Purification of a vasodilator-regulated phosphoprotein from human platelets

Area of Science:

• Biochemistry
• Molecular Biology
• Pharmacology

Background:

• Cyclic-nucleotide-elevating vasodilators inhibit vascular smooth muscle contraction and platelet aggregation.
• These vasodilators activate protein phosphorylation mediated by cAMP- and cGMP-dependent protein kinases in human platelets.

Purpose of the Study:

• To elucidate the molecular mechanism of cyclic-nucleotide-elevating vasodilators.
• To purify and characterize a 50-kDa phosphoprotein regulated by these vasodilators.

Main Methods:

• Purification of the 50-kDa phosphoprotein using chromatography.
• SDS-PAGE analysis to determine protein size before and after phosphorylation.
• Limited proteolysis, tryptic fingerprinting, and phosphoamino acid analysis for protein identification.

Main Results:

• The 50-kDa phosphoprotein was purified to homogeneity.
• The purified protein is a substrate for both cAMP- and cGMP-dependent protein kinases.
• Phosphorylation increases the protein's apparent molecular weight from 46 kDa to 50 kDa.
• The protein contains at least two phosphorylation sites, each preferentially phosphorylated by either kinase.

Conclusions:

• The purified 50-kDa phosphoprotein is identical to the vasodilator-regulated protein in platelets.
• This protein plays a role in the mechanism of action of cyclic-nucleotide-elevating vasodilators.
• Availability of the purified protein enables further functional studies.