Isolation and characterization of three protein proteinase isoinhibitors from the granular fraction of horse neutrophilic granulocytes

Three cathodically migrating protein protease isoinhibitors were isolated from the granule-rich fraction of equine neutrophilic granulocytes by means of FPLC chromatography, in addition to two previously described anodically migrating inhibitors. The three isoinhibitors had an identical enzyme specificity which was equal to the two previously described isoinhibitors; they inhibited exclusively proteinase K and subtilisin. The inhibitors retained their activity between pH 1 and 12. They also were heat stable at 100 degrees C for 20 min. Neither the biological function of isoinhibitors nor the fundamental role of granular protease inhibitors of such narrow and peculiar enzyme specificity are known.     

Isolation and characterization of two proteinase inhibitors from the male reproductive tract of mice

Low molecular weight, acid-stable proteinase inhibitors from epididymal and seminal vesicle homogenates were isolated and characterized. The isolation procedure consisted of gel filtration, trypsin affinity, and ion exchange chromatography. The inhibitor from seminal vesicle homogenates has a molecular weight of approximately 6,200, and that of the epididymal inhibitor was estimated at 4,000. Antiserum directed against the seminal vesicle inhibitor did not react with epididymal components. The epididymal inhibitor shows competitive, whereas the seminal vesicle inhibitor shows noncompetitive inhibition against trypsin on double reciprocal plots. Both inhibitors are effective against trypsin and acrosin but not against chymotrypsin, kallikrein, thrombin, or plasmin. To verify site of origin and to investigate androgen dependency of the epididymal inhibitor, mice were efferentiectomized, orchiectomized, or orchiectomized with androgen supplementation. Gel filtration profiles of acid-treated epididymal homogenates from normal and efferentiectomized animals show inhibitor peaks in the same regions. The concentration of acid-stable inhibitor from epididymal homogenates decreased with orchiectomy but returned to normal values when exogenous androgen was supplied. These observations suggest that the low molecular weight inhibitor in the epididymal homogenates is distinct from that in the seminal vesicles. Furthermore, the inhibitor associated with epididymal homogenates is androgen-dependent, and the epididymis is the site of origin of this inhibitor.     

Isolation and characterization of a proteinase K-sensitive PrPSc fraction

Recent studies have shown that a sizable fraction of PrPSc present in prion-infected tissues is, contrary to previous conceptions, sensitive to digestion by proteinase K (PK). This finding has important implications in the context of diagnosis of prion disease, as PK has been extensively used in attempts to distinguish between PrPSc and PrPC. Even more importantly, PK-sensitive PrPSc (sPrPSc) might be essential to understand the process of conversion and aggregation of PrPC leading to infectivity. We have isolated a fraction of sPrPSc. This material was obtained by differential centrifugation at an intermediate speed of Syrian hamster PrPSc obtained through a conventional procedure based on ultracentrifugation in the presence of detergents. PK-sensitive PrPSc is completely degraded under standard conditions (50 mug/mL of proteinase K at 37 degrees C for 1 h) and can also be digested with trypsin. Centrifugation in a sucrose gradient showed sPrPSc to correspond to the lower molecular weight fractions of the continuous range of oligomers that constitute PrPSc. PK-sensitive PrPSc has the ability to convert PrPC into protease-resistant PrPSc, as assessed by the protein misfolding cyclic amplification assay (PMCA). Limited proteolysis of sPrPSc using trypsin allows for identification of regions that are particularly susceptible to digestion, i.e., are partially exposed and flexible; we have identified as such the regions around residues K110, R136, R151, K220, and R229. PK-sensitive PrPSc isolates should prove useful for structural studies to help understand fundamental issues of the molecular biology of PrPSc and in the quest to design tests to detect preclinical prion disease.     

Isolation and characterization of a low-molecular-weight immunoglobulin-binding protein from Yersinia pseudotuberculosis

A low-molecular-weight immunoglobulin-binding protein (IBP) bound with the cell envelope has been isolated from Yersinia pseudotuberculosis cells and partially characterized. This IBP is a hydrophilic protein with a high polarity index of 55.3%. The molecular weight of the protein has been determined by MALDI-TOF mass spectrometry as 14.3 kD. CD spectroscopy showed that the IBP has high contents of the beta-structure and random coil structure. The IBP contains glycine as the N-terminal amino acid. The protein can be stored for a long time at acidic pH values but aggregates and loses activity at alkaline and neutral pH. The IBP binds rabbit IgG with optimum at pH of 6.0-7.5. The IBP interacts with IgG molecule in the Fc-fragment region. The protein retains activity after heating at 100 degrees C in the presence of SDS.     

