Cathepsin B and aminopeptidase in the posterior midgut of Phymata wolffii Stål (Hemiptera: Phymatidae)

1985 ◽  
Vol 63 (6) ◽  
pp. 1288-1291 ◽  
Author(s):  
Jon G. Houseman ◽  
P. E. Morrison ◽  
A. E. R. Downe
Keyword(s):  
Molecular Weight ◽  
Trypsin Inhibitor ◽  
Cathepsin B ◽  
Ethylenediaminetetraacetic Acid ◽  
Soybean Trypsin Inhibitor ◽  
Aminopeptidase Activity ◽  
Chloromethyl Ketone ◽  
Posterior Midgut ◽  
Hydrolysis Of

The posterior midgut of the phymatid Phymata wolffii Stål contains cathepsin B and aminopeptidase activity. Identification of cathepsin B was based on maximal hydrolysis of benzoyl-DL-arginine-2-naphthylamide and benzoyl-DL-arginine-p-nitroanilide at pH 5.8 and 5.5, respectively. Cathepsin B hydrolysis of the tested substrates was activated by thiol chemicals and ethylenediaminetetraacetic acid (EDTA) and inhibited by tosyl-L-lysine chloromethyl ketone, iodoacetamide, and soybean trypsin inhibitor. Aminopeptidase hydrolyzed leucine-p-nitroanilide maximally at pH 7.8 and hydrolysis of the substrate was activated by magnesium and inhibited by EDTA, dithiothreitol, glutathione, and cysteine. The molecular weight of cathepsin B was 40 000 and was greater than 150 000 for aminopeptidase.

CATHEPSIN B AND AMINOPEPTIDASE ACTIVITY IN THE POSTERIOR MIDGUT OF EUSCHISTUS EUSCHISTOIDES (HEMIPTERA: PENTATOMIDAE)

1984 ◽  
Vol 116 (10) ◽  
pp. 1393-1396 ◽  
Author(s):  
Jon G. Houseman ◽  
W. K. MacNaughton ◽  
A. E. R. Downe
Keyword(s):  
Cathepsin B ◽  
Blood Feeding ◽  
Aminopeptidase Activity ◽  
Posterior Midgut ◽  
Hydrolysis Of

AbstractThe posterior midgut of a seed-feeding pentatomid, Euschistus euschistoides (Vollenhoven), contains the proteinases cathepsin B and aminopeptidase. Cadiepsin B hydrolysis of benzoyl-DL-arginine-2-naphthylamide is activated by thiol chemicals and EDTA. Aminopeptidase hydrolysis of leucine-p-nitroanilide is activated by MgCl2 and inhibited by cysteine, glutathione, EDTA, and CaCl2. These results are similar to those obtained for cathepsin B and aminopeptidase from blood-feeding Hemiptera and support the hypothesis that catheptic proteinases are unique to this order.

Partial purification and characterization of cysteine proteinases in eccrine sweat

1987 ◽  
Vol 252 (6) ◽  
pp. R1119-R1129
Author(s):  
H. Yokozeki ◽  
T. Hibino ◽  
K. Sato
Keyword(s):  
Concanavalin A ◽  
Cathepsin B ◽  
Ethylenediaminetetraacetic Acid ◽  
Sweat Glands ◽  
Partial Purification ◽  
Aminopeptidase Activity ◽  
Specific Substrate ◽  
Cysteine Proteinases ◽  
Eccrine Sweat

