共查询到20条相似文献,搜索用时 15 毫秒
1.
A membrane-bound protease induced by sulfur mustard in cultured normal human epidermal keratinocytes (NHEK) was purified and partially characterized. Maximum enzyme stimulation occurred at 16 hr after normal human epidermal keratinocytes were exposed to 300 microM sulfur mustard. Purification to homogeneity of the protease was accomplished by Triton X-100 solubilization, ultracentrifugation, and dialysis, followed by ion-exchange chromatography through DEAE-cellulose and finally hydrophobic column chromatography through phenyl Sepharose. Analysis of the purified enzyme by SDS-PAGE revealed a single polypeptide at the 80 kDa region. Further investigation of biochemical properties showed that a synthetic serine-specific Chromozym TRY peptide and the physiological protein laminin were good substrates for this enzyme. Moreover, this enzyme was inhibited mostly by the serine-protease inhibitors leupeptin and di-isopropyl fluorophosphate and not by the cysteine protease inhibitor E-64 or the metalloprotease inhibitor 1,10-phenanthroline (Component H, CH), indicating the serine protease nature of this enzyme. This enzyme had a pH optimum in the range of 7.0 to 8.0. Amino acid sequencing of the purified enzyme revealed that this enzyme belongs to the endopeptidase family (serine protease), and is homologous with a mammalian-type bacterial serine endopeptidase that can preferentially cleave K-X, including K-P. These results suggest that serine-protease stimulation may be one of the mechanisms of mustard-induced skin blister formation, and that some specific serine-protease inhibitors may be useful for the treatment of this sulfur mustard toxicity. 相似文献
2.
V Chesneau AR Pierotti N Barré C Créminon C Tougard P Cohen 《Canadian Metallurgical Quarterly》1994,269(3):2056-2061
A metalloendopeptidase that selectively cleaves doublets of basic amino acids on the amino-terminal side of arginine residues was purified to homogeneity from rat testes and analyzed further. Two catalytically active forms with apparent relative molecular masses of 110,000 and 140,000 Da, respectively, were present in the purified preparation of the enzyme. Antibodies raised against the purified testis endopeptidase revealed by immunoblot both the 110- and 140-kDa forms in both rat testis and brain cortex extracts. The isolated enzyme was inhibited by metal chelators and divalent cations. Its activity, lost after preincubation with EDTA, was restored by low concentrations of Zn2+ and Mn2+, thus demonstrating the metallopeptidase nature of the enzyme. This endopeptidase also exhibited a high sensitivity to amastatin (100% inhibition at 20 microM), an aminopeptidase inhibitor. A substrate specificity study using physiologically important or synthetic peptides containing a processing dibasic site indicated that cleavage occurred selectively at the amino-terminal side of an arginine residue, independent of the nature of the basic doublet. The enzyme produced such a cleavage at the Arg-Lys doublet of somatostatin 28 (Km = 43 microM), at the Arg-Arg doublet of dynorphin A (Km = 6.45 microM) and atrial natriuretic factor (Km = 6.25 microM), and at the Lys-Arg doublet of preproneurotensin-(154-170) (Km = 17.3 microM). Moreover, cleavage efficiency was found to be higher for the larger substrates. The distinctive properties of this endopeptidase imply that this protein is a member of a novel class of proteolytic enzymes that may be involved in the endoproteolytic maturation of hormonal precursors. 相似文献
3.
