共查询到20条相似文献,搜索用时 36 毫秒
1.
L J Hyland B D Dayton M L Moore A Y Shu J R Heys T D Meek 《Analytical biochemistry》1990,188(2):408-415
A rapid, high-throughput radiometric assay for HIV-1 protease has been developed using ion-exchange chromatography performed in 96-well filtration plates. The assay monitors the activity of the HIV-1 protease on the radiolabeled form of a heptapeptide substrate, [tyrosyl-3,5-3H]Ac-Ser-Gln-Asn-Tyr-Pro-Val-Val-NH2, which is based on the p17-p24 cleavage site found in the viral polyprotein substrate Pr55gag. Specific cleavage of this uncharged heptapeptide substrate by HIV-1 protease releases the anionic product [tyrosyl-3,5-3H]Ac-Ser-Gln-Asn-Tyr, which is retained upon minicolumns of the anion-exchange resin AG1-X8. Protease activity is determined from the recovery of this radiolabeled product following elution with formic acid. This facile and highly sensitive assay may be utilized for steady-state kinetic analysis of the protease, for measurements of enzyme activity during its purification, and as a routine assay for the evaluation of protease inhibitors from natural product or synthetic sources. 相似文献
2.
We have developed an assay using peptide arrays based on phase transition from the glass substrate to the liquid for monitoring quantitative protease activity in real-time. Peptide arrays were fabricated using a bifunctional cross-linker, N-[γ-maleimidobutyryloxy] sulfosuccinimide ester, and a substrate peptide containing two functional groups, cysteine and tetramethyl-6-carboxyrhodamine (TAMRA) on the C- and N-terminus, respectively. The phase transition-based peptide arrays were characterized by analyzing the substrate peptide cleaved from the solid substrate by matrix metalloproteinase-3 (MMP-3). We successfully used this assay to determine the quantitative proteolytic activity of MMP-3 in a dose-dependent manner. In addition, parameters including Michaelis constant (K(m)), maximum rate of enzymatic reaction (V(max)), and half maximal inhibitory concentration (IC(50)) were determined by analyzing the concentrations of substrate peptide cleaved by MMP-3. Therefore, this new assay has potential for the quantitative analysis of enzyme kinetics of protease and informs research developments in drug discovery utilizing kinetic studies. 相似文献
3.
4.
A new, rapid assay for 1 alpha-hydroxylase has been developed using 25-hydroxy-[1 alpha-3H]vitamin D3 as the substrate. Using the solubilized and reconstituted chick 1 alpha-hydroxylase, conversion of this substrate to 1,25-dihydroxyvitamin D3 causes the release of tritium into the aqueous medium. This 3H2O can be easily separated from the labeled substrate by passing the reaction mixture through a reverse-phase silica cartridge. The release of tritium is stereospecific as evidenced by the lack of 3H2O formed when 25-hydroxy-[1 beta-3H]vitamin D3 is used as the substrate. In parallel reactions containing the 25-hydroxy-[26,27-3H]vitamin D3 substrate, production of labeled 1,25-dihydroxyvitamin D3 was assessed by extraction and high-performance liquid chromatography and found to agree very closely with the amount of 3H2O produced from 25-hydroxy-[1 alpha-3H]vitamin D3, validating the accuracy of the new assay. Finally, a major advantage of the tritium-release assay for 1 alpha-hydroxylase is that the results are not affected by further metabolism of the 1,25-dihydroxyvitamin D formed in the incubations. 相似文献
5.
A highly sensitive, mixed-phase assay for chloramphenicol acetyltransferase activity in transfected cells 总被引:8,自引:0,他引:8
We describe a simple, rapid yet extremely sensitive assay for chloramphenicol acetyltransferase (CAT) activity in extracts from transfected eukaryotic cells. Using our modified reaction conditions and the mixed-phase assay, less than 0.000010 unit of CAT activity in transfected cells can be reliably detected. The mixed-phase assay is based on the inability of the polar [3H]-acetyl-Coenzyme A (CoA) substrate to partition out of a urea containing aqueous phase into the nonpolar scintillation fluor, while the [3H]chloramphenicol reaction products partition into the toluene scintillation fluor and are quantitated by scintillation counting. The increased sensitivity of this assay is due to the optimization of the acetyl-CoA concentration, to a urea-containing aqueous phase which lowers the assay background, and to the use of extract blanks. The mixed-phase assay is simpler, is quantitative, uses less costly substrates, and is far more sensitive than the most widely used CAT assays, which require solvent extraction followed by thin-layer chromatography to separate the unreacted substrate from product. 相似文献
6.
