Purification and partial characterization of an α-chymotrypsin-like protease of rat peritoneal mast cells

Everitt, Michael T.; Neurath, Hans

doi:10.1016/s0300-9084(79)80163-7

Cited by 24 publications

(17 citation statements)

References 46 publications

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“…There are numerous chymotrypsin-like proteinases suggested to have a physiological role in protein turnover and in tissue-localized angiotensin II formation: peritoneal mast cell proteinase I (41), skeletal muscle mast cell proteinase 11 (42), cytoplasmic mast cell chymase (43), salivary gland tonin (44,45), skin chymotrypsin-like proteinase (45,46), and neutrophil cathepsin G (28,(45)(46)(47)(48). * Although there are differences in the structural, functional, and catalytic activities of these chymotrypsin-like enzymes compared to chymotrypsin (41)(42)(43)(44)(45)(46)(47)(48), one of these proteinases may be inhibited by heparin cofactor II (with or without glycosaminoglycan) at a physiologically important rate. …”

Section: Resultsmentioning

confidence: 99%

Inhibition of chymotrypsin by heparin cofactor II.

Church¹,

Noyes²,

Griffith³

1985

Proc. Natl. Acad. Sci. U.S.A.

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Human heparin cofactor II is a plasma protein that is known to inhibit thrombin. We have recently determined that the reactive-site sequence (P1-P'l) in heparin cofactor II is Leu-Ser (9). Therefore, the reactive-site sequence in heparin cofactor II resembles that found in a1-antichymotrypsin (10) but not that in antithrombin III, which is Arg-Ser (11). Chymotrypsin Aa has a substrate specificity primarily directed toward P1 amino acid residues that have large or aromatic hydrophobic side chains (12). The present investigation was undertaken to determine whether or not heparin cofactor II inhibits chymotrypsin and if the reaction rate is enhanced by glycosaminoglycans. We report here that heparin cofactor II inhibits chymotrypsin more rapidly than thrombin, but the reaction rate is not affected by glycosaminoglycans. EXPERIMENTAL PROCEDURESMaterials. Human plasma heparin cofactor II was isolated as described (13). Chymotrypsin Aa, heparin (ammonium salt), dermatan sulfate, and succinyl-Ala-Ala-Pro-Phe-p-nitroanilide were obtained from Sigma. Nutritional Biochemicals supplied N-acetyl-a-azaphenylalanine p-nitrophenyl ester; Polybrene and 2,3-butanedione were purchased from Aldrich. All buffers used in this study contained 0.1% (wt/vol) polyethylene glycol (Mr = 8000) to limit protein adsorption to various surfaces.Enzyme and Inhibitor Assays. Chymotrypsin activity was measured by using succinyl-Ala-Ala-Pro-Phe-p-nitroanilide (14). Heparin cofactor II activity was determined by measuring the rate of chymotrypsin inhibition in the absence and presence of heparin or dermatan sulfate. Stock chymotrypsin solutions (100 AM) were dissolved in 1 mM HCl and kept at 4°C. Inhibition reactions were started by adding chymotrypsin (final concentration, -15-35 nM) to a solution containing heparin cofactor II (final concentration, about 0.05-2 ,uM) in 50 mM triethanolamine acetate, pH 8.0/100 mM NaCl. Portions (0.1 ml) were removed at intervals, added to a substrate solution (0.8 ml) containing 100 AM succinyl-AlaAla-Pro-Phe-p-nitroanilide in 50 mM triethanolamine acetate, pH 8.0/100 mM NaCl, and incubated at ambient temperature. Polybrene (0.5 mg/ml) was included in the substrate solution when inhibition assays were performed in the presence of heparin or dermatan sulfate. Substrate hydrolysis was terminated after a suitable time, usually 3-5 min, by the addition of glacial acetic acid (0.1 ml). pNitroaniline released during the assay was measured at 400 nm and the amount of substrate hydrolyzed was proportional to the chymotrypsin concentration. Antithrombin activity of heparin cofactor II was determined as described (13,15).Under the reaction conditions used in which heparin cofactor II concentration was in excess of the chymotrypsin concentration, apparent pseudo-first-order kinetics was followed. Under these circumstances, the rate of disappearance of free chymotrypsin (E) may be described by ln(E/Eo) = -k2(IO) t, [11 where the residual activity (%) is E/Eo at time t, I0 is the initial concentration of heparin cofac...