In this study we describe the partial purification and characterization of the HeLa cell oligopeptidase M or endopeptidase 3.4.24.16. The HeLa enzyme was isolated initially by its ability to hydrolyse a nonapeptide substrate (P9) which was cognate to the N-terminal cleavage site of preproTGF alpha. The enzyme was shown to be a metalloprotease as it was inhibited by Zn(2+)-chelating agents and DTT, and had an approximate molecular weight of 55-63 kD determined by gel filtration. Neurotensin, dynorphin A1-17 and GnRH1-9 were rapidly degraded by the enzyme while GnRH1-10 and somatostatin were not. Neurotensin was cleaved at the Pro10-Tyr11 bond, leading to the formation of neurotensin (1-10) and neurotensin (11-13). The K(m) for neurotensin cleavage was 7 microM and the Ki for the specific 24.16 dipeptide inhibitor (Pro-ile) was 140 microM which were similar to those observed from the human brain enzyme [Vincent et al. (1996): Brain Res 709:51-58]. Through the use of specific antibodies, the purified HeLa enzyme was shown to be oligopeptidase M. This enzyme and its closely related family member thimet oligopeptidase were shown to co-elute during the isolation procedure but were finally separated using a MonoQ column. Oligopeptidase M is located mainly in mitochondria though it was detected on the plasma membrane in an inactive form. The results obtained demonstrate the first recorded instance of this enzyme in human tissue cultured cells, and raise the issue of its function therein. 相似文献
4.
Alzheimer's disease (AD) is a slowly progressing neurodegenerative disease characterized by the loss of neurons and formation of amyloid plaques, often surrounded by reactive astrocytes. Astrocytes are important regulators of the normal neuronal environment, and changed astrocyte function may lead to increased neuronal vulnerability. The slow onset of the disease with a gradual increase in the beta-amyloid peptide (beta-AP) concentrations may alter astrocyte function long before any visible symptoms of the disease are observed. We, therefore, studied in vitro the effects of small amounts of beta-AP(1-40) and -(25-35) on rat cortical astrocyte function observing changes in cell morphology, intracellular calcium levels (Cai), and ion channel activity. Incubation with 10 and 200 nM beta-APs caused increased process formation and hypertrophy. Stellation was also detected when astrocyte cultures were incubated with 1 microM AlCl3 alone, or together with beta-APs. Fura-2AM-loaded astrocytes were used to test whether the morphological changes were connected to changes in Cai levels. 1 microM beta-AP(1-40) induced transient Cai increase in approximately 17%, and beta-AP(25-35) in approximately 36% of astrocytes. In patch-clamp studies, increased K+ and Cl- channel activity was detected with 10-100 nM beta-AP(1-40). With large amounts (20 microM) of beta-AP(1-40), an additional giant channel activity emerged. These beta-AP-induced changes in astrocyte function may eventually be critical for the neuronal survival in Alzheimer's disease. 相似文献
5.
MA Medeiros MS Fran?a G Boileau L Juliano KM Carvalho 《Canadian Metallurgical Quarterly》1997,30(10):1157-1162
Two intramolecularly quenched fluorogenic peptides containing o-aminobenzoyl (Abz) and ethylenediamine 2,4-dinitrophenyl (EDDnp) groups at amino- and carboxyl-terminal amino acid residues, Abz-DArg-Arg-Leu-EDDnp (Abz-DRRL-EDDnp) and Abz-DArg-Arg-Phe-EDDnp (Abz-DRRF-EDDnp), were selectively hydrolyzed by neutral endopeptidase (NEP, enkephalinase, neprilysin, EC 3.4.24.11) at the Arg-Leu and Arg-Phe bonds, respectively. The kinetic parameters for the NEP-catalyzed hydrolysis of Abz-DRRL-EDDnp and Abz-DRRF-EDDnp were K(m) = 2.8 microM, kcat = 5.3 min-1, kcat/K(m) = 2 min-1 microM-1 and K(m) = 5.0 microM, kcat = 7.0 min-1, kcat/K(m) = 1.4 min-1 microM-1, respectively. The high specificity of these substrates was demonstrated by their resistance to hydrolysis by metalloproteases [thermolysin (EC 3.4.24.2), angiotensin-converting enzyme (ACE; EC 3.4.24.15)], serineproteases [trypsin (EC 3.4.21.4), alpha-chymotrypsin (EC 3.4.21.1)] and proteases present in tissue homogenates from kidney, lung, brain and testis. The blocked amino- and carboxyl-terminal amino acids protected these substrates against the action of aminopeptidases, carboxypeptidases and ACE. Furthermore, DR amino acids ensured total protection of Abz-DRRL-EDDnp and Abz-DRRF-EDDnp against the action of thermolysin and trypsin. Leu-EDDnp and Phe-EDDnp were resistant to hydrolysis by alpha-chymotrypsin. The high specificity of these substrates suggests their use for specific NEP assays in crude enzyme preparations. 相似文献
6.