Isolation and characterization of four serine proteinase inhibitors (serpins) from hemolymph of Manduca sexta

Four serine proteinase inhibitors have been isolated from hemolymph of fifth instar larvae of Manduca sexta. One of these, an inhibitor specific for elastase, has been previously shown to be a member of the serpin family of serine proteinase inhibitors. Of the three remaining inhibitors, two are specific for chymotrypsin and one for trypsin. The four inhibitors have molecular weights of approx. 47,000 and isoelectric points between 4.4 and 4.8. The four proteins have very similar amino acid compositions, and NH₂-terminal sequence analysis suggests that they represent members of a gene family.     

Isolation and characterization of two new saponins from Lysimachia capillipes

Two new saponins, capilliposide K (1) and capilliposide L (2), were isolated from the whole plants of Lysimachia capillipes. Their structures were established by spectral and chemical techniques.     

Isolation and characterization of a proteinase from white gourd

A proteinase from the sarcocarp of Benincasa cerifera was purified. ItsMW was estimated by two different methods to be about 50000. The maximum activity was found in the alkaline pH region against casein as a substrate. The enzyme was strongly inhibited by di-isopropyl fluorophosphate and not inhibited by EDTA and p-chloromercuribenzoic acid.     

Purification and molecular characterization of two inhibitors of yeast proteinase B.

A rapid purification procedure for large scale preparations of yeast proteinase B inhibitors 1 and 2 (IB1 and IB2) is described. By disc gel electrophoresis, amino acid analysis, and end-group determinations, each of the inhibitors is homogeneous. Both inhibitors are polypeptides with molecular weights of 8,500, containing 74 residues. No components other than amino acids could be detected. There is no significant difference in the amino acid compositions of the two inhibitors as analyzed after acid hydrolysis. Both polypeptides are characterized by the total absence of arginine, tryptophan, and sulfur-containing amino acid residues. The proteinase B inhibitors of yeast, therefore, differ fundamentally from proteinase inhibitors of many other organisms, which generally contain a large number of disulfide bridges. Both proteinase B inhibitors have threonine as the NH2-terminal residue and -Val-His-Thr-Asn-COO- as the COOH-terminal sequence. Comparison of peptide maps after tryptic digestion reveals that the two inhibitors differ definitely in only a few tryptic peptides. The inhibitors are rapidly inactivated by digestion with carboxypeptidase A from bovine pancreas at pH 8.5. Inactivation occurs stoichiometrically with the release of threonine, the penultimate residue at the COOH-terminal end of both inhibitors.     

Neutral proteinase inhibitors in PMN leukocytes. II. Isolation and characterization of a neutral proteinase inhibitor from porcine PMN leukocytes

An inhibitor of neutral proteinases was purified from porcine PMN leukocytes by gel filtration on Sephadex G-75 superfine and ion-exchange chromatography on Mono S. Thus an inhibitor preparation with a specific inhibitory activity against chymotrypsin of 10 IU/mg was obtained. In dodecyl sulfate gel electrophoresis a single protein band with an apparent molecular mass of 40 kDa was found under reducing conditions. Under non-reducing conditions the inhibitor forms higher molecular mass aggregates. On isoelectric focusing several protein bands with isoelectric points between pH 7.0 and 7.5 could be separated. The amino-acid composition of the inhibitory protein was determined. The inhibition mechanism was studied and association rate constants (kon) were measured and calculated for the reaction with chymotrypsin as well as leukocyte and pancreatic elastase. In Western blot analysis and in enzyme immunoassay studies crossreactivity between antibodies directed against porcine leukocyte neutral proteinase inhibitor and the corresponding inhibitor of bovine PMN leukocytes could be demonstrated.     

Isolation and characterization from potato tubers of two polypeptide inhibitors of serine proteinases

Two polypeptides, isolated to electrophoretic homogeneity from Russet Burbank potato tubers, are powerful inhibitors of pancreatic serine proteinases. One of the inhibitors, called polypeptide trypsin inhibitor, PTI, has a molecular weight of 5100, and inhibits bovine trypsin. The inhibitor is devoid of methionine, histidine, and tryptophan and contains eight half-cystine residues as four disulfide bridges. The second inhibitor, polypeptide chymotrypsin inhibitor II, PCI-II, has a molecular weight of 5700 and powerfully inhibits chymotrypsin. This inhibitor is also devoid of methionine and tryptophan but it contains only six of half-cystines as three disulflde bonds. Both polypeptides strongly inhibit pancreatic elastase. In immunological double diffusion assays, polypeptide trypsin inhibitor and polypeptide chymotrypsin inhibitor II exhibit a high degree of immunological identity (a) with each other, (b) with a polypeptide chymotrypsin inhibitor (PCI-I, M_r 5400) previously isolated from potato tubers, and (c) with inhibitor II, a larger (monomer M_r ~ 12,000) inhibitor of both trypsin and chymotrypsin which has also been previously isolated from potato tubers. The four polypeptide proteinase inhibitors now isolated from Russet Burbank potato tubers cumulatively inhibit all five major intestinal digestive endo- and exoproteinases of animals. The inhibitors are thought to be antinutrients that are present as part of the natural chemical defense mechanisms of potato tubers against attacking pests.     

