Identification of the PR Prealbumin Proteins in Horse Serum

The Pr protein, which is one of the major equine acidic prealbumins and which consists of a large number of phenotypes, has been studied with regard to its chemical identity. Serum samples of known Pr phenotype which had been treated with varying amounts of bovine trypsin were subjected to starch gel electrophoresis at pH 4.8. When a certain amount of trypsin was used, the Pr protein was markedly affected, whereas the other acidic prealbumins retained their normal electrophoreitic pattern. Extracts from three different regions of the acidic prealbumin field were tested by the casein precipitating inhibition test (CPI-test). Marked antitrypsin effect appeared against the extract from the Pr zone but not against the extracts from the two other acidic prealbumin zones. When acidic starch gel electrophoresis was combined with the CPI-test, a broad inhibitory zone appeared in the area of the Pr proteins. Pr protein was isolated by the use of agarose gel electrophoresis and sepharose column chromatography. The isolated protein which was tested for purity by gel electrophoresis had a molecular weight of about 60,000. It is concluded that the equine Pr protein corresponds to αi-antitrypsin.     

Serum complex of trypsin 2 and alpha 1 antitrypsin as diagnostic and prognostic marker of acute pancreatitis: clinical study in consecutive patients.

OBJECTIVE--To estimate the usefulness of serum concentrations of the complex of trypsin 2 and alpha 1 antitrypsin in diagnosing and assessing the severity of acute pancreatitis in comparison with serum C reactive protein, amylase, and trypsinogen 2 concentrations (reference markers). DESIGN--Markers were measured in consecutive patients admitted with acute abdominal pain that was either due to pancreatitis or to other disease unrelated to the pancreas (controls). SETTING--Department of surgery of a teaching hospital in Helsinki. SUBJECTS--110 patients with acute pancreatitis and 66 with acute abdominal diseases of extrapancreatic origin. On the basis of the clinical course, acute pancreatitis was classified as mild (82 patients) or severe (28 patients). MAIN OUTCOME MEASURES--Clinical diagnosis of acute pancreatitis and severity of the disease. RESULTS--At admission all patients with acute pancreatitis had clearly raised concentrations of trypsin 2-alpha 1 antitrypsin complex (32 micrograms/l), whereas only three of the controls had such values. Of the markers studied, trypsin 2-alpha 1 antitrypsin complex had the largest area under the receiver operating curve, both in differentiating acute pancreatitis from extrapancreatic disease and in differentiating mild from severe disease. CONCLUSIONS--Of the markers studied, trypsin 2-alpha 1 antitrypsin complex was the most accurate in differentiating between acute pancreatitis and extrapancreatic disease and in predicting a severe course for acute pancreatitis.     

Regulation of GH binding to specific cellular receptors in vitro--a new model of growth regulation in vivo

A new theory on the complex growth hormone (GH) mediated promotion of tissue growth has been developed on the basis of in vitro studies of growth hormone receptors on human peripheral mononuclear cells (PMC). It is hypothesized that GH acts through its tissue receptors and regulates its own receptors through two different pathways: first GH directly downregulates GH binding, second a partially GH-dependent serum factor (the so-called SM-B) enhances GH binding. Some experimental evidence for this hypothesis is presented using a new method to investigate GH receptors in circulating human blood cells: The effect of trypsin, antitrypsin, growth hormone, somatomedin-B (SM-B) and anti-SM-B-antiserum on GH binding to PMC was studied. Trypsinization of cells leads to a decrease both of specific binding and of binding affinity (affinity constant after 60 minutes of trypsinization 0.5 X 10(6) M-1 versus 1.5 X 10(6) M-1 in untreated control cells). Exposure of PMC to antitrypsin activities was followed by an increase of binding affinity and specific binding (affinity constants with 10 KIU 1.9 X 10(6) M-1, with 100 KIU 2.4 X 10(6) M-1, with 1000 KIU 3.6 X 10(6) M-1). This antitrypsin effect exceeds the binding values expected after blocking trypsin activities possibly being present in the incubation medium. In a subset of experiments the partially GH-dependent serum factor SM-B was used as the antitrypsin moiety and was shown to increase specific GH binding to PMC in a similar manner as did antitrypsin (with 1000 ng SM-B affinity constant 12.0 X 10(6) M-1, specific binding 9.7%).(ABSTRACT TRUNCATED AT 250 WORDS)     

Binding of alpha 1 antitrypsin (alpha 1 protease inhibitor) to human lymphocytes

Radioiodinated α₁ antitrypsin has been found to bind to human lymphocytes. This binding is fast and reversible, and the cells can be saturated. Each lymphocyte can bind a maximum of approximately 1.2 × 10⁶ molecules of α₁ antitrypsin with an association constant of 0.7 × 10⁶ M^?1×l. The binding is inhibited by the addition of cold α₁ antitrypsin or Soybean trypsin inhibitor, and partially by α₂ macroglobulin. The data suggest that α₁ antitrypsin is likely to bind to a cell surface-associated protease. The addition of cell-free supernatants from lymphocytes incubated at 37°C was found to decrease the binding of α₁ antitrypsin, suggesting that the receptor is released from the cell surface.     