LH Pope MW Shotton T Forsyth DJ Hughes RC Denny W Fuller 《Canadian Metallurgical Quarterly》1998,70(2):161-172
The neurotoxic beta-amyloid (Abeta) peptide fragment Abeta(25-35) has been suggested to exert its deleterious effects on cells via production of hydrogen peroxide. In human platelets and in the presence of DMSO to prevent production of hydroxyl radicals from hydrogen peroxide, both Abeta(25-35) and hydrogen peroxide were found to increase intracellular calcium levels. Hydrogen peroxide in addition reduced the calcium response to thrombin, whereas this was not seen with Abeta(25-35). A similar pattern of effects to those seen with hydrogen peroxide were also seen with the neurotoxic aldehyde lipid peroxidation product 4-hydroxy-2-nonenal (HNE). The initial increase in calcium produced by hydrogen peroxide was not affected by EGTA, but was partially prevented by dithiothreitol. The calcium response to Abeta(25-35) [which was also seen with Abeta(1-40) and Abeta(1-42) but not with the inactive peptide Abeta(40-1)] consisted of an EGTA-sensitive and an EGTA-resistant component, of which the latter was also sensitive to DTT. Hydrogen peroxide increased basal phosphoinositide breakdown in rat brain miniprisms and decreased the responses to noradrenaline, carbachol and veratrine. The specific binding of [3H]inositol-1,4,5-trisphosphate ([3H]Ins(1,4,5)P3) to its receptor recognition site in human platelet membranes was increased by Abeta(25-35) but remained unchanged following hydrogen peroxide treatment. It is concluded that under conditions where production of hydroxyl radicals from hydrogen peroxide is blocked, hydrogen peroxide and Abeta(25-35) produce their effects on calcium by affecting the mobilisation of intracellular calcium. The qualitative differences in the calcium responses of these two agents can be explained (a) by an additional effect of Abeta(25-35) upon calcium entry and (b) by differences in their effects upon the Ins(1,4,5)P3 receptor. 相似文献
7.
Recently, several studies have proposed models describing the mechanisms of Alzheimer's beta-amyloid fibril formation in vitro. However, these models are somewhat controversial and no exact kinetic analyses measuring the polymerization velocity as an indicator of the reaction, have thus far been available. We first formed beta-amyloid fibrils from a synthetic peptide, beta-amyloid(1-40), and determined the optimum conditions for quantitative fluorometry of these beta-amyloid fibrils with thioflavine T. Optimum fluorescence measurements of beta-amyloid fibrils were obtained at the excitation and emission wavelengths of 446 and 490 nm, respectively, with the reaction mixture containing 5 microM thioflavine T and 50 mM of glycine-NaOH buffer, pH 8.5. We then focused our study on the extension phase of beta-amyloid fibril formation in vitro. When beta-amyloid fibrils were incubated with monomeric beta-amyloid(1-40) in conditions where de novo seed formation does not occur, the extension of beta-amyloid fibrils was observed with electron microscopy. Quantitative fluorometry revealed that: (a) extension of amyloid fibrils proceeded by a pseudo-first-order exponential increase as measured by the fluorescence of thioflavine T; (b) the rate of extension was maximum around pH 7.5, and was dependent on the incubation temperature. Between 20 and 37 degrees C, good linearity was observed between the common logarithm of the initial rate and the reciprocal of the absolute temperature; (c) the rate of polymerization was found to be proportional to the product of beta-amyloid fibrils number concentration and the beta-amyloid(1-40) concentration; (d) the net rate of extension was the sum of the rates of polymerization and depolymerization. These results show that beta-amyloid fibril formation can be explained by a first-order kinetic model: i.e., the extension of beta-amyloid fibrils proceeds via the consecutive association of beta-amyloid(1-40) onto the ends of existing fibrils. 相似文献
8.
