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Biomedical subjects

S Iwanaga

Publications and source records attributed to S Iwanaga.

At least 361 records · Page 20Linked to original sources
PubMed1980

New fluorogenic peptide substrates for plasmin.

Fluorogenic peptides, peptidyl-4-methylcoumaryl-7-amides (MCA), containing COOH-terminal lysine residues, were newly synthesized and tested as substrates for plasmin. Among six peptidyl-MCA's, Boc-Val-Leu-Lys-MCA and Boc-Glu-Lys-Lys-MCA were found to be useful for the specific and sensitive assay of plasmin. The Km values estimated from Line-weaver-Burk plots for these substrates using human and bovine plasmins were in the region of 10(-4) M. Boc-Glu-Lys-Lys-MCA was slightly hydrolyzed by bovine plasma kallikrein, and Boc-Val-Leu-Lys-MCA was slightly hydrolyzed by human and hog urinary kallikreins and hog pancreatic kallikrein. However, both of the fluorogenic peptides were essentially unaffected by urokinase, alpha-thrombin, Factor Xa, Factor IXa, Factor XIa, and Factor XIIa. It was confirmed that plasmin hydrolyzed Boc-Glu-Lys-Lys-MCA, cleaving the lysyl-MCA bond, but not the lysyl-lysyl bond. These fluorogenic peptides were resistant to human plasmin activated by streptokinase. Boc-Glu-Lys-Lys-MCA was not hydrolyzed by human plasmin or plasminogen in the presence of more than a 5-fold molar excess of streptokinase. The sensitivity of Boc-Val-Leu-Lys- of more than a 5-fold molar excess of streptokinase. The sensitivity of Boc-Val-Leu-Lys-MCA to human plasmin was also reduced, but plasmin retained 35% of the maximum activity even in the presence of a 20-fold molar excess of streptokinase. These results suggest that streptokinase-plasmin complex has essentially no activity towards Boc-Glu-Lys-Lys-MCA.

Animals↗
PubMed1980

A new fluorogenic substrate method for the estimation of kallikrein in urine.

Kallikrein in human urine was measured using a fluorogenic peptide substrate, proly-phenyl-alanyl-arginine-4-methylcoumaryl-7-amide (Pro-Phe-Arg-MCA). Using 20 microliters of normal urine, kallikrein activity was quantitatively assayed by incubation with a final concentration of 10(-4) M Pro-Phe-Arg-MCA at 37 degrees C for 90 min. The reaction was terminated by adding 50 units of Trasylolr or 20 microliters of 10% acetic acid. Using this method, kallikrein activity was measured in urine from normal subjects and patients with glomerulonephitis and Bartter's syndrome. These values were comparable with the values obtained by radioimmunoassay using bovine low-molecular-weight kininogen. When normal urine was applied to a column of arginine-Sepharose 4B, two activities, Pro-Phe-Arg-MCA hydrolyzing activity and kinin-releasing activity toward bovine low-molecular-weight kininogen, were eluted in the same fraction. These results indicate that the present method is useful for the estimation of kallikrein in urine, in terms of specificity, sensitivity, and simplicity.

Acetates↗
PubMed1979

The sugar chains of cold-insoluble globulin. A protein related to fibronectin.

