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Quantitative immunoassays for components of the human plasma kinin-forming system
Vol . 16, No . 5
Conference Abstzacts
79 9
addition, between tissue kallikreina from different glands of the same species . These observations, as well as the results of X-ray crystallographic and kinetic studies obtained recently, show that the affinity of a protein or polypeptide inhibitor to its antagonist (proteinase) is determined by the shape (tertiary Molecular parameters structure) of the reactive siEes (areas) of both partners . responsible for complex formation will be discussed, too. Interesting properties of known and recently described kallikrein inhibitors will be presented, including those isolated from sea anemones, snails, snake toxins, earth worms, cuttle fishes, potatoes, peanuts, and from microorganisms . PROTEINASE INHIBITORS OF SNARE VENOMS : ISOLATION, CHARACTERIZATION AND AMINO ACID SEQUENCE S . Iwanaga, H. Takahashi, T. Tatsuki, Y. Hokama, and T . Suzuki Institute for Protein Research, Osaka University, Suits, Osaka-565, Japan In the course of our studies on the kallikrein-kinin system of mammalian blood plasma, we found some potent kallikrein inhibitors in several snake venoms . The inhibitors were mainly distributed in venoms belonging to Viperidae and Elapidae families . Using the venoms of Vipern rusaeli, Hemachatus haemachatus and Na a nivea , seven proteinase inhibitors were isolated as gel-electrophoretically pure substances . One of the inhibitors, RW-inhibitor II, isolated from _V . ruasellü venom, had a strong inhibitory effect on so-called serine-proteinases like bovine pancreatic trypain inhibitor (Kunitz type) . However, it had no effect on the activities of thrombin, reptilase, venom kininogenase, thiolproteinase, metal-proteinase, and exopeptidases . RW-inhibitor II inactivated bovine trypain probably by formation of an enzyme-inhibitor complex in a molar ratio of 1 :1 . Moreover, inhibition constants (Ki) of the inhibitor, measured using synthetic ester substrate, were 2 .9 x 10 -10 M for bovine plasma kallikrein, 7.6 x 10 -10 M for bovine trypsin, and 1.4 x 10 -10 M for bovine a-chymotrypaia . All the inhibitors isolated were basic polypeptides with molecular weights of about 6,500 . The amino acid sequences of three of them were determined completely or partially through the use of the Edman degradation and standard enzymatic and chemical techniques . The overall primary structure of these inhibitors were quite similar to those of the Kunitz type inhibitor and cow colostrum trypsin inhibitor, indicating about 55 percent sequence homology . Moreover, the structural portions, which are found in the interior and reactive site and tend to stabilize the unique structure of the Kunitz type inhibitor, contained an extremely high sequence homology in the snake venom inhibitors . Thus, the tertiary structure of the venom inhibitor appears very similar to that of the Runitz inhibitor. The results are interesting when one considers the different descent of the animals species . QUANTITATIVE Itß4UNOASSAYS FOR COMPONENTS OF THE HUMAN PLASMA KININ-FORMING SYSTEM Jocelyn Spragg Department of Medicine, Ilarvard Medical School and Robert B . Brigham Hospital, Boston, Zfassachusetts 02120
80 0
Conference Abstracts
Vol . 16, No . 5
The development in recent yearn of quantitative immunoassays for components of the plasma kinia-forming system has provided a aeries of sensitive and specific methods applicable to basic and clinical studies of this system . A radial immunodiffusion assay (1) and a radioimmunoassay (2) for human Hageman factor have indicated that the normal plasma levels of this protein are 15-47 or 30-40 ugm/ml, respectively . Earlier immunoassays of plasminogen and plasmin using an immunogen and standard purified from Cohn Fraction III2 3, of plasma have reported plasma levels of 206 ug/ml (3) and 200-400 ug/ml (4), wile the more recent purification of a plasminogen immuaogen and standard from fresh human plasma by the rapid affinity chromatography method (5) has led to the development of a radial immunodiffusion assay in which a mean plasma plasminogen level of 464 Kg/ml was determined for a series of 30 normal donors (6) . Purification of human plasma kininogen with a molecular weight of 70,000 ae calculat®d from experimentally determined diffusion constant, sedimentation coefficient and partial specific volume, and the preparation of monospecific antiserum shown to be specific for such kininogen (7,8) were used in a radial immunodiffusion assay for this protein. The mean level determined in plasma samples from 50 normal individuals was 674 ug/ml and a good correlation (r-0 .91) was found by comparing immunoassay and functional values (9) . Radioimmunoassays for bradykinin described by several groups have all employed antibradykinin elicited with bradykinin coupled to ovalbumin and the labeled hapten (1251) tyr8-bradykinin (10,11,12) . Reported plasma levels have ranged from 0.07 to 5 mug/ml ; the range of values appears to depend upon methodologic differences in collection and processing as well as in the assay itself . S.D . REVAK, C .G . COCHRANE, A.R . JOHNSTON and T .E . HUGLI, J. CZin .
