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At least 19 recordsLinked to original sources
PubMed1977

[About degradation products of the inter-alpha-trypsin inhibitor in serum. I. The inter-alpha-trypsin inhibitor as precursor of the acid stable trypsin-plasmin-inhibitor of the serum (author's transl)].

The humoral inter-alpha-trypsin inhibitor is to define as precursor of the acid stable trypsin-plasmin-inhibitor in the serum. The inhibitor is filtrated by the glomerulum and excreted in the urine. The serum level of the inhibitor is increased in nephropathy. Using a new assay for the intact precursor it was found that during inflammation the decreased precursor level indicates an increased turnover, though the glomerular filtration of the acid-stable inhibitor is within normal range. The increase of the precursor level during nephropathy indicates that the kidney is the main degradation organe for the inter-alpha-trypsin inhibitor. Nevertheless, an increase of the acid-stable inhibitor is to be seen. This fact is only to explain if it is assumed that the inter-alpha-trypsin inhibitor is permanently degraded everywhere in the organism.

Fibrinolysin↗
PubMed1979

Kunitz-type proteinase inhibitors derived by limited proteolysis of the inter-alpha-trypsin inhibitor, III. Sequence of the two Kunitz-type domains inside the native inter-alpha-trypsin inhibitor, its biological aspects and also of its cleavage products.

The human inhibitor HI-14 consists of two Kunitz-type domains covalently connected. They are liberated from the human ITI by limited tryptic proteolysis. The inhibitor HI-14 is formed via a trypsin inhibitor complex. We have reported the amino acid sequences of the domain with antitryptic activity and the homologous domain without activity. Here we present the sequence of the domains as present in ITI. The domain lacking antitryptic activity is the N-terminal part of the inhibitor HI-14, whereas the domain with antitryptic activity represents the C-terminal part of HI-14 and probably the C-terminus of the ITI-molecule, too.

Alpha-Globulins↗
PubMed1976

Isolation and characterization of alpha2-plasmin inhibitor from human plasma. A novel proteinase inhibitor which inhibits activator-induced clot lysis.

A procedure is presented for purifying a novel proteinase inhibitor in human plasma whose apparent unique biological property is to inhibit efficiently the lysis of fibrin clots induced by plasminogen activator. The final product is homogeneous as judged by disc gel electrophoresis, and immunoelectrophoresis. Its molecular weight estimated by sodium dodecyl sulfate gel electrophoresis or sedimentation equilibrium is 67,000 and 63,000, respectively. The inhibitor is a glycoprotein consisting polypeptide chain containing 11.7% carbohyrate. It migrates in the alpha2-globulin region in immunoelectrophoresis. The inhibitor is chemically and immunologically different from all the other known inhibitors in plasma. Inhibition of plasmin by the inhibitor is almost instantaneous even at 0 degrees, in contrast to the slow inhibition of urokinase (plasminogen activator in urine). Plasminogen activation by urokinase-induced clot lysis is inhibited by the inhibitor mainly through a mechanism of instantaneous inhibition of plasmin formed and not through the inhibition of urokinase. The inhibitor also inhibits trypsin. Consequently, it is suggested that this newly identified inhibitor is named alpha2-plasmin inhibitor or alpha2-proteinase inhibitor. A specific antibody directed against the inhibitor neutralizes virtually all inhibitory activity of plasma to activator-induced clot lysis. Immunochemical quantitation of the inhibitor was specific antiserum to the inhibitor and the purified inhibitor as a standard indicates that the concentration of the inhibitory in the serum of a healthy man is in or near the range of 5 to 7 mg/100 ml, which is the lowest concentration among the concentration of the proteinase inhibitors in plasma. The inhibitor and plasmin, trypsin, or urokinase form a complex which cannot be dissociated with denaturing and reducing agents. The formation of the enzyme-inhibitor complex occurs on a 1:1 molar basis and is associated with the cleavage of a unique peptide bone, which is most clearly demonstrated in the interaction of the inhibitor and beta-trypsin. In the complex formation between the inhibitor and plasmin, the inhibitor is cross-linked with the light chain which contains the active site of plasmin. It is suggested that, in a fashion analogous to complex formation between alpha1-antitrypsin and trypsin, the cross-links are formed between the active site serine of the enzyme and the newly formed COOH-terminal residue of the inhibitor, with cleavage of a peptide bond.