We have investigated the production, growth and inactivation of gastrin-releasing peptide (GRP)-like peptides in human breast cancer cell lines. Radioimmunoassay detected GRP-like immunoreactivity (GRP-LI) in T47D breast cancer cells but not in the conditioned medium, indicating rapid clearance. No GRP-LI was found in the ZR-75-1 or MDA-MB-436 cells or their conditioned medium. High-performance liquid chromatography (HPLC) analysis of the GRP-LI in the T47D cells revealed a major peak, which co-eluted with GRP(18-27), and a minor more hydrophilic peak. In vitro stimulation of T47D cell growth by bombesin (BN) was enhanced to 138% of control levels (bombesin alone) by the addition of the selective endopeptidase EC 3.4.24.11 inhibitor phosphoramidon (0.1 ng ml(-1)). Fluorogenic analysis using whole cells confirmed low levels of this phosphoramidon-sensitive enzyme on the T47D cells. This enzyme, previously unreported in human breast cancer cells, significantly modulates both T47D growth and its response to BN-induced growth. 相似文献
9.
An endo-acting proline-specific oligopeptidase (prolyl oligopeptidase [POPase], EC 3.4.21.26) was purified to homogeneity from the Triton X-100 extracts of cells of Treponema denticola ATCC 35405 (a human oral spirochete) by a procedure that comprised five successive fast protein liquid chromatography steps. The POPase is a cell-associated 75- to 77-kDa protein with an isoelectric point of ca. 6.5. The enzyme hydrolyzed (optimum pH 6.5) the Pro-pNA bond in carbobenzoxy-Gly-Pro-p-nitroanilide (Z-Gly-Pro-pNA) and bonds at the carboxyl side of proline in several human bioactive peptides, such as bradykinin, substance P, neurotensin, angiotensins, oxytocin, vasopressin, and human endothelin fragment 22-38. The minimum hydrolyzable peptide size was tetrapeptide P3P2P1P'1, while the maximum substrate size was ca. 3 kDa. An imino acid residue in position P1 was absolutely necessary. The hydrolysis of Z-Gly-Pro-pNA was potently inhibited by the following, with the Ki(app) (in micromolar) in parentheses: insulin B-chain (0.7), human endothelin-1 (0.5), neuropeptide Y (1.7), substance P (32.0), T-kinin (4.0), neurotensin (5.0), and bradykinin (16.0). Chemical modification and inhibition studies suggest that the POPase is a serine endopeptidase whose activity depends on the catalytic triad of COOH ... Ser ... His but not on a metal. The amino acid sequence around the putative active-site serine is Gly-Gly-Ser-Asn-Pro-Gly. The enzyme is suggested to contain a reactive cysteinyl residue near the active site. Amino acid residues 4 to 24 of the first 24 N-terminal residues showed a homology of 71% with the POPase precursor from Flavobacterium meningosepticum and considerable homology with the Aeromonas hydrophila POPase. The ready hydrolysis of human bioactive peptides at bonds involving an imino acid residue suggests that enzymes like POPase may contribute to the chronicity of periodontal infections by participating in the peptidolytic processing of those peptides. 相似文献
10.
K Holzer M Thiel S Moritz U Kreimeier K Messmer 《Canadian Metallurgical Quarterly》1996,81(1):341-348
The activity of endopeptidase EC 3.4.24.15 (thimet oligopeptidase, EP 24.15), as measured by cleavage of a quenched fluorescent substrate, 7-methoxycoumarin-4-acetyl-Pro-Leu-Gly-Pro-D-Lys (2,4-dinitrophenyl), was increased 2-3 fold by the addition of 1 mM Mn2+ or of 10 mM Ca2+. The inhibitory capability of a specific EP. 24.15 inhibitor, N-[1-(R,S)-carboxy-3-phenylpropyl]-Ala-Ala-Tyr-p-aminobenzoate, was also increased at similar concentrations of these metal ions. However, the hydrolysis of naturally-occurring peptides, thought to be the physiological substrates for EP 24.15, was not affected by either Mn2+ or Ca2+. These results suggest that the binding of synthetic analogs to the enzyme may differ significantly from the binding, and thus hydrolysis, of natural peptide substrates and caution against drawing conclusions about substrate interactions with the active site from data obtained with modified peptide ligands. 相似文献
11.