Isolation and structure characterization of two new diterpenoids from Clerodendrum bungei

Two new diterpenoids, 3β-(β-d-glucopyranosyl)isopimara-7,15-diene-11α,12α-diol (1), and 16-O-β-d-glucopyranosyl-3β-20-epoxy-3-hydroxyabieta-8,11,13-triene (2), along with three known ones were isolated from the roots of Clerodendrum bungei. These compounds were purified, and their structures were elucidated by extensive spectroscopic analyses, especially 2D NMR experiments. All compounds were evaluated for cytotoxicity against several tumor cell lines.     

Isolation and partial characterization of cysteine proteinase from sparganum.

A proteolytic enzyme was purified from the tissue extract of spargana (plerocercoids of Spirometra erinacei) by DEAE-Trisacryl M ion exchange chromatography and thiopropyl-sepharose affinity chromatography resulted in a 21-fold purification. The proteinase activity was assayed with a synthetic fluorescent substrate, carbobenzoxy-phenylalanyl-7-amino-4-trifluoromethyl-coumarin. SDS-polyacrylamide gel electrophoresis of the purified materials revealed a single 28,000 dalton band. Inhibitor profiles of the band indicated that it belonged to cysteine endopeptidases. It exhibited identical pH curves with optimum at pH 5.5, and 50% activity from pH 4.7 to 8. It could completely degrade collagen chains to three identical products. It also showed some activity on hemoglobin. Furthermore, the band on immunoblots was reactive to the sera of sparganosis patients. These results suggest that the proteolytic enzyme belongs to cysteine proteinase which plays a role in the tissue penetration. Also it may be used as the antigen for diagnosis of active sparganosis.     

Isolation and characterization of a proteinase inhibitor from marama beans

A protease inhibitor was purified from the African marama bean (Tylosema esculenturm). The inhibitor is present in large amounts, representing about 10.5% of the total protein. The molecular weight is slightly larger than soybean trypsin inhibitor and was estimated at 23,000 by SDS-gel electrophoresis or 24,500 by amino acid analysis. The amino acid composition was atypical of most other plant inhibitors with a cysteine content of only one or possibly two residues/mole and a blocked amino terminus. Inhibition studies indicated virtually no inhibition of chymotrypsin activity. Elastase, however, was inhibited to the same extent as trypsin, requiring about 2 moles of inhibitor for complete inhibition of the enzyme.     

Characteristics of RNA and protein synthesis in the polymorphonuclear neutrophilic granulocytes of wounds

RNA and protein synthesis was comparatively studied in blood and wound neutrophils by electron microscopic autoradiography. It has been shown that the penetration of neutrophils into the wound was associated with a significant rise in RNA and a decrease in protein synthesis.     

Guinea pig plasma trypsin inhibitors. Purification and characterization of two functionally distinct proteinase inhibitors.

Two trypsin inhibitors (TI-1, TI-2) were isolated from guinea pig plasma and purified to homogeneity. In amino-acid composition as well as molecular masses, TI-1 (Mr 58,000) and TI-2 (Mr 57,000) are similar to each other and to human and mouse alpha 1-proteinase inhibitors, and mouse con-trapsin. The two inhibitors form equimolar complexes with proteinases. The effectiveness of the inhibitors was characterized by association rate constants under second-order rate conditions. The inhibitory action of TI-1 was rapid for bovine trypsin, porcine pancreatic elastase and guinea pig plasma kallikrein, but slow for bovine thrombin and guinea pig plasmin and not detectable for bovine chymotrypsin and porcine pancreatic kallikrein. The inhibitory action of TI-2 was rapid for trypsin and chymotrypsin, but slow for guinea pig plasma kallikrein and not detectable for other proteinases. These results show that TI-1 and TI-2 are physicochemically similar but functionally distinct from each other and from human alpha 1-proteinase inhibitor that inhibits trypsin, chymotrypsin and elastase.