2. 3. 4. 5. 6. 7. 8. 9. 10 . 11 .
Invest . 54 619 (1974) .
H . SAITO, O .D . RATNOFF and J . PENSKY, Fed . Proc . 33 226 abs . (1974) . S.F . RABINER, I.D . GOLDFINE, A . HART, L. SUMMARIA and K.C . ROBBINS, J. Lab . Clin . Med . 7 4 265 (1969) . K. STÖRIKO, BZut Z6 200 (1968) . D .G . DEUTSCH and E .T . MERTZ, Science Z70 1095 (1970) . E.H . MAGOON, K.F . AUSTEN and J . SPRAGG, Clin . E~p . ImrmmoZ . Z7 345 (1974) . J . SPRAGG, E. HABER and R .F . AUSTEN, J. Immunol . Z04 1348 (1970) . J. SPRAGG and K .F . AUSTEN, J. ImmunoZ . Z07 1512 (1971) . R .C . TALAMO, E . HABER and K .F . AUSTEN, J. Lab . CZin . hfed. 7 4 816 (1969) . M .L . MASHFORD and M .L . ROBERTS, Biochem . Pharme~coZ . 2Z 2727 (1972) . ß .U . WEINTRAUB, J . SPRAGG, D .J . STECHSCHULTE and R.F . AUSTEN, Mechanism in AZZergy : Reagin Mediated Hypersensitivity . p . 495, Marcel Dekker, New York (1973) .
THE USE OF TYRl-KALLIDIN, TYRS-BRADYKININ, AND TYRE-BRADYKININ IN THE EVALUATION OF BRADYKININ ANTISERA FOR USE IN RADIOIPß~RTNOASSAY Charles E. Odya, Theodore L . Goodfriend, Clare Pëna and John M. Stewart Department of Pharmacology, University of Wisconsin Medical School Madison, Wisconsin and
Conference Abstzacts
79 9
addition, between tissue kallikreina from different glands of the same species . These observations, as well as the results of X-ray crystallographic and kinetic studies obtained recently, show that the affinity of a protein or polypeptide inhibitor to its antagonist (proteinase) is determined by the shape (tertiary Molecular parameters structure) of the reactive siEes (areas) of both partners . responsible for complex formation will be discussed, too. Interesting properties of known and recently described kallikrein inhibitors will be presented, including those isolated from sea anemones, snails, snake toxins, earth worms, cuttle fishes, potatoes, peanuts, and from microorganisms . PROTEINASE INHIBITORS OF SNARE VENOMS : ISOLATION, CHARACTERIZATION AND AMINO ACID SEQUENCE S . Iwanaga, H. Takahashi, T. Tatsuki, Y. Hokama, and T . Suzuki Institute for Protein Research, Osaka University, Suits, Osaka-565, Japan In the course of our studies on the kallikrein-kinin system of mammalian blood plasma, we found some potent kallikrein inhibitors in several snake venoms . The inhibitors were mainly distributed in venoms belonging to Viperidae and Elapidae families . Using the venoms of Vipern rusaeli, Hemachatus haemachatus and Na a nivea , seven proteinase inhibitors were isolated as gel-electrophoretically pure substances . One of the inhibitors, RW-inhibitor II, isolated from _V . ruasellü venom, had a strong inhibitory effect on so-called serine-proteinases like bovine pancreatic trypain inhibitor (Kunitz type) . However, it had no effect on the activities of thrombin, reptilase, venom kininogenase, thiolproteinase, metal-proteinase, and exopeptidases . RW-inhibitor II inactivated bovine trypain probably by formation of an enzyme-inhibitor complex in a molar ratio of 1 :1 . Moreover, inhibition constants (Ki) of the inhibitor, measured using synthetic ester substrate, were 2 .9 x 10 -10 M for bovine plasma kallikrein, 7.6 x 10 -10 M for bovine trypsin, and 1.4 x 10 -10 M for bovine a-chymotrypaia . All the inhibitors isolated were basic polypeptides with molecular weights of about 6,500 . The amino acid sequences of three of them were determined completely or partially through the use of the Edman degradation and standard enzymatic and chemical techniques . The overall primary structure of these inhibitors were quite similar to those of the Kunitz type inhibitor and cow colostrum trypsin inhibitor, indicating about 55 percent sequence homology . Moreover, the structural portions, which are