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1.
Eran J. Yavin Lin Yan Dominic M. Desiderio Michel Pontet Mati Fridkin 《Letters in Peptide Science》1997,4(3):157-166
Extended peptides that derive from the primary sequence of the acute phase reactant C-reactive protein (CRP) are shown to inhibit in vitro the enzymatic activities of human leukocyte elastase (hLE) and human leukocyte cathepsin G (hCG), which are associated with the tissue damage that occurs during the course of several chronic inflammatory conditions. Major inhibitory activity was observed in the peptides CRP70-98 and CRP50-98 towards hLE (Ki = 4.0 µM) and hCG (Ki = 1.4 µM), respectively. In contrast to the inability of intact CRP pentamers to inhibit both enzymes, CRP subunits (monomers) inhibited hLE (3.0 µM) and hCG (3.6 µM) activity. 相似文献
2.
Summary Several metabolic compounds have been found to be competitive inhibitors of the anomerase activity of phosphoglucose isomerase
(EC 5.3.1.9).Ki values for erythrose 4-phosphate, 6-phosphogluconate, and fructose 1,6-bisphosphate for the anomerase reaction are 0.32 μM,
21 μM, and 84 μM respectively at 0° and pH 8.2. A significant difference between the fructose, 1,6-bisphosphate inhibition
constants for both activities was found (ki(isomerase)=800 μM and Ki(anomerase)=84 μM). Also the Km values for both activities were found to be significantly different (Km(isomerase)=140 μM and Km(anomerase)=3.6 μM).
Attempts to independently alter the anomerase to isomerase activity ratio through protein modification yielded mixed results.
While several modifying reagents destroyed the catalytic activities at identical rates, inactivation by iodoacetamide or pyridoxal
5′ phosphate sensitized photo-oxidation displayed differential initial effects on the two activities with the anomerase activity
being the less affected. These data support the theory that an imidazole residue is catalytically important for isomerization,
but less so for anomerization. 相似文献
3.
Agnieszka Markowska Irena Bruzgo Wojciech Miltyk Krystyna Midura-Nowaczek 《International journal of peptide research and therapeutics》2011,17(1):47-52
We synthesized and tested ten peptides with the molecular structure being H–d-Ser–AA–Arg–OH for their effect on the amidolytic activities against urokinase, thrombin, trypsin, plasmin, tissue plasminogen
activator and kallikrein. The inserted amino acid in each peptide was either leucine, norleucine, izoleucine, valine, norvaline,
α-metyloalanine, α-aminobutanoic acid, homoleucine, tert-leucine or neoglycine. H–d-Ser–NVal–Arg–OH (compound 4) was the most active inhibitor of urokinase plasminogen activator with a Ki value of 0.85 μM. Compound 4 showed cytotoxic effect against MDA-MB-231 and DLD cell lines, respectively, with IC50 values of 25 and 19 μM. Synthesised compounds did not have activity against MCF-7 cancer cells. These peptides were nontoxic
against pig’s erythrocytes in vitro. 相似文献
4.
In order to clearly establish the properties of the enzymes responsible for hexose phosphorylation we have undertaken the
separation and characterization of these enzymes present in tomato fruit (Martinez-Barajas and Randall 1996). This report
describes the partial purification and characterization of glucokinase (EC. 2.7.1.1) from young green tomato fruit. The procedure
yielded a 360-fold enrichment of glucokinase. Tomato fruit glucokinase is a monomer with a molecular mass of 53 kDa. Glucokinase
activity was optimal between pH 7.5 and 8.5, preferred ATP as the phosphate donor (K
m = 0.223 mM) and exhibited low activity with GTP or UTP. The tomato fruit glucokinase showed highest affinity for glucose
(K
m = 65 μM). Activity observed with glucose was 4-fold greater than with mannose and 50-fold greater than with fructose. The tomato
fruit glucokinase was sensitive to product inhibition by ADP (K
i = 36 μM). Little inhibition was observed with glucose 6-phosphate (up to 15 mM) at pH 8.0; however, at pH 7.0 glucokinase
activity was inhibited 30–50% by physiological concentrations of glucose 6-phosphate.
