ABSTRACT
Determination of crystal structures of chicken cystatin and human stefin B complexed with papain revealed a novel model of protease inhibition and also structural differences between two cysteine proteinase inhibitor (CPI) families. According to the 3D alignment, stefins have an extension of 9 amino acids on their carboxy terminus in comparison with cystatins. The extension was not expected to make a major contribution to interaction with the enzyme. A deletion mutant of stefin B, corresponding in length to the carboxy terminal sequence of chicken cystatin, was constructed by the use of polymerase chain reaction (PCR). This (C3S, delta 89-98) human stefin B, 10 amino acids shorter, inhibited papain with a Ki of 0.012 nM which is comparable to the Ki of 0.03 nM for authentic, nondeleted recombinant stefin B. This finding thus confirms the tertiary structure-based alignment.
Subject(s)
Cystatins/metabolism , Papain/metabolism , Amino Acid Sequence , Animals , Base Sequence , Binding Sites , Chromosome Deletion , Cystatin B , Cystatins/genetics , DNA/genetics , Humans , In Vitro Techniques , Kinetics , Molecular Sequence Data , Mutation , Structure-Activity RelationshipABSTRACT
The amino acid sequence of a cathepsin D inhibitor isolated from potato is described. It was determined by analysis of peptides generated by use of the glycine-specific proteinase PPIV. The order of the peptides was established by examination of tryptic peptides derived from the two cyanogen bromide peptides. The inhibitor comprises 187 amino acid residues, and has a calculated Mr of 20,450.
Subject(s)
Cathepsin D , Protease Inhibitors/analysis , Solanum tuberosum/enzymology , Amino Acid Sequence , Cathepsin D/antagonists & inhibitors , Chromatography, Gel , Chromatography, High Pressure Liquid , Molecular Sequence DataABSTRACT
A new intracellular inhibitor of plasmin and trypsin was isolated from porcine leukocytes by ion exchange chromatography and affinity chromatography. In dodecyl sulphate gel electrophoresis a single protein band with an apparent molecular mass of 15 kDa was found under reducing conditions. On isoelectric focusing three protein bands with isoelectric points between pH 4.0 and 4.5 were found. The association rate constants and the inhibition constants were determined for porcine plasmin and bovine trypsin. The inhibitor shows no immunologic cross-reactivity with any of the tested leukocyte inhibitors. On the basis of its N-terminal amino-acid sequence a great degree of similarity to Kunitz-type inhibitors was observed.
Subject(s)
Fibrinolysin/antagonists & inhibitors , Leukocytes/enzymology , Trypsin Inhibitors/isolation & purification , Amino Acid Sequence , Animals , Aprotinin , Cattle , Molecular Sequence Data , Swine , Trypsin Inhibitors/analysisABSTRACT
A new low-molecular mass cysteine proteinase inhibitor (CPI) was purified from the cytosol of peripheral pig leukocytes. The isolation procedure included DEAE chromatography, Sephadex G-100 gel filtration and fast-protein liquid chromatography on Mono Q. The procedure resulted in the isolation of a homogeneous protein with a molecular mass of approximately 12 kDa and a pI of 4.8. The amino terminus is blocked. The amino-acid composition and the sequence of the C-terminal part of the molecule are suggestive of a new family of cystatins. The CPI was found to be a tight-binding inhibitor of both papain and cathepsin L, with Ki values of 0.1 nM and 1 nM, respectively.
Subject(s)
Amino Acids/analysis , Cysteine Proteinase Inhibitors/isolation & purification , Leukocytes/analysis , Amino Acid Sequence , Animals , Chickens , Cysteine Proteinase Inhibitors/analysis , Cysteine Proteinase Inhibitors/metabolism , Humans , Methods , Molecular Sequence Data , SwineABSTRACT
The primary structure of a pig leucocyte cysteine proteinase inhibitor, also called cathelin, was determined. The sequence was obtained from analyses of peptides isolated from the chymotryptic, endoproteinase Lys-C and protease V8 digests, and by analysis of the peptides derived from the hydrolysis of the aspartyl-prolyl bond of the carboxymethylated inhibitor. The inhibitor consists of 96 residues. The N-terminal residue of the inhibitor is pyrrolidone-carboxylic acid. The amino acid sequence of cathelin suggests the appearance of a new family of cysteine proteinase inhibitors.