THE ANTIPROTEOLYTIC ACTIVITY OF SERUM : II. PHYSIOLOGICAL SIGNIFICANCE. THE INFLUENCE OF PURIFIED TRYPSIN INHIBITOR ON THE COAGULATION OF THE BLOOD

1. Serum antitrypsin and pancreatic trypsin inhibitor inhibited the coagulation of plasma in vitro. 2. This could be largely prevented by trypsin. 3. The anticoagulant action of the trypsin inhibitor was apparently due to its antiprothrombic action. It had no appreciable antithrombic action. 4. Examination of the blood of two hemophiliacs indicated that the prolonged coagulation time of their blood is not due to an excess of trypsin inhibitor. 5. Examination of the blood of heparinized dogs indicated that heparin does not appreciably contribute to the antiproteolytic activity of the serum.     

alpha 1-Antitrypsin Christchurch, 363 Glu----Lys: mutation at the P'5 position does not affect inhibitory activity

alpha 1-Antitrypsin Christchurch was isolated from the plasma of a Cambodian woman who was heterozygous for this variant and for the normal M protein. Tryptic peptide maps revealed that the inhibitory-site peptide, 359-365 Ser-Ile-Pro-Pro-Glu,Val,Lys, was missing and replaced by two new peptides Ser-Ile-Pro-Pro,Lys and Val-Lys, indicating a mutation of 363 Glu----Lys. There was no obvious clinical condition associated with this new antitrypsin. Competition experiments showed that antitrypsin Christchurch reacted at the same rate as normal antitrypsin in the presence of limiting amounts of trypsin, chymotrypsin, thrombin and neutrophil elastase. Both inhibitors were inactivated by catalytic amounts of papain. This inactivation was due to cleavage at the phenylalanine residue at the P7 position, seven residues towards the N-terminal of the inhibitory site. A one-step ethanol extraction procedure is described for isolating the papain cleavage products.     

Inactivation of tryptic activity by a human-derived strain of Bacteroides distasonis in the large intestines of gnotobiotic rats and mice.

Tryptic activity disappeared and trypsin was no longer detected with an antitrypsin antiserum in the large intestines of gnotobiotic rats and mice monoassociated with a human-derived strain of Bacteroides distasonis, whereas tryptic activity was not modified in the small intestines. This function was shown to be strain specific.     

Active site of α1-antitrypsin: Homologous site in antithrombin-III

Examination of peptides resulting from reaction of bovine trypsin and human α₁-antitrypsin in near-equimolar amounts showed anomalous cleavage of antitrypsin at a Met-Ser bond 37 residues from the C-terminus, giving evidence that this is the active site for trypsin inhibition. Alignment of the C-terminal 141 residues of α₁-antitrypsin with the C-terminal 147 residues of human antithrombin-III showed homology with 30% identity and allowed the identification of a homologous active site in antithrombin.     

Chemical synthesis of new trypsin, chymotrypsin and elastase inhibitors by amino-acid substitutions in a trypsin inhibitor from squash seeds (CMTI III)

Five new analogues of squash trypsin inhibitor CMTI III were obtained by the solid-phase method, exhibiting antitrypsin, antichymotrypsin or antielastase activity. Modification in the reactive site region changes dramatically the specificity, whereas substitution in non-contact positions facilitates the refolding of the reduced form of the peptides and does not have a significant influence on the association equilibrium constants of the inhibitor analogues with cognate enzymes.     

A turbidimetric method for measuring the activity of trypsin and its inhibition

Microscopic poly(styrene-divinylbenzene) beads coated with a monomolecular film of fibrinogen agglutinate when stirred in the presence of thrombin, a consequence of interbead fibrin formation. Trypsin, by digesting bead-bound fibrin, dissociates bead aggregates at a rate proportional to the amount of enzyme activity. The agglutination of beads and the dissociation of bead aggregates can be monitored turbidimetrically using a platelet aggregometer or other photometric device equipped with a stirred cell. We have exploited the behavior of aggregates of fibrin-coated beads to develop a rapid, sensitive, and accurate method for measuring the activity of trypsin and its inhibition, in aqueous media, including serum. The new method yields serum antitrypsin activity levels that correlate well with immunological levels of alpha1-antitrypsin and, thus, may prove useful for assessing antitrypsin activity in clinical specimens.     