Cold-insoluble globulin isolated from bovine plasma contains six asparagine-linked sugar chains in 1 molecule (a dimeric form). These sugar chains were released from the polypeptide backbone by hydrazinolysis and labeled by reduction with NaB[3H]4. Most of these sugar chains contain N-acetylneuraminic acid and can be separated by paper electrophoresis. By combination of sequential exoglycosidase digestion and methylation study, their structures were elucidated as Gal beta 1 leads to 4GlcNAc beta 1 leads to 2Man alpha 1 leads to 6(NeuAc alpha 2 leads to 6Gal beta 1 leads to 4GlcNAc beta 1 leads to 2Man alpha 1 leads to 3)Man beta 1 leads to 4GlcNAc beta 1 leads to 4GlcNAc, NeuAc alpha 2 leads to 6 or 4Gal beta 1 leads to 4GlcNAc beta 1 leads to 2Man alpha 1 leads to 6(NeuAc alpha 2 leads to 4 or 6Gal beta 1 leads to 4GlcNAc beta 1 leads to 2Man alpha 1 leads to 3)Man beta 1 leads to 4GlcNAc beta 1 leads to 4GlcNAc, NeuAc alpha 2 leads to 6Gal beta 1 leads to 4GlcNAc beta 1 leads to 2Man alpha 1 leads to 6[NeuAc alpha 2 4Gal beta 1 leads to 3(NeuAc alpha 2 leads to 6)GlcNAc beta 1 leads to 2Man alpha 1 leads to 3]-Man beta 1 leads to 4GlcNAc beta 1 leads to 4GlcNAc and NeuAc alpha 2 leads to 4Gal beta 1 leads to 3(NeuAc alpha 2 leads to 6)GlcNAc beta 1 leads to 2Man alpha 1 leads to 6[NeuAc alpha 2 leads to 4Gal beta 1 leads to 3(NeuAc alpha 2 leads to 6)GlcNAc beta 1 leads to 2Man alpha 1 leads to 3]man beta 1 leads to 4GlcNAc beta 1 leads to 4GlcNAc.

Animals↗
PubMed1979

Occurrence of Leu-Lys-bradykinin and histidine-rich peptide in high-molecular-weight kininogen isolated from horse plasma.

On incubation of purified horse plasma high-molecular-weight kininogen with purified plasma kallikrein, three new peptides, named fragment 1.2, fragment 1 and fragment 2, were released, in addition to the vasopeptide, bradykinin. Fragment 2 contained an extremely high level of histidine, in which eleven residues out of the total 48 residues were characterized. Thus the result proves the existence of the histidine-rich region in horse high-molecular-weight kininogen, which is similar to the region previously identified in bovine high-molecular-weight kininogen. Moreover, we have identified a new kinin derivative, Leu-Lys-bradykinin, in horse high-molecular-weight kininogen.

Amino Acid Sequence↗
PubMed1979

Primary structure of human fibrinogen and fibrin. Structural studies on NH2-terminal part of B beta chain.

The cyanogen bromide fragment, N-DSK, containing the NH2-terminal portions of the three chains of fibrinogen, was found to exist in dimeric and polymeric forms. These different forms gave rise to identical chain fragments on reduction and alkylation. The B beta chain of N-DSK from fibrinogen and the beta chain of N-DSK from fibrin were isolated and characterized. The B beta chain fragment has a blocked NH2-terminal residue, and fibrinopeptide B is released on digestion with thrombin. The beta chain fragment has glycine as NH2-terminal residue. The molecular weight of the B beta chain fragment is 12200 as determined by ultracentrifugal analysis. Gel electrophoresis in sodium dodecyl sulphate gave the molecular weights of 14000 and 13000 for the B beta chain and beta chain fragments, respectively. The NH2-terminal B beta chain fragment consists of 118 amino acid residues and the beta chain fragment of 104 residues. The amino acid sequence of beta chain fragment is identical to B beta chain fragment except for the fibrinopeptide B portion. The isolation of a B beta-related fragment (B beta +), with a molecular weight of 30000, is also reported. The presence of B beta + was explained on the basis of incomplete cleavage at the Met-118 residue during treatment with cyanogen bromide. Some functional aspects of the B beta chain fragment are discussed.

Amino Acid Sequence↗
PubMed1979

Relation between structure and correcting activity of bovine high molecular weight kininogen upon the clotting time of Fitzgerald-trait plasma.

