scholarly journals Sheep mast cell proteinase-1, a serine proteinase with both tryptase- and chymase-like properties, is inhibited by plasma proteinase inhibitors and is mitogenic for bovine pulmonary artery fibroblasts

1997 ◽  
Vol 323 (3) ◽  
pp. 719-725 ◽  
Author(s):  
Alan D. PEMBERTON ◽  
Christopher M. BELHAM ◽  
John F. HUNTLEY ◽  
Robin PLEVIN ◽  
Hugh R. P. MILLER
Keyword(s):  
Mast Cell ◽  
Pulmonary Artery ◽  
Proteinase Inhibitors ◽  
Gel Filtration ◽  
Serine Proteinase ◽  
Gastrointestinal Nematode ◽  
Α2 Macroglobulin ◽  
Sds Page ◽  
Bean Trypsin Inhibitor ◽  
Β Chain

Sheep mast cell proteinase-1 (sMCP-1), a serine proteinase with dual chymase/tryptase activity, is expressed in gastrointestinal mast cells, and released systemically and on to the mucosal surface during gastrointestinal nematode infection. The potential for native plasma proteinase inhibitors to control sMCP-1 activity was investigated. Sheep α1-proteinase inhibitor (α1PI) inhibited sMCP-1 slowly, with second-order association rate constant (kass) 1.1×103 M-1·s-1, whereas sheep contrapsin inhibited trypsin (kass 2.2×106 M-1·s-1) but not sMCP-1. Western-blot analysis and gel filtration showed that when added to serum or plasma, sMCP-1 was partitioned between α1PI and α2-macroglobulin. The possibility that significant cleavage of plasma proteins could occur before sMCP-1 was inhibited was investigated using gel filtration and SDS/PAGE after adding sMCP-1 to plasma. Cleavage of ovine fibrinogen occurred in the presence of excess α1PI and α2-macroglobulin, the α-chain being cleaved C-terminally and the β-chain at the putative Lys-27. In addition, sMCP-1 was found to be mitogenic for bovine pulmonary artery fibroblasts, but was not mitogenic in the presence of soya-bean trypsin inhibitor. In terms of fibrinogen cleavage and fibroblast stimulation, sMCP-1 shows functional similarities to mast cell tryptase.

scholarly journals A high-molecular-mass neutral endopeptidase-24.5 from human lung

1987 ◽  
Vol 241 (1) ◽  
pp. 129-135 ◽  
Author(s):  
R Zolfaghari ◽  
C R Baker ◽  
P C Canizaro ◽  
A Amirgholami ◽  
F J Bĕhal
Keyword(s):  
Metal Ions ◽  
Human Lung ◽  
Proteinase Inhibitors ◽  
Gel Filtration ◽  
Serine Proteinase ◽  
Neutral Endopeptidase ◽  
Exchange Chromatography ◽  
Polyacrylamide Gel ◽  
Ph Optimum ◽  
Apparent Homogeneity

A high-Mr neutral endopeptidase-24.5 (NE) that cleaved bradykinin at the Phe5-Ser6 bond was purified to apparent homogeneity from human lung by (NH4)2SO4 fractionation, ion-exchange chromatography and gel filtration. The final enzyme preparation produced a single enzymically active protein band after electrophoresis on a 5% polyacrylamide gel. Human lung NE had an Mr of 650,000 under non-denaturing conditions, but after denaturation and electrophoresis on an SDS/polyacrylamide gel NE dissociated into several lower-Mr components (Mr 21,000-32,000) and into two minor components (Mr approx. 66,000). The enzyme activity was routinely assayed with the artificial substrate Z-Gly-Gly-Leu-Nan (where Z- and -Nan represent benzyloxycarbonyl- and p-nitroanilide respectively). NE activity was enhanced slightly by reducing agents, greatly diminished by thiol-group inhibitors and unchanged by serine-proteinase inhibitors. Human lung NE was inhibited by the univalent cations Na+ and K+. No metal ions were essential for activity, but the heavy-metal ions Cu2+, Hg2+ and Zn2+ were potent inhibitors. With the substrate Z-Gly-Gly-Leu-Nan a broad pH optimum from pH 7.0 to pH 7.6 was observed, and a Michaelis constant value of 1.0 mM was obtained. When Z-Gly-Gly-Leu-Nap (where -Nap represents 2-naphthylamide) was substituted for the above substrate, no NE-catalysed hydrolysis occurred, but Z-Leu-Leu-Glu-Nap was readily hydrolysed by NE. In addition, NE hydrolysed Z-Gly-Gly-Arg-Nap rapidly, but at pH 9.8 rather than in the neutral range. Although human lung NE was stimulated by SDS, the extent of stimulation was not appreciable as compared with the extent of SDS stimulation of NE from other sources.

