Acid-stable trypsin-plasmin inhibitors formed enzymatically from plasma precursor protein

Enzymatic formation of acid-stable trypsin-plasmin inhibitors (ASTPIs) in human plasma with several proteinases, particularly SH-proteinases, was demonstrated. The maximal activity obtained with bromelain was 40 U/ml plasma, which corresponded to about a 10-fold increase as compared to the untreated control plasma (4.2 U/ml). Gel filtration revealed at least two ASTPI activity peaks of molecular weight 16,000 (main peak) and 8000 (minor peak). The main ASTPI was further purified by trypsin-Sepharose affinity chromatography, isoelectric focusing and gel filtration on Sephadex G-75 superfine. The purified inhibitor was found to be identical to the active fragment of plasma ASTPI or urinary trypsin inhibitor (UTI) formed by bromelain treatment. It had an isoelectric point (pI) of 3.7, a molecular weight of 16,000 by SDS-polyacrylamide gel electrophoresis and was a glycine- and glutamic acid-rich protein lacking histidine. The NH2-terminal amino acid sequence was H2N-(Lys)-Glu-Asp-Ser-X-Gln-Leu-Gly-Tyr-Ser-Ala-Gly-Pro-X-Met-Gly-Met-Th r-X-Arg - Tyr-Phe-Tyr-... COOH, which was homologous to the Lys22-Met36 part (or Glu23-Met36 part; 30% of the total) of the plasma ASTPI or UTI molecule (molecular weight 70,000-80,000 by gel filtration). The purified ASTPI displayed the same antigenicity as UTI and exerted strong inhibitory effects on trypsin, chymotrypsin and plasmin amidolysis, but had a much lesser effect on plasmin fibrinolysis. It also strongly inhibited non-plasmic fibrinolysis with human leukocyte proteinase and earthworm proteinase.     

Purification of a proteinase and proteinase inhibitor from Tetrahymen a pyriformis

• 1.1. A cysteine proteinase and cysteine proteinase inhibitor have been purified from Tetrahymena.
• 2.2. The proteinase was purified by ammonium sulphate fractionation, gel filtration, ion exchange chromatography and affinity chromatography, and appeared homogeneous by gel filtration and electrophoresis (mol. wt approx 28,000). It hydrolysed BAPNA, degraded azocasein, and converted 80S ribosomes to subunits. Thiol reagents inhibited these activities.
• 3.3. The inhibitor was purified by heat treatment, ammonium sulphate fractionation and ion exchange chromatography, and appeared homogeneous by gel filtration and electrophoresis (mol. wt approx 12.500). The inhibitor was heat stable and it inhibited papain, as well as the Tetrahymena proteinase.

     

Purification and properties of streptococcal nicotinamide adenine dinucleotide glycohydrolase.

Highly purified streptococcal nicotinamide adenine dinucleotide glycohydrolase (NADase) was obtained by utilizing disodium tetrathionate to protect the enzyme by blocking the sulfhydryl groups of streptococcal proteinase. This was followed by two-step ion-exchange chromatography. The pure enzyme, demonstrated as a single band on sodium dodecyl sulfate/polyacrylamide gel electrophoresis, had a specific activity of 11,200 NADase units per mg of protein and was devoid of hemolytic activity. NADase had a molecular weight of about 55,000 as determined by gel filtration, by summation of amino acid residues, and by sodium dodecyl sulfate/gel electrophoresis. The purified enzyme had optimal activity at pH 7.3 and at 40 C and a calculated Km of 5.1 times 10- minus 4 mM. It was inhibited by alpha-iodoacetamide.     