Received: 4 October 1997 / Accepted: 10 January 1998 相似文献
5.
M. V. Potapovich V. P. Kurchenko D. I. Metelitza O. I. Shadyro 《Applied Biochemistry and Microbiology》2011,47(4):346-355
Inhibition efficiency (antioxidant activity) of 26 oxygen-containing aromatic compounds was studied in methemalbumin-H2O2-o-phenylenediamine (PDA) or tetramethylbenzidine (TMB) pseudoperoxidase system at 20°C in buffered physiological solution (pH
7.4) containing 6% DMF and 0.25% DMSO. The inhibitor’s efficiency was quantitatively characterized by the inhibition constants
(K
i, μM) or the inhibition degree (%). K
i values varied in the range of 4 to 500 μM and were influenced by a substrate, the structure of an inhibitor, hydroxyl groups,
electron-donating substituents in aromatic ring, and steric hindrances. The type of inhibition at cooxidation of eight pairs
was noncompetitive, and that of five pairs was mixed and determined by the substrate nature and the inhibitor structure. Lignin
phenolic compounds of guaiacyl and syringal series exhibited high antioxidant activity (K
i in the range of 10–300 μM), and their efficiency decreased in the following order: caffeic acid > synapaldehyde > syringic
acid > coniferyl aldehyde > para-hydroxycoumaric acid. 相似文献
6.
A series of 5-oxaproline peptide derivatives was synthesized and evaluated for its ability to inhibit the prolyl 4-hydroxylasein vitro. Structure-activity studies show that the 5-oxaproline sequences, prepared by the 1,3-dipolar cycloaddition of the C-methoxycarbonyl-N-mannosyl
nitrone in the presence of the ethylene, are more active than the corresponding proline derivatives. Prolyl 4-hydroxylase
belongs to a family of Fe2+-dependent dioxygenase, which catalyzes the formation of 4-hydroxyproline in collagens by the hydroxylation of proline residues
in-Gly-Xaa-Pro-Gly- of procollagen chains. In this paper we discover the more selective N-Cbz-Gly-Phe-Pro-Gly-OEt (K
m=520 μM) sequences which are showed stronger binding than othersin vitro. Therefore, we set out to investigate constrained tetrapeptide that was designed to mimic the proline structure of peptides
for the development of prolyl 4-hydroxylase inhibitor. From this result, we found that the most potent inhibitor is N-Dansyl-Gly-Phe-5-oxaPro-Gly-OEt
(K
i=1.6 μM). This has prompted attempts to develop drugs which inhibit collagen synthesis. Prolyl 4-hydroxylase would seem a
particularly suitable target for antifibrotic therapy. 相似文献
7.
《Peptides》2015
The enzyme dipeptidyl-peptidase IV (DPP-IV) is recognized to be a promising target for the management of type 2 diabetes. Over the last decade, numerous synthetic molecules and more recently, peptides from dietary proteins, have been reported to be able to inhibit DPP-IV activity. Most studies that have investigated the in vitro effect of these inhibitors have used porcine or human DPP-IV. Although structurally alike, it is unclear whether these two species display similar inhibition patterns. Therefore, the objective of this study was to compare the effects of protein-derived peptides on the activity of porcine and recombinant human DPP-IV. The two species showed different inhibition susceptibility to 43 of the 62 peptide sequences investigated. While 37 protein-derived peptides were more effective at inhibiting the porcine DPP-IV, only six caused a stronger inhibition of the activity of the human enzyme. Although the peptides WR, IPIQY and WCKDDQNPHS were found to be among the most potent inhibitors of both species, the inhibitory effect was greater on the porcine enzyme than on human DPP-IV (αKi or Ki = 11.5, 13.4, 13.3 μM and 31.4, 28.2, 75.0 μM for porcine and human DPP-IV, respectively). Investigation into the mode of action of the most effective inhibitory peptides revealed that both species were inhibited in a similar manner by short fragments (≤5 amino acid residues), but that some of the longer peptides acted differently on the enzymes. This study shows that porcine DPP-IV is generally inhibited with greater potency by protein-derived peptides than is the human enzyme. 相似文献
8.