JR Somoza AG Skillman NR Munagala CM Oshiro RM Knegtel S Mpoke RJ Fletterick ID Kuntz CC Wang 《Canadian Metallurgical Quarterly》1998,37(16):5344-5348
All parasitic protozoa obtain purine nucleotides solely by salvaging purine bases and/or nucleosides from their host. This observation suggests that inhibiting purine salvage may be a good way of killing these organisms. To explore this idea, we attempted to block the purine salvage pathway of the parasitic protozoan Tritrichomonas foetus. T. foetus is a good organism to study because its purine salvage depends primarily on a single enzyme, hypoxanthine-guanine-xanthine phosphoribosyltransferase (HGXPRTase), and could provide a good model for rational drug design through specific enzyme inhibition. Guided by the crystal structure of T. foetus HGXPRTase, we used structure-based drug design to identify several non-purine compounds that inhibited this enzyme without any detectable effect on human HGPRTase. One of these compounds, 4-[N-(3, 4-dichlorophenyl)carbamoyl]phthalic anhydride (referred to as TF1), was selected for further characterization. TF1 was shown to be a competitive inhibitor of T. foetus HGXPRTase with respect to both guanine (in the forward reaction; Ki = 13 microM) and GMP (in the reverse reaction; Ki = 10 microM), but showed no effect on the homologous human enzyme at concentrations of up to 1 mM. TF1 inhibited the in vitro growth of T. foetus with an EC50 of approximately 40 microM. This inhibitory effect was associated with a decrease in the incorporation of exogenous guanine into nucleic acids, and could be reversed by supplementing the growth medium with excess exogenous hypoxanthine or guanine. Thus, rationally targeting an essential enzyme in a parasitic organism has yielded specific enzyme inhibitors capable of suppressing that parasite's growth. 相似文献
12.
RF Cowburn B Wiehager E Trief M Li-Li E Sundstr?m 《Canadian Metallurgical Quarterly》1997,22(12):1437-1442
The neurotoxic fragment corresponding to residues 25-35 of the beta-amyloid (A beta) peptide [A beta-(25-35)] has been shown to exert effects on (+)-[3H]5-methyl-10, 11-dihydro-5H-dibenzo[a,d]-cyclohepten-5,10-imine maleate ([3H]MK-801) binding to the cation channel of the N-methyl-D-aspartate (NMDA) receptor. In the present study, we investigated whether the amidated and carboxylic acid C-terminated forms of A beta-(25-35) [A beta-(25-35-NH2) and A beta-(25-35-COOH), respectively] exert effects on other excitatory amino acid receptor and cation channel types in rat cortical membranes. Both A beta-(25-35-NH2) and A beta-(25-35-COOH) gave statistically significant dose-dependent inhibitions of [3H]glutamate and [3H]glycine binding to the agonist recognition sites of the NMDA receptor. Ten microM A beta-(25-35-NH2) and A beta-(25-35-COOH) gave 25% and 20% inhibitions of [3H]glutamate binding and 75% and 70% inhibitions of [3H]glycine binding, respectively. A beta-(25-35-NH2), but not A beta-(25-35-COOH), gave a small (ca. 17% at 10 microM) statistically significant increase of [3H]amino-3-hydroxy-5-methylisoxazole-4-propionate ([3H]AMPA) binding. [3H]kainate binding was not significantly affected by either peptide. Similarly, neither peptide affected either the maximal level or EC50 value for calcium stimulation of [3H]nitrendipine binding. It is concluded that A beta-(25-35) shows slight affinity for the agonist recognition sites of the NMDA receptor, but not for other excitatory amino acid receptor types or for L-type voltage-dependent calcium channels. 相似文献
13.