Proteinases of small intestine enterocytes of swine. Purification and properties of aspartyl proteinase similar to cathepsin D

A new aspartic proteinase was isolated from porcine intestine mucosa by affinity chromatography on pepstatin-Sepharose 4B and gel filtration on Sephadex G-100. The enzyme was purified 1600-fold and appeared homogeneous upon polyacrylamide gel electrophoresis. The proteinase has a Mr 60 000 +/- 4000 Da. During sodium dodecyl sulfate polyacrylamide gel electrophoresis the enzyme produced a single protein band (Mr 30 000 +/- 3000 Da). Isoelectric focusing revealed that the enzyme has several multiple forms (pI 6.9, 7.5, 8,0). The enzyme is a glycoprotein containing 5.9% of carbohydrates; the mannose to galactose ratio is 1:3. The amino acid composition of the enzyme was studied. The proteinase splits an oxidized insulin B-chain and synthetic substrates. The pH optimum is 3.2. The enzyme is immunologically identical to porcine spleen cathepsin D.     

Clitocypin, a new type of cysteine proteinase inhibitor from fruit bodies of mushroom clitocybe nebularis

A novel inhibitor of cysteine proteinases has been isolated from fruit bodies of a mushroom Clitocybe nebularis. The inhibitor was purified to homogeneity by affinity chromatography and gel filtration, followed by reverse-phase high pressure liquid chromatography. The active inhibitor has an apparent molecular mass of about 34 kDa by gel filtration and by SDS-polyacrylamide gel electrophoresis without prior boiling of the sample. Boiling in 2.5% SDS or incubation in 6 m guanidine hydrochloride resulted in a single band of 17 kDa, indicating homodimer composition with no intersubunit disulfide bonds. The inhibitor in nondenaturing buffer is resistant to boiling in water, retaining its activity and dimer composition. The mushroom protein is a tight binding inhibitor of papain (K(i) = 0.59 nm), cathepsin L (K(i) = 0.41 nm), cathepsin B (K(i) = 0.48 micrometer), and bromelain (K(i) = 0.16 micrometer) but is inactive toward cathepsin H, trypsin, and pepsin. Its isoelectric point is 4.4, and sugar analysis indicates the absence of carbohydrate. A single protein sequence of 150 amino acids, containing no cysteine or methionine residues, was obtained by amino acid sequencing. The calculated molecular mass of 16854 Da corresponds well with the value obtained by mass spectrometry. A major part of this sequence was verified by molecular cloning. The monomer sequence is clearly devoid of typical cystatin structure elements and has no similarity to any other known cysteine proteinase inhibitors but bears some similarity to a lectin-like family of proteins from mushrooms. The inhibitor, which is present in at least two other members of the Clitocybe genus, has been named clitocypin (Clitocybe cysteine proteinase inhibitor).     

Purification and characterization of plasma T-kininogen from Wistar and brown Norway rats

A rapid and convenient three-step purification scheme has been developed for the purification of T-kininogen (alpha 1-cysteine proteinase inhibitor) from rat plasma. The purification process includes chromatography on hydroxyapatite, immunoaffinity chromatography and gel filtration. This procedure is applied to plasma from the brown Norway rat which is known to be deficient in high and low molecular weight kininogens. The method furnished large amounts of T-kininogen from turpentine-treated Wistar rats as well as from untreated and turpentine-treated deficient brown Norway rats. The amino acid and hexose content of the three T-kininogens has been determined. While the composition of the molecules isolated from both injured rats was similar, the neutral sugar content of T-kininogen purified from untreated brown Norway rats was lower and its amino acid composition showed slight differences. The three molecules have identical behaviour and similar physicochemical and immunological properties when analysed by SDS electrophoresis, isoelectrofocusing and two-dimensional immunoelectrophoresis.     

Clonorchis sinensis: purification and characterization of a cysteine proteinase from adult worms

1. Adult Clonorchis sinensis, the Chinese liver fluke, is known to migrate to the bile ducts of its mammalian host and cause significant pathology. 2. An acidic, thiol-dependent proteinase with a native mol. wt of approximately 18,500 was purified to homogeneity using ion-exchange chromatography and gel filtration chromatography. By SDS-polyacrylamide gel electrophoresis, the mol. wt of the enzyme was estimated to be 15,000. 3. The enzyme was similar to cathepsin B-like cysteine proteinases based on pH optimum, substrate specificity, and inhibitor sensitivity. 4. Antisera from human clonorchiasis and C. sinensis-infected rabbits reacted in immunoblots with the partially purified proteinase. The C. sinensis proteinase may be useful for serodiagnosis of clonorchiasis.     