Metabolic transformations of two substrates for human cytochrome P450 (CYP450) 2C9, tolbutamide and diclofenac, were investigated
in hepatic microsomes from Atlantic salmon (Salmo salar L.). Tolbutamide hydroxylation followed Michaelis–Menten kinetics. Mean apparent Michaelis–Menten constant (Km) and maximum reaction velocity (Vmax) values for 4-hydroxytolbutamide (TBOH) formation were 0.09 ± 0.031 mM and 49.5 ± 6.03 pmol/min/mg, respectively. Addition
of sulfaphenazole, an inhibitor for mammalian CYP2C9, in a range from 1 to 200 μM decreased formation of TBOH in a concentration-dependent
manner, but not to 50%. Neither fluconazole, an inhibitor of human CYP2C9, nor ketoconazole, inhibitor of CYP1A and CYP3A
in fish, affected TBOH formation. In contrast ellipticine, an inhibitor of CYP1A in fish inhibited TBOH formation with the
IC50 value of 12.1 μM. The rate of TBOH formation was competitively inhibited by 100 μM of sesamin in the incubations, but the
degree of inhibition did not increase with increased sesamin concentration. Ethoxyresorufin hydroxylase (EROD) activity was
inhibited by tolbutamide in a non-competitive manner (inhibition constant Ki = 218 μM). Our data suggest that tolbutamide is metabolized by salmon microsomes with formation of TBOH. CYP1A might be involved
in this reaction as suggested by decreased TBOH formation in the presence of ellipticine and decreased EROD activity in the
presence of tolbutamide. Incubation of diclofenac with the microsomes yielded no metabolite formation, suggesting that salmon
does not possess diclofenac-metabolizing activity. 相似文献
9.
E. V. Navolotskaya T. A. Zargarova T. N. Lepikhova V. L. Turobov R. I. Nurieva N. V. Malkova V. P. Zav’yalov V. M. Lipkin 《Russian Journal of Bioorganic Chemistry》2000,26(1):27-33
The antiproliferative and immunosuppressivein vitro effects ofimmunocortin, a synthetic adrenocorticotropin-like (ACTH-like) decapeptide H-Val-Lys-Lys-Pro-Gly-Ser-Ser-Val-Lys-Val-OH, whose sequence
corresponds to segment 11–20 of the variable part of the human IgG1 heavy chain, were studied. At concentrations of 10−11−10−7 M, immunocortin was found to inhibit the growth of the human MT-4 T-lymphoblastoid cell line, to suppress the blast transformation
of thymocytes, and to decrease the spontaneous mobility of peritoneal macrophages and their bactericidal action toward the
virulent strainSalmonella typhimurium 415. By using a125I-labeled “addressing” fragment of ACTH {[125I]ACTH (13–24)}, we showed that MT-4 cells express specific receptors for ACTH (K
d 97 pM). Immunocortin and human ACTH (but not the heavy chain of IgG1) competitively inhibited the binding of [125I]ACTH-(13–24) to these receptors withK
i1 of 0.38 andK
i2 of 0.34 nM, respectively. Specific receptors for ACTH (K
d 5.8 nM) on mouse thymocytes were detected and characterized. The unlabeled immunocortin was shown to compete with labeled
ACTH-(13–24) for binding to these receptors (K
i=1.8 nM), and this binding of immunocortin to receptors on thymocytes activates adenylate cyclase from these cells and increases
the intracellular concentration of cAMP. 相似文献
10.
Zi-gang Tian Tian-tang Dong Da Teng Ya-lin Yang Jian-hua Wang 《Applied microbiology and biotechnology》2009,82(6):1097-1103
The increasing problem of antibiotic resistance among pathogenic bacteria requires development of new antimicrobial agents.
The pivotal assets of the antimicrobial peptide include potential for rapid bactericidal activity and low propensity for resistance.