Interaction of silkworm larval hemolymph antitrypsin and bovine trypsin

Antitrypsin isolated from silkworm larval hemolymph can inhibit bovine trypsin. The apparent molar ratio of silkworm antitrypsin (sw-AT) to trypsin at extrapolated null trypsin activity was determined to be 1.3 by titration of trypsin with sw-AT. The undissociability of the complex between sw-AT and trypsin on sodium dodecyl sulfate-polyacrylamide gel electrophoresis was confirmed by immunoblotting and fluorescence labeling techniques. Chemical analysis of the complex elucidated that it contained equimolar sw-AT and trypsin. Densitometric analysis of electrophoretic patterns obtained during the titration of trypsin by sw-AT suggested the presence of a suicide product formed from sw-AT. This was the reason why excess sw-AT was needed for complete inhibition of trypsin. In the complex, the sw-AT molecule was cut at one site but the fragments produced were still joined together. Trypsin in the complex was released by treatment at pH 10.0, and it was deduced that the complex formation involved acyl-bond formation between sw-AT and trypsin. The sw-AT component obtained from the alkali-treated complex possessed two kinds of NH2-terminal amino acid sequences. Non-covalent forces may bind the two fragments of sw-AT, which could be separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Amino-terminal amino acid sequence analysis of the large fragment gave a sequence identical with that of intact sw-AT. This indicated that the reactive site of sw-AT with trypsin was located at the COOH-terminal region of the molecule. These characteristics resemble those of inhibitors belonging to the serpin family.     

Inhibitory effect of methyl esters of arginine-containing oligopeptides on thrombin and trypsin

Stereoisomeric oligopeptides were studied for their inhibitory effect on the hydrolysis of benzoyl-L-arginine methyl ester catalyzed by thrombin and trypsin, as well as on the thrombin-fibrinogen reaction. Comparison of the peptide structures, their conformational flexibility and inhibitory effects on thrombin and trypsin shows the availability of the essential differences in organization and functioning of the subsites S3, S2 and S'1 of these enzymes. In contrast to trypsin, thrombin is shown to be characterized by more pronounced secondary stereospecificity. This manifests in the more vigorous dropping of the catalytic constants of thrombin-catalyzed esterolysis than those of trypsin-catalyzed hydrolysis of the substrates, containing D-amino acids at the subsite P2. It is revealed that the peptide Tos-D-Val-D-Ala-D-Agr-D-Phe-OCH3 is the most powerful inhibitor among studied compounds. It is noteworthy that its antithrombin effect is almost an order of magnitude higher than its antitrypsin effect.     

Precipitating antibody against protease of Corynebacterium pyogenes in pigs.

Antibody in sera from pigs carrying an abscess associated with Corynebacterium pyogenes and healthy pigs was examined by the agar gel diffusion test. In the test, the concentrated culture fluid containing the protease of C. pyogenes was used as antigen. As a result, precipitating antibody was demonstrated in sera from 25 of 30 abscessed pigs and a few of the healthy pigs. When the relationship between precipitating antibody and protease was examined by the immunoelectrophoresis and gel filtration of the concentrated culture fluid, the antibody was shown in the same position as the protease. From the result, it was clear that the precipitating antibody was against the protease of C. pyogenes. All the proteases produced by 27 strains of C. pyogenes of porcine and bovine origin were serologically identical with one another. They were, however, serologically different from those of Staphylococcus aureus, Bacillus cereus, and B. subtilis. In the inhibition test, the proteolytic activity of C. pyogenes was inhibited by the serum of the abscessed pig. It was also inhibited by healthy pig serum. From the results, it seems that the determination of precipitating antibody may be useful for the diagnosis of C. pyogenes infection.     

Isolation of a protease inhibitor from tissues resistant to tumor invasion

We have purified to homogeneity the major trypsin inhibitors from both bovine cartilage and aorta, two tissues reported to be highly resistant to invasion. The two inhibitors appear to be identical and they resemble the Kunitz inhibitor with respect to molecular weight, amino acid composition, range of susceptible proteases, and antigenicity. Each of these inhibitors accounts for 100% of the antitrypsin activity found in extracts of bovine cartilage and aorta.     

Interaction between duodenase, a proteinase with dual specificity, and soybean inhibitors of Bowman-Birk and Kunitz type

The interaction between duodenase, which belongs to a group of Janus-faced proteinases, and classical Bowman--Birk (BBI) and Kunitz (STI) type inhibitors from soybean was investigated. Duodenase was shown to interact only with the antichymotrypsin site (Leu-Ser) of BBI, whereas the antitrypsin site (Lys-Ser) of the inhibitor appeared to be vacant and capable of interaction with trypsin. The inhibition constants of duodenase by BBI, the BBI--trypsin complex, and STI were 4, 400, and 40 nM, respectively.     