Detection of proteolytic bond cleavage was achieved by taking advantage of the bioluminescence emission generated by the photoprotein aequorin. A genetically engineered HIV-1 protease substrate was coupled with a cysteine-free mutant of aequorin by employing the polymerase chain reaction to produce a fusion protein that incorporates an optimum natural protease cleavage site. The fusion protein was immobilized on a solid phase and employed as the substrate for the HIV-1 protease. Proteolytic bond cleavage was detected by a decrease in the bioluminescence generated by the aequorin fusion protein on the solid phase. A dose-response curve for HIV-1 protease was constructed by relating the decrease in bioluminescence signal with varying amounts of the protease. The system was also used to evaluate two competitive and one noncompetitive inhibitor of the HIV-1 protease. Among the advantages of this assay is that by using recombinant methods a complete bioluminescently labeled protease recognition site can be designed and produced. The assay yields very sensitive detection limits, which are inherent to bioluminescence-based methods. An application of this system may be in the high-throughput screening of biopharmaceutical drugs that are potential inhibitors of a target protease. 相似文献
7.
A single-vial liquid extraction assay for choline acetyltransferase that uses [3H]choline as the labeled substrate has been devised. [3H]Choline is incubated with an excess of acetyl-CoA in a small reaction vial which also serves as a scintillation vial. After a suitable reaction period, unreacted [3H]choline is quickly and quantitatively converted to phosphoryl-[3H]choline by the addition of an excess of choline kinase. This treatment is followed by the addition of scintillation fluid containing sodium tetraphenylboron after which the vial is capped, shaken, and counted. A two-phase system is produced in which product [3H]acetylcholine is selectively extracted into the scintillation fluid, where it is counted. Phosphoryl-[3H]choline remains in the aqueous phase and is not counted. This assay is rapid, simple, and quite sensitive. In comparison to assays using acetyl-CoA as the labeled substrate, it is less sensitive to interference by other enzymes and thus more suitable for measuring choline acetyltransferase in crude extracts and in the initial stages of purification. Similar single-vial radiometric assays are described for choline kinase and acetyl-CoA hydrolases. 相似文献
8.
D B Kohn M J Weber P L Carl J A Katzenellenbogen P K Chakravarty 《Analytical biochemistry》1979,97(2):269-276
A peptidyl derivative of [3H]aniline, Gly-Gly-Arg-[3H]anilide, can be used as a substrate in a convenient and sensitive assay procedure for trypsin, urokinase, and plasminogen activator from transformed cells. The extent of hydrolysis can be determined simply by selective extraction of the product [3H]aniline into an organic phase containing a scintillant. (The uncleaved peptide is not appreciably soluble in this phase and is not counted.) The reaction is of comparable sensitivity to fluorimetric assays, but has the advantage that no cleanup of the biological sample is required, since it is far less subject to interference from fluorescence quenching. Other peptidyl anilides should be useful for assaying proteolytic enzymes with widely varying specificities. 相似文献
9.
A simple procedure is presented for the enzymatic preparation of [2-3H]mannose 6-phosphate (Man 6-P) with purified yeast hexokinase and unlabeled ATP. The enzymatically synthesized [2-3H]Man 6-P is utilized as the radiolabeled substrate in a new rapid assay for glucose 6-phosphate (Glc 6-P) phosphatase. The principle of the assay procedure is that the unreacted substrate, [2-3H]Man 6-P, is retained by the anion-exchange resin, AG 1-X8 (acetate), while the enzymatic product, [2-3H]-mannose, is eluted directly into a scintillation counting vial. When Glc 6-P phosphatase activity associated with mouse liver endoplasmic reticulum (ER) vesicles is assayed by the new chromatographic assay, the same characteristic latency and properties are observed, as determined by the commonly used colorimetric assay of inorganic phosphate produced. The anion-exchange radioassay described should be useful for a variety of topological studies on enzymes associated with membrane vesicles derived from liver and kidney ER. 相似文献
10.