Amino Acids↗
PubMed1976

Snake venom proteinase inhibitors. III. Isolation of five polypeptide inhibitors from the venoms of Hemachatus haemachatus (Ringhal's corbra) and Naja nivea (Cape cobra) and the complete amino acid sequences of two of them.

Five proteinase inhibitors which all inhibit the activity of bovine trypsin [EC 3.4.21.4] were isolated from African Elapid venoms of Hemachatus haemachatus (HHV, Ringhal's cobra) and Naja nivea (NNV, Cape cobra). All the inhibitors were essentially homogeneous by polyacrylamide gel electrophoresis in the presence or absence of sodium dodecylsulfate. Amino acid analysis and terminal analysis also supported their chemical homogeneities, except for one of the two inhibitors from Hemachatus haemachatus venom. The isolated inhibitors had a molecular weight of about 6,500, consisting of 52 to 57 amino acid residues, and they were all devoid of tryptophan. However, their amino acid compositions differed from each other. One of the three inhibitors isolated from Naja nivea venom, designated NNV inhibitor Ia, was unique, in that 4 half-cystinyl residues per mole fof the polypeptide were present, whereas all the others contained six residues. Of the isolated proteinase inhibitors, the complete amino acid sequences of two major inhibitors were established by manual and automatic Edman degradations and standard enzymatic techniques. Each of the inhibitors, designated HHV inhibitor II and NNV inhibitor II, consisted of 57 amino acid with arginine and glycine at the NH2- and COOH-termini, respectively. Both contained six half-cystines in disulfide linkages, and their overall amino acid sequences were similar, showing 91% homology. The two inhibitors differed in sequence by only five amino acid replacements, Asp-3 to Arg; Tyr-17 to Arg; Leu-25 to Arg; Gln-32 to Glu; and Arg-52 to His, in the 57 residue peptide chain. Comparing the amino acid sequences of these two cobra venom inhibitors with those of Russell's viper venom inhibitor II and bovine pancreatic trypsin inhibitor (BPTI), about 50% homology was found in their sequences. The 6 half-cystinyl residues of these inhibitors were in the same linear positions. Moreover, the regions which are structurally and functionally important in the well-known BPTI molecule were found with extremely high sequence homology in the cobra venom inhibitors. These findings strongly suggest that the cobra venom inhibitors as well as Russell's viper inhibitor II have very similar conformations to that established for BPTI.

Amino Acid Sequence↗
PubMed1976

[The inter-alpha-trypsin inhibitor as precursor of the acid-stable proteinase inhibitors in human serum and urine].

A small amount of antitryptic activity is detectable in the supernatant of deproteinized human serum. Preincubation of serum with trypsin causes an increase in acid-stable antitryptic activity. This rise in activity depends on the inter alpha-trypsin inhibitor concentration. The native inhibitor present in normal sera, and in higher concentrations in sera of patients with nephropathies, and the trypsin-liberated inhibitor show immunological cross reaction with antibodies to the serum inter-alpha-trypsin inhibitor. The two inhibitors differ in molecular weight and electrophoretic mobility. The physiological inhibitor (I-34), with a molecular weight of 34 000 and a high carbohydrate content, can be transformed by trypsin into an inhibitor (I-17) with a molecular weight of 17 000. This inhibitor is identical with the inhibitors liberated by trypsin from serum or from purified inter-alpha-trypsin inhibitor. The acid-stable inhibitor from urine is identical with the physiological serum inhibitor. Analogously, this inhibitor is transformed by trypsin into the inhibitor with a molecular weight of 17 000. We conclude that the inter-alpha-trypsin inhibitor is the precursor of both the physiological and the trypsin-liberated inhibitor. By a mechanism as yet unknown, but most likely a limited proteolysis, the secreted inhibitor is liberated from the high molecular weight precursor. In contrast to the monospecific trypsin-inhibiting precursor, the physiological and artificially liberated inhibitors are trypsin/chymotrypsin/plasmin inhibitors.

Humans↗
PubMed1978

Proteinase inhibitors from the excretory gland cells of Stephanurus dentatus. Purification and properties of three secretory proteinase inhibitors.

