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.

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.

Affinity labeling the bovine gallbladder cholecystokinin receptor using a battery of probes

1988 ◽  
Vol 255 (5) ◽  
pp. G579-G586
Author(s):  
B. Schjoldager ◽  
S. P. Powers ◽  
L. J. Miller
Keyword(s):  
Plasma Membranes ◽  
Biochemical Characterization ◽  
Peptide Hormone ◽  
Molecular Probes ◽  
Affinity Labeling ◽  
Molecular Heterogeneity ◽  
Cck Receptors ◽  
Amino Terminal ◽  
Binding Subunit

Although the gallbladder was the first recognized target of the peptide hormone cholecystokinin (CCK) and is a physiologically important target, only one preliminary report of the biochemical characterization of this receptor exists. Recently, a series of molecular probes for the affinity labeling of different domains of the pancreatic CCK receptor have been developed. In this work we report the application of several of those probes toward the biochemical characterization of the bovine gallbladder muscularis receptor. These include "long" (125I-Bolton-Hunter-CCK-33) and "short" (125I-D-Tyr-Gly-[Nle28,31)CCK-(26-33)]) probes chemically cross-linkable through their amino-terminal amino groups and monofunctional probes with their photolabile moieties at their amino terminus (2-diazo-3,3,3-trifluoropropionyl-125I-D-Tyr-Gly-[(Nle28,31) CCK-(26-33)]) and carboxyl terminus (125I-D-Tyr-Gly-[(Nle28,31,pNO2-Phe33)CCK-(26-33)]), that span the receptor-binding region. Each of these bound specifically and saturably to a preparation enriched in plasma membranes from bovine gallbladder muscularis (mean inhibitor constants: 5.2, 1.1, 0.8, and 1.8 nM, respectively). A major relative molecular weight (Mr) 70,000-85,000 band was specifically and reproducibly labeled with the appropriate apparent affinity by each of the probes, whereas labeling of minor bands of Mr 40,000-50,000, Mr 92,000, Mr 120,000, and Mr 200,000 was dependent on cross-linker type or concentration. These observations support the identification of the Mr 70,000-85,000 protein as the bovine gallbladder CCK-binding subunit and, since this is a different size from the pancreatic CCK-binding subunit, provide biochemical evidence for molecular heterogeneity of peripheral CCK receptors.

scholarly journals The type 1 CD10/neutral endopeptidase 24.11 promoter: functional characterization of the 5′-untranslated region

2003 ◽  
Vol 123 (1) ◽  
pp. 177-183 ◽  
Author(s):  
Nobuo Sezaki ◽  
Fumihiko Ishimaru ◽  
Takayuki Tabayashi ◽  
Itaru Kataoka ◽  
Koichi Nakase ◽  
...  
Keyword(s):  
Untranslated Region ◽  
Functional Characterization ◽  
Neutral Endopeptidase ◽  
Endopeptidase 24.11

Biochemical characterization of GSK1070916, a potent and selective inhibitor of Aurora B and Aurora C kinases with an extremely long residence time1

2009 ◽  
Vol 420 (2) ◽  
pp. 259-265 ◽  
Author(s):  
Kelly Anderson ◽  
Zhihong Lai ◽  
Octerloney B. Mcdonald ◽  
J. Darren Stuart ◽  
Eldridge N. Nartey ◽  
...  
Keyword(s):  
Biochemical Characterization ◽  
Enzyme Inhibitor ◽  
Competitive Inhibitor ◽  
Tumour Regression ◽  
Aurora B ◽  
Aurora Kinases ◽  
Chromosome Alignment ◽  
Xenograft Models ◽  
Protein Enzyme

The Aurora kinases AurA, B and C are serine/threonine protein kinases that play essential roles in mitosis and cytokinesis. Among them, AurB is required for maintaining proper chromosome alignment, separation and segregation during mitosis, and regulating a number of critical processes involved in cytokinesis. AurB overexpression has been observed in a variety of cancer cell lines, and inhibition of AurB has been shown to induce tumour regression in mouse xenograft models. In the present study we report the enzymatic characterization of a potent and selective AurB/AurC inhibitor. GSK1070916 is a reversible and ATP-competitive inhibitor of the AurB–INCENP (inner centromere protein) enzyme. It selectively inhibits AurB–INCENP (Ki*=0.38±0.29 nM) and AurC–INCENP (Ki*=1.5±0.4 nM) over AurA–TPX2 (target protein for Xenopus kinesin-like protein 2) (Ki=490±60 nM). Inhibition of AurB–INCENP and AurC–INCENP is time-dependent, with an enzyme-inhibitor dissociation half-life of >480 min and 270±28 min respectively. The extremely slow rate of dissociation from the AurB and AurC enzymes distinguishes GSK1070916 from two other Aurora inhibitors in the clinic, AZD1152 and VX-680 (also known as MK-0457).