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Section: Resultsmentioning

confidence: 99%

Inhibition of chymotrypsin by heparin cofactor II.

Church¹,

Noyes²,

Griffith³

1985

Proc. Natl. Acad. Sci. U.S.A.

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“…In the 50 NH2-terminal residues, factor D and this mast cell-derived protease have 40% identity; the other proteases shown range from 28% to 34% identity. Other rat mast cell proteases recently described (27,28) also have approximately 40% identity with the NH2-terminal sequence of factor D. Based on homologies with the other proteases, position 41 would be expected to be a histidine residue. This histidine is analogous to histidine-57 of chymotrypsin and is required for the charge-relay system of serine proteases.…”

Section: Discussionmentioning

confidence: 98%

Active site amino acid sequence of human factor D.

Davis¹

1980

Proc. Natl. Acad. Sci. U.S.A.

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C3 and C5 (3-6). Factor B has recently been characterized as a serine protease (11). Factor D is thus required for alternative pathway activation (4,(12)(13)(14). Its activity is inhibited by diisopropyl phosphorofluoridate (iPr2P-F) (7-9), and its NH2-terminal amino acid sequence is homologous with the NH2-terminal sequences of the serine proteases (15, 16). However, this enzyme differs from other plasma proteases in several characteristics. Factor D probably circulates in its active proteolytic form. The data of Fearon et al. (7) suggest that a zymogen exists, but others suggest that it does not and that only the active proteolytic form is present in plasma (8). Factor.D has restricted substrate specificity. It probably does not cleave peptide bonds other than the susceptible bond in factor B (9). Among the synthetic substrates hydrolyzed by most serine proteases, factor D inefficiently hydrolyzes only the chromogenic substrate N-benzoylisoleucylglutamylglycylarginine-p-nitroanilide hydrochloride (9, 15). Although high concentrations of iPr2P-F inhibit factor D functional activity (7, 9), stoichiometric incorporation of iPr2P-F into the active site of factor D has not been demonstrated. In addition, factor D is not inhibited by the plasma protease inhibitors (9). Determination of the primary structure of factor D should clarify some of these unusual characteristics.The present study characterized the cyanogen bromide peptides of factor D and their NH2-terminal amino acid sequences, which includes the active site serine residue. MATERIALS AND METHODSFresh frozen human plasma was obtained from the American Red Cross Blood Services, Northeast Region. CM-Sephadex C-50 and Sephadex G-75 were purchased from Pharmacia. Hydroxylapatite (Bio-Gel HT) and NaDodSO4 were purchased from Bio-Rad. Amicon positive-pressure ultrafiltration cells and PM10 ultrafiltration membranes werepurchased from Amicon (Lexington, MA). Acrylamide, N,N'-rnethylenebisacrylamide, N,N,NlNl-tetramethylethylenediamine, iodoacetic acid, and CNBr were purchased from Eastman. Molecular weight markers for NaDodSO4/polyacrylamide gel electrophoresis were insulin, lysozyme, bovine trypsinogen, carbonic anhydrase, and ovalbumin, all obtained from Sigma. All sequencing reagents (5% phenylisothiocyanate in heptane, 0.1 M Quadrol buffer, and heptafluorobutyric acid) and solvents (benzene, ethyl acetate, and butyl chloride) were obtained from Pierce. Polybrene was also from Pierce.Protein Purification. Factor D was isolated from 4-8 liters of fresh frozen human plasma essentially as described (15) The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.

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“…To solve these discrepancies, we examined the effects of various protease inhibitors including some esters of ^«^-(guanidinomethyOcyclohexanecarboxylic acid (GMCHA) on tryptase and on chymase. Chymase was purified from rat mast cells by Yurt and Austen^7 1 , Everitt and Neurath [8] , and Okuno-Kaneda et al 191 . Tryptase was first purified by Schwartz et al llo] from human pulmonary mast cells, and recently, Kido et al [11] purified a tryptase from rat peritoneal mast cells.…”

Section: Tryptase In Mastzellen Der Ratte: Eigenschaften Und Hemmbarkmentioning

confidence: 99%