The enzyme N-acetylglucosamine-1-phosphodiester alpha-N-acetylglucosaminidase (EC 3.1.4.45; uncovering enzyme) catalyzed the removal of N-acetylglucosamine from the N-acetylglucosamine-alpha-phospho-mannose portion of selected lysosomal enzyme oligosaccharide chains, thereby forming the mannose 6-phosphate signal which is responsible for the targeting of these lysosomal enzymes for transport into lysosomes. The uncovering enzyme has been purified approximately 7000-fold to electrophoretic homogeneity from Epstein-Barr virus-transformed human lymphoblast cells. The purification sequence involves solubilizing this membrane-bound enzyme with Tergitol NP-10, affinity chromatography on Lentil lectin-Sepharose 4B, ion-exchange chromatography on DEAE-Sephacel, chromatography on zinc(II)-IDA-Sepharose 6B, and preparative SDS-PAGE electrophoresis. The purified enzyme migrated as a single band of 114 kDa which was coincident with enzyme activity on analytical SDS-PAGE electrophoresis. Characterization studies of the purified enzyme demonstrated that catalytic activity was maximal at pH 6.95 and that the enzyme retained full activity following incubation for 10 min at 60 degrees C. No requirement was found for a divalent cation, but Zn2+, Hg2+, and Cu2+ were found to reduce the enzyme's activity by 30-40%. The highest catalytic efficiency was observed with N-acetylglucosamine-phospho-methylmannoside as a substrate while uridine diphosphate-N-acetylglucosamine, N-acetylglucosamine-phosphomannose-uteroferrin, and N-acetylglucosamine-phosphate were also cleaved by the enzyme with decreasing efficiency. Acetamino-deoxycastanospermine was a potent inhibitor of the human enzyme with a Ki of 0.35 microM, while N-acetylglucosamine phosphate (Ki 1.58 mM) and N-acetylglucosamine (Ki 5.1 mM) inhibited the enzyme to a lesser degree. 相似文献
14.
S Guérin B Mari E Fernandez N Belhacene ML Toribio P Auberger 《Canadian Metallurgical Quarterly》1997,11(12):1003-1011
Thymic hormones such as thymopoietin (TP) have been shown to regulate thymocyte differentiation and lymphocyte activation. However, it is not known whether thymopoietin affects thymic epithelial cell (TEC) functions. In this study we have examined the effect of a five amino acid active peptide (TP5), corresponding to amino acids 32-36 of TP, on the proliferation of nontransformed clones of human TEC. Our results indicate that TP5 induced reinitiation of DNA synthesis and potentiated fetal calf serum (FCS)-induced cell growth in postnatal and fetal-derived human TEC. We also found that TEC lines express high levels of endopeptidase 24.11, a cell-surface metallopeptidase also known as the CD10 antigen. We show that TP5 is cleaved by CD10 at the surface of TEC lines, indicating that this endopeptidase may regulate TP5-induced TEC proliferation. Phosphoramidon, a specific endopeptidase 24.11 inhibitor, consistently acts in synergy with TP5 to enhance FCS-induced TEC growth. Hence, we conclude that 1) TP5 alone or in combination with FCS supports the growth of TEC lines, and 2) TEC lines express high levels of CD10, which regulates TP5-induced TEC proliferation by acting as a thymic peptide degrading enzyme. 相似文献
15.
M Raiteri G Bonanno P Paudice P Cavazzani G Schmid 《Canadian Metallurgical Quarterly》1996,278(2):747-751
The release of cholecystokinin-like immunoreactivity (CCK-LI) in human brain was investigated using synaptosomes prepared from neocortical specimens removed during neurosurgery. CCK-LI basal release from superfused synaptosomes was increased 3 to 4-fold during depolarization with 15 mM KCI. The K(+)-evoked overflow of CCK-LI was strictly Ca(++)-dependent. The gamma-aminobutyric acidB (GABA(B)) receptor agonist (-)baclofen (0.3-100 microM) inhibited CCK-LI overflow in a concentration-dependent manner (EC50 = 2.20 microM; maximal effect: 45%). The novel GABA(B) receptor ligand CGP 47656 mimicked (-)baclofen (EC50 = 2.45 microM; maximal effect: 50%), whereas the GABA(A) agonist muscimol was ineffective up to 100 microM. The inhibitory effect of 10 microM (-)baclofen on the CCK-LI overflow was concentration-dependently prevented by two selective GABA(B) receptor antagonists, CGP 35348 (IC50 = 13.91 microM) and CGP 52432 (IC50 = 0.08 microM). The effect of 10 microM CGP 47656 was abolished by 1 microM CGP 52432. In experiments on [3H]GABA release, CGP 47656 behaved as an antagonist at the GABA(B) autoreceptors: added at 10 microM, it prevented the inhibitory effect of 10 microM (-)baclofen on the K+ (15 mM)-evoked release of [3H]GABA from human synaptosomes. We conclude that 1) the release of CCK-LI evoked from human brain tissue appears of neuronal origin; 2) the CCK-releasing terminal possess inhibitory presynaptic GABA(B) receptors; 3) these receptors differ pharmacologically from human neocortex GABA(B) autoreceptors, which are CGP 35348-insensitive (Fassio et al., 1994) but can be blocked by CGP 47656; 4) because cholecystokinin has been implicated in anxiety, the GABA(B) receptors here characterized may represent targets for novel anxiolytic agents. 相似文献
16.