Three proteinase inhibitors designated as I, II, and III were isolated from the excretory gland cells of the swine kidney worm, Stephanurus dentatus. The inhibitors, which were trichloroacetic acid-soluble, were purified by affinity chromatography and ion exchange chromatography. The homogeneity of each inhibitor was shown by polyacrylamide gel electrophoresis and electrofocusing. The molecular weights of the inhibitors estimated by sodium dodecyl sulfate gel electrophoresis fell within a limited range of 9300 to 9700, and the isoelectric points were 6.45, 6.20, and 5.34 for Inhibitors I, II, and III, respectively. The inhibitors formed complexes with trypsin having apparent dissociation constants (Ki) of 2.9 X 10(-11), 7.6 X 10(-11), and 6.4 X 10(-11) M, respectively. Each inhibitor inhibits the esterolytic and proteolytic activities of both trypsin and chymotrypsin. A proteinase inhibitor present in the reproductive organs, intestines, body walls, and esophagi was identical with Inhibitor II found in the excretory gland cells. Culture medium collected after 24-h incubation with adult worms contained the same three inhibitors as the excretory gland cells. These data suggest that the gland cells may secrete the inhibitors internally and externally.

Amino Acids↗
PubMed1977

On the interaction of alpha2-plasmin inhibitor and proteases. Evidence for the formation of a covalent crosslinkage and non-covalent weak bondings between the inhibitor and proteases.

alpha2-plasmin inhibitor is a proteinase inhibitor in plasma which efficiently inhibits the lysis of fibrin clots induced by plasminogen activator. The nature of the binding of the inhibitor to trypsin or plasmin was studied by the chemical treatment of the enzyme-inhibitor complex with 7.5 M hydrazine at pH 10.0. With the hydrazine treatment, the complexes were degraded to proteins corresponding to the respective enzyme and inhibitor moieties. These results indicate that the covalent bond between the inhibitor and the enzymes is a carboxylic ester. The binding reaction of the inhibitor to active site-modified trypsin was also studied. The inhibitor formed complexes with anhydrotrypsin and carboxyamidomethylated trypsin. The complexes were dissociated in the presence of 1% sodium dodecyl sulfate, to the individual components: the respective enzyme and inhibitor moieties. The inhibitor, however, did not form a complex with diisopropylphosphorylated trypsin regardless of the presence or absence of the denaturing reagent. These results suggest the contribution of non-covalent interactions to the complex formation between the inhibitor and native enzymes.

Binding Sites↗
PubMed1977

Synthesis and characterization of a pancreatic trypsin inhibitor homologue and a model inhibitor.

The synthesis and characterization of protein proteinase inhibitor homologues with variations in the amino acid composition in the vicinity of the reactive site should aid the understanding of the mechanism by which inhibition of enzymatic activity occurs. A homologue inhibitor in which the reactive-site residue Ala-16 of basic pancreatic trypsin inhibitor (Kunitz) (BPTI) is replaced by Phe has been synthesized to study the effect of this replacement on the dissociation constants of the enzyme-inhibitor complexes. The replacement of Ala-16 by Phe causes a dramatic increase in the K1 value of the trypsin-BPTI complex while that of the chymotrypsin-BPTI complex remains essentially the same. This cannot be explained simply in terms of increased steric crowding. The Phe replacement probably causes a small change in the local conformation of the reactive site of the inhibitor which leads to a large decrease in the stability of the very tight trypsin-BPTI complex. This conformation change apparently can be tolerated in the less tightly bound chymotrypsin-BPTI complex. On the basis of the known structure of BPTI, a cyclic heptadecapeptide containing one disulfide bond was synthesized as a model inhibitor in order to determine if a smaller peptide can be designed to act as a highly efficient inhibitor for trypsin. This heptadecapeptide which contains all of the amino acid residues of BPTI taking part in the interaction of the proteinase inhibitor with trypsin binds 3 X 10(7) time more weakly to the enzyme than native BPTI does. It thus appears that even though only a small part of the inhibitor molecule enters directly into interaction with the enzyme, the remaining portions of the molecule which hold the structure of the inhibitor rigid are essential for the strong interaction.

Amino Acid Sequence↗
PubMed1976

The amino acid sequences of two soybean double headed proteinase inhibitors and evolutionary consideration on the legume proteinase inhibitors.