Subject(s)
Cysteine Proteinase Inhibitors/blood , Leukocytes/metabolism , Amino Acid Sequence , Animals , Cysteine Proteinase Inhibitors/isolation & purification , Molecular Sequence Data , Sequence Homology, Nucleic Acid , SwineSubject(s)
Leukocytes/analysis , Plasminogen Activators/antagonists & inhibitors , Plasminogen Inactivators/blood , Amino Acid Sequence , Animals , Blood Proteins/genetics , Blotting, Western , Humans , Immunodiffusion , Molecular Sequence Data , Molecular Weight , Plasminogen Inactivators/genetics , Plasminogen Inactivators/isolation & purification , Sequence Homology, Nucleic Acid , SwineABSTRACT
A plasminogen activator inhibitor (PA-I) which inhibits primarily plasminogen activator of the urokinase type (u-PA) was isolated from the cytosol of human peripheral leukocytes. The inhibitor was isolated using ion exchange chromatography, gel filtration and FPLC. This inhibitor has an apparent molecular weight of 45 kDa, determined by SDS-PAGE, and a pI of 5.5-5.7. The inhibitor is a fast reacting inhibitor, is thermally unstable and is inactivated outside the pH range 7-9. Treatment of cytosol to pH 9 for 30 min at 37 degrees C resulted in a large increase in inhibitory activity. Antibodies against human placental UK-I completely quenched the inhibitory activity of human leucocyte UK-I.
Subject(s)
Blood Proteins , Glycoproteins/blood , Leukocytes/metabolism , Urokinase-Type Plasminogen Activator/antagonists & inhibitors , Blood Proteins/immunology , Cross Reactions , Female , Humans , In Vitro Techniques , Isoelectric Point , Molecular Weight , Placenta/metabolism , Plasminogen Inactivators , PregnancyABSTRACT
A protein inhibitor of cysteine proteinases, "stefin", was purified from cytosol of human polymorphonuclear granulocytes. Affinity chromatography on carboxymethylated papain-Sepharose was used as the first step, followed by ion exchange chromatography on DEAE-Sephacel, which resolved four inhibitory peaks. The main peak, comprising approx. 80% of total inhibitory activity was characterized. It was found to be a homogenous protein with an apparent molecular mass slightly lower than that of cytochrome c and with an isoelectric point of 4.65. The inhibitor inhibits papain at a molar ratio of 1:1 as well as cathepsin B and H, but it does not inhibit serine and aspartic proteinases. It is stable at elevated temperature and in alkaline pH, but looses its activity in acid pH. Oxidized glutathione has no effect on its inhibitory activity.
Subject(s)
Cystatins , Neutrophils/enzymology , Protease Inhibitors/isolation & purification , Proteins/isolation & purification , Chromatography, Affinity , Cystatin B , Cytosol/metabolism , Humans , Hydrogen-Ion Concentration , Kinetics , Proteins/physiologyABSTRACT
Procedures are described for extraction or release, assay and purification of cerebrocystatin an inhibitor of brain cathepsin B or of papain. Neurosecretory regions of rat brain contained significantly higher amounts of cerebrocystatin compared to cortex, cerebellum, mid- and lower brain regions, and spinal cord. Inhibitor was purified to apparent homogeneity by alkaline treatment of rat brain cytosol, followed by gel-filtration and affinity chromatography on Reacti-gel coupled to alkylated papain. Purified cerebrocystatin was a single polypeptide of Mr 12,500 as shown by gel-electrophoresis on urea-SDS slab gels. Cerebrocystatin inhibited the hydrolysis of BANA by papain (Ki, 1 nM) or by purified rat brain cathepsin B (Ki, 10 nM) and suppressed the hydrolysis of myelin basic protein (MBP) by cathepsin B (I50, 0.8 microM) and prevented its cleavage to form polypeptides of Mr 15,000-17,000.
