Purification and properties of human platelet P235. A high molecular weight protein substrate of endogenous calcium-activated protease(s).
Two major high molecular weight proteins of human platelets are highly susceptible to proteolytic degradation by endogenous calcium-activated protease activities. Of the two proteins, one has been identified as filamin (Mr = 250,000 subunit); the second, a Mr = 235,000 subunit protein contributing 3-8% of the total platelet proteins, has not been previously characterized. We have now purified this protein, designated P235, to apparent homogeneity (greater than 95%). P235 was extracted by a Triton X-100 and EDTA containing buffer at pH 9.0 and purified by a series of DEAE-cellulose, phosphocellulose, and gel filtration chromatographies. Purified P235 is a dimer of Mr = 235,000 subunit. Its Stokes radius (67 A) and frictional ratio of 1.3 suggest that P235 is approximately globular. Despite this similarity in subunit and molecular weight of P235 to filamin, spectrin, fibronectin, and myosin, its amino acid composition, immunological properties, and peptide map are distinctly different and showed no precursor-product relationship to these proteins. Calcium-activated protease(s) in crude platelet extract rapidly degrade P235 into a Mr = 200,000 stable fragment. Upon prolonged storage at 4 degrees C, purified P235 partially degrades into a Mr = 220,000 and a Mr = 200,000 fragment. This degradation pattern suggests that P235 contains a large Mr = 200,000 protease-resistant domain. The availability of pure P235 will be useful in elucidating the functional role of this platelet protein, as well as the role of calcium-activated proteases in platelet function.
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000936:Antigen-Antibody Complex; D001792:Blood Platelets / Q000032:analysis*; D002118:Calcium / Q000494:pharmacology*; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003285:Contractile Proteins / Q000302:isolation & purification*; D000939:Epitopes / Q000032:analysis; D064448:Filamins; D006801:Humans; D008840:Microfilament Proteins; D008970:Molecular Weight; D010446:Peptide Fragments / Q000032:analysis; D010447:Peptide Hydrolases / Q000378:metabolism*; D011487:Protein Conformation; D013045:Species Specificity; D013329:Structure-Activity Relationship; D016608:Talin
D000596:Amino Acids; D000936:Antigen-Antibody Complex; D003285:Contractile Proteins; D000939:Epitopes; D064448:Filamins; D008840:Microfilament Proteins; D010446:Peptide Fragments; D016608:Talin; D010447:Peptide Hydrolases; D002118:Calcium