cGMP has been implicated in modulation of numerous mammalian systems pertinent to pathophysiology, including smooth muscle tone in blood vessels and other tissues, vision, and platelet aggregation. The objective of this proposal is to examine the regulation of a cGMP-binding phosphodiesterase (B-PDE) which is a probable target of agents that affect the cGMP cascade. This will be done by purifying the bovine lung enzyme and determining its structure and regulatory features. The functions of the B-PDE may be regulated both by an allosteric mechanism and by phosphorylation. The evidence suggests that the enzyme contains at least two functional domains--a highly specific cGMP binding site and a separate hydrolytic site for cGMP. While there is evidence for communication between these putative domains, there is no dir""""""""ct evidence for a function of the binding site. The quaternary structure of B-PDE, number of cGMP binding domains and cyclic nucleotide binding properties of these domains will be ascertained. Effects of binding site- or hydrolytic site-specific cGMP analogs on the enzyme will be tested. Purified protein kinases will be used to study phosphorylation of the enzyme and to determine the mechanism whereby phosphorylation is regulated in a substrate-directed manner by cGMP binding to B-PDE. Preliminary results indicate that the B-PDE is a relatively specific and potent substrate of cGMP-dependent protein kinase, an enzyme for which a physiological substrate has not yet been established. The functional domains of B-PDE will also be studied by generating proteolytic fragments which contain binding activity, hydrolytic activity, or the phosphorylation site. Fragments will be labeled by prior phosphorylation or (32P)cGMP photoaffinity- labeling of B-PDE, and characterized in order to orient the functional domains within the B-PDE. Partial amino acid sequencing of the fragments may reveal features required for function or phosphorylation. Peptides modeled after the sequences of phosphorylation site(s) will be synthesized and tested as substrates for protein kinases in order to identify determinants important for phosphorylation. Oligonucleotide probes based on sequences of peptide fragments, and polyclonal antibodies generated against B-PDE, will be used to screen a bovine lung cDNA library for purposes of cloning. This will permit deduction of the complete primary sequence, assessment of the domain structure, and may indicate evolutionary homologies with other proteins.
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