Subject(s)
Brain Chemistry , Cytosol/metabolism , Myelin Basic Protein/metabolism , Nerve Tissue Proteins/pharmacology , Protease Inhibitors , Animals , Cattle , Cysteine Endopeptidases , Nerve Tissue Proteins/isolation & purification , RatsABSTRACT
The antiserum was raised in rabbits against intracellular inhibitors I-1, I-2 and I-3 isolated from the soluble phase of disrupted pig peripheral leucocytes. It was demonstrated with double immunodiffusion and with immunoelectrophoresis that the isolated inhibitors with different biochemical characteristics are three different, specific and unrelated proteins. With the techniques used, it was clearly confirmed that the inhibitors were isolated in a pure form and that they are located in cytoplasm and nucleus. The suppression of inhibitors by antiinhibitors antibodies was also demonstrated.
Subject(s)
Leukocytes/analysis , Protease Inhibitors/immunology , Animals , Antibodies/immunology , Cell Nucleus/analysis , Cytoplasm/analysis , Immunodiffusion , Immunoelectrophoresis , Protease Inhibitors/isolation & purification , SwineABSTRACT
Parenteral administration of sericystatin A, an intracellular leukocyte inhibitor of the proinsulin-splitting enzyme cathepsin B, prolongs the mean survival time of mice with insulin-dependent myeloid leukemia. Sericystatin A reduces the supranormal blood levels of substances detectable by insulin-specific radioimmunoassay in these animals; this effect is abolished by concomitant insulin administration. It is concluded that sericystatin A reduces tumor proliferation indirectly, i.e. by reducing circulating insulin levels.
Subject(s)
Leukemia, Myeloid/drug therapy , Leukemia, Myeloid/metabolism , Protease Inhibitors/therapeutic use , Proteins/therapeutic use , Animals , Cells, Cultured , Cystatin A , Cystatins , Insulin/blood , Insulin/pharmacology , Leukemia, Experimental/drug therapy , Leukemia, Experimental/metabolism , Leukemia, Experimental/mortality , Leukemia, Myeloid/mortality , Male , Mice , Mice, Inbred Strains , Proteins/antagonists & inhibitors , Spleen/metabolism , Thymidine/metabolismABSTRACT
The proteolytic activity in homogenates and extracts of subcellular fractions prepared from subcutaneous Lewis lung carcinoma was determined using proteins and synthetic peptides as substrates. The presence of cathepsin D, plasminogen activator, cathepsin B-, cathepsin G- and elastase-like enzymes was observed. No difference was revealed between the proteolytic activity in homogenates of Lewis lung carcinoma, at the growth stage examined, and in homogenates of normal lung. High specific activities were found in the lysosomal extract, whereas decreasing activities were found in the nuclear extract, the homogenate and the postlysosomal mitochondrial supernatant; no active or trypsin-activatable collagenase activity was detected. The presence in the tumor tissue of these enzymatic activities is in agreement with their proposed role in the process of metastasis. The lack of differences between homogenates of tumor and normal lung tissue suggests that the use of whole cells is required to selectively study tumor proteinases specifically involved in tumor malignancy.
Subject(s)
Lung Neoplasms/enzymology , Peptide Hydrolases/analysis , Animals , Mice , Microbial Collagenase/analysis , Neoplasms, Experimental/enzymologyABSTRACT
Pig leucocytes contain inhibitors of neutral and thiol proteinases. These proteins could be isolated from post-granule supernatant fraction as well as from nuclear extract using ion exchange chromatography, gel chromatography and affinity chromatography. Inhibitors differ in molecular weight, isoelectric point, immunologically and their inhibition ability against tested enzymes.