Purification and partial characterization of a plasma inhibitor of tonin

A plasma inhibitor of tonin activity in the rat, was purified by ammonium sulfate precipitation, ion-exchange of chromatography, and gel filtration. Its purity was investigated by analytical electrophoresis on polyacrylamide gel and by ultracentrifugation sedimentation velocity. The molecular weight (360 000) of the purified inhibitor was determined by sodium dodecyl sulfate electrophoresis and its isoelectric point (4.5) by gel isoelectrofocusing. The Stokes radius (640 nm) was evaluated by gel filtration studies and a frictional ratio (f/fo) of 1.95 was calculated from the molecular weight and Stokes radius. Kinetic studies using angiotensin I as substrate showed that the inhibition of tonin by the purified inhibitor was noncompetitive and does not exceed 70%. Electrophoresis showed the same mobility for [125I]tonin bound to plasma proteins and for [125I]tonin bound to the purified inhibitor. The inhibitor may be a protein resembling half of the dimeric protease inhibitor rat alpha 1-macroglobulin or human alpha 2-macroglobulin.     

Isolation from opossum serum of a metalloproteinase inhibitor homologous to human alpha 1B-glycoprotein.

Fractionation of opossum (Didelphis virginiana) serum with (NH4)2SO4, followed by chromatography on DEAE-Sepharose, phenyl-Sepharose, and Mono Q HR 5/5, has resulted in the isolation in homogeneous condition of a metalloproteinase inhibitor designated oprin (opossum proteinase inhibitor). Oprin is a single-chain glycoprotein (26% carbohydrate) with an estimated Mr = 52,000, pI = 3.5, and E(1%/1 cm) = 11. Oprin inhibited snake venom metalloproteinases, but showed no activity on venom serine proteinases or on bacterial metalloproteinases. Incubation of Crotalus atrox alpha-proteinase (EC 3.4.24.1) with oprin, and analysis of the reaction products by chromatography on Mono Q HR 5/5 and by electrophoresis under nondenaturing conditions, indicated formation of an inactive enzyme/inhibitor complex. The complex dissociated during SDS/polyacrylamide gel electrophoresis. An opossum liver cDNA library was immunoscreened, and clones containing cDNA encoding for part of the open reading frame for oprin were isolated. The cDNA inserts contained nucleotide sequences corresponding to two internal amino acid sequences of oprin which had been separately determined by protein sequence analysis. Protein database screening using a 211 amino acid sequence deduced from one of the cDNA inserts showed no significant homology to known proteinase inhibitors. There was, however, a 36% identity with human alpha 1B-glycoprotein, a plasma protein of unknown function related to the immunoglobulin supergene family. In addition, the amino-terminal sequence of oprin showed 46% identity with human alpha 1B-glycoprotein in a 26 amino acid residue overlap.(ABSTRACT TRUNCATED AT 250 WORDS)     

Purification and characterization of peptidylarginine deiminase from rabbit skeletal muscle

The preceding paper described the identification and some properties of peptidylarginine deiminase, which catalyzes the deimination of arginyl residues in protein, from rabbit skeletal muscle, kidney, brain, and lung. In the present work we purified peptidylarginine deiminase from rabbit skeletal muscle with a 16% yield by 7 steps. The purification involved ion-exchange chromatography on DEAE-Sephacel, gel filtration on Bio-Gel A-0.5 m, and affinity chromatography on soybean trypsin inhibitor-Sepharose 4B and aminohexyl-Sepharose 4B. The purified enzyme was homogeneous on polyacrylamide gel electrophoresis with and without sodium dodecyl sulfate. The molecular weight of the enzyme was estimated to be about 83,000 by sodium dodecyl sulfate polyacrylamide gel electrophoresis and 130,000-140,000 by gel filtration on Sephadex G-200. The isoelectric point was 5.3 and the amino acid composition was also determined. The enzyme preferably catalyzed the formation of citrulline derivatives from arginine derivatives in which both the amino and carboxyl groups were substituted and showed the highest activity towards Bz-L-Arg-O-Et among the arginine derivatives tested. The Km value for Bz-L-Arg-O-Et was found to be 0.50 X 10(-3) M. The enzyme also showed marked activities towards native protein substrates, such as protamine sulfate, soybean trypsin inhibitor, histone and bovine serum albumin.     

