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.

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.

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.

A preliminary characterization of alkaline digestive proteases in the posterior midgut of the face fly, Musca autumnalis De Geer

1987 ◽  
Vol 65 (3) ◽  
pp. 635-639 ◽  
Author(s):  
F. C. Campbell ◽  
Jon G. Houseman ◽  
P. E. Morrison
Keyword(s):  
Aminopeptidase Activity ◽  
Tetraacetic Acid ◽  
Carboxypeptidase A ◽  
Phenyllactic Acid ◽  
Carboxypeptidase B ◽  
Posterior Midgut ◽  
The Face ◽  
Musca Autumnalis ◽  
Hydrolysis Of

Alkaline proteases in the posterior midgut of the face fly, Musca autumnalis De Geer, were identified using specific synthetic substrates and nonspecific substrate azocasein. Identification was confirmed with potential protease activators and inhibitors. Optimal azocasein hydrolysis occurred at pH 8.0 and substrate hydrolysis was inhibited by EGTA (ethyleneglycol-bis-(2-aminoethyl ether)-N,N-tetraacetic acid), tosyl-L-lysine chloromethyl ketone (TLCK), and soybean trypsin inhibitor (STI). Tryptic proteolysis was identified by maximal hydrolysis, at pH 8.0, of trypsin specific substrates BAPNA (benzoyl-DL-arginine-p-nitroanilide) and TAME (tosyl-L-arginine methyl ester). TAME hydrolysis was inhibited by PMSF (phenylmethylsulfonyl fluoride), STI, pepstatin A, and dithiothreitol (DTT), but BAPNA hydrolysis was inhibited only by STI and DTT. Chymotrypsin was demonstrated by maximal hydrolysis of BTEE (benzoyl-L-tyrosine ethyl ester) at pH 8.0 and DTT, PMSF, and STI inhibited hydrolysis of this substrate. Aminopeptidase activity was demonstrated by maximal hydrolysis of leucine-p-nitro-anilide at pH 8.0 and the peptidase was inhibited by o-phenanthroline. Carboxypeptidase A-like enzyme hydrolyzed hippuryl-DL-phenyllactic acid maximally at pH 7.5 and carboxypeptidase B showed maximum hydrolysis of hippuryl-L-arginine at pH 7.0. Both carboxypeptidases were inhibited by EDTA (ethylene diamine tetraacetic acid), EGTA, and DTT and carboxypeptidase A was also inhibited by PMSF.

Quantification of small-scale heterogeneity in aquatic aminopeptidase activity

2020 ◽  
Vol 84 ◽  
pp. 127-140
Author(s):  
BM Gaas ◽  
JW Ammerman
Keyword(s):  
Chlorophyll Fluorescence ◽  
Leucine Aminopeptidase ◽  
Hudson River ◽  
Aminopeptidase Activity ◽  
Small Scale ◽  
Analysis Instrument ◽  
Sequential Samples ◽  
Degree Of Confidence ◽  
The Mean ◽  
Hydrolysis Of

Leucine aminopeptidase (LAP) is one of the enzymes involved in the hydrolysis of peptides, and is sometimes used to indicate potential nitrogen limitation in microbes. Small-scale variability has the potential to confound interpretation of underlying patterns in LAP activity in time or space. An automated flow-injection analysis instrument was used to address the small-scale variability of LAP activity within contiguous regions of the Hudson River plume (New Jersey, USA). LAP activity had a coefficient of variation (CV) of ca. 0.5 with occasional values above 1.0. The mean CVs for other biological parameters—chlorophyll fluorescence and nitrate concentration—were similar, and were much lower for salinity. LAP activity changed by an average of 35 nmol l-1 h-1 at different salinities, and variations in LAP activity were higher crossing region boundaries than within a region. Differences in LAP activity were ±100 nmol l-1 h-1 between sequential samples spaced <10 m apart. Variogram analysis indicated an inherent spatial variability of 52 nmol l-1 h-1 throughout the study area. Large changes in LAP activity were often associated with small changes in salinity and chlorophyll fluorescence, and were sensitive to the sampling frequency. This study concludes that LAP measurements in a sample could realistically be expected to range from zero to twice the average, and changes between areas or times should be at least 2-fold to have some degree of confidence that apparent patterns (or lack thereof) in activity are real.

scholarly journals Determination of digestive proteolytic profile in the larvae of Dyspessa palidata (Staudinger) (Lepidoptera: Cossidae)

2015 ◽  
Vol 47 (3) ◽  
pp. 91
Author(s):  
M. Mardani-Talaee ◽  
A. Zibaee
Keyword(s):  
Proteolytic Activity ◽  
Cathepsin B ◽  
Posterior Midgut ◽  
Ph Dependency ◽  
Metalloproteinase Inhibitors ◽  
Optimal Value ◽  
Important Pest ◽  
Proteolytic Activities ◽  
Membrane Bound