The four new antimicrobial hybrid peptides were designed based on peptides LFB15(W4,10), HP(2-20), and cecropin A according
to the structure–activity relationship of the amphipathic and cationic antimicrobial peptides. Their structural parameters
were accessed by bioinformatics tools, and then two hybrids with the most potential candidates were synthesized. The hybrid
peptide LH28 caused an increase in antibiotic activity (MIC50 = 1.56–3.13 μM) against given bacterial strains and did not cause obvious hemolysis of rabbit erythrocytes at concentration
of 3.13 μM with effective antimicrobial activity. The results demonstrate that evaluating the structural parameters could
be useful for designing novel antimicrobial peptides.
Zi-gang Tian and Tian-tang Dong contributed equally to this paper 相似文献
11.
Mary Chebib Navnath Gavande Kit Yee Wong Anna Park Isabella Premoli Kenneth N. Mewett Robin D. Allan Rujee K. Duke Graham A. R. Johnston Jane R. Hanrahan 《Neurochemical research》2009,34(10):1704-1711
GABAC receptors play a role in myopia, memory-related disorders and circadian rhythms signifying a need to develop potent and selective
agents for this class of receptors. Guanidino analogs related to glycine, β-alanine and taurine were evaluated at human ρ1GABAC receptors expressed in Xenopus oocytes using 2-electrode voltage clamp methods. Of the 12 analogs tested, 8 analogs were active as antagonists and the remaining
were inactive. (S)-2-Guanidinopropionic acid (IC50 = 2.2 μM) and guanidinoacetic acid (IC50 = 5.4 μM; K
B = 7.75 μM [pK
B = 5.11 ± 0.06]) were the most potent being competitive antagonists at this receptor. In contrast, the β-alanine and GABA
guanidino analogs showed reduced activity, indicating the distance between the carboxyl carbon and terminal nitrogen of the
guanidino group is critical for activity. Substituting the C2-position of guanidinoacetic acid with various alkyl groups reduced
activity indicating that steric effects may impact on activity. The results of this study contribute to the structure–activity-relationship
profile required in developing novel therapeutic agents. 相似文献
12.
Rocío Montoya-Pérez Alfredo Saavedra-Molina Xóchitl Trujillo Miguel Huerta Felipa Andrade Enrique Sánchez-Pastor Mónica Ortiz 《Journal of bioenergetics and biomembranes》2010,42(1):21-27
Cell intermediary metabolism and energy production succeeds by means of mitochondria, whose activity is in relation to transmembrane
potential and/or free radical production. Adenosine triphosphate (ATP)-dependent potassium channels (KATP) in several cell types have shown to couple cell metabolism to membrane potential and ATP production. In this study, we explore
whether oxygen consumption in isolated skeletal-muscle mitochondria differs in the presence of distinct respiration substrates
and whether these changes are affected by KATP-channel inhibitors such as glibenclamide, 5-Hydroxydecanoate (5-HD), and KATP channel activators (pinacidil and diazoxide). Results demonstrate a concentration-dependent diminution of respiration rate
by glibenclamide (0.5–20 μM), pinacidil (1–50 μM), and diazoxide (50–200 μM), but no significant differences were found when
the selective mitochondrial KATP-channel inhibitor (5-HD, 10–500 μM) was used. These results suggest that these KATP-channel agonists and antagonists exert an effect on mitochondrial respiration and that they could be acting on mito-KATP or other respiratory-chain components. 相似文献
13.
Human C-reactive protein (CRP) is a clinically important classical acute phase protein. Although CRP has been reported to
bind with many nucleated cells, the direct binding of CRP to erythrocytes in diseases remains largely unexplored. The main
focus of the present study was to investigate the binding of disease-specific CRP to erythrocytes of same patients. Distinct
molecular variant of disease-specific CRP was affinity purified from sera of malaria patients (CRPMal). This CRP showed strong binding with malaria erythrocytes (RBCMal) as confirmed by flow cytometric analysis (FACS), enzyme-linked immunosorbent assays (ELISA), and radio binding assays. Calcium
and phosphoryl choline (PC) were found to be essential for this interaction. A 2.3-fold increased binding of induced CRP to
RBCMal as compared to normal erythrocytes (RBCN) confirmed disease-specificity. Preincubation of RBCMal with unconjugated CRP showed 3–5 fold inhibition. The association constant of CRP and RBCMal was 4.7 × 106 cpm/μg with the corresponding number of receptors/cell being 4.3 × 105. The effector function of CRPMal has been demonstrated by its potency to activate the complement pathway. An optimal dose of 10 μg/ml of CRP induced three-fold
higher hemolysis of patient erythrocytes as compared to RBCN. These studies provide direct evidence for an important phagocytic functional interaction of this acute-phase protein by
triggering the CRP-complement pathway after the binding of CRPMal with RBCMal. Hemolysis as triggered by this pathway may be one of the causative factors of anemia, a common clinical manifestation of
this disease. 相似文献
14.
