Isolation and partial characterisation of a thiol proteinase inhibitor from human plasma.

A thiol proteinase inhibitor has been isolated from human plasma by ion exchange, salt-mediated hydrophobic and ion chelation chromatography. It was found to be electrophoretically heterogeneous (in both its native state and after isolation) giving a bimodal arc with an α₁ and α₂ peak in bidimensional immunoelectrophoresis. It was a good inhibitor of papain but only partially inhibited human kidney cathepsin Bl and did not inhibit the bacterial thiol proteinase, clostripain. Its mean protein concentration in adults sera was 42.5 ± 6.8 mg per dl.     

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.     

Isolation and partial characterization of a Taenia taeniaeformis metacestode proteinase inhibitor

Suquet C., Green-Edwards C. and Wes Leid R. 1984. Isolation and partial characterization of a Taenia taeniaeformis metacestode proteinase inhibitor. International Journal for Parasitology14: 165–172. A proteinase inhibitor from the metacestode of Taenia taeniaeformis was purified 136-fold to apparent homogeneity as evidenced by one Coomassie Blue protein staining band on 10% SDS slab gels under both reducing and non-reducing conditions. The apparent molecular weight under dissociating conditions was 19,500. This parasite protein inhibited esterolysis of TAME and BTEE by bovine pancreatic trypsin and chymotrypsin respectively in a time and dose-dependent manner. Proteolysis of casein by both enzymes was also inhibited in a time and dose-dependent manner. The parasite inhibitor was stable from pH 2.2 to 10.5 and was fully active after heating at 56 °C for 3 h. The proteases pronase and thermolysin, at concentrations of 1 mg ml^?1, completely inactivated the metacestode inhibitor. Two sulfhydryl proteases, papain and chymopapain, used at concentrations of 1 mg ml^?1 were without effect. The irreversible proteinase inhibitors TLCK, TPCK and PMSF at concentrations up to 10 mM had no effect on the parasite inhibitor.     

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 partial characterization of a low molecular weight trypsin inhibitor from human urine

A low molecular weight glycoprotein which completely inhibited trypsin at a 1 : 1 molar ratio was isolated from human urine. It was generated from a precursor molecule which in turn derived from plasma inter-alpha-trypsin inhibitor. It had one polypeptide chain with a molecular weight of about 20 000 and a high content of half-cystine residues. Its amino-terminal amino-acid sequence was Val-Thr-Glu-Val-Thr-X-Leu-Glu-Asp-.     

Isolation and characterization of proteinase inhibitor I from etiolated tobacco leaves

Proteinase Inhibitor I was induced to accumulate in tobacco (Nicotiana tabaccum) leaves by placing plants in darkness for 10 days at 27 degrees C. The inhibitor was isolated using ammonium sulfate precipitation, Sephadex G-75 chromatography, heating, and affinity chromatography with a chymotrypsin-Sepharose column. Inhibitor I was purified 232-fold with a yield of 34 mg from 2.5 kg of leaves. Affinity-purified tobacco Inhibitor I was shown to be homogeneous by gel electrophoresis in both nondissociating and dissociating buffers. The inhibitor has a molecular weight of 39,000 +/- 1000 determined by gel filtration and, like its potato and tomato counterparts, is composed of five subunits of molecular weight 8100. The tobacco Inhibitor I strongly inhibits chymotrypsin and weakly inhibits trypsin. The chemical, physical, and immunological properties of tobacco Inhibitor I indicate that it is structurally very similar to potato tuber Inhibitor I and tomato leaf Inhibitor I, although the synthesis and accumulation of the three inhibitors in their respective tissues are all under different developmental or environmental regulation.     

