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Circulating inhibitors of human plasma kallikrein
Vol . 16, No . 5
Conference Abstracts
79 7
SOLUTION STRUCTURE OF BRADYKININ D.I . Marlborough and J .W . Ryan Papanicolaou Cancer Research Institute ând University of Miami School of Medicine, Ptiami, Florida The nonapeptide, bradykinin (H-Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg-OH), has been implicated as a mediator of conditions ranging from functional vasodilation to acute inflammation (1) . Bradykinin (BK) is unusual for a small peptide hor mone for the large percentage of proline residues comprising the structure and the known ateric properties of this residue would suggest a limit to the number of possible conformations open to the peptide . Hence, it might be expected that BR would show some degree of order in solution . Initial spectroscopic studies on BK were interpreted as indicating random disordered structure (2,3) . However, two further publications reinterpreted the data as showing partial hydrogen-bonding in the secondary structure of the peptide, with hydrogen-bonds bridging the proline residues 7 and 3, and the phenylalanine residue 8 (4,5) . It is probable, however, that the pharmacologically active solution structure of the peptide hormone will result from environmental influences acting upon it in the vicinity of its receptor site . Hence, investigation of the conformative response of the peptide to different environmental perturbations might lead to information regarding the stronger conformational influences on the peptide . It is postulated that these stronger conformational perturbations might be those responsible for maintaining the peptide in its pharmacologically active solution structure . The circular dichroism spectrum of BK was followed by titrating against urea, a hydrogen-bond breaking reagent, and 2,2,2-trifluoroethanol (TFE), a solvent with order-producing potential . Whereas urea has only a very small influence on the spectrum, TFE has a relatively more marked influence promoting (presumed) ordered structure . Furthermore, addition of 4 M urea to the partially ordered solution structure in 50 percent aqueous TFE induces a larger change in magnitude of the ellipticity than 4 M urea in aqueous solution alone . This suggests that more ordered structure exists in the 50 percent aqueous TFE solution than in aqueous solution . Hence, it would appear that hydrogen-bonding is only a very minor influence on the solution structure of BK, whereas the hydrophobic influence (as demonstrated by the action of TFE) plays a more predominant role in the conformational behavior of BK . Thus, it could be considered that interaction of BK with its receptor site may be mediated more through the ordering influences of a hydrophobic environment than possible hydrogen-bonding in the structure.
2. 3. 4. 5.
E .G . ERDÖS, Advances in Ph¢m+acology, p . 1, Academic Press, New York (1966) . A . BODANSZRY, t4 . BODANSZKY, E .J . JORPES, V . MUTT and M .A . ONDETTI, Experientia 26 948-950 (1970) . A .H . BRADY, J .W . RYAN and J .M . STEWART, Biochem. J. Z2Z 179-184 (1971) . J .R . CANN, Biochemistry ZZ 2654-2659 (1972) . J .R . CANN, J .M . STEWART and G.R . MATSUEDA, Biochwmistry Z2 3780-3788 (1973) . CIRCULATING INHIBITORS OF HUMAN PLASMA RALLIRREIN P.C . Harpel The New York Hospital-Cornell Medical Center, New York, New York
~9g
Conference Abstracts
Vol . 16, No .
