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J. Biol. Chem., Vol. 261, Issue 26, 12047-12052, Sep, 1986
GN DeMartino, CA Huff and DE Croall
Calcium-dependent protease II (CDP-II) from bovine heart is a heterodimer
with subunit molecular weights of 80,000 and 26,000. Previous studies have
demonstrated that the protease requires 350 microM Ca2+ for half-maximal
activity and that the large subunit contains both the catalytic and Ca2+
binding functions of the enzyme. The function of the small subunit has been
unclear. We have examined the effect of Ca2+ on structural and catalytic
properties of CDP-II in the presence and absence of substrate proteins.
When incubated with Ca2+ in the absence of substrate, CDP-II undergoes a
series of autoproteolytic cleavages that sequentially reduce the small
subunit's molecular weight from 26,000 to 24,000 to 22,000 to 17,000.
During this time there is no detectable change in the 80-kDa subunit, which
remains associated with the autolyzed small subunit. The rate of
autoproteolysis is dependent on temperature and on the concentration of
Ca2+ (half-maximal rate at approximately 600 microM Ca2+). The first
cleavage appears to be unimolecular because its rate is unaffected by
CDP-II concentration or by the presence of exogenous protein substrates.
Subsequent cleavages result in the formation of the 80- kDa/17-kDa
heterodimer and appear to occur by bimolecular reactions; rates of these
reactions were slowed by decreasing CDP-II concentrations and by the
presence of protein substrates. Autoproteolysis of the small subunit has
two distinct functional consequences, each of which is associated with
different forms of the autolyzed protease. Our results indicate that the
80-kDa/26-kDa form of CDP-II represents an inactive proenzyme and that the
initial Ca2+- dependent cleavage of the 26-kDa subunit results in
activation of the protease. The activated enzyme hydrolyzes protein
substrates with a Ca2+ concentration requirement of 350 microM for
half-maximal rates. The further autoproteolysis, which results in the
formation of the 80- kDa/17-kDa heterodimer, serves to reduce the Ca2+
concentration requirement for protease activity by 25-fold. Thus, these
results provide evidence for specific roles of the small subunit in the
regulation of CDP-II activity.
Autoproteolysis of the small subunit of calcium-dependent protease II activates and regulates protease activity
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