Isolation and partial characterization of a proteinase with elastolytic activity from mouse blood leukocytes

Leukocyte elastase has been implicated in the etiology of pulmonary emphysema. Recently, two genetic models of emphysema have been described, in mouse, which may enhance our understanding of the pathogenesis of emphysema. We therefore sought to purify mouse leukocyte elastase in order to characterize its biochemical properties. Leukocyte enzyme has been purified by a two-step procedure involving salt extraction of granular fraction, followed by preparative isoelectric focusing on Sephadex G-75 Superfine. The enzyme hydrolyses elastin and synthetic substrates for elastase, even if to a different extent. Inhibition studies indicates that the enzyme is a serine proteinase. Mouse elastase has a single isoelectric point of 8.65 and it behaves on sodium dodecyl sulphate polyacrylamide gel electrophoresis as a major band (molecular weight 29,000) and two minor bands (molecular weight 27,000 and 25,800, respectively.     

Purification and characterization of a serine proteinase inhibitor from human articular cartilage

An inhibitor of serine proteinases from human articular cartilage was purified to homogeneity by sequential ultrafiltration and ion exchange chromatography on CM-Sephadex C-50. The apparent molecular weight of the cationic glycoprotein (pI > 10) was determined to be 16.5 · 10³ by SDS gel electrohoresis. The inhibitor blocked the activity of leukocyte elastase, cathepsin G and trypsin but not leukocyte collagenase. In kinetic studies for the interactions with leukocyte elastase a firm enzyme-inhibitor binding was obtained. Amino acid analyses did not reveal homologies with other serine proteinase inhibitors already purified from human tissues.     

Isolation and partial characterization of ribonuclease inhibitor from goat liver

Ribonuclease inhibitor (RI), a 50 kDa protein, has been found both in mammalian and nonmammalian tissues. We have isolated RI from goat liver and partial characterization has been accomplished. For the isolation of RI, DEAE cellulose column chromatography followed by affinity chromatography using CNBr activated Sepharose 4B was performed. The inhibition of ribonucleolytic activity of Ribonuclease A has been checked by an agarose gel based assay. The antiangiogenic property of the protein was tested by the chorioallantoic membrane (CAM) assay. Results indicate inhibition of angiogenesis.     

Purification and characterization of human bronchial proteinase inhibitor

We describe a purification procedure for the human bronchial proteinase inhibitor which involves trichloroacetic acid precipitation of sputum followed by ion-exchange and gel filtration chromatography. The inhibitor shows a major band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, but exhibits microheterogeneity on high-resolution chromatography. It has a molecular mass of 15.5-16 kDa as determined by electrophoresis and gel filtration and is 90% active against leukocyte elastase. The amino acid sequence of the N-terminal portion of the inhibitor was determined and was found to be identical (through 29 amino acids) to that recently reported for the human seminal plasma proteinase inhibitor I (Seemuller et al. (1986) FEBS Lett. 199, 43-48).     

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.     

Isolation and characterization of a translation inhibitor from human term placenta

An inhibitor of protein synthesis has been isolated from free cytoplasmic ribonucleoprotein particles of human term placenta. The inhibitor is resistant to phenol, DNase, proteinase K, and heating at 100 degrees C, but is sensitive to alkaline hydrolysis. These data suggest that the inhibitor is RNA. Experiments provide evidence that this preparation contains no RNase contaminant and does not induce an RNase in this assay system. Three lines of evidence suggest that the inhibitor acts at the initiation of protein synthesis in the wheat germ translation system. First, a lag occurs before cessation of translation when the inhibitor is added to translating polyribosomes. This lag is identical to that seen upon the addition of aurintricarboxylic acid, a known inhibitor of initiation. Second, sucrose gradient analyses demonstrate that, when the inhibitor is present at the start of translation, 40 S complexes form, but neither 80 S complexes nor polyribosomes are seen. Third, gradient analyses show that, when the inhibitor is added to translating polyribosomes, 40 S complexes accumulate with a progressive loss of polyribosomes. Finally, the extent of inhibition depends upon the amount of wheat germ extract added to the reaction mixture and not the amount of mRNA present. This suggests an interaction between the inhibitor and a component of the wheat germ extract.     

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 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.     