Olga A. Akimova Johanne Tremblay James W. Van Huysse Pavel Hamet Sergei N. Orlov 《Apoptosis : an international journal on programmed cell death》2010,15(1):55-62
Mechanisms underlying the tissue-specific impact of cardiotonic steroids (CTS) on cell survival and death remain poorly understood.
This study examines the role of Na+,K+-ATPase α subunits in death of Madin-Darby canine kidney (MDCK) cells evoked by 24-h exposure to ouabain. MDCK cells expressing
a variant of the α1 isoform, CTS-sensitive α1S, were stably transfected with a cDNA encoding CTS-resistant α1R-Na+,K+-ATPase, whose expression was confirmed by RT–PCR. In mock-transfected and α1R-cells, maximal inhibition of 86Rb influx was observed at 10 and 1000 μM ouabain, respectively, thus confirming high abundance of α1R-Na+,K+-ATPase in these cells. Six-hour treatment of α1R-cells with 1000 μM ouabain led to the same elevation of the [Na+]i/[K+]i ratio that was detected in mock-transfected cells treated with 3 μM ouabain. However, in contrast to the massive death of
mock-transfected cells exposed to 3 μM ouabain, α1R-cells survived after 24-h incubation with 1000 μM ouabain. Inversion of
the [Na+]i/[K+]i ratio evoked by Na+,K+-ATPase inhibition in K+-free medium did not affect survival of α1R-cells but increased their sensitivity to ouabain. Our results show that the α1R
subunit rescues MDCK cells from the cytotoxic action of CTS independently of inhibition of Na+,K+-ATPase-mediated Na+ and K+ fluxes and inversion of the [Na+]i/[K+]i ratio. 相似文献
15.
The bacterium Oenococcus oeni employs the heterolactic fermentation pathway (products lactate, ethanol, CO2) during growth on fructose as a substrate, and the mannitol pathway when using fructose as an electron acceptor. In this study, [U-13C]glucose, [U-13C]fructose, HPLC, NMR spectroscopy, and enzyme analysis were applied to elucidate the use of both pathways by the hexoses. In the presence of glucose or pyruvate, fructose was metabolized either by the mannitol or the phosphoketolase pathways, respectively. Phosphoglucose isomerase, which is required for channeling fructose into the phosphoketolase pathways, was inhibited by a mixed-type inhibition composed of competitive (K
i=180 M) and uncompetitive (Ki=350 M) inhibition by 6-phosphogluconate. Erythrose 4-phosphate inhibited phosphoglucose isomerase competitively (K
i=1.3 M) with a low contribution of uncompetitive inhibition (Ki=13 M). The cellular 6-phosphogluconate content during growth on fructose plus pyruvate (<75 M) was significantly lower than during growth on fructose alone or fructose plus glucose (550 and 480 M). We conclude that competitive inhibition of phosphoglucose isomerase by 6-phosphogluconate (and possibly erythrose 4-phosphate) is responsible for exclusion of fructose from the phosphoketolase pathway during growth on fructose plus glucose, but not during growth on fructose plus pyruvate. 相似文献
16.
