Ca2+-dependent
proteases isolated from chicken gizzard and bovine aortic smooth muscle were compared with respect to subunit
autolysis and the role of
autolysis in modulating
enzyme activity. The
protease isolated from chicken gizzard was a heterodimer consisting of 80,000- and 30,000-dalton subunits. The
protease isolated under identical conditions from bovine aorta consisted of 75,000- and 30,000-dalton subunits. In the presence of Ca2+, both
enzymes underwent
autolysis of their 30,000-dalton subunits with conversion to an 18,000-dalton species. In addition, the 80,000-dalton subunit of the gizzard
protease was degraded to a 76,000-dalton form. The Ca2+ concentrations required for
autolysis of the 30,000-dalton subunits were different for the two
enzymes (i.e. gizzard: K0.5 Ca2+ = 335 microM; aortic: K0.5 Ca2+ = 1,250 microM) although in both cases, stimulation of
autolysis by Ca2+ exhibited positive cooperativity. When compared with respect to kinetics of substrate degradation, the native forms of the smooth muscle Ca2+-dependent
proteases (gizzard, GIIa = 80,000/30,000-dalton heterodimer; bovine aortic, IIa = 75,000/30,000-dalton heterodimer) exhibited a lag phase in product appearance. On the other hand, the autolyzed forms (gizzard, GIIb = 76,000/18,000-dalton heterodimer; bovine aortic, IIb = 75,000/18,000-dalton heterodimer) exhibited linear rates of substrate degradation. These results were analyzed in terms of
autolysis of the 30,000-dalton subunits as determined by the conversion of this subunit to its 18,000 dalton form. For both
enzymes, the time course for the autolytic transition, 30,000----18,000 daltons, and Ca2+-dependence of the apparent rate constants for this transition were found to correlate well with the lag phase in enzymatic activity. No such correlation could be established for the 80,000----76,000 dalton autolytic transition of the high molecular mass subunit of the gizzard
protease. Our results suggest that catalytic activity of the Ca2+-dependent
proteases isolated from gizzard and bovine aortic smooth muscle requires
autolysis of the 30,000-dalton subunit. The native or unautolyzed forms of these
enzymes appear to be
proenzymes that can be activated by
autolysis.