A new serum component which specifically inhibits thiol proteinases

A serum proteinase inhibitor specific for thiol proteinases was prepared in a functionally pure state by Sephadex G-200 gel filtration, starch block electrophoresis and immunoaffinity chromatography. This component was distinct from the known serum proteinase inhibitors. It was demonstrated by immuno-electrophoresis that the incubated mixture of thiol proteinase and this inhibitor produced a soluble complex possessing both antigenicities. The molecular weight of the inhibitor was found to be 90,000 by gel filtration on Sephadex G-150 column, and the electrophoretic mobility was in the α₂-region. A tentative term, α₂-thiol proteinase inhibitor, was given because of its mobility and inhibition spectrum.     

Purification and characterisation of a novel 34,000-Mr cell-associated proteinase from the dermatophyte Trichophyton rubrum

Abstract A novel cell-associated proteinase was purified to homogeneity from cytoplasmic antigen preparations of Trichophyton rubrum by sequential isoelectric focusing and gel filtration chromatography. The enzyme exhibited relative molecular masses of 34,000- M _r (non-reduced sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE)), 15,000- M _r (reduced SDS-PAGE) and 37,000- M _r (substrate SDS-PAGE). It had a pH optimum of 7.5 and a p I of 4.5. The proteinase exhibited broad substrate specificity and it was strongly inhibited by the serine proteinase inhibitors phenylmethylsulfonyl fluoride and chymostatin. The N-terminal amino acid sequence of the 34,000- M _r proteinase shared 50% homology with the deduced amino acid sequence of a Coccidioides immitis wall-associated chymotrypsin-type serine proteinase. This is the first cell-associated proteinase to be purified and characterised from T. rubrum and it would appear to be related to the chymotrypsin-type serine proteinases, a class of enzymes that have rarely been isolated from fungi. The function of the proteinase remains speculative although it may play a role in the development and subsequent proliferation of the fungus in vivo.     

Purification and physical-chemical properties of acetyl-CoA:arylamine N-acetyltransferase from pigeon liver

Acetyl-CoA:arylamine N-acetyltransferase (EC 2.3.1.5) from pigeon liver was purified by protamine sulfate precipitation, ion exchange chromatography on DEAE-A-25 Sephadex, gel filtration on Sephadex G-75, amethopterin-AH-Sepharose 4B affinity chromatography, and finally, gel filtration on Sephadex G-100. The enzyme preparation was homogeneous as judged by ultracentrifugation studies, SDS-polyacrylamide gel electrophoresis and gel filtration. The N-terminal amino acid was detected to be histidine and the complete amino acid composition is reported. The enzyme contains one disulfide bridge and two cysteine residues/mol monomer. The isoelectric point was estimated to be 4.8. The molecular weight was determined to be 32900 by high-speed sedimentation equilibrium analysis, 33000 by Sephadex G-100 gel filtration and 31600 by SDS-disc gel electrophoresis. The sedimentation coefficient from conventional sedimentation velocity runs was 3.1 S observed by ultraviolet optics. 'Active enzyme centrifugation' showed a sedimentation constant of 5.0 and 4.8 S for the purified enzyme and crude extract from pigeon liver, respectively, indicating that the enzyme forms a dimer under conditions of catalysis. It could be demonstrated that the inhibitor amethopterin was noncompetitive with respect to the acetyl donor and the acetyl acceptor. Acetyl-CoA:arylamine N-acetyltransferase was examined in different organs of pigeon. The enzyme was not inducible by 1,3-phenylenediamine and hexobarbital in vivo.     