Cathespin B has been purified 750-fold to apparent homogeneity from human and bovine brain cortex using ammonium sulfate fractionation
(30–70%), chromatography on Sephadex G-100, CM-Sephadex C-50, and concanavalin A-Sepharose. Enzyme was assayed fluorometrically
at pH 4.0 with pyridoxyl-hemoglobin in the presence of 1 mM DTT and 1 mM EDTA. Properties of the enzyme from the two sources
proved to be similar. On disc PAGE the purified preparation produced two bands associated with proteinase activity that are
due to existence of two multiple forms of brain cathepsin B with pI 6.1 and 6.8. The enzyme is completely inactivated by thiol-blocking
reagents, leupeptin, E-64, and demands thiol compounds for its ultimate activity. Z-Phe-Ala-CHN2 is a potent inhibitor of the enzyme (K
2nd=1280 M−1s−1) in contrast to Z-Phe-Phe-CHN2 (K
2nd=264 M−1s−1). pH optimum in the reaction of hydrolysis of Pxy-Hb is 4.0–6.0,K
M(app.) =10−5 M. Cathepsin B splits azocasein: pH optimum 5.0–6.0,K
M(app.)=2.2·10−5 M, but inclusion of urea in the incubation medium depresses the azocaseinolytic activity of the enzyme 1.5-fold. It does
not split Lys-NNap, Arg-NMec and is not inhibited by bestatin. The specific activity of brain cathepsin B with Z-Arg-Arg-NNapOMe
at pH 6.0 is 10-fold higher than with Bz-Arg-NNap, Z-Gly-Gly-Arg-NNap is a poor substrate. With Z-Arg-Arg-NMec and Bz-Phe-Val-Arg-NMec
the specific acitivity is 80 and 35%, respectively of that with Z-Phe-Arg-NMec.
Special Issue dedicated to Dr. Eugene Kreps. 相似文献
17.
M. V. Potapovich D. I. Metelitza O. I. Shadyro 《Applied Biochemistry and Microbiology》2012,48(3):250-256
The inhibition efficiency (antioxidant activity) of hydroxy derivatives of coumarin, such as esculetin, dicumarol, and fraxetin,
was studied in the methemalbumin-H2O2-tetramethylbenzidine (TMB) pseudoperoxidase system at 20°C in a buffered physiological solution (pH 7.4) containing 6% DMF
and 0.25% DMSO. The inhibitor’s efficiency was quantitatively characterized by the inhibition constants (K
i, μM) and the inhibition degree (%). The K
i values for esculetin, dicumarol, and fraxetin were 9.5, 15, and 26 μM, respectively. Esculetin and fraxetin inhibited pseudoperoxidase
oxidation of TMB in a noncompetitive manner; dicumarol, in a mixed manner. The inhibiting activity of esculetin in peroxidase-catalyzed
TMB oxidation at pH 6.4 is characterized by a K
i value equal to 1.15 μM, and the inhibition process is competitive. Esculetin was found to be the most effective antioxidant
of plant origin among all derivatives previously studied in model biochemical systems. 相似文献
18.
Rohanah Hussain Christopher L. Joannou Giuliano Siligardi 《International journal of peptide research and therapeutics》2006,12(3):269-273
Microbes are increasingly developing defensive mechanisms against known drugs via mutations. There are signs of emergence of superbugs immune to most known antibiotics available. The need for a new class of drugs to counteract this problem is of paramount importance for continued general well being of mankind. A new class of drugs, antimicrobial peptides, has not been fully exploited primarily due to high cytotoxicity, poor lipophilicity preventing systemic distribution and stability. We have synthesised 9-amino acid residue cationic peptides RH01 and RH02 lipidated with myristoyl and octyl groups respectively. These peptides exhibited potent antimicrobial activity and low cytotoxicity. The lipopeptide RH01 has antimicrobial activity against a broad range of microorganisms including bacteria, yeast and filamentous fungi with greatest activity toward Gram-positive bacteria, including S. aureus MRSA stain, MIC’s ranging between 2–8 μM. The MIC for Gram-negative bacteria was higher ranging from between 30–250 μM. RH01 also had antimicrobial activity towards fungi showing good activity against the pathogenic yeast Candida albicans but was less active towards the filamentous fungi Aspergillus niger. The antimicrobial activity of RH01 as a measure of Ki(50) for E. coli and S. aureus was 35–60 μM and 3–7 μM, respectively. In-house data showed the compound is bactericidal even at higher bacteria concentration. The octylated lipopeptide RH02 has similar activities towards S. aureus (3.3 μM) and E coli (53.3 μM) as the myristolated RH01. There was no haemolytic activity of the lipopeptide RH01 towards human blood. Acute intravenous toxicity study in mice showed that both RH01 and RH02 induced no macroscopic abnormalities at their highest non-lethal dose of 75 mg/kg and 150 mg/kg bodyweight, respectively.Australian Peptide Conference Issue. 相似文献
19.
