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P C Harpel

Publications and source records attributed to P C Harpel.

9 recordsLinked to original sources
PubMed1979

Heat-induced fragmentation of human alpha 2-macroglobulin.

Previous studies have demonstrated that human plasma alpha 2-macroglobulin (alpha 2 M) possesses a single subunit chain (Mr approximately 185,000) when incubated with dodecyl sulfate and dithiothreitol at 37 degrees C and analyzed by dodecyl sulfate-gel electrophoresis. The present study details the observation that heating alpha 2 M to 90 degrees C under identical conditions produces at least two additional polypeptide chains, termed bands II and III, with apparent molecular weights of 125,00 and 62,000. The generation of these fragments is enhanced by increasing the time of incubation. The appearance of band II composition of the buffer, dodecyl sulfate concentrations, or alpha 2 M protein concentration in the incubation mixture. The electrophoretic bands II and III of alpha 2 M have dissimilar 125I-labeled tryptic peptide digests and also differ in their amino acid composition. The heat-induced fragmentation of alpha 2M is not affected by the inclusion of a variety of low molecular weight protease inhibitors, suggesting that the appearance of bands II and III is not due to enzyme-catalyzed hydrolysis. When the subunit chain of alpha 2M is first cleaved by trypsin into the previously described Mr = 85,000 derivative, neither band II nor III material, nor other lower molecular weight products are generated by heat treatment. Furthermore, preincubation of alpha 2M with methylamine prevents fragmentation of the subunit chain. These results indicate that these fragments are neither pre-existing subunits of alpha 2M nor derivatives formed prior to treatment for gel analysis. These data provide evidence that a covalent bond in the alpha 2M molecule is unusually susceptible to heat-induced cleavage.

Amino Acids↗
PubMed1977

Plasmin inhibitor interactions. The effectiveness of alpha2-plasmin inhibitor in the presence of alpha2-macroglobulin.

alpha2-Plasmin inhibitor and alpha2-macroglobulin were allowed to compete for the protease plasmin. The binding of the enzyme to these inhibitors was assessed by two different but comparable methods. The interactions were completed in 10 s of incubation, and transfer of plasmin from one inhibitor to the other did not occur. Almost as much plasmin was bound to alpha2-plasmin inhibitor in mixtures containing a large molar excess of alpha2-macroglobulin relative to plasmin or alpha2-plasmin inhibitor, as was bound in mixtures not containing alpha2-macroblobulin. These studies demonstrate directly the effectiveness of alpha2-plasmin inhibitor in binding and inhibiting plasmin in the presence of alpha2-macroglobulin, and suggest that the alpha2-plasmin inhibitor may be the major circulating plasmin inhibitor.

Binding, Competitive↗
PubMed1976

Platelet alpha2-macroglobulin and alpha1-antitrypsin.

Subcellular membrane and granule fractions derived from human platelets contain immunologically identifiable alpha2-macroglobulin and alpha1-antitrypsin. These platelet-derived inhibitors show a reaction of immunologic identity when compared to alpha2-macroglobulin and alpha1-antitrypsin purified from human plasma. Further, the platelet protease inhibitors possessed a similar subunit polypeptide chain structure to their plasma counterparts as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis. Studies of the binding of radiolabeled trypsin to the various solubilized platelet subcellular fractions suggest that the granule-associated alpha2-macroglobulin and alpha1-antitrypsin, as well as membrane-associated alpha2-macroglobulin were functionally active. Quantitatively, circulating platelets contain relatively small concentrations of these inhibitors as compared to platelet-associated fibrinogen and factor VIIIAGN. Platelet protease inhibitors may modulate the protease-mediated events involved in the formation of hemostatic plugs and thrombi.