A technique is described for the assay of phosphatidate phosphohydrolase using 1,2-[9,10-3H]dioleoyl-sn-glycero-3-phosphate as a substrate. This substrate was prepared enzymatically using mouse liver microsomes washed with 0.5 M NaCl, which synthesize minimal amounts of neutral lipids at high enzyme concentrations. Measurement of the product, 1,2-[9,10-3H]dioleoylglycerol, was 10-fold more sensitive than the usual colorimetric assay for inorganic phosphate release. In addition, the assay provides information about the relative contribution of other activities which limit the availability of diacylglycerols for further esterification to triacylglycerols and/or phospholipids. 相似文献
11.
A tridecapeptide containing tritium-labelled lysine and corresponding closely to residues 98 to 110 of the alpha chain of type I collagen was synthesized by the solid-phase method. Gly-Leu-Hyp-Gly-Nle-[4,5-3H]Lys-Gly-His-Arg-Gly-Phe-Ser-Gly was used as a substrate of human protocollagen lysyl hydroxylase (peptidyllysine, 2-oxoglutarate: oxygen 5-oxidoreductase, EC 1.14.11.4) obtained from dermal fibroblasts. L-[4,5-3H]Lysine was converted to N alpha-t-butyloxycarbonyl-N epsilon-o-chlorobenzyloxycarbonyl [3H]lysine which was incorporated during stepwise synthesis of the peptide. The chemical and radiochemical purities and specific activity of the completed peptide were characterized. A non-radiolabelled analogue of the peptide inhibited the hydroxylation of [3H]lysine-containing protocollagen by human lysyl hydroxylase, indicating that the synthetic peptide interacted with the enzyme. The peptide containing [3H]lysine was a substrate for lysyl hydroxylase and permitted direct measurement of enzyme activity in relatively crude cell extracts by a tritium-release assay. Extracts of cultured fibroblasts from a patient with an autosomal recessive pattern of inheritance for Ehlers-Danlos syndrome type VI had activities for tritium release from either the radiolabelled synthetic peptide or from [3H]lysine-containing protocollagen that were only 30% of those from control cells. These data indicate that a stable, well-defined synthetic peptide containing [3H]lysine is a useful substrate for studies of genetically variant lysyl hydroxylase from cultured human cells. 相似文献
12.
New spectrophotometric and radiochemical assays for acetyl-CoA: arylamine N-acetyltransferase applicable to a variety of arylamines 总被引:3,自引:0,他引:3
Simple and sensitive spectrophotometric and radiochemical procedures are described for the assay of acetyl-CoA:arylamine N-acetyltransferase (NAT; EC 2.3.1.5), which catalyzes the reaction acetyl-CoA + arylamine----N-acetylated arylamine + CoASH. The methods are applicable to crude tissue homogenates and blood lysates. The spectrophotometric assay is characterized by two features: (i) NAT activity is measured by quantifying the disappearance of the arylamine substrate as reflected by decreasing Schiff's base formation with dimethylaminobenzaldehyde. (ii) During the enzymatic reaction, the inhibitory product CoASH is recycled by the system acetyl phosphate/phosphotransacetylase to the substrate acetyl-CoA. The radiochemical procedure depends on enzymatic synthesis of [3H]acetyl-CoA in the assay using [3H]acetate, ATP, CoASH, and acetyl-CoA synthetase. NAT activity is measured by quantifying N-[3H]acetylarylamine after separation from [3H]acetate by extraction. Product inhibition by CoASH is prevented in this system by the use of acetyl-CoA synthetase. 相似文献
13.