Characterization of serine proteinase inhibitors in dry seeds of cultivated pasture grass species

1994 ◽  
Vol 4 (3) ◽  
pp. 335-345 ◽  
Author(s):  
Mohammed Tasneem ◽  
Clive A. Cornford ◽  
Michael T. McManus
Keyword(s):  
Festuca Arundinacea ◽  
Proteinase Inhibitors ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
Serine Proteinase ◽  
Grass Species ◽  
Pasture Grass ◽  
Molecular Weights ◽  
Trypsin Inhibition ◽  
Pasture Grasses

AbstractA survey of proteinaceous inhibitors of the serine proteinases, bovine trypsin and chymotrypsin, that are extractable from dry seeds of several cultivars of pasture grasses has been undertaken. Using crude extracts, most cultivars screened contained inhibitors of chymotrypsin, whereas trypsin inhibition was not detectable. Seeds from four cultivars, Lolium perenne L. cv. Grasslands Ruanui, Lolium × boucheanum cv. Grasslands Greenstone, Festuca arundinacea Schreb. cultivars Grasslands Roa and Grasslands Garland, that contained more potent chymotrypsin inhibition were purified further. After gel filtration chromatography, both trypsin and chymotrypsin inhibition could be observed in all four cultivars, and each separated into two discrete native molecular weights; one of ca. 20–22 kDa and one of ca. 8–10 kDa. However, activity staining, after polyacrylamide gel electrophoresis, revealed an array of iso-inhibitors with molecular weights that ranged from ca. 3 kDa to 20 kDa. One of these, a dual trypsin/chymotrypsin inhibitor of ca. 12 kDa that is present in all four cultivars examined, was purified to homogeneity from F. arundinacea cv. Grasslands Garland using anhydro-trypsin affinity chromatography and reverse-phase HPLC. The protein was found to comprise two closely related peptides and N-terminal amino acid sequencing revealed highest identity with a trypsin inhibitor identified in rye (Secale cereale) seeds.

scholarly journals Identification of monomeric α-macroglobulin proteinase inhibitors in birds, reptiles, amphibians and mammals, and purification and characterization of a monomeric α-macroglobulin proteinase inhibitor from the American bullfrog Rana catesbiana

1993 ◽  
Vol 290 (1) ◽  
pp. 85-95 ◽  
Author(s):  
D S Rubenstein ◽  
I B Thøgersen ◽  
S V Pizzo ◽  
J J Enghild
Keyword(s):  
Proteinase Inhibitor ◽  
Rana Catesbeiana ◽  
Proteinase Inhibitors ◽  
Gel Filtration ◽  
Primary Amines ◽  
Octopus Vulgaris ◽  
American Bullfrog ◽  
Sds Page ◽  
Small Primary