Bovine high molecular weight kininogen (bHMWK) partially corrects the activated plasma thromboplastin time (aPTT) of Fitzgerald trait plasma which is congenitally deficient in HMWK. The relationship between the structure and activity of HMWK was clarified by studying the effects of different fragments of bHMWK on the aPTT of Fitzgerald-trait plasma. The peptides studied were lys-bradykinin-free HMWK, bradykinin-fragment 1-2-free HMWK, heavy chain, fragment 1-2-light chain, and light chain. All fragments were tested in equimolar concentrations. Bradykinin-fragment 1-2-free HMWK, heavy chain, and light chain have little or no correcting activity upon Fitzgerald-trait plasma aPTr. Fragment 1-2 light chain has the same correcting activity as intact bHMWK, while that of lys-bradykinin-free HMWK appears to be higher. Both fragment 1-2 and fragment 2 inhibit the clotting time of normal human plasma. When compared on a molar basis, fragment 2 is a more active inhibitor than fragment 1-2. When the effects of bovine plasma kallikrein upon bHMWK and hHMWK were studied, it was found that it released kinins from both kininogens. However, while the correcting activity of bHMWK was completely destroyed after 60 min of incubation, that of hHMWK was fully retained. These data suggest that: (a) the active part of bHMWK is comprised of the fragment 1-2 light chain portion; (b) fragment 1-2 or fragment 2 is the binding site to negatively charged surfaces, while the light chain interacts with other components of the surface-mediated reactions; and (c) bovine plasma kallikrein releases kinins, but probably does not cause the release of fragment 1-2 from human HMWK.

Animals↗
PubMed1979

A new method for purification of staphylocoagulase by a bovine prothrombin-Sepharose column.

Staphylocoagulase was isolated from a culture filtrate of Staphylococcus aureus, strain st-213, by a two step purification procedure of chromatography on a bovine prothrombin-Sepharose 4B affinity column and gel filtration on Sephadex G-25. The yield of the coagulase activity ranged from 75--83% and the purified preparation gave a single precipitin line in immunodiffusion tests against anti-crude and anti-purified staphylocoagulase sera. However, the final product was shown to contain one major and two minor components by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Chemical analysis of the material indicated that it does not contain any cystine residues and that its NH2-terminal residue is a single isoleucine.

Amino Acids↗
PubMed1979

Inhibition of porcine glandular kallikreins by structurally homologous proteinase inhibitors of the Kunitz (Trasylol) type. Significance of the basic nature of amino acid residues in subside positions for kallikrein inhibition.

The newly synthesized chromogenic substrate D ValLeuArgNHNp was employed to study the inhibition strength of Trasylol-like inhibitors from bovine lung (TKI), sea anemone (SAI), snake venoms (NNV and HHV), snails (HPI) and cow colostrum (CTI) against porcine pancreatic, submandibular and urinary kallikreins. The dissociation constants of the corresponding kallikrein-inhibitor complexes were found close to Ki = 1.5 x 10(-9)M (TKI, SAI, NNV) or to Ki = 10--210 x 10(-9)M (HHV, HPI). CTI does not inhibit the three porcine glandular kallikreins. Comparison of the inhibitory active areas of the inhibitors with their affinities to the three kallikreins shows that kallikrein inhibition is observed only if basic amino acid residues are present in distinct positions of the inhibitory active sites.

Amino Acid Sequence↗
PubMed1978

Bovine plasma cold-insoluble globulin: gross structure and function.

Bovine plasma CIg, like human CIg, is a glycoprotein with a molecular weight of approximately 450,000 daltons and consists of two homologous subunits, the alpha and beta chains. These subunits are covalently linked through disulfide bridges in their carboxyl terminal domains. The carboxyl terminal regions are presumed to contain the fibrin-reactive transamidation site. The covalent incorporation of CIg into fibrin has been conclusively demonstrated by isolation of the S-carboxymethyl derivative of the CIg-fibrin-alpha chain complex and by determination of its terminal amino acid sequences. Cold-insoluble globulin has been shown to exert a stimulatory effect on the urokinase-mediated activation of bovine plasminogen to plasmin.

Amino Acid Sequence↗