Two proteinase inhibitors, C-II and D-II, were isolated from soybeans. C-II was shown to be an inhibitor of bovine trypsin [EC 3.4.21.4], bovine alpha-chymotrypsin [EC 3.4.21.1], and porcine elastase [EC 3.4.21.11], whereas D-II inhibited only trypsin. The complete amino acid sequences of the two inhibitors establishors. On the basis of the specificities of the inhibitors and their homologies with other double-headed inhibitors, the reactive sites of C-II seems to be alanine-22 for elastase and arginine-49 for trypsin (and probably also for chymotrypsin). D-II was quite unique because its both reactive sites are arginine residues and it only inhibits trypsin. It is suggested that D-II might be a primitive form of double-headed inhibitor and that the prototype single-headed inhibitor was a trypsin inhibitor with an arginine residue as the reactive site.

Amino Acid Sequence↗
PubMed1977

Natural plant enzyme inhibitors. V. A trypsin/chymotrypsin inhibitor from Alocasia macrorhiza tuber.

A trypsin/chymotrypsin inhibitor was isolated from the tubers of Alocasia macrorhiza by extraction at pH 7.6, heat treatment at 80 degrees C, ammonium sulphate precipitation and successive column chromatography on CM-cellulose, DEAE-Sephadex A-50 and Sephadex G-100. The inhibitor was pure by cellulose acetate electrophoresis. The molecular weight was approximately 32 000 as determined by gel filtration on Sephadex G-100. The inhibitor acted on bovine trypsin, human trypsin and bovine chymotrypsin. It had no action on human chymotrypsin, subtilisin BPN', pronase, Aspergillus oryzae protease, human and porcine pepsins. The binding sites for bovine trypsin and chymotrypsin are not mutually exclusive. The inhibitor was stable over a pH range of 1--10. The purified inhibitor was far more thermostable than the crude inhibitor. The purified inhibitor lost only 33% of activity on heat treatment at 95 degrees C for 2 h. Trinitrobenzene sulphonate treatment resulted in the loss of antichymotryptic activity faster than the antitrypsin activity of the inhibitor.

Chymotrypsin↗
PubMed1978

Studies on soybean trypsin inhibitors, XII. Linear sequences of two soybean double-headed trypsin inhibitors, D-II and E-I.

Soybean inhibitor D-II is an inhibitor of bovine trypsin. Sequence analysis was carried out on the reduced and S-carboxymethylated protein by conventional methods to establish the complete amino acid sequence. The sequence of D-II indicated high homology with other legume inhibitors, but it was unique because of the occurrence of identical residues (arginine) at both of the reactive sites. This structure is thought to reflect that of a prototype double-headed inhibitor. The possible evolutionary process of the legume double-headed inhibitors is discussed on this basis. Comparison with another soybean inhibitor C-II suggested that a single methionine (C-II)-glutamine (D-II) replacement at the P2'position resulted in the loss of alpha-chymotrypsin inhibitory activity of D-II. The results of a hydrogen peroxide oxidation experiment on C-II supported this suggestion. The sequence of the amino-terminal 21 residues of inhibitor E-I was determined using a sequentor. It was shown that this inhibitor lacks the amino-terminal nine residues of D-II.

Amino Acid Sequence↗
PubMed1975

Intracellular distribution of neutral proteinases and inhibitors in pig leucocytes. Isolation of two inhibitors of neutral proteinases.

Granule and post-granular-supernatant fractions were obtained from pig leucocyte cells by differential centrifugation in 0.34 M sucrose. Granule extract possesses proteinase activity at acid and at neutral pH. Three groups of neutral and a group of acid proteinases were isolated from granule extracts by chromatography on DEAE-cellulose. In the first group are present elastase-like and plasminogen-activator proteinases, that are inhibited by diisopropylphosphorofluoridate, alpha1-antitrypsin, intracellular leucocyte inhibitor and partly with p-aminomethylbenzoic acid and Trasylol. The second group of neutral proteinases is unstable under the conditions of isolation used the third group of neutral proteinases comprises collagenases that are inhibited by ethylenediamine tetraacetic acid disodium salt, alpha1-antitrypsin and leucocyte inhibitor. The acid proteinases are inhibited only with pepstatin, up to 90%. In the post-granular supernatant was found the acid proteinase activity towards hemoglobin and casein, and non-stable neutral proteolytic activity towards bovine serum albumin and serum gamma globulin. In the post-granular supernatant also the inhibitors of neutral proteinases were found. By gel filtration on Sephadex G-100 and ion-exchange chromatography on CM-cellulose two inhibitors of neutral proteinases were isolated. The majority of the inhibitor capacity (about 80%) of post-granular supernatant was eluted together with ovalbumin (Mr 43000) and the remainder with cytochrome c (12300). These inhibitors inhibit the granule neutral proteinases, acting on all substrates used, but do not inhibit granule acid proteinase. Inhibition effects of post-granular-supernatant inhibitors on trypsin and chymotrypsin were obtained only when bovine serum albumin was used as substrate. Inhibitors of post-granular supernatant are stable at pH 6-8, but unstable in the pH rnage 2-5 and are thermolabile.