The alpha-macroglobulins are classified as broad-spectrum inhibitors because of their ability to entrap proteinases of different specificities and catalytic class. Tetrameric and dimeric alpha-macroglobulins have been identified in a wide variety of organisms including those as primitive as the mollusc Octopus vulgaris; however, monomeric alpha-macroglobulin proteinase inhibitors have been previously identified only in rodents. The monomeric alpha-macroglobulin proteinase inhibitors are believed to be analogous to the evolutionary precursor of the multimeric members of this family exemplified by the tetrameric human alpha 2-macroglobulin. Until now, monomeric alpha-macroglobulin proteinase inhibitors have only been identified in rodents and have therefore been considered an evolutionary anomaly. However, in this report we have utilized several sensitive assays to screen various plasmas and sera for the presence of monomeric alpha-macroglobulins, and our results suggest that monomeric alpha-macroglobulin proteinase inhibitors are present in organisms belonging to the avian, reptilian, amphibian and mammalian classes of the chordate phylum. This indicates that these proteins are more widespread than previously recognized and that their presence in rodents is not an anomaly. To demonstrate further that the identified proteins were indeed monomeric alpha-macroglobulin proteinase inhibitors, we purified the monomeric alpha-macroglobulin from the American bullfrog Rana catesbeiana. We conclude that this protein is a monomer of 180 kDa on the basis of its behaviour on (i) pore-limit gel electrophoresis, (ii) non-reducing and reducing SDS/PAGE and (iii) gel-filtration chromatography. In addition, we demonstrate that this protein is an alpha-macroglobulin proteinase inhibitor by virtue of (i) its ability to inhibit proteinases of different catalytic class, (ii) the presence of a putative internal beta-cysteinyl-gamma-glutamyl thioester and (iii) an inhibitory mechanism characterized by steric protection of the proteinase active site and by sensitivity to small primary amines. The frog monomeric alpha-macroglobulin is structurally and functionally similar to the well-characterized monomeric alpha-macroglobulin proteinase inhibitor rat alpha 1-inhibitor-3.

scholarly journals Purification and characterization of a tetrameric α-macroglobulin proteinase inhibitor from the gastropod mollusc Biomphalaria glabrata

1996 ◽  
Vol 316 (3) ◽  
pp. 893-900 ◽  
Author(s):  
Randall C. BENDER ◽  
Christopher J. BAYNE
Keyword(s):  
Quaternary Structure ◽  
Proteinase Inhibitors ◽  
Cysteine Proteinase ◽  
Serine Proteinase ◽  
Biomphalaria Glabrata ◽  
Sedimentation Equilibrium ◽  
Flight Mass Spectrometry ◽  
Isolation And Characterization ◽  
Α2 Macroglobulin

The α-macroglobulin proteinase inhibitors (αMs) are a family of proteins with the unique ability to inhibit a broad spectrum of proteinases. Whereas monomeric, dimeric and tetrameric αMs have been identified in vertebrates, all invertebrate αMs characterized so far have been dimeric. This paper reports the isolation and characterization of a tetrameric αM from the tropical planorbid snail Biomphalaria glabrata. The sequence of 18 amino acids at the N-terminus indicates homology with other αMs. The subunit mass of approx. 200 kDa was determined by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and SDS/PAGE. The quaternary structure was determined by sedimentation equilibrium centrifugation and native pore-limit electrophoresis. Evidence for a thioester is provided by the fact that methylamine treatment prevents the autolytic cleavage of the snail αM subunit and results in the release of 4 mol of thiols per mol of snail αM. The snail αM inhibited the serine proteinase trypsin, the cysteine proteinase bromelain and the metalloproteinase thermolysin. The spectrum of proteinases inhibited, together with the demonstration of steric protection of the proteinase active site and a ‘slow to fast’ conformational change after reacting with trypsin, all suggest that the inhibitory mechanism of the snail αM is similar to the ‘trap mechanism’ of human α2-macroglobulin.

scholarly journals SUBSTRATES OF HAGEMAN FACTOR

1974 ◽  
Vol 140 (6) ◽  
pp. 1615-1630 ◽  
Author(s):  
Louis W. Heck ◽  
Allen P. Kaplan
Keyword(s):  
Trypsin Inhibitor ◽  
Gel Electrophoresis ◽  
Antithrombin Iii ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Lima Bean ◽  
Hageman Factor ◽  
Α2 Macroglobulin ◽  
Coagulant Activity ◽  
Bean Trypsin Inhibitor

Unactivated partial thromboplastin antecedent (PTA) has been purified by sequential chromatography of plasma on quaternary aminoethyl Sephadex, sulphoprophyl Sephadex, Sephadex G-150, and passage over an anti-IgG immunoadsorbant. The preparation gave a single band after alkaline disc gel electrophoresis, sodium dodecyl sulfate (SDS) gel electrophoresis and isoelectric focusing in acrylamide gels and was found to have a mol wt of 175,000 by gel filtration, 163,000 by SDS gel electrophoresis, and an isoelectric point of 8.8–9.4 (peak 9.0–9.1). Pre-PTA was activated directly by activated Hageman factor or by Hageman factor prealbumin fragments. Its coagulant activity was inhibited by DFP, soybean trypsin inhibitor and trasylol but not by lima bean trypsin inhibitor or ovomucoid trypsin inhibitor indicating that activated PTA possesses the same inhibition profile utilizing these reagents as does plasma kallikrein. A major plasma inhibitor of activated PTA was found to be a 65,000 mol wt α-globulin which was isolated free of α1-chymotrypsin inhibitor, inter α-trypsin inhibitor, α2-macroglobulin, and the other known inhibitors of activated PTA, the activated first component of complement (C1 INH), and antithrombin III. Its physicochemical properties were identical to α1-antitrypsin, and it was absent in α1-antitrypsin-deficient plasma thereby identifying this PTA inhibitor as α1-antitrypsin.