Digestive proteolytic profile was determined in the larvae of <em>Dyspessa palidata</em> (Staudinger), which is the most important pest of Alliaceae in Europe and Iran. Compartmentalisation of the proteolytic activities by considering soluble and membrane-bound fractions revealed that soluble fractions of the whole midgut preparations had higher general proteolytic activity than membrane-bound fractions. Also, four proteolytic bands were observed in the soluble fraction of the total midgut preparation in electrophoresis. Compartmentalisation of the specific proteases revealed presence of trypsin, elastase, aminoand carboxy peptidases in posterior midgut but the highest activities of other proteases were found in anterior midgut. The highest activity of general protease was found at pHs of 6 and 8. Also, pH dependency of trypsin, chymotrypsin and elastase were found at values of 8, 7-8 and 9 but cathepsins had the optimal pH at 6. Exopeptidases showed the optimal value at pH of 7 although carboxypeptidase showed same activity at values of 6 and 7. The inhibitory concentrations 50% (IC<sub>50</sub>) of AEBSF.HCL on trypsin, chymotrypsin and elastase proteases were found to be 3.69, 3.31 and 4.09 mM, respectively. IC<sub>50</sub> concentrations of TLCK, SBTI and TPCK significantly inhibited trypsin and chymotrypsin activities. IC<sub>50</sub> of E-64 were 3.67 and 4.16 mM on cathepsin B and L but cystatin revealed 5.22 and 4.48 mM concentrations on cathepsin B and L, respectively. EDTA and phenathroline as metalloproteinase inhibitors had IC<sub>50</sub> of 3.25 and 3.91 mM on general proteolytic activity. Results of the current study revealed larvae of <em>D. palidata</em> utilised different proteases to increase digestive efficiency when they fed on the host plants containing several toxic molecules.

Confocal Analysis of the Distribution and Persistence of Sindbis Virus (TaV-GFP) Infection in Midguts of Aedes aegypti Mosquitoes

2020 ◽  
Vol 26 (2) ◽  
pp. 267-274
Author(s):  
Jason J. Saredy ◽  
Florence Y. Chim ◽  
Zoë L. Lyski ◽  
Yani P. Ahearn ◽  
Doria F. Bowers
Keyword(s):  
Aedes Aegypti ◽  
Sindbis Virus ◽  
Fluorescent Protein ◽  
Blood Feeding ◽  
Target Tissue ◽  
Secondary Target ◽  
Posterior Midgut ◽  
Infected Cells ◽  
Green Fluorescent ◽  
Global Threat

AbstractBiological transmission of arthropod-borne viruses (arboviruses) to vertebrate hosts by hematophagous insects poses a global threat because such arboviruses can result in a range of serious public health infectious diseases. Sindbis virus (SINV), the prototype Alphavirus, was used to track infections in the posterior midgut (PMG) of Aedes aegypti adult mosquitoes. Females were fed viremic blood containing a virus reporter, SINV [Thosea asigna virus-green fluorescent protein (TaV-GFP)], that leaves a fluorescent signal in infected cells. We assessed whole-mount PMGs to identify primary foci, secondary target tissues, distribution, and virus persistence. Following a viremic blood meal, PMGs were dissected and analyzed at various days of post blood-feeding. We report that virus foci indicated by GFP in midgut epithelial cells resulted in a 9.8% PMG infection and a 10.8% dissemination from these infected guts. The number of virus foci ranged from 1 to 3 per individual PMG and was more prevalent in the PMG-middle > PMG-frontal > PMG-caudal regions. SINV TaV-GFP was first observed in the PMG (primary target tissue) at 3 days post blood-feeding, was sequestered in circumscribed foci, replicated in PMG peristaltic muscles (secondary target tissue) following dissemination, and GFP was observed to persist in PMGs for 30 days postinfection.