A simple assay for phosphatidylserine decarboxylase is described. Following incubation of a mitochondrial fraction from Saccharomyces cerevisiae with purified, exogenous phosphatidyl[3H]serine, the lipid extract is applied to a small DEAE-cellulose column equilibrated in CHCI3-CH3OH (1:1). The unreacted substrate, phosphatidyl[3H]serine, is quantitatively bound by the ion-exchange column while the product, phosphatidyl[3H]ethanolamine, is eluted by sequential washing with CHCI3-CH3OH (1:1) and CH3OH. The organic solvents are evaporated, and the amount of radiolabeled phosphatidyl[3H]ethanolamine formed by enzymatic decarboxylation is determined by liquid scintillation spectrometry. The reliability of this assay was established by showing that several enzymatic properties of the yeast enzyme, defined by the new assay, were essentially identical to the properties characterized by a more tedious paper chromatographic assay described previously. Virtually identical rates of enzymatic decarboxylation of phosphatidyl[3H]serine were also obtained for mitochondrial fractions from pig brain and rat liver when the activities were compared by the column and paper chromatographic methods. 相似文献
14.
The assay of fibroblast and leukocyte-N-acetylglucosaminylphosphotransferase with alpha-methylmannoside acceptor and commercially available UDP-[3H or 14C]N-acetylglucosamine donor was modified to yield low background and consequently high sensitivity and reliability comparable to those obtained with the synthetically made [beta-32P]UDP-N-acetylglucosamine donor. This was achieved by an additional elution step that removed free [3H or 14C]N-acetylglucosamine which appeared to be the breakdown product responsible for the high background. In addition, the [3H or 14C]N-acetylglucosamine-1-phospho-6-alpha-methylmannoside product of the transfer reaction was then isolated and, following desalting, could serve as a substrate for the assay of alpha-N-acetylglucosaminyl phosphodiesterase. Cell preparations of patients with I-cell disease and pseudo-Hurler polydystrophy demonstrated severe to moderate deficiency of transferase activity and normal phosphodiesterase activity toward the respective substrates labeled with 3H or 14C in the glucosamine moiety. 相似文献
15.
M L Fonda 《Analytical biochemistry》1986,155(1):14-22
The alternate procedures used in the tyrosine apodecarboxylase assays for pyridoxal 5'-phosphate were evaluated to determine optimal conditions. Two preparations of tyrosine apodecarboxylase from Streptococcus faecalis were used: a cell suspension and a partially purified cell-free form. The activity of the decarboxylase was measured in two different assays using [14C]tyrosine or [3H]tyrosine as substrate. The presence of serum proteins caused greater inhibition of the assay for serum pyridoxal phosphate using [14C]tyrosine as substrate than the assay with [3H]tyrosine. In contrast, addition of deproteinized serum extract did not appear to inhibit either assay. The rate of reconstitution of the apodecarboxylase in the cell suspension was at least four times slower than that of the cell-free enzyme. The rate of reconstitution of the cell-free enzyme was faster in acetate than in citrate buffer. Inorganic sulfate or phosphate, at normal plasma concentrations, did not alter either the reconstitution rate of tyrosine decarboxylase or the final activity obtained in the assays using either substrate. The tyrosine apodecarboxylase assay for pyridoxal phosphate can be optimized by using deproteinized sera or plasma and incubating the cell-free apoenzyme with the coenzyme in acetate buffer for a time sufficient to obtain maximum reconstitution. 相似文献
16.
A direct assay method is described for L-pipecolate oxidase. The assay uses NaHSO3 to trap the L-alpha-amino [3H]adipate delta-semialdehyde (AAS) formed as a direct reaction product of L-pipecolate oxidase from L-[3H]pipecolic acid. The adduct so formed was separated from the substrate on Dowex 50 (H+) column. The product was identified as [3H]AAS by amino acid analysis after breaking down the adduct by boiling under acidic conditions. The assay is simpler and more specific than fluorometric methods; it is also more sensitive, requiring at most 16 micrograms of liver peroxisome-enriched protein per assay. We have used this assay procedure to detect L-pipecolate oxidase in skin fibroblasts obtained from a control subject and from patients of hyperpipecolic acidaemia and Zellweger syndrome and found that this enzyme activity is present in the control, but absent or decreased in the patients with the