Functional proteomics of kallikrein-related peptidases in ovarian cancer ascites fluid

2010 ◽  
Vol 391 (4) ◽  
Author(s):  
Katerina Oikonomopoulou ◽  
Ihor Batruch ◽  
Chris R. Smith ◽  
Antoninus Soosaipillai ◽  
Eleftherios P. Diamandis ◽  
...  
Keyword(s):  
Ovarian Cancer ◽  
Proteinase Inhibitors ◽  
Serine Proteinase ◽  
Cancer Cell Line ◽  
Malignant Ascites ◽  
Ascites Fluid ◽  
Clinical Samples ◽  
Serine Proteinases ◽  
Α2 Macroglobulin

Abstract Kallikrein-related peptidases (KLKs) are secreted serine proteinases with trypsin or chymotrypsin-like activity. Several family members, such as KLKs 6 and 10, are potential ovarian cancer biomarkers. Recently, using a newly developed assay for active KLK6, we found that only a very small proportion of immunoreactive KLK6 in tumor-derived clinical samples (malignant ascites fluid), in cerebrospinal fluid, and in cancer cell line supernatants is enzymatically active. We therefore hypothesized that a proportion of other immunoreactive KLKs in such samples could be present, but might be partly complexed to endogenous serine proteinase inhibitors. Using a combination of immunological isolation of the enzymes, activity-based probe analysis and proteomics, we identified active KLK10 in ovarian cancer ascites and we provide preliminary data that the activity of other KLKs present in these samples can be decreased by known proteinase inhibitors (e.g., α2-macroglobulin, α1-antitrypsin). Our data suggest that the enzymatic activity of ovarian cancer-released KLKs that are detected by regular immunoassays is low in vivo and very likely regulated by proteinase inhibitors.

scholarly journals Structural and functional analysis of the spontaneous re-formation of the thiol ester bond in human α2-macroglobulin, rat α1-inhibitor-3 and chemically modified derivatives

1996 ◽  
Vol 318 (2) ◽  
pp. 539-545 ◽  
Author(s):  
Hanne GRØN ◽  
Ida B. THØGERSEN ◽  
Jan J. ENGHILD ◽  
Salvatore V PIZZO
Keyword(s):  
Room Temperature ◽  
Elevated Temperatures ◽  
Proteinase Inhibitors ◽  
Gel Filtration ◽  
Complement Components ◽  
Thiol Ester ◽  
Chemically Modified ◽  
Inhibitory Capacity ◽  
Α2 Macroglobulin ◽  
The Complement System

The α-macroglobulins are proteinase inhibitors that form part of a superfamily along with components of the complement system. Internal β-cysteinyl–γ-glutamyl thiol ester bonds are an important structural feature of most α-macroglobulins and several complement components. We have studied the reversibility of thiol ester cleavage caused by NH3 or CH3NH2 in tetrameric human α2-macroglobulin (α2M) and monomeric rat α1-inhibitor-3 (α1 I3). When employing NH3 as the nucleophile, the thiol ester in α1I3 re-formed spontaneously at room temperature after gel filtration to remove excess nucleophile, and an active proteinase inhibitor was regained. When CH3NH2 was employed as the nucleophile, thiol ester reversibility was more energy-demanding. With either nucleophile, α2M once inactivated did not regain proteinase-inhibitory capacity at room temperature. At elevated temperatures, however, the reaction between α2M and NH3 or CH3NH2 was reversible and the inhibitory capacity could be recovered. Modification of the cysteinyl groups from the thiol ester prevented its re-formation but did not prevent the heat-induced retrieval of inhibitory capacity, suggesting that conformational features rather than the thiol ester are essential for α2M to function as an inhibitor. As demonstrated by non-denaturing PAGE, the conformation of native α2M is restored when the proteinase-inhibitory capacity is recovered.

