ABSTRACT
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
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 , SwineABSTRACT
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 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
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
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/enzymologySubject(s)
Cathepsins/analysis , Chromatography, Paper , Microchemistry , Animals , Cattle , Chemistry, Clinical , Hemoglobins/analysis , In Vitro Techniques , Peptides/analysis , SolutionsSubject(s)
Cathepsins/immunology , Cytoplasmic Granules/immunology , Leukocytes , Protease Inhibitors/immunology , Animals , Cattle , Cross Reactions , Hydrogen-Ion Concentration , Immune Sera , Immunodiffusion , Immunoelectrophoresis , Molecular Weight , Precipitins , Rabbits , alpha 1-Antitrypsin/immunologySubject(s)
Leukocytes , Protease Inhibitors , Animals , Cathepsins/antagonists & inhibitors , Cathepsins/metabolism , Cell Transformation, Neoplastic , Chemical Phenomena , Chemistry , Chromatography, Gel , Chymotrypsin/antagonists & inhibitors , Hydrogen-Ion Concentration , Neoplasm Metastasis , Pancreatic Elastase/antagonists & inhibitors , Protease Inhibitors/metabolism , Protease Inhibitors/pharmacology , 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
Two inhibitors of urokinase were isolated from the extracts of the nuclei of peripheral pig leucocytes, by ion exchange chromatography and by gel filtration on Sephadex G-100 and G-150. Only the inhibitor with the higher molecular weight was isolated in the homogenous form, in a single polypeptide chain with molecular weight of 68,000, designated Inhibitor-3. This inhibitor has an isoelectric point from 4.4 to 4.5 and is stable in buffer solutions from pH 3 to 8, and belongs to the type of so-called fast reacting inhibitors.
Subject(s)
Leukocytes , Protease Inhibitors , Urokinase-Type Plasminogen Activator/antagonists & inhibitors , Animals , Chromatography, Affinity , Chromatography, Gel , Dialysis , Hydrogen-Ion Concentration , Isoelectric Focusing , Molecular Weight , Osmolar Concentration , Peptide Hydrolases/pharmacology , SwineABSTRACT
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.
Subject(s)
Blood Proteins/physiology , Leukocytes/enzymology , Peptide Hydrolases/blood , Animals , Chymotrypsin/antagonists & inhibitors , Cytoplasmic Granules/physiology , Drug Stability , Hydrogen-Ion Concentration , Kinetics , Leukocytes/drug effects , Protease Inhibitors , Subcellular Fractions/physiology , Swine , Temperature , Trypsin Inhibitors/pharmacologyABSTRACT
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
Two different types of neutral proteinase inhibitors were isolated from postmicrosomal supernatant of bovine spleen. Inhibitor A with molecular weight 40,000 inhibits elastases and chymotrypsin-like neutral proteinases from bovine spleen, whereas inhibitor B with estimated molecular weight 20 000 inhibits only chymotrypsin-like neutral proteinase.
Subject(s)
Protease Inhibitors/isolation & purification , Spleen/physiology , Animals , Cattle , Chymotrypsin/antagonists & inhibitors , Kinetics , Molecular Weight , Pancreatic Elastase/antagonists & inhibitorsABSTRACT
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 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.
Subject(s)
Blood Proteins/antagonists & inhibitors , Cathepsins/pharmacology , Leukocytes/enzymology , Animals , Cathepsin D , Cattle , Dose-Response Relationship, Drug , Electrophoresis, Polyacrylamide Gel , Hydrogen-Ion Concentration , Immunodiffusion , Immunoelectrophoresis , Kinetics , Molecular Weight , Peptide Fragments , SwineABSTRACT
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.