Immunogold labelling of neutral endopeptidase 24.11 (enkephalinase), on human neutrophils and neutrophil cytoplasts

1989 ◽  
Vol 47 ◽  
pp. 920-921
Author(s):  
V. Kriho ◽  
B. Wagner ◽  
E.G. Erdos ◽  
R.P. Becker
Keyword(s):  
Electron Microscopy ◽  
Plasma Membrane ◽  
Cell Surface ◽  
Plasma Membranes ◽  
Intact Cell ◽  
Neutral Endopeptidase ◽  
Immunogold Labelling ◽  
Human Neutrophils ◽  
Endopeptidase 24.11 ◽  
Cell Surface Structure

We have documented the presence of neutral endopeptidase 24.11 (NEP), on the surface of human neutrophils (PMN) and PMN cytoplasts. Cytoplasts are whole cell preparations which contain cytomatrix, but lack internal membranes and organelles ,such as nuclei and lysosomal granules. These structures have been extracted mechanically, leaving the plasma membrane “outside-out” topology intact. Cytoplasts are very useful in correlative studies of cell surface structure and function. Biochemically, the membrane component of cytoplasts is predominantly plasma membrane; structurally, chemical activity may be localized to domains of the intact cell surface. NEP is a membrane-bound metalloendopeptidase present in human PMN' s. We have marked NEP on the plasma membranes of PMNs and PMN cytoplasts via pre-embedding iramunocytochemistry. We used scanning electron microscopy (SEM) with backscattered electron imaging (BEI) to visualize Au labelled anti-NEP on the surface of a large number of cells. Transmission electron microscopy (TEM) was used to confirm the presence of the enzyme on PMN's and PMN cytoplasts.Suspensions of PMN or PMN cytoplasts (2 x 106 cells/ml) were fixed for 8 min at room temp. in 0.25% glutaraldehyde in phosphate buffered saline (PBS) pH 7.2 rinsed in PBS, treated with 0.1% glycine in PBS for 10 rain and then incubated for 15 min in 5% normal goat serum (NGS) in 0.1% bovine serum albumin dissolved in PBS (BSA/PBS). Following this step, cells were incubated for 20 min in anti- NEP antibody, rinsed in BSA/PBS, incubated in goat anti-rabbit IgG coupled to 15nm colloidal Au particles (GARG15) for 1 h and again rinsed in PBS. Postfixation for 30 min in 2.5% glutaraldehyde and PBS rinsing followed. For SEM a drop of cell suspension was put on a polylysine- treated Formvar-carbon-coated Au grid and cells were allowed to settle and attach for 30 min. The grid was rinsed in water, dehydrated and critical point dried. Cells were coated with carbon before viewing by SEM. For TEM, following immunolabelling, cells were post-fixed in OsO4, rinsed, dehydrated and embedded in Epon for sectioning.

scholarly journals Isolation and biochemical characterization of the α- and β-subunits of glycoprotein IIb of human platelet plasma membrane

1986 ◽  
Vol 240 (1) ◽  
pp. 155-161 ◽  
Author(s):  
J J Calvete ◽  
J González-Rodríguez
Keyword(s):  
Plasma Membrane ◽  
Plasma Membranes ◽  
Biochemical Characterization ◽  
Human Platelet ◽  
Size Exclusion ◽  
Beta Subunits ◽  
Exclusion Chromatography ◽  
Stepwise Reduction ◽  
Acidic Nature

The alpha- and beta-subunits of glycoprotein IIb (GPIIb) of human platelet plasma membrane were isolated in fully reduced, partially reduced and alkylated, and fully alkylated forms, by size-exclusion chromatography after reduction of pure GPIIb. The sugar moiety of GPIIb alpha accounts for 16.4% of its total weight, whereas that of GPIIb beta accounts for only 10.2%. The molar percentages (per 100 mol of total amino acids) of neuraminic acid and galactose in the alpha-subunit more than double those in the beta-subunit, whereas galactosamine is present only in GPIIb alpha. From the amino acid and sugar compositions the acidic nature of both subunits was confirmed. The Mr values obtained, 114,000 for GPIIb alpha and 22,200 for GPIIb beta, are in very good agreement with those obtained by physical methods. We found by stepwise reduction of pure GPIIb with dithioerythritol that GPIIb alpha and GPIIb beta are joined by a single interchain disulphide bridge, while the remaining half-cystine residues participate in intrachain bonds, six in GPIIb alpha and one in GPIIb beta, the intersubunit disulphide bond being that reduced first. Neither of the two subunits is liberated from isolated plasma membranes when this GPIIb interchain bond is reduced in isolated membranes.

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