5
a2-macroglobulin and C1 inactivator (C1 1N) are the two major circulating inhibitors of plasma kallikrein . These inhibitors have a broad specificity, and in addition to their role in controlling the generation of kinine, they interact with enzymes in the coagulation, complement, and fibrinolytic pathways . Although the inhibitory spectrum of these human plasma inhibitors has been relatively well-defined, the sequence of the molecular events which occur during the reaction between these inhibitors and the enzymes they inhibit has not previously been defined . In the studies to be reported, the interactions between trypain, plasmin, plasma kallikrein, thrombin, sad a2reiacroglobulin, as we_11 as between active C1 subunit (Cls), plasmin, plasma kallikrein, trypsin and C1 1N have been examined . a2-macroglobulin has been found to be proteolytically cleaved by the enzymes which it inhibits . Furthermore, a2-macroglobulin preparations degrade fibrinogen indicating that in addition to its role as an enzyme inhibitor, a2 macroglobulia may modulate enzyme substrate interactions . C1 iN was found to interact with each enzyme studied in a different manner . In association with enzyme inhibition, C1 1N formed a at_able 1 :1 molar complex with kallikrein, Cls, and plasmin. The light chain of Cls and plasmin provided the binding site for the inhibitor . The plasmin-C1 1N reaction was characterized by the production of several lower molecular weight derivatives, one of which _retained the ability to complex with plasmin . Trypsin failed to complex with C1 1N sad produced derivatives, similar to that produced by plasmin, but which were unable to participate is complex formation. Further studies indicated that conformational factors played a major role in the inhibitory activity of both a2-macroglobulin and C1 1N . GLANDULAR AND PLANT KALLIRREIN INHIBITORS Hans Fritz Institute of Clinical Chemistry and Clinical Biochemistry D-8 Munich 2, Nussbaum Str. 20 Germany The main characteristic of a proteinase inhibitor is its inhibition specificity . Thus far, only protein proteinase inhibitors of broad specificity were found to inhibit kallikraina from glands (pancreas, aubmandibularie, kidney, and urine) and sera ; kallikrein-specific inhibitors were "not described until now . Recently, several new inhibitors were discovered in cow colostrum, sea anemones, snails, and snake venoms . The structures of these broad specificity inhibitors turned out to be homologues to the structure of the basic inhibitor from bovine organs (Kunitz type) which inhibits besides trypsin, chymotrypsin, and plasmin, also plasma as well as glandular kallikreina from various species . However, inhibition of kallikreins by the other mentioned inhibitors ie observed only if basic amino acid residues are located in certain positions of the reactive sits sequences . This special structural feature of kallikrein inhibitors reflects the restricted specificity of kallikreina, especially the glandular kallikreins, to protein substrates compared to trypain, chymotrypain, and plasmin. Proteinsee inhibitors with different inhibition apecificities can be used to discriminate between plasma kallikreine and glandular kallikreins and, in
Conference Abstracts
79 7
SOLUTION STRUCTURE OF BRADYKININ D.I . Marlborough and J .W . Ryan Papanicolaou Cancer Research Institute ând University of Miami School of Medicine, Ptiami, Florida The nonapeptide, bradykinin (H-Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg-OH), has been implicated as a mediator of conditions ranging from functional vasodilation to acute inflammation (1) . Bradykinin (BK) is unusual for a small peptide hor mone for the large percentage of proline residues comprising the structure and the known ateric properties of this residue would suggest a limit to the number of possible conformations open to the peptide . Hence, it might be expected that BR would show some degree of order in solution . Initial spectroscopic studies on BK were interpreted as indicating random disordered structure (2,3) . However, two further publications reinterpreted the data as showing partial hydrogen-bonding in the secondary structure of the peptide, with hydrogen-bonds bridging the proline residues 7 and 3, and the phenylalanine residue 8 (4,5) . It is probable, however, that the pharmacologically active solution structure of the peptide hormone will result from environmental influences acting upon it in the vicinity of its receptor site . Hence, investigation of the conformative response of the peptide to different environmental perturbations might lead to information regarding the stronger conformational influences on the peptide . It is postulated that these stronger conformational perturbations might be those responsible for maintaining the peptide in its pharmacologically active solution structure . The circular dichroism spectrum of BK was followed by titrating against urea, a hydrogen-bond breaking reagent, and 2,2,2-trifluoroethanol (TFE), a solvent with order-producing potential . Whereas urea has only a very small influence on the spectrum, TFE has a relatively more marked influence promoting (presumed) ordered structure . Furthermore, addition of 4 M urea to the partially ordered solution structure in 50 percent aqueous TFE induces a larger change in magnitude of the ellipticity than 4 M urea in aqueous solution alone . This suggests that more ordered structure exists in the 50 percent aqueous TFE solution than in aqueous solution . Hence, it would appear that hydrogen-bonding is only a very minor influence on the solution structure of BK, whereas the hydrophobic influence (as demonstrated by the action of TFE) plays a more predominant role in the conformational behavior of BK . Thus, it could be considered that interaction of BK with its receptor site may be mediated more through the ordering influences of a hydrophobic environment than possible hydrogen-bonding in the structure.
