Cofactors of the adenovirus proteinase: measuring equilibrium dissociation constants and stoichiometries of binding.

Abstract

Human adenovirus proteinase (AVP) is required for the synthesis of infectious virus. AVP is synthesized in an inactive form; it is unusual in that it requires cofactors for activation of enzyme activity. Inside nascent virions, an 11-amino-acid peptide and the viral DNA are cofactors for activation; this enables the enzyme to cleave virion precursor proteins, rendering the virus particle infectious. In the cytoplasm, actin is a cofactor for activation, and an actin-AVP complex can cleave cytokeratin 18 and actin itself; this may prepare the infected cell for lysis. Experimental protocols are presented to determine stoichiometries of binding and equilibrium dissociation constants, Kd values, for the binding of pVc, DNA, or actin to AVP by changes in enzyme activity. Techniques are also presented for measuring stoichiometries of binding and Kd values for the binding of various lengths of DNA to AVP by changes in fluorescence polarization. Finally, the binding of different size classes of polymers of glutamic acid to AVP, the Kd values, and stoichiometries of binding are characterized by fluorescence polarization in an indirect assay involving competition with fluorescein-labeled DNA.