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.

scholarly journals Cathepsin B, the lysosomal thiol proteinase of calf liver

1969 ◽  
Vol 114 (4) ◽  
pp. 673-678 ◽  
Author(s):  
O. Snellman
Keyword(s):  
Cathepsin B ◽  
Ph Dependence ◽  
Gel Filtration ◽  
Chelating Agent ◽  
Thiol Group ◽  
Maximum Activity ◽  
Active Centre ◽  
Peptide Bonds ◽  
Hydrolysis Of ◽  
Thiol Proteinase

Cathepsin B from calf liver was obtained by a method involving preparation of a lysosomal–mitochondrial pellet and treatment of this pellet with acetone. The material was extracted with an acid buffer, pH4·0, and then precipitated from the extract with acetone. The precipitate was dissolved in phosphate buffer, pH7·4, and subjected to gel filtration on Sephadex G-200 and G-100. The cathepsin B emerged in a range of molecular weight much lower than 50000 as a well-defined component. The purity of this material was checked by electrophoresis. To obtain maximum activity the enzyme had to be activated with a chelating agent and a reducing agent (i.e. EDTA and cysteine). A number of different substrates were used. The enzyme was active for the hydrolysis of both peptide bonds and ester bonds and had approximately equal reactivity in the two cases. The pH-dependence of the hydrolysis was the same with both substrates. The binding of the substrates was half-maximal at pH4·5 and at pH6·8. A thiol group occurred in the active centre but this group ought to have a much higher pK than that found in this enzyme.

Ultrastructural changes in posterior midgut cells associated with blood feeding in adult female Rhodnius prolixus Stal (Hemiptera: Reduviidae)

1983 ◽  
Vol 61 (11) ◽  
pp. 2574-2586 ◽  
Author(s):  
P. F. Billingsley ◽  
A. E. R. Downe
Keyword(s):  
Endoplasmic Reticulum ◽  
Rough Endoplasmic Reticulum ◽  
Blood Meal ◽  
Apical Cell ◽  
Blood Feeding ◽  
Rhodnius Prolixus ◽  
Ultrastructural Changes ◽  
Dense Matrix ◽  
Apical Surface ◽  
Posterior Midgut

Modifications of posterior midgut cells of Rhodnius prolixus following a meal of rabbit blood are described. Prominent stacks and whorls of rough endoplasmic reticulum become redistributed following a blood meal but later reform during the postfeeding period. Lysosomes undergo internal structural changes and apparent fluctuations in their number per cell as a result of blood meal ingestion. Before blood feeding, the apical surface of the midgut cells show a typical arrangement of a plasma membrane covered on the lumenal surface by a glycocalyx. After a blood meal, a more complex organisation appears, consisting of two plasma membranes separated by an electron-dense matrix. The lumenal apical membrane proliferates during the digestion period to form loosely organised extracellular membrane layers which may function as a peritrophic membrane. Changes in the rough endoplasmic reticulum and lysosomes and modifications to the apical cell surface appear to coincide with previously described proteinase activity cycles in the posterior midgut of R. prolixus. The implications of these results are discussed and are compared with similar ultrastructural events from haematophagous Diptera.

scholarly journals Human kidney cathepsins B and L. Characterization and potential role in degradation of glomerular basement membrane

1988 ◽  
Vol 252 (1) ◽  
pp. 301-304 ◽  
Author(s):  
W H Baricos ◽  
Y Zhou ◽  
R W Mason ◽  
A J Barrett
Keyword(s):  
Basement Membrane ◽  
Glomerular Basement Membrane ◽  
Cathepsin B ◽  
Cathepsin L ◽  
Polyacrylamide Gel Electrophoresis ◽  
Human Kidney ◽  
Ph Optimum ◽  
Dose Dependent ◽  
Hydrolysis Of

Cathepsins B and L were purified from human kidney. SDS/polyacrylamide-gel electrophoresis demonstrated that cathepsins B and L, Mr 27000-30000, consist of disulphide-linked dimers, subunit Mr values 22000-25000 and 5000-7000. The pH optimum for the hydrolysis of methylcoumarylamide (-NHMec) substrates (see below) is approx. 6.0 for each enzyme. Km and kcat. are 252 microM and 364s-1 and 2.2 microM and 25.8 s-1 for the hydrolysis of Z-Phe-Arg-NHMec (where Z- represents benzyloxycarbonyl-) by cathepsins B and L respectively, and 184 microM and 158 s-1 for the hydrolysis of Z-Arg-Arg-NHMec by cathepsin B. A 10 min preincubation of cathepsin B (40 degrees C) or cathepsin L (30 degrees C) with E-64 (2.5 microM) results in complete inhibition. Under identical conditions Z-Phe-Phe-CHN2 (0.56 microM) completely inhibits cathepsin L but has little effect on cathepsin B. Incubation of glomerular basement membrane (GBM) with purified human kidney cathepsin L resulted in dose-dependent (10-40 nM) GBM degradation. In contrast, little degradation of GBM (less than 4.0%) was observed with cathepsin B. The pH optimum for GBM degradation by cathepsin L was 3.5. Cathepsin L was significantly more active in degrading GBM than was pancreatic elastase, trypsin or bacterial collagenase. These data suggest that cathepsin L may participate in the lysosomal degradation of GBM associated with normal GBM turnover in vivo.

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