found in the interior and reactive site and tend to stabilize the unique structure of the Kunitz type inhibitor, contained an extremely high sequence homology in the snake venom inhibitors . Thus, the tertiary structure of the venom inhibitor appears very similar to that of the Runitz inhibitor. The results are interesting when one considers the different descent of the animals species . QUANTITATIVE Itß4UNOASSAYS FOR COMPONENTS OF THE HUMAN PLASMA KININ-FORMING SYSTEM Jocelyn Spragg Department of Medicine, Ilarvard Medical School and Robert B . Brigham Hospital, Boston, Zfassachusetts 02120
80 0
Conference Abstracts
Vol . 16, No . 5
The development in recent yearn of quantitative immunoassays for components of the plasma kinia-forming system has provided a aeries of sensitive and specific methods applicable to basic and clinical studies of this system . A radial immunodiffusion assay (1) and a radioimmunoassay (2) for human Hageman factor have indicated that the normal plasma levels of this protein are 15-47 or 30-40 ugm/ml, respectively . Earlier immunoassays of plasminogen and plasmin using an immunogen and standard purified from Cohn Fraction III2 3, of plasma have reported plasma levels of 206 ug/ml (3) and 200-400 ug/ml (4), wile the more recent purification of a plasminogen immuaogen and standard from fresh human plasma by the rapid affinity chromatography method (5) has led to the development of a radial immunodiffusion assay in which a mean plasma plasminogen level of 464 Kg/ml was determined for a series of 30 normal donors (6) . Purification of human plasma kininogen with a molecular weight of 70,000 ae calculat®d from experimentally determined diffusion constant, sedimentation coefficient and partial specific volume, and the preparation of monospecific antiserum shown to be specific for such kininogen (7,8) were used in a radial immunodiffusion assay for this protein. The mean level determined in plasma samples from 50 normal individuals was 674 ug/ml and a good correlation (r-0 .91) was found by comparing immunoassay and functional values (9) . Radioimmunoassays for bradykinin described by several groups have all employed antibradykinin elicited with bradykinin coupled to ovalbumin and the labeled hapten (1251) tyr8-bradykinin (10,11,12) . Reported plasma levels have ranged from 0.07 to 5 mug/ml ; the range of values appears to depend upon methodologic differences in collection and processing as well as in the assay itself . S.D . REVAK, C .G . COCHRANE, A.R . JOHNSTON and T .E . HUGLI, J. CZin .
2. 3. 4. 5. 6. 7. 8. 9. 10 . 11 .
Invest . 54 619 (1974) .
H . SAITO, O .D . RATNOFF and J . PENSKY, Fed . Proc . 33 226 abs . (1974) . S.F . RABINER, I.D . GOLDFINE, A . HART, L. SUMMARIA and K.C . ROBBINS, J. Lab . Clin . Med . 7 4 265 (1969) . K. STÖRIKO, BZut Z6 200 (1968) . D .G . DEUTSCH and E .T . MERTZ, Science Z70 1095 (1970) . E.H . MAGOON, K.F . AUSTEN and J . SPRAGG, Clin . E~p . ImrmmoZ . Z7 345 (1974) . J . SPRAGG, E. HABER and R .F . AUSTEN, J. Immunol . Z04 1348 (1970) . J. SPRAGG and K .F . AUSTEN, J. ImmunoZ . Z07 1512 (1971) . R .C . TALAMO, E . HABER and K .F . AUSTEN, J. Lab . CZin . hfed. 7 4 816 (1969) . M .L . MASHFORD and M .L . ROBERTS, Biochem . Pharme~coZ . 2Z 2727 (1972) . ß .U . WEINTRAUB, J . SPRAGG, D .J . STECHSCHULTE and R.F . AUSTEN, Mechanism in AZZergy : Reagin Mediated Hypersensitivity . p . 495, Marcel Dekker, New York (1973) .
THE USE OF TYRl-KALLIDIN, TYRS-BRADYKININ, AND TYRE-BRADYKININ IN THE EVALUATION OF BRADYKININ ANTISERA FOR USE IN RADIOIPß~RTNOASSAY Charles E. Odya, Theodore L . Goodfriend, Clare Pëna and John M. Stewart Department of Pharmacology, University of Wisconsin Medical School Madison, Wisconsin and