2. 3. 4. 5.
E .G . ERDÖS, Advances in Ph¢m+acology, p . 1, Academic Press, New York (1966) . A . BODANSZRY, t4 . BODANSZKY, E .J . JORPES, V . MUTT and M .A . ONDETTI, Experientia 26 948-950 (1970) . A .H . BRADY, J .W . RYAN and J .M . STEWART, Biochem. J. Z2Z 179-184 (1971) . J .R . CANN, Biochemistry ZZ 2654-2659 (1972) . J .R . CANN, J .M . STEWART and G.R . MATSUEDA, Biochwmistry Z2 3780-3788 (1973) . CIRCULATING INHIBITORS OF HUMAN PLASMA RALLIRREIN P.C . Harpel The New York Hospital-Cornell Medical Center, New York, New York
~9g
Conference Abstracts
Vol . 16, No .
5
a2-macroglobulin and C1 inactivator (C1 1N) are the two major circulating inhibitors of plasma kallikrein . These inhibitors have a broad specificity, and in addition to their role in controlling the generation of kinine, they interact with enzymes in the coagulation, complement, and fibrinolytic pathways . Although the inhibitory spectrum of these human plasma inhibitors has been relatively well-defined, the sequence of the molecular events which occur during the reaction between these inhibitors and the enzymes they inhibit has not previously been defined . In the studies to be reported, the interactions between trypain, plasmin, plasma kallikrein, thrombin, sad a2reiacroglobulin, as we_11 as between active C1 subunit (Cls), plasmin, plasma kallikrein, trypsin and C1 1N have been examined . a2-macroglobulin has been found to be proteolytically cleaved by the enzymes which it inhibits . Furthermore, a2-macroglobulin preparations degrade fibrinogen indicating that in addition to its role as an enzyme inhibitor, a2 macroglobulia may modulate enzyme substrate interactions . C1 iN was found to interact with each enzyme studied in a different manner . In association with enzyme inhibition, C1 1N formed a at_able 1 :1 molar complex with kallikrein, Cls, and plasmin. The light chain of Cls and plasmin provided the binding site for the inhibitor . The plasmin-C1 1N reaction was characterized by the production of several lower molecular weight derivatives, one of which _retained the ability to complex with plasmin . Trypsin failed to complex with C1 1N sad produced derivatives, similar to that produced by plasmin, but which were unable to participate is complex formation. Further studies indicated that conformational factors played a major role in the inhibitory activity of both a2-macroglobulin and C1 1N . GLANDULAR AND PLANT KALLIRREIN INHIBITORS Hans Fritz Institute of Clinical Chemistry and Clinical Biochemistry D-8 Munich 2, Nussbaum Str. 20 Germany The main characteristic of a proteinase inhibitor is its inhibition specificity . Thus far, only protein proteinase inhibitors of broad specificity were found to inhibit kallikraina from glands (pancreas, aubmandibularie, kidney, and urine) and sera ; kallikrein-specific inhibitors were "not described until now . Recently, several new inhibitors were discovered in cow colostrum, sea anemones, snails, and snake venoms . The structures of these broad specificity inhibitors turned out to be homologues to the structure of the basic inhibitor from bovine organs (Kunitz type) which inhibits besides trypsin, chymotrypsin, and plasmin, also plasma as well as glandular kallikreina from various species . However, inhibition of kallikreins by the other mentioned inhibitors ie observed only if basic amino acid residues are located in certain positions of the reactive sits sequences . This special structural feature of kallikrein inhibitors reflects the restricted specificity of kallikreina, especially the glandular kallikreins, to protein substrates compared to trypain, chymotrypain, and plasmin. Proteinsee inhibitors with different inhibition apecificities can be used to discriminate between plasma kallikreine and glandular kallikreins and, in