Isolation and partial characterization of keratan sulphate from human brain

A sulphated glycoconjugate was isolated from adult human brain from a glycosaminoglycan fraction which was not precipitated with 1% cetylpyridinium chloride or ethanol below 50% concentration. It appeared heterogeneous on gel filtration, exhibiting a molecular weight range from about 7000 to over 10 000. Its main covalent structure was shown to contain sulphated, repeating disaccharide units of (beta-D-galactose-(1----4)-N-acetyl-D-glucosamine-(1----3)). In addition, it was susceptible to degradation by keratan sulphate endo-beta-galactosidase and thus was assumed to be keratan sulphate.     

Purification and partial characterization of a cysteine proteinase from Trypanosoma rangeli

Abstract Epimastigotes of the American Trypanosome Trypanosoma rangeli contain a very low cysteine proteinase (CP) activity. The enzyme was purified to homogeneity by affinity chromatography on ConA-Sepharose and Cystatin-Sepharose. This CP had a similar apparent molecular mass and an identical N-terminal sequence (15 amino acids) as compared with cruzipain from Trypanosoma cruzi ; cross-reacted immunologically with the latter enzyme, it was inhibited by E-64 and TLCK, but not by PMSF, o-phenanthroline or Pepstatin, and was able to use the same substrates, although with different order of effectiveness and optimum pH.     

Purification and characterization of a low molecular mass cysteine proteinase inhibitor from human amniotic fluid

We have purified the human low molecular mass cysteine proteinase inhibitor in good yield from amniotic fluid, using ultrafiltration through 100-kDa and 1-kDa cut-off filters, chromatography on Ultrogel AcA 54, and affinity chromatography on alkylated papain-agarose. Approximately 1-4 mg/l of this inhibitor are present in amniotic fluid. The purified inhibitor had an apparent molecular mass of 10.5-12 kDa, as judged by its electrophoretic behavior. Amino acid analysis showed it to be rich in acidic and aliphatic residues and in cysteine. No carbohydrate side-chains could be demonstrated. The purified inhibitor inhibited papain, ficin, cathepsins B, C, and H, the cathepsin B-like enzyme from B16 melanoma cells, and a bovine chromaffin granule enkephalin-converting activity. No inhibition of Ca2-dependent neutral cysteine proteinase, serine- or metallo-proteinases was seen. Analysis of the purified inhibitor by isoelectric focusing revealed 7 major bands with pI values of 7.95, 7.0, 6.7, 6.55, 6.25, 5.5, and 5.2, all of which inhibited papain.     

Isolation and characterization of a membrane-bound proteinase from rat liver.

The proteinase previously found in chromatin prepared from a total rat liver homogenate was purified from the rat liver mitochondrial fraction. The membrane-bound enzyme is solubilized in either 0.6% digitonin or 0.5 m phosphate buffer. After a 1330-fold purification, the enzyme appears homogeneous by acrylamide-gel electrophoresis. Sucrose density gradient centrifugation indicated a molecular weight of 22,500, a molecular weight of 23,500 ± 10% has been estimated by acrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The enzyme showed a high substrate specificity. Among several proteins tested, only glucagon, nonhistone chromosomal proteins, and histones are good substrates. A limited proteolysis was found for the very-lysine-rich histone H1, which was split into a high molecular weight fragment (M_r 13,000). The highly phosphorylated histone H1 isolated from regenerating rat liver 24 h after partial hepatectomy exhibited the same susceptibility to the proteinase as H1 from normal liver. Large polypeptides of a nonhistone chromosomal protein fraction were degraded more rapidly than the small ones. N-Acetyl-l-tyrosine ethyl ester was used with alcohol dehydrogenase and NAD in a coupled enzyme assay for the proteinase. The apparent Michaelis constant for the hydrolysis of N-acetyl-l-tyrosine ethyl ester is 5.0 × 10^?3m. The proteinase has catalytic properties simlar to trypsin and chymotrypsin. The pH optimum was around 8, soybean trypsin inhibitor depressed the enzymatic activity, and the serine modifying reagents diisopropyl phosphofluoridate and phenylmethanesulfonyl fluoride inactivated the enzyme. The affinity reagent for chymotrypsin-like active sites, l-1-tosylamido-2-phenylethyl chloromethyl ketone, inactivated the proteinase.