrophoresis, respectively. The isoeletric point was determined to be 4.3 by isoelectric focusing. The synthetic substrates including N-benzoyl l-arginine ethylester and acetyltyrosine ethylester were not hydrolysed by this protease. Among the endogenous myofibrillar proteins, the contractile proteins (myosin and actin) were not degraded, while the regulatory proteins, tropomyosin and troponin were degraded only in the presence of Ca 2+. Of three components of troponin, both troponin-T and troponin-I were susceptible to the proteolytic action of calcium-activated neutral protease, while troponin-C was well preserved, as verified by the electrophoretic pattern in sodium dodecyl-sulfate-polyacrylamide gel. The breakdown of the regulatory proteins was also functional, as demonstrated in natural actomyosin pretreated by calcium-activated neutral protease by measurement of Ca 2+-sensitivity of superprecipitation. Although the physiological feature of calcium-activated neutral protease is still unknown, this enzyme might act as an aggravating factor in the course of myocardial necrosis, when the intracellular concentration of free Ca 2+ is considered to be elevated to millimolar levels.