Subject(s)
Leukocytes/enzymology , Protease Inhibitors/blood , Animals , Electrophoresis, Polyacrylamide Gel , Humans , Leukocytes/ultrastructure , Protease Inhibitors/isolation & purification , Subcellular Fractions/enzymology , Substrate SpecificityABSTRACT
By acid extraction, ethanol precipitation, affinity chromatography on 4-phenylbutylamine-Sepharose 4B and gel filtration on Sephadex G-100, calf liver neutral proteinase was purified. The purified enzyme was electrophoretically homogeneous and over 2000 times more active than the starting homogenate. The molecular weight, determined by SDS electrophoresis, was calculated as 27000. The pH optimum of the enzyme for whole calf thymus histones and N-benzoyltyrosine, ethyl ester (BTEE) was at 7.0 and 7.0-7.5. The Km value for histones was 2% and for BTEE 1.66 mM. The enzyme was strongly inhibited by soya-bean trypsin inhibitor and leucocyte intracellular I-1A inhibitor and less by alpha 1-antitrypsin and leucocyte inhibitor I-1B. The enzyme hydrolyzed only selected protein substrates, such as total thymus histones, Lys-rich histones, nucleoprotein and substance P, but not Arg-rich histones, hemoglobin and casein. The enzyme showed chymotrypsin-like properties by cleavage of substance P at the carboxyl groups of phenylalanine and leucine.
Subject(s)
Endopeptidases/isolation & purification , Liver/enzymology , Animals , Cattle , Chromatography, Affinity , Endopeptidases/metabolism , Histones/metabolism , Kinetics , Molecular Weight , Protease Inhibitors/pharmacology , Sepharose/analogs & derivatives , Serine Endopeptidases , Substrate Specificity , Trypsin Inhibitors/pharmacologyABSTRACT
Leucocytes and spleen contain four different types of protein proteinase inhibitors. Two of them can be inactivated by cathepsin D. In this work biochemical and immunological studies of the inactivation of I-2 by cathepsin D are presented. Polyacrylamide gel electrophoretic examinations indicate that cathepsin D inactivates I-2 by hydrolysis of the inhibitor molecule. The conversion of the active inhibitor into inactive protein proceeds catalytically. The studies on the inhibitor mechanism of the isoinhibitors of I-1 type explain the unusual inhibitor property of this type of inhibitor to inhibit two different types of proteinases, cysteine and serine. The evidence suggests that the inhibitory mechanism is based on an active sulfhydryl group of the inhibitor which may interact with the disulfide bridge of the inhibited proteinase.
Subject(s)
Leukocytes/enzymology , Protease Inhibitors/metabolism , Spleen/enzymology , Animals , Cathepsin B , Cathepsin D , Cathepsins/antagonists & inhibitors , Cathepsins/pharmacology , Cysteine/pharmacology , Cysteine Endopeptidases , Hydrogen-Ion Concentration , Mice , Mice, Inbred C57BL , Neoplasms, Experimental/pathology , Papain/antagonists & inhibitors , Protease Inhibitors/antagonists & inhibitors , Protease Inhibitors/pharmacology , Serine Endopeptidases , SwineSubject(s)
Lung Neoplasms/secondary , Protease Inhibitors/therapeutic use , Animals , Dose-Response Relationship, Drug , Female , Lung Neoplasms/metabolism , Lung Neoplasms/prevention & control , Mice , Mice, Inbred Strains , Neoplasm Transplantation , Neoplasms, Experimental/drug therapy , Neoplasms, Experimental/metabolismABSTRACT
Studies are reported on the inhibitory mechanism of an endogenous inhibitor, which has the ability of inhibiting both cysteine and serine proteinases. On the basis of this unusual inhibitory property, we propose the name "sericystatin". The cysteine proteinases (cathepsin B and papain) can be reactivated from their complex with sericystatin by the action of oxidized glutathione or by chymotrypsin-like neutral proteinases. The inhibitory activity of sericystatin can be inactivated by treatment with oxidized glutathione. The results indicate that sericystatin inhibits these enzymes by a reversible thiol-disulphide exchange mechanism.
Subject(s)
Protease Inhibitors/antagonists & inhibitors , Proteins/metabolism , Animals , Endopeptidases , Glutathione/pharmacology , Kinetics , Leukocytes/analysis , Serine Endopeptidases , SwineABSTRACT
Leucocytes contain an urokinase inhibitor, that can be inactivated by cathepsin D. In this work biochemical and immunological studies on the inactivation of this inhibitor by cathepsin D are presented. Examinations by polyacrylamide gel electrophoresis and SDS electrophoresis indicate that cathepsin D inactivates urokinase inhibitor by hydrolysis of the inhibitor molecule and that the degradation needed for total inactivation is different from that for formation of the precipitin line with antibodies. The conversion of active inhibitor into inactive protein proceeds catalytically.