scholarly journals Identification and characterization of aminopeptidases from Aplysia californica

1992 ◽  
Vol 286 (3) ◽  
pp. 967-975 ◽  
Author(s):  
W Bawab ◽  
E Querido ◽  
P Crine ◽  
L DesGroseillers
Keyword(s):  
Enzyme Inhibitor ◽  
Aplysia Californica ◽  
Chelating Agent ◽  
Neutral Endopeptidase ◽  
Aminopeptidase Activity ◽  
Bivalent Cation ◽  
Enzyme Preparations ◽  
Membrane Bound ◽  
Hydrolysis Of

Aminopeptidase activities were identified in extracts of kidney, ovotestis, head ganglia, heart and haemolymph of Aplysia californica. These enzyme preparations hydrolysed [3H][Leu]enkephalin at the Try-1-Gly-2 bond as determined by h.p.l.c. analysis of cleavage products. In all these tissues, enkephalin-degrading aminopeptidase activities were present both in membrane-bound and cytosolic fractions. The bivalent-cation-chelating agent, 1,10-phenanthroline, inhibited kidney membrane aminopeptidase activity with an IC50 of 30 microM, suggesting that this enzyme is a metalloproteinase. The aminopeptidase inhibitor amastatin was the most potent inhibitor of [Leu]enkephalin degradation (IC50 25 nM) by membrane-bound aminopeptidase, and bacitracin, bestatin and puromycin were about 100-1000 times less potent. In contrast with membrane-bound aminopeptidase, the cytosolic form is sensitive to puromycin. Angiotensin-converting enzyme inhibitor had no effect on [Leu]enkephalin degradation by kidney membranes, while the neutral endopeptidase inhibitors were poor inhibitors of the enzymes in this preparation. The Km values of the aminopeptidase in the kidney membranes and cytosolic fractions for the [Leu]enkephalin substrate were 2.4 and 7.4 microM respectively. The aminopeptidase present in the kidney membranes also hydrolysed endogenous Phe-Met-Arg-Phe-amide peptide at the Phe-1-Met-2 bond as well as synthetic alanine p-nitroanilide and leucine p-nitroanilide. When used in a competition assay, these substrates inhibited hydrolysis of [3H][Leu]enkephalin, suggesting that the same enzyme degraded all these substrates. Taken together, these results suggest that Aplysia tissues contain both a membrane-bound aminopeptidase related to the mammalian aminopeptidase N and a cytosolic puromycin-sensitive aminopeptidase.

scholarly journals Identification and characterization of a neutral endopeptidase activity in Aplysia californica

1993 ◽  
Vol 296 (2) ◽  
pp. 459-465 ◽  
Author(s):  
W Bawab ◽  
R S Aloyz ◽  
P Crine ◽  
B P Roques ◽  
L DesGroseillers
Keyword(s):  
Plasma Membranes ◽  
Biochemical Characterization ◽  
Enzyme Inhibitor ◽  
Molecular Form ◽  
Aplysia Californica ◽  
Neutral Endopeptidase ◽  
Bivalent Cation ◽  
Endopeptidase 24.11 ◽  
Endopeptidase Activity

Kidney plasma membranes of Aplysia californica were shown to contain an endopeptidase activity which cleaved [Leu]enkephalin (Tyr-Gly-Gly-Phe-Leu) and [Leu]enkephalinamide (Tyr-Gly-Gly-Phe-Leu-NH2) at the Gly3-Phe4 bond, as determined by reverse-phase h.p.l.c. analysis of metabolites. The optimal pH was shown to be 6.5. The bivalent cation chelating agent, 1,10-phenanthroline protected [Leu]enkephalin from degradation, suggesting that this enzyme is a metallopeptidase. The degradation of [Leu]enkephalin was also abolished by the neutral endopeptidase-24.11 inhibitors RB104 (2-[(3-iodo-4-hydroxyl)-phenylmethyl]-4-N-[3-(hydroxyamino-3-oxo-1- phenylmethyl)-propyl]amino-4-oxobutanoic acid), HABCO-Gly [(3-hydroxy-aminocarbonyl-2-benzyl-1-oxypropyl)glycine], phosphoramidon and thiorphan, with IC50 values of 1 nM, 1 microM, 20 microM and 30 microM respectively. By contrast, the angiotensin-converting enzyme inhibitor captopril and the serine proteinase inhibitor phenylmethanesulphonyl fluoride were without effect. Phase separation experiments using Triton X-114 showed that about 64% of the neutral endopeptidase activity in the Aplysia kidney membrane corresponds to an integral membrane protein. A specific radioiodinated inhibitor ([125I]RB104) was shown to bind the Aplysia endopeptidase with high affinity; the KD and Bmax. values were 21 +/- 5 pM and 20.3 +/- 5 fmol/mg of proteins respectively. This inhibitor was used to determine the molecular form of the enzyme, after separation of solubilized membrane proteins on SDS/PAGE and transfer on to nitrocellulose membranes. A single protein band with an apparent molecular mass of 140 kDa was observed. The labelling was abolished by specific neutral endopeptidase inhibitors. This study provides the first biochemical characterization of an endopeptidase with catalytic properties similar to those of neutral endopeptidase-24.11 in the mollusc Aplysia californica.