scholarly journals Purification and characterization of human brain prolyl endopeptidase

1991 ◽  
Vol 276 (1) ◽  
pp. 237-244 ◽  
Author(s):  
S Kalwant ◽  
A G Porter
Keyword(s):  
Human Brain ◽  
Proteinase Inhibitors ◽  
Polyclonal Antibodies ◽  
Deae Cellulose ◽  
Serine Proteinase ◽  
Prolyl Endopeptidase ◽  
Blue Staining ◽  
Coomassie Blue Staining ◽  
Coomassie Blue ◽  
Sds Page

Prolyl endopeptidase (EC 3.4.21.26) was purified from human brain by a series of column-chromatographic steps using DEAE-cellulose DE-52, hydroxyapatite, phenyl-Sepharose, Sephacryl S-200 and f.p.l.c. (Mono Q). The enzyme was purified by a factor of 943 and was homogeneous in a SDS/polyacrylamide gel as judged by Coomassie Blue staining. The Mr estimated by SDS/PAGE is 79,600, and under native conditions on Sephacryl S-200 it is 85,600. Therefore the enzyme exists as a monomer. With benzyloxycarbonylglycylproline p-nitroanilide as substrate, the optimum pH of the enzyme is 6.8, and with the substrate concentration between 0.059 mM and 0.37 mM the Km is 9.0 x 10(-4) M. The pI of the enzyme is 4.75. The enzyme is classified as a serine proteinase, as it is strongly inhibited by di-isopropyl fluorophosphate. However, other serine proteinase inhibitors do not inhibit the enzyme significantly, suggesting that the active site of prolyl endopeptidase differs from that of classical serine proteinases such as trypsin. Polyclonal antibodies were raised against purified human brain prolyl endopeptidase in rabbits. Western-blot analysis, enzyme-inhibition assays, antibody binding and immunoprecipitation experiments indicated that the polyclonal antibodies are both specific and inhibitory to the enzyme activity.

scholarly journals Human mast cell tryptase isoforms: separation and examination of substrate-specificity differences

1995 ◽  
Vol 307 (2) ◽  
pp. 341-346 ◽  
Author(s):  
S S Little ◽  
D A Johnson
Keyword(s):  
Mast Cell ◽  
Molecular Mass ◽  
Peptide Mapping ◽  
Proteinase Inhibitors ◽  
High Molecular Mass ◽  
Mass Separation ◽  
Human Mast Cell ◽  
Mast Cell Tryptase ◽  
Sds Page ◽  
Mass Form

Tryptases are trypsin-like enzymes found in mast cell granules that appear to exist as tetramers. These enzymes are not controlled by blood plasma proteinase inhibitors and only cleave a few physiological substrates in vitro, including high-molecular-mass kininogen (HMMK) and vasoactive intestinal peptide (VIP). Purified human lung mast cell tryptase (HLT) contained two bands of approx. molecular mass 29 and 33 kDa on SDS/PAGE. These two forms of HLT have been separated by chromatography on a cellulose phosphate column, with the high-molecular-mass form (high-HLT) being eluted with 10 microM heparin and the low-molecular-mass form (low-HLT) subsequently eluted with 1 M NaCl. Removal of asparagine-linked carbohydrate caused both isoforms to run as single sharp bands on SDS/PAGE, differing slightly in molecular mass. Separation of these two isoforms of tryptase shows that tetramers consist of four homologous subunits rather than mixtures of the two isoforms. Using HMMK and VIP as substrates, these two forms of HLT were found to differ with regard to specificity and rate of cleavage. High-HLT initially cleaved HMMK at Arg-431 within the C-terminal anionic binding region of the molecule, whereas low-HLT cleaved HMMK simultaneously at multiple sites within the C-terminal portion of the molecule. On the basis of HPLC peptide mapping, each isoform also cleaved VIP at different sites. Comparison of cleavage rates based on the active-site concentrations of titrated isoforms showed that low-HLT cleaved HMMK more rapidly than did high-HLT. These two isoforms may represent different gene products or they may result from post-translational modification.

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