Attempts were made to purify and characterize cysteine proteinases in human eccrine sweat and further clarify their origin. Benzoyl-DL-arginine-beta-naphthylamide (BANA) and L-leucine beta-naphthylamide (LeuNA) hydrolases in thermally induced sweat were sequentially purified by Sephacryl S-200 chromatography and chromatofocusing, which yielded two major peaks of BANA hydrolase activity, BANA-I and BANA-II. Both enzymes are cysteine proteinases as evidenced by stimulation of enzymic activity by dithiothreitol and ethylenediaminetetraacetic acid and its inhibition by iodoacetic acid, (PCMB), and trans-epoxysuccinyl-L-leucylamido-(4-guanidino)-butane (E-64). Unlike BANA-II, BANA-I showed an additional aminopeptidase activity, an affinity to concanavalin A-Sepharose but no affinity to organomercurial sepharose and failed to hydrolyze benzyloxycarbonyl-phenylalanyl-arginine 4-methyl 7-coumarylamide (Z-Phe-Arg-NMec), a specific substrate for cathepsin B, which is poorly sensitive to leupeptin [inhibitor constant (Ki) = 1 X 10(-5) M] and relatively heat resistant. These and other characteristics such as its isoelectric points (PI) (= 5.8) and the Km for Arg-NMec (0.1 mM) and BANA (0.71 mM) all support the possibility that BANA-I is closely related to cathepsin H. In contrast, BANA-II is sensitive to Zn2+, leupeptin (Ki = 5.5 X 10(-9) M), is not adsorbed by concanavalin A- (Con-A)Sepharose, but is bound to organomercurial sepharose. It has a specificity to Z-Phe-Arg-NMec but not to Arg-NMec, has the molecular weight of 27, PI of 5.2, the pH optima for BANA (6.0), and the Km for BANA of 3.3 mM and the Km for Z-Phe-Arg-NMec of 0.1 mM. These features resemble those of liver cathepsin B. Leupeptin-sensitive BANA hydrolase was observed in the glandular extract of isolated sweat glands, which was increased after stimulation with methacholine and isoproterenol in vitro. The data are consistent with the notion that cathepsins B- and H-like enzymes are present in eccrine sweat and the former may be derived from the sweat gland.

Purification and characterization of trypsin from the Greenland cod (Gadus ogac). 1. Kinetic and thermodynamic characteristics

1984 ◽  
Vol 62 (9) ◽  
pp. 894-900 ◽  
Author(s):  
B. K. Simpson ◽  
N. F. Haard
Keyword(s):  
Trypsin Inhibitor ◽  
Polyacrylamide Gel Electrophoresis ◽  
Thermodynamic Characteristics ◽  
Soybean Trypsin Inhibitor ◽  
Turnover Number ◽  
Bovine Trypsin ◽  
Hydrolytic Reaction ◽  
Entropy Of Activation ◽  
Sepharose 4B ◽  
Hydrolysis Of

Trypsinogen was isolated from the pyloric ceca of Greenland cod by ammonium sulfate fractionation followed by acetone precipitation, and the trypsin(ogen) thus obtained was purified by affinity chromatography on soybean trypsin inhibitor – Sepharose 4B. The purified trypsin migrated as a single zone during polyacrylamide gel electrophoresis and its identity as trypsin (EC 3,4.21.4) was established by its catalytic specificity for amide or ester bonds involving the carboxyl group of arginine, its sensitivity to serine protease inhibitors and soybean trypsin inhibitor, and its molecular weight of 23 500. With tosylarginine methyl ester (TAME) as substrate, the turnover number of the hydrolytic reaction was about three times greater for the cod trypsin than for bovine trypsin at 5 °C. The Michaelis–Menten constant (Km,app) for cod trypsin and TAME increased from 0.14 mM at 5 °C to 0.26 mM at 35 °C, while the Km,app for bovine trypsin – TAME was about 0.05 mM at all assay temperatures. The free energy of activation (ΔG*) for the hydrolysis of TAME was about 600 cal/mol (1 cal = 4.1868 J) lower for the cod trypsin than for bovine trypsin at 5 °C. The contribution of enthalpy of activation (ΔH*) and entropy of activation (ΔS*) to ΔG* differed considerably for the two enzymes. The "physiological efficiency" (Vmax/Km,app) of the two enzymes with TAME was similar at 5 °C, but was much greater for bovine trypsin than cod trypsin at warmer temperatures. With N-α-benzoylarginine-p-nitroanilide (BAPA) as substrate, the turnover number was about eight times greater for the cod trypsin at 25 °C. The Km,app for cod trypsin – BAPA increased from 1.67 mM at 25 °C to 1.84 mM at 35 °C, whereas the Km,app for bovine trypsin – BAPA decreased from 0.97 mM at 25 °C to 0.90 mM at 35 °C. The ΔG* for hydrolysis of BAPA was about 1800 cal/mol lower for cod trypsin than it was for bovine trypsin at 25 °C. Vmax/Km,app was three to four times greater for cod trypsin than for bovine trypsin at 25 and 35 °C. These results show that Greenland cod trypsin is a better catalyst than bovine trypsin at low temperatures and that catalysis by the fish trypsin is less responsive to temperature change than is catalysis by bovine trypsin.