A rapid, enzymatic assay for the measurement of inorganic pyrophosphate in animal tissues 总被引:2,自引:0,他引:2
A simple, rapid enzymatic assay for the determination of inorganic pyrophosphate in tissue and plasma has been developed using the enzyme pyrophosphate--fructose-6-phosphate 1-phosphotransferase (EC 2.7.1.90) which was purified from extracts of Propionibacterium shermanii. The enzyme phosphorylates fructose-6-phosphate to produce fructose-1,6-bisphosphate using inorganic pyrophosphate as the phosphate donor. The utilization of inorganic pyrophosphate is measured by coupling the production of fructose-1,6-bisphosphate with the oxidation of NADH using fructose-bisphosphate aldolase (EC 4.1.2.13), triosephosphate isomerase (EC 5.3.1.1), and glycerol-3-phosphate dehydrogenase (NAD+)(EC 1.1.1.8). The assay is completed in less than 5 min and is not affected by any of the components of tissue or plasma extracts. The recovery of pyrophosphate added to frozen tissue powder was 97 +/- 1% (n = 4). In this assay the change in absorbance is linearly related to the concentration of inorganic pyrophosphate over the curvette concentration range of 0.1 microM to 0.1 mM. 相似文献
17.
Alzheimer's disease (AD) is a multifactorial disease in which beta-amyloid peptide (betaAP) plays a critical role. We report here that the soluble fraction 1-40 of betaAP differentially degrades protein kinase C-alpha and -gamma (PKCalpha and PKCgamma) isoenzymes in normal (age-matched controls, AC) and AD fibroblasts most likely through proteolytic cascades. Treatment with nanomolar concentrations of betaAP(1-40) induced a 75% decrease in PKCalpha, but not PKCgamma, immunoreactivity in AC fibroblasts. In the AD fibroblasts, a 70% reduction of the PKCgamma, but not PKCalpha, immunoreactivity was observed after betaAP treatment. Preincubation of AC or AD fibroblasts with 50 microM lactacystine, a selective proteasome inhibitor, prevented beta-AP(1-40)-mediated degradation of PKCalpha in the AC cells, and PKCgamma in the AD fibroblasts. The effects of betaAP(1-40) on PKCalpha in AC fibroblasts were prevented by inhibition of protein synthesis and reversed by PKC activation. A 3-hr treatment with 100 nM phorbol 12-myristate 13-acetate restored the PKCalpha signal in treated AC cells but it did not reverse the effects of betaAP(1-40) on PKCgamma in the AD fibroblasts. Pretreatment with the protein synthesis inhibitor, cycloheximide (CHX, 100 microM), inhibited the effects of betaAP(1-40) on PKCalpha and blocked the rescue effect of phorbol 12-myristate 13-acetate in AC fibroblasts but did not modify PKCgamma immunoreactivity in AD cells. These results suggest that betaAP(1-40) differentially affects PKC regulation in AC and AD cells via proteolytic degradation and that PKC activation exerts a protective role via de novo protein synthesis in normal but not AD cells. 相似文献
18.
