Properties of regulatory subunit of cyclic AMP-dependent protein kinase (peak I) from rabbit skeletal muscle prepared by urea treatment of the holoenzyme

Schwechheimer, K.; Hofmann, F.

Journal of Biological Chemistry 252(21): 7690-7696


ISSN/ISBN: 0021-9258
PMID: 199596
Accession: 068523787

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Chromatography of essentially pure preparations of cyclic (Peak I) from rabbit skeletal muscle on Sephadex CM-50 and DEAE-cellulose in the presence of 4.2 M urea dissociated the holoenzyme into its subunits. Regulatory subunit was eluted from the DEAE-cellulose by 150 mM NaCl with a yield of 20%. Urea treatment did not change relative mobility of regulatory subunit on sodium dodecyl sulfate gel electrophoresis. On sucrose density gradients, isolated regulatory subunit sedimented with an s20,w value of 3.2. Regulatory subunit added at stoichiometric concentration inhibited catalytic subunit almost completely in the absence, but not in the presence, of cAMP. Regulatory and catalytic subunit recombined in the absence of MgATP. After sucrose density gradient centrifugation, a cAMP-dependent enzyme with an s20,w value of 7.0 was obtained. The regulatory subunit bound 1 mol of cAMP/mol of subunit. The apparent dissociation constant (Kd) for the binding of cAMP increased from 0.0029-0.29 .mu.M when the concentration of regulatory subunit was increased from 0.001-0.92 .mu.M. An increase in the apparent Hill coefficient from 1.15-1.5 was observed. These values did not change when Mg2+ or MgATP were added. Addition of catalytic subunit at stoichiometric concentrations to regulatory subunit (20 nM each) did not change the apparent Kd value. In the presence of MgATP, the apparent Kd value for cAMP increased about 20-fold. Addition of catalytic subunit at 4-fold higher concentration than regulatory subunit yielded 2- and 4-fold higher apparent Kd values in the absence and presence of MgATP, respectively. Binding of MgATP with high affinity was observed only when equal concentraions of catalytic and regulatory subunit were used. In the phosphotransferase assay, an apparent activation constant (KA) for cAMP of 0.23 .mu.M was obtained when regulatory and catalytic subunit were mixed at stoichiometric concentrations (20 nM each). Addition of 4-fold higher concentrations of each subunit yielded an apparent KA value of 0.6 .mu.M. Addition of regulatory subunit in a 4-fold excess over catalytic subunit (20 nM) increased the apparent KA value further to 1 .mu.M. Apparent Hill coefficients for activation of protein kinase mixed from regulatory and catalytic subunit at 1:1 and 4:1 ratio were 1.0 and 1.5, respectively.