Rapid Determination of Antifibrinolysin (Antiplasmin) in Plasma

1966 ◽  
Vol 15 (01/02) ◽  
pp. 273-283 ◽  
Author(s):  
L. B Nanninga ◽  
M. M Guest
Keyword(s):  
Molecular Weight ◽  
Trypsin Inhibitor ◽  
Rapid Determination ◽  
Soybean Trypsin Inhibitor

SummaryA nephelometric assay of antifibrinolysin in plasma is described. This assay is based on the inhibition of fibrinogenolysis. An equation is derived to calculate the antifibrinolysin content. Only the activity of the rapid acting antifibrinolysin is obtained. The values for antifibrinolysin are given in mol/L based on a molecular weight 57.000 (1). All values are based (standardized) on the inhibition of fibrinolysin by crystalline soybean trypsin inhibitor.

Evidence that hatching enzyme of the sea urchin Strongylocentrotus purpuratus is a chymotrypsin-like protease

1988 ◽  
Vol 66 (11) ◽  
pp. 1200-1209 ◽  
Author(s):  
Laura L. Post ◽  
Regina Schuel ◽  
Herbert Schuel
Keyword(s):  
Ethyl Ester ◽  
Proteolytic Activity ◽  
Sea Urchin ◽  
Trypsin Inhibitor ◽  
Strongylocentrotus Purpuratus ◽  
Soybean Trypsin Inhibitor ◽  
Chloromethyl Ketone ◽  
Hatching Enzyme ◽  
Sea Urchin Eggs ◽  
Chymotrypsin Inhibitors

The sea urchin blastula secretes a hatching enzyme (HE) that dissolves the fertilization envelope. HE was collected from the supernatant seawater of cultures of hatched Strongylocentrotus purpuratus blastulae, and concentrated 20 times by ultrafiltration. The proteolytic activity of HE using casein as substrate was inhibited by the chymotrypsin inhibitors, chymostatin and N-tosyl-L-phenylalanine chloromethyl ketone. The activity was not inhibited by inhibitors (antipain, elastatinal, pepstatin, phosphoramidon, soybean trypsin inhibitor, and Nα-p-tosyl-L-lysine chloromethyl ketone) of other types of proteases. HE did not hydrolyze the synthetic trypsin substrate, α-N-benzoyl-L-arginine ethyl ester, but did hydrolyze the synthetic substrate of chymotrypsin, N-benzoyl-L-tyrosine ethyl ester (BTEE). The BTEEase activity of HE was completely inhibited by the chymotrypsin inhibitors chymostatin and 2-nitro-4-carboxyphenyl N,N-diphenylcarbamate (NCDC). Chymostatin inhibited the natural hatching of sea urchin blastulae. Application of HE to freshly fertilized sea urchin eggs, 2 h after insemination, caused premature dispersal of the hardened fertilization envelope. Chymostatin and NCDC inhibited HE-induced lysis of the fertilization envelope, while inhibitors of other types of proteases were ineffective. These data suggest that sea urchin HE is a chymotrypsin-like protease we call "chymostrypsin."