Animals↗
PubMed1979

Kunitz-type proteinase inhibitors derived by limited proteolysis of the inter-alpha-trypsin inhibitor, I. Determination of the amino acid sequence of the antitryptic domain by solid-phase Edman degradation.

The acid-stable trypsin inhibitor of human serum and urine is released in vivo by limited proteolysis from the high molecular weight, acid-labile inter-alpha-trypsin inhibitor. When complexed with trypsin, both this acid-stable, active derivative and the inter-alpha-trypsin inhibitor can be degraded in vitro by prolonged digestion with trypsin to a low molecular weight "minimal" inhibitor. This minimal trypsin inhibitor was sequenced and found to be homologous to the known Kunitz-type inhibitors (e.g. the basic trypsin-kallikrein inhibitor from bovine organs). This indicates that the antitryptic activity of the big inter-alpha-trypsin inhibitor is due to a Kunitz-type domain.

Alpha-Globulins↗
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↗
PubMed1979

Protease inhibitors in porcine serum and their immunological relationships to human protease inhibitors.

A close molecular relationship exists between the protease inhibitors of porcine serum and those of human serum as shown by studying their immunological cross-reactivities with gel diffusion and immunoelectrophoretic methods. On studying seven different antisera to human protease inhibitors, five were found to cross-react with porcine serum, and on this bisis it was possible to identify alpha 2 -macroglobulin f, alpha 2 -macroglobulin s, alpha 1 -protease inhibitor, inter-alpha-trypsin inhibitor, antithrombin and alpha 2 -antiplasmin in porcine serum. Antisera to four of these porcine serum inhibitors (alpha 2 -macroglobulin f, alpha 2 -macroglobulin s, alpha 1 -protease inhibitor and inter-alpha-trypsin inhibitor) were produced and were shown to react immunologically with their human serum protease inhibitor counterparts.

Animals↗
PubMed1978

Oligonucleotide inhibitor of protein synthesis made in extracts of interferon-treated chick embryo cells: comparison with the mouse low molecular weight inhibitor.

Cytoplasmic extracts of interferon-treated primary chick embryo cells contain an enzyme activity that synthesized an inhibitor of chick cell-free protein synthesis. The same activity was detected in extracts of cells treated with mock preparations of interferon, but at <0.3% of the level found in interferon-treated cell extracts. The enzyme was activated by double-stranded RNA and could be isolated by binding to columns of poly(I)-poly(C)-agarose. In the column-bound state, the enzyme reacted with ATP to synthesize the inhibitor, which could then be continuously eluted from the column. The inhibitor was purified and its structure and function were compared with those of the low molecular weight inhibitor of protein synthesis made by an enzyme from interferon-treated mouse L cells. The avian and mammalian inhibitors comigrated on thin layers of polyethyleneimine-cellulose during chromatography in three different solvent systems, and they coeluted as a series of peaks from columns of DEAE-cellulose during sodium chloride gradient elution. Digestion with bacterial alkaline phosphatase or snake venom phosphodiesterase yielded products that similarly comigrated. Functionally, the two inhibitors were interchangeable: both inhibited protein synthesis in extracts of mammalian and avian cells, producing 50% inhibition at a concentration of about 0.3 nM (AMP equivalents). We conclude that the chick cell-derived oligonucleotide inhibitor has a structure that is closely related or identical to that of the inhibitor made in the mouse system, and that both preparations inhibit cell-free protein synthesis in a non-species-specific manner.

Adenosine Triphosphate↗