Highly acidic proteins from human brain: purification and properties of Glu-50 protein

Three extremely acidic proteins were isolated from human brain and purified to apparent homogeneity. One of them, Glu-50 protein, contained much glutamic acid (about 50% of the total amino acids). Its purification involved ammonium sulfate fractionation, DEAE-Sephadex A-50 chromatography, and gel filtration on Sephadex G-100 and G-75. Its molecular weight was determined to be 11,000 by SDS polyacrylamide gel electrophoresis and 34,000-36,000 by gel filtration on Sephadex G-75, suggesting that it consists of three identical polypeptide chains. Its isoelectric point was pH 3.9. Its N-terminal amino acid sequence was NH2-Asp-Glu-Pro-Pro-Asp-Glu and its C-terminal amino acid was Lys. It contained no detectable carbohydrate.     

Naturally occurring protein crystals in the potato : inhibitor of papain, chymopapain, and ficin

Protein crystals isolated from potato tubers were found to consist of a proteinase inhibitor active against the cysteine proteinases papain, chymopapain, and ficin. The molecular weight as determined by gel filtration at pH 4.3 or by gel electrophoresis in the presence of dodecylsulfate was 80 kilodaltons. When the inhibitor was evaluated at pH 8.4 in a linear concentration (4-30% polyacrylamide) under nondenaturing conditions, it appeared as two bands of approximately 320 to 350 kilodaltons indicating that the inhibitor forms tetrameric aggregates in neutral or weakly alkaline media, while the monomeric form predominates under acidic conditions. Gel filtration in the presence of varying amounts of papain suggested that the monomer combines with four papain molecules. The inhibitor contains no cystine.     

Trypsin-like proteinase from Actinomyces fradiae 119

Using electrophoresis and ultracentrifugation, a homogeneous proteinase was isolated from protofradine, a protease Act. fradiae 119 preparation. The purification procedure included filtration on DEAE-cellulose, gel filtration through Arcylex P-10, CM-chromatography and desalting on Sephadex G-15. The proteinase under study is an endopeptidase which hydrolyzes low molecular weight synthetic trypsin substrates as well as casein and denaturated collagen. Diisopropylfluorophosphate and soya bean trypsin inhibitor completely inhibit the proteinase activity, whereas pCMB and EDTA have no such effect. The stability maximum is observed at pH of 2.5-3.5, the action maximum at pH 8.7-9.5. The amino acid composition of the enzyme is similar to that of trypsin from Str. griseus. The molecular weights of the proteinase as determined by gel filtration and sedimentation equilibrium method are equal to 25400 and 26500, respectively. The isoelectric point lies at 5.3. The data obtained suggest that the proteinase can be attributed to the family of trypsin proteinases.     

Type I procollagen carboxyl-terminal proteinase from chick embryo tendons. Purification and characterization

Procollagen carboxyl-terminal proteinase, the enzyme which cleaves the carboxyl-terminal propeptides from type I procollagen, was extensively purified in a yield of 25% from pooled culture media of 17-day-old chick embryo tendons using a procedure which involved chromatography on Green A Dye matrix gel, concanavalin A-Sepharose and heparin-Sepharose, and filtration gels of Sephacryl S-300 and S-200. The purified enzyme is a neutral, Ca2+-dependent proteinase which is inhibited by metal chelators, but not by inhibitors for serine and cysteine proteinases. Calcium in a concentration of 5-10 mM is required for optimal activity. The molecular weight of the enzyme was determined to be 97,000-110,000 by gel filtration and by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. Other properties of the carboxyl-terminal proteinase are: 1) the Km for the type I procollagen is 96 nM at pH 7.5 and 35 degrees C; 2) the activation energy for the reaction with type I procollagen is 21,000 cal mol-1; 3) amino acid sequencing of the released carboxyl-terminal propeptide indicated the enzyme specifically cleaves an -Ala-Asp- bond in both the pro-alpha 1(I) and pro-alpha 2(I) chains; 4) the enzyme specifically cleaves the carboxyl-terminal propeptides of a homotrimer of pro-alpha 1(I) chains and type II and III procollagens, but it does not cleave type IV procollagen. The results suggest that the enzyme is involved in the processing of type I procollagen in vivo.     