Identification and partial characterization of digestive proteinases from two species of bedbug (Hemiptera: Cimicidae)

1982 ◽  
Vol 60 (8) ◽  
pp. 1837-1840 ◽  
Author(s):  
Jon G. Houseman ◽  
A. E. R. Downe
Keyword(s):  
Cathepsin B ◽  
Horse Serum ◽  
Cimex Lectularius ◽  
Aminopeptidase Activity ◽  
Blood Sucking ◽  
Digestive Proteinases ◽  
Cimex Hemipterus ◽  
Hydrolysis Of ◽  
Optimal Ph

The digestive midgut of Cimex hemipterus (Fabr.) and Cimex lectularius L. contains cathepsin B, aminopeptidase, and an acidic proteinase that hydrolyzes haemoglobin at an optimal pH of 3.4. Cathepsin B was demonstrated by hydrolysis of benzoyl-DL-arginine-β-napthylamine at an optimal pH of 5.4. Hydrolysis of the substrate was activated by thiol chemicals and EDTA and inhibited by iodoacetamide (IAA) and horse serum. Maximum aminopeptidasehydrolysis of leucine-p-nitroanilide occurred at pH 8.4. Aminopeptidase activity was activated by MgCl2 and inhibited by EDTA, thiol chemicals, and CaCl2. Only C. hemipterus aminopeptidase was inhibited by IAA. The digestive proteinases in bedbugs are similar to those reported for other blood-sucking hemipteran insects.

Two satisfactory methods for purification of human acrosin

1986 ◽  
Vol 64 (10) ◽  
pp. 1020-1024 ◽  
Author(s):  
Lisette Leyton ◽  
Alfredo De Ioannes ◽  
Horacio B. Croxatto ◽  
Elise J. Graham ◽  
John S. Elce
Keyword(s):  
Molecular Weight ◽  
Affinity Chromatography ◽  
Trypsin Inhibitor ◽  
Active Form ◽  
Human Sperm ◽  
The Other ◽  
Soybean Trypsin Inhibitor ◽  
Sperm Cells ◽  
Acid Extraction ◽  
Alternative Procedures

Acrosin has been purified from human sperm cells by two alternative procedures which give purer products and in higher yields than could be achieved previously. The products were characterized by their molecular weight, catalytic action, sensitivity to inhibitors, and reaction with a polyclonal anti-acrosin antibody. After acid extraction of the cells, one method involves removal of acrosin inhibitors by vacuum dialysis, followed by affinity chromatography on a soybean trypsin inhibitor (SBTI) column, and therefore requires that the acrosin be in an active form capable of binding to the inhibitor. The other method involves affinity chromatography on a column of a monoclonal anti-acrosin antibody (MAb) and can be used to provide either active or proenzyme forms of acrosin, by choice of extraction conditions and inclusion of appropriate inhibitors. The yield of human acrosin from the SBTI method was 104% and from the MAb column was 75%. It is hoped that these procedures will make the very scarce human acrosin more readily available for further study.

α-Glutamic acid-β-naphthylamidase from Neisseria catarrhalis

1969 ◽  
Vol 15 (11) ◽  
pp. 1293-1300 ◽  
Author(s):  
Sidney T. Cox ◽  
Francis J. Behal
Keyword(s):  
Molecular Weight ◽  
Amino Acid ◽  
Glutamic Acid ◽  
Metal Ion ◽  
Gel Filtration ◽  
Aminopeptidase Activity ◽  
Amino Acid Residues ◽  
Ph Optimum ◽  
Hydrolysis Of ◽  
Derivatives Of

A second bacterial peptidase-like enzyme, arylamidase-II, has been isolated from cell free extracts of Neisseria catarrhalis. Arylamidase-II action is limited primarily to the hydrolysis of α-glutamic acid and α-aspartic acid derivatives of β-naphthylamine and short peptides of glutamic acid. The enzyme was purified 450-fold by gel filtration, ion exchange, and calcium phosphate chromatography. Its pH optimum and molecular weight were 7.7 and 170 000, respectively. Aside from its restricted substrate specificity, arylamidase-II has been found to be closely related in its mechanism of action, molecular weight, pH optimum, and metal ion dependence to arylamidase-I, which catalyzes the hydrolysis of neutral amino acid derivatives of β-naphthylamine. Arylamidase-II exhibits aminopeptidase activity, requiring the amino acid residues in the N-terminal and penultimate position to be of the L-configuration in order for hydrolysis to occur.

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