Blood Platelets↗
PubMed1976

alpha2-Macroglobulin on human vascular endothelium.

alpha2-Macroglobulin (alpha2M) has been identified on the luminal surface of endothelial cells in sections of normal human arteries, veins, and lymphatics by the indirect immunofluorescent technique. The specificity of the immunofluorescent reaction was confirmed by immunoabsorption studies. Prior absorption of the anti-alpha2M antiserum by purified alpha2M at equivalence completely inhibited endothelial surface as well as hepatic parenchymal cell staining. Endothelial cells in blood vessels were not stained when sections were treated with rabbit antisera toward alpha1-antitrypsin, antithrombin III, IgG, IgA, IgM, C3, or fibrinogen. The location of alpha2M at the surface of the vessel wall suggests that this protease inhibitor may protect the vascular endothelium from potentially injurious intravascular proteases.

Blood Vessels↗
PubMed1976

C1 inactivator.

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Amino Acids↗
PubMed1975

Studies on human plasma C1 inactivator-enzyme interactions. I. Mechanisms of interaction with C1s, plasmin, and trypsin.

This study has explored the nature of the molecular events which occur when C1 inactivator, a human plasma inhibitor of the complement, kinin-forming, coagulation, and fibrinolytic enzyme systems, interacts with C1s, plasmin, and trypsin. Purified inhibitor preparations demonstrated two bands, when examined by acrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS). The molecular weights of the major and minor bands were 105,000 and 96,000 daltons, respectively. The minor component appeared to be immunologically and functionally identical to the main C1 inactivator component. Loss of C1s and plasmin functional activity was associated with the formation of a 1:1 molar complex between the inhibitor and each enzyme. These complexes were stable in the presence of SDS and urea. The light chain of both these enzymes provided the binding site for C1 inactivator. Complex formation and enzyme inhibition occurred only with native and not with an inhibitor preparation denatured by acid treatment, thereby demonstrating the importance of conformational factors in the enzyme-inhibitor reaction. Although peptide bond cleavage of the C1 inactivator molecule by C1s was not documented, plasmin was found to degrade the inhibitor with the production of several characteristic derivatives. At least one of these products retained the ability to complex with C1s and plasmin. Trypsin, which failed to form a complex with C1 inactivator, degraded the inhibitor in a limited and sequential manner with the production of nonfunctional derivatives one of which appeared structurally similar to a plasmin-induced product. These studies therefore, provide new information concerning the molecular interactions between C1 inactivator and several of the proteases which it inhibits.

Animals↗
PubMed1975

Studies on human plasma C1 inactivator-enzyme interactions. II. Structural features of an abnormal C1 inactivator from a kindred with hereditary angioneurotic edema.

The function and several of the structural features of the C1 inactivator protein isolated from the plasma of a mother and daughter with the variant form of hereditary angioneurotic edema have been examined. These abnormal inhibitors shared immunologic identity with the normal C1 inactivator protein; however, they were inactive in inhibiting the functional activity of C1s. Analysis of the abnormal inhibitors by sodium dodecyl sulfate (SDS) acrylamide gel electrophoresis suggested that each consisted of a single polypeptide chain, the mobility of which was slower than that of the normal C1 inactivator. The apparent molecular weight of the patients' inhibitors was 109,000 daltons as contrasted to 105,000 daltons, that of the normal C1 inactivator. The abnormal inhibitors failed to form a complex with C1s or plasmin as analyzed by SDS-acrylamide gels. The large proteolytic derivatives resulting from the plasmin- and trypsin-induced degradation of the abnormal inhibitors were approximately 3,000 daltons heavier than the corresponding products derived from normal C1 inactivator. Thus, the structural abnormality identified appeared to be a property of the core molecule. Treatment of the inhibitors with neuraminidase failed to demonstrate a difference between the normal and patient-derived C1 inactivator molecule. Neither were major differences found between the amino acid composition of the defective and normal inhibitors; however, the acidic amino acids tended to be higher in the patients' inhibitors, and the phenylalanine content lower. Thus, these studies have identified both structural and functional abnormalities in the C1 inactivator protein isolated from two related patients with hereditary angioneurotic edema. Examination of the interaction between endopeptidases and the inhibitors has further delineated the abnormal structural features.

Amino Acids↗