Prevention of Staphylococcal Bacteriophage Activity by Antigen A Precipitins in Human Sera

Antigen A precipitins in human sera prevented plaque formation and propagation of staphylococcal bacteriophages. Over 20% of total IgG was removed from human sera by absorption with staphylococci containing antigen A. The specific precipitating antibody in rabbit antisera formed lines of idenity with antigen A precipitins in lower dilutions of human sera but formed lines of nonidenity with antigen A precipitins in higher dilutions of the same sera, suggesting both specific and nonspecific antigen A precipitins in human sera. The specific and nonspecific antigen A precipitins in human sera may prevent the in vivo activity of staphylococcal bacteriophages which have been demonstrated previously in animals whose sera do not contain either specific or nonspecific antigen A precipitins.     

Low-molecular-weight trypsin inhibitors of microbial origin

Three hundred actinomyces cultures newly isolated from the soil of different regions of the Soviet Union were tested for their ability to produce inhibitors of trypsin-like proteases. Seven previously not known to produce trypsin inhibitors (Streptomyces bikiniensis 17-5, S. sporoclivatus 28-1, S. filamentosus 32-11, S. diastatochromogenes 20-4, S. lavendulae 29-4, S. violacens 52-8, and Streptoverticillium cinnamoneum 36-8) were found to possess high antitrypsin activity. The morphological and cultural properties of the strains and the dynamics of inhibitor production were investigated. S. bikiniensis 17-5 was studied in greatest detail. Its culture filtrate contained several inhibitors for trypsin and one for chymotrypsin. A mixture of oligopeptides with Mr of 300-500 was obtained by the described procedure which included the adsorption of the culture fluid filtrate on charcoal followed by ion-exchange chromatography on CM-cellulose. Four trypsin inhibitors (Sb-IT1, Sb-IT2, Sb-IT3, and Sb-IT4) were isolated from the mixture in a highly purified state by reversed-phase high-performance liquid chromatography. Sb-IT2 has been recognized as formylhistidylvaline with an Mr of 282. No trypsin inhibitor of this structure has been described previously.     

Immunoreactive trypsins in sera from dogs before and after induction of experimental pancreatitis

Radioimmunoassays for anionic and cationic dog trypsins are described. Characterization of the immunoreactivities in sera from fasting dogs demonstrated the presence of the two proenzymes only. Fasting sera from 10 dogs contained anionic and cationic trypsinogen in concentrations between 17-110 micrograms/l and 7-19 micrograms/l, respectively. Induction of experimental pancreatitis in dogs was accompanied by a large increase of immunoreactive anionic and cationic trypsins in the circulation. During the progress of the pancreatitis, immunoreactive trypsin with larger molecular weight than trypsinogen appeared. This high molecular weight immunoreactive trypsin was not seen in serum after intravenous injection of pancreatic juice in dogs. The high molecular weight immunoreactive trypsin probably consists of trypsin in complex with protease inhibitors. In vitro studies showed that the immunoreactivity of trypsin decreased considerably after binding to alpha 1-antitrypsin or alpha-macroglobulins.     

Toxocara canis: Some characteristics of larval precipitating antibodies in rat serum

1972. Toxocara canis: Some characteristics of larval precipitating antibodies in rat serum. International Journal for Parasitology 2: 471–479. Serum from rats infected with 1000 T. canis eggs had larval precipitating antibodies present at least between 21 and 183 days post infection. The precipitating antibodies in whole serum were stable to heating at 60°C for 1 h and excretory pore precipitates developed in serum treated with 2-mercaptoethanol.

Fractionation of serum on G-200 and DEAE-cellulose and analysis of the fractions by immunoelectrophoresis indicated the fraction containing fast 7S globulin was primarily responsible for precipitating activity. Purified precipitating antibodies were stable to heating at 70°C for 15 min.

No skin fixing antibodies were detected in immune sera employing both a homologous and heterologous PCA text.     

Actinomycetes producing trypsin inhibitors

The ability to produce inhibitors of trypsin-like proteases was tested in 300 cultures of actinomycetes freshly isolated from different soils of the USSR. A high antitrypsin activity was found in seven cultures which had not been known before as those producing trypsin inhibitors: Streptomyces sporoclivatus 28 (1), S. lavendulae 29 (4), S. diastatochromogenes 20 (4), S. violascens 52 (8), S. bikiniensis 17 (5), S. filamentosus 32 (11), and Streptoverticillium cinnamoneum 86 (8). The morphological and cultural characteristics of the strains were studied as well as their production of trypsin inhibitors.