SB Bleyl BR Mumford MC Brown-Harrison LT Pagotto JC Carey TJ Pysher K Ward TK Chin 《Canadian Metallurgical Quarterly》1997,72(3):257-265
Cerebellar granule cells (CGC) at different stages of maturation in vitro (1 or 6 DIV), were treated with beta 25-35 and acetyl-L-carnitine arginine amide (ST857) in presence of 25 mM KCl in the culture medium, and neuronal viability was assessed. Three days of treatment slightly modified the survival of 1 DIV-treated cells, which degenerate and die five days later beta-amyloid matching. Similarly, a significative neurotoxic effect was observed on 6 DIV treated-cells after 5 days of exposure to the peptide, while the death occurred within 8 days. ST857 coincubated with beta 25-35 was able to rescue neurons from beta 25-35-induced neurotoxicity. We also studied the changes in Ca2+ homeostasis following glutamate stimulation, in control and beta-amyloid treated single cells, either in presence or in absence of ST857. beta 25-35 did not affect basal [Ca2+]i, while modified glutamate-induced [Ca2+]i increase, causing a sustained plateau phase of [Ca2+]i, that persisted after the removal of the agonist. ST857 pretreatment completely reverted this effect suggesting that, in CGC chronically treated with beta 25-35, ST857 could protect the cells by neurotoxic insults of the peptide likely interfering with the cellular mechanisms involved in the control of Ca2+ homeostasis. 相似文献
19.
We have identified on the membranes of the locomotory muscle of Ascaris suum an amastatin-sensitive aminopeptidase that hydrolyses the bioactive neuropeptides AF1 (KNEFIRF-NH2) and AF2 (KHEYLRF-NH2), by cleavage of the Lys1-Asn2 and Lys1-His2 peptide bonds, respectively. AF2 (1.2 nmol of HEYLRF-NH2 formed min[-1] (mg protein[-1])) was hydrolysed at a faster rate compared to AF1 (0.2 nmol of NEFIRF-NH2 formed min[-1] (mg protein[-1])). AF1 hydrolysis by the aminopeptidase was inhibited by the amastatin (IC50, 9.0 microM), leuhistin (IC50, 1.25 microM) but was insensitive to puromycin, indicating a similarity to mammalian aminopeptidase N. The enzyme was also inhibited by arphamenine B (IC50, 9.0 microM), (2S, 3R)-3-amino-2-hydroxy-4-(4-nitrophenyl)butanoyl-L-leucine (IC50, 8.0 microM), bestatin (IC50, 15.0 microM) and 1 mM 1-10 bis-phenanthroline. The detergent Triton X-100 solubilised enzyme had a pI of 5.0 and after 1000-fold purification by ion-exchange chromatography, appeared to have a Mr of around 240,000 by SDS-PAGE. The purified aminopeptidase had a Km of 534 microM for the hydrolysis of AF1 and cleaved Phe1 from FMRF-NH2, but was unable to hydrolyse DFMRF-NH2 or FDMRF-NH2. The aminopeptidase that we have described in this report might have a role in the extracellular metabolism and inactivation of neuropeptides acting on the locomotory muscle of A. suum. 相似文献
20.
Aminopeptidase N (EC 3.4.11.2) is an important enzyme that is involved in the degradation of regulatory peptides including enkephalins. We report here that purified and native membrane-bound aminopeptidase N will sequentially and completely hydrolyze both Leu-enkephalin and Met-enkephalin from the amino terminus. Both purified pig aminopeptidase N and the enzyme on live HL60 cells displayed similar Km values for enkephalin. The naturally occurring neuropeptides substance P and bradykinin, and the morphine agonist, morphiceptin, were not hydrolyzed by aminopeptidase N and each inhibited the enzymatic activity. Each of these peptides contains a proline at the second residue. The Ki values for substance P (0.44 microM), bradykinin (9.4 microM), and morphiceptin (169 microM) were obtained with the enzyme on live HL60 cells. The values for the purified enzyme from pig were similar. The potent inhibition of aminopeptidase N by substance P and bradykinin suggests that these peptides may be natural inhibitors of the enzyme. 相似文献