O-Linked N-acetylglucosamine (O-GlcNAc) modification of serines/threonines on cytoplasmic proteins is a significant signal regulating cellular processes
such as cell cycle, cell development, and cell apoptosis. O-GlcNAcase (OGA) is responsible for the removal of O-GlcNAc, and it thus plays a critical role in O-GlcNAc metabolism. Interestingly, OGA can be cleaved by caspase-3 into two fragments during apoptosis, producing an N-terminal
fragment (1–413 a.a.), termed nOGA. Here, using 4-MU-GlcNAc (4-methylumbelliferyl 2-acetamido-2-deoxy-β-D-glucopyranoside)
as substrate, we found that the nOGA fragment retains high glycosidase activity. To probe the role of nOGA in apoptosis, it
is essential to develop a potent and specific nOGA inhibitor. However, many reported inhibitors active at nanomolar concentrations
(including PUGNAc, STZ, GlcNAc-statin, and NAG-thiazoline) against full-length OGA were not potent for nOGA. Next, we screened
a small triazole-linked carbohydrate library and first identified compound 4 (4-pyridyl-1-(2′-deoxy-2′-acetamido-β-D-glucopyranosyl)-1,2,3-triazole) as a potent and competitive inhibitor for nOGA. This
compound shows 15-fold selectivity for nOGA (K
i = 48 μM) over the full-length OGA (K
i = 725 μM) and 10-fold selectivity over human lysosomal β-hexosaminidase A&B (Hex A&B) (K
i = 502 μM). These results reveal that compound 4 can be used as a potent and selective inhibitor for probing the role of nOGA in biological systems. 相似文献
20.
Muscarinic receptor-linked G protein, G
i
, can directely activate the specific K+ channel (I
K(ACh)) in the atrium and in pacemaker tissues in the heart. Coupling of G
i
to the K+ channel in the ventricle has not been well defined. G protein regulation of K+ channels in isolated human ventricular myocytes was examined using the patch-clamp technique. Bath application of 1 μm acetylcholine (ACh) reversibly shortened the action potential duration to 74.4 ± 12.1% of control (at 90% repolarization,
mean ±sd, n= 8) and increased the whole-cell membrane current conductance without prior β-adrenergic stimulation in human ventricular
myocytes. The ACh effect was reversed by atropine (1 μm). In excised inside-out patch configurations, application of GTPγS (100 μm) to the bath solution (internal surface) caused activation of I
K(ACh) and/or the background inwardly-rectifying K+ channel (I
K1) in ventricular cell membranes. I
K(ACh) exhibited rapid gating behavior with a slope conductance of 44 ± 2 pS (n= 25) and a mean open lifetime of 1.8 ± 0.3 msec (n= 21). Single channel activity of GTPγS-activated I
K1 demonstrated long-lasting bursts with a slope conductance of 30 ± 2 pS (n= 16) and a mean open lifetime of 36.4 ± 4.1 msec (n= 12). Unlike I
K(ACh), G protein-activated I
K1 did not require GTP to maintain channel activity, suggesting that these two channels may be controlled by G proteins with
different underlying mechanisms. The concentration of GTP at half-maximal channel activation was 0.22 μm in I
K(ACh) and 1.2 μm in I
K1. Myocytes pretreated with pertussis toxin (PTX) prevented GTP from activating these channels, indicating that muscarinic
receptor-linked PTX-sensitive G protein, G
i
, is essential for activation of both channels. G protein-activated channel characteristics from patients with terminal heart
failure did not differ from those without heart failure or guinea pig. These results suggest that ACh can shorten the action
potential by activating I
K(ACh) and I
K1 via muscarinic receptor-linked G
i
proteins in human ventricular myocytes.
Received: 23 September 1996/Revised: 18 December 1996 相似文献