Characterization of lactococci and lactobacilli isolated from semihard goats' cheese

1991 ◽  
Vol 58 (1) ◽  
pp. 137-145 ◽  
Author(s):  
Teresa Requena ◽  
Carmen Peláez ◽  
Michel J. Desmazeaud
Keyword(s):  
Leucine Aminopeptidase ◽  
Sodium Dodecyl ◽  
Skim Milk ◽  
Titratable Acidity ◽  
Aminopeptidase Activity ◽  
Whole Cells ◽  
Alanine Aminopeptidase ◽  
Triton X ◽  
Hydrolysis Of

SummarySeveral strains ofLactococcus lactissubsp.lactis, Lactobacillus caseiandLactobacillus plantarumisolated from traditional goats' cheese have been studied for titratable acidity, proteolysis in milk and enzymic activities. Aminopeptidasc activities were measured with whole cells and cells permeabilized with Triton X-100. Caseinolytic activity was investigated using electrophoresis in polyacrylamide gel with sodium dodecyl sulphate.Lc. lactissubsp.lactishad a level of proteolytic activity in skim milk greater than that ofLb. casei, while this activity inLb. plantarumwas very low. Alanine aminopeptidase activity was almost non-existent for all strains tested, while lysine aminopeptidase activity appeared to be of fundamentally intracellular origin. Leucine aminopeptidase activity was also greater in cells that had been permeabilized than in whole cells forLb. caseiandLb. plantarum. Lc. lactissubsp.lactisleucine aminopeptidase activity was greater in whole cells. No significant hydrolysis of casein was found withLb. caseiI FPL 725 andLb. plantarumIFPL 722 permeabilized with Triton X-100 after 24 h incubation with whole bovine casein.

scholarly journals Characterization of Proteolytic and Collagenolytic Enzymes from the Larvae of Lucilia cuprina, the Sheep Blowfly

1988 ◽  
Vol 41 (2) ◽  
pp. 269 ◽  
Author(s):  
VM Bowles ◽  
PR Carnegie ◽  
RM Sandeman
Keyword(s):  
Ethylenediaminetetraacetic Acid ◽  
Proteolytic Enzymes ◽  
Chelating Agent ◽  
Collagenolytic Activity ◽  
Enzyme Preparations ◽  
Molecular Weights ◽  
Highly Active ◽  
Wide Range ◽  
Secretory Enzyme

Isoelectric focusing was used to characterize proteolytic enzymes in homogenate and excretory-secretory preparations of the larvae of L. cuprina, the sheep blowfly. Zymogram overlays showed that the larvae produce a number of highly active proteases which have a wide range of isoelectric points and molecular weights. The alkaline and neutral pI proteases were inhibited by phenylmethyl-sulfonylfluoride, leupeptin and aprotinin; this indicated the presence of serine in the active site. Pepstatin and the metal chelating agent ethylenediaminetetraacetic acid had no effect Oil the activity of any of the proteases. Optimal pH for activity of the proteases was between 7 and 8. In addition, the proteases were found to be heat labile. Digestion of collagen fibrils confirmed the existence of collagenolytic activity in the excretory-secretory enzyme preparations. It is suggested that these enzymes may be involved in the nutrition of the larvae and in the pathogenesis of the lesion on the skin.

Characterization of Membrane-Bound Neuropeptide-Degrading Activities in Aplysia Californica

1993 ◽  
Vol 44 (3-4) ◽  
pp. 451-462
Author(s):  
Philippe Crine ◽  
Luc Desgroseillers ◽  
Raquel S. Aloyz ◽  
Wafa Bawab
Keyword(s):  
Aplysia Californica ◽  
Membrane Bound

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.

Purification and characterization of a membrane-bound neutral endopeptidase from human placenta

1987 ◽  
Vol 37 (1) ◽  
pp. 22-30 ◽  
Author(s):  
Yasuhiro Watanabe ◽  
Yuka Kumagai ◽  
Yoshimitsu Shimamori ◽  
Yukio Fujimoto
Keyword(s):  
Human Placenta ◽  
Neutral Endopeptidase ◽  
Purification And Characterization ◽  
Membrane Bound

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 Pretreatment of Switchgrass for Production of Glucose via Sulfonic Acid-Impregnated Activated Carbon

Processes ◽  
2021 ◽  
Vol 9 (3) ◽  
pp. 504
Author(s):  
Yane Ansanay ◽  
Praveen Kolar ◽  
Ratna Sharma-Shivappa ◽  
Jay Cheng ◽  
Consuelo Arellano
Keyword(s):  
Activated Carbon ◽  
Sulfonic Acid ◽  
Solid Acid ◽  
Methanesulfonic Acid ◽  
Biofuel Production ◽  
Acid Catalysts ◽  
Effects Of Temperature ◽  
Carbon Catalysts ◽  
Hydrolysis Of

In the present research, activated carbon-supported sulfonic acid catalysts were synthesized and tested as pretreatment agents for the conversion of switchgrass into glucose. The catalysts were synthesized by reacting sulfuric acid, methanesulfonic acid, and p-toluenesulfonic acid with activated carbon. The characterization of catalysts suggested an increase in surface acidities, while surface area and pore volumes decreased because of sulfonation. Batch experiments were performed in 125 mL serum bottles to investigate the effects of temperature (30, 60, and 90 °C), reaction time (90 and 120 min) on the yields of glucose. Enzymatic hydrolysis of pretreated switchgrass using Ctec2 yielded up to 57.13% glucose. Durability tests indicated that sulfonic solid-impregnated carbon catalysts were able to maintain activity even after three cycles. From the results obtained, the solid acid catalysts appear to serve as effective pretreatment agents and can potentially reduce the use of conventional liquid acids and bases in biomass-into-biofuel production.

Sign in / Sign up

Export Citation Format

Share Document