Isolation of an extracellular neutral proteinase from Pseudomonas fragi.

A single proteolytic enzyme (EC 3.4.4.-) was isolated from culture supernatants of Pseudomonas fragi with 20% yielded and 60-fold purification by means of stepwise DEAE-Sephadex batch adsorption, ammonium sulfate precipitation, gel filtration and DEAE-cellulose chromatography. The enzyme was Zn-2+ activated and Ca-2+ stabilized, had optimum activity at pH 6.5--8.0 and 40 degrees C. The molecular weight range was 40 000--50 000 as determined by dodecylsulfate gel electrophoresis, gel filtration and Zn assay. This proteinase has properties similar to other extracellular bacterial neutral proteinases.     

Purification and some physicochemical properties of staphylococcal enterotoxin D.

A method was developed for the isolation of staphylococcal enterotoxin D in highly purified form from cultures of Staphylococcus aureus strain 1151m. The method involves removal of the toxin from the culture supernatant fluid with the ion-exchange resin CG-50 followed by chromatography on carboxymethylcellulose (twice) and by gel filtration on Sephadex G-75 (twice). The purified toxin is homogeneous by polyacrylamide gel and sodium dodecyl sulfate-polyacrylamide gel electrophoresis and double gel diffusion tests. It is a simple, colorless, antigenic protein with an isoelectric point of 7.4 as determined by isoelectric focusing. Its molecular weight was determined to be 27 300 +/- 700 by molecular sieve chromatography on Sephadex G-100 and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Its serological activity is stable over a wide range of pH values (1.2--10.7). The enterotoxin consists of 236 amino acid residues and contains no free sulfhydryl groups. End-group analysis showed serine to be the NH2-terminal amino acid and lysine to be the COOH-terminal amino acid.     

In vivo metabolism of inter-alpha-trypsin inhibitor and its proteinase complexes: evidence for proteinase transfer to alpha 2-macroglobulin and alpha 1-proteinase inhibitor

Inter-alpha-trypsin inhibitor was purified by a modification of published procedures which involved fewer steps and resulted in higher yields. The preparation was used to study the clearance of the inhibitor and its complex with trypsin from the plasma of mice and to examine degradation of the inhibitor in vivo. Unlike other plasma proteinase inhibitor-proteinase complexes, inter-alpha-trypsin inhibitor reacted with trypsin did not clear faster than the unreacted inhibitor. Studies using 125I-trypsin provided evidence for the dissociation of complexes of proteinase and inter-alpha-trypsin inhibitor in vivo, followed by rapid removal of proteinase by other plasma proteinase inhibitors, particularly alpha 2-macroglobulin and alpha 1-proteinase inhibitor. Studies in vitro also demonstrated the transfer of trypsin from inter-alpha-trypsin inhibitor to alpha 2-macroglobulin and alpha 1-proteinase inhibitor but at a much slower rate. The clearance of unreacted 125I-inter-alpha-trypsin inhibitor was characterized by a half-life ranging from 30 min to more than 1 h. Murine and human inhibitors exhibited identical behavior. Multiphasic clearance of the inhibitor was not due to degradation, aggregation, or carbohydrate heterogeneity, as shown by competition studies with asialoorosomucoid and macroalbumin, but was probably a result of extravascular distribution or endothelial binding. 125I-inter-alpha-trypsin inhibitor cleared primarily in the liver. Analysis of liver and kidney tissue by gel filtration chromatography and sodium dodecyl sulfate gel electrophoresis showed internalization and limited degradation of 125I-inter-alpha-trypsin inhibitor in these tissues. No evidence for the production of smaller proteinase inhibitors from 125I-inter-alpha-trypsin inhibitor injected intravenously or intraperitoneally was detected, even in casein-induced peritoneal inflammation. No species of molecular weight similar to that of urinary proteinase inhibitors, 19,000-70,000, appeared in plasma, liver, kidney, or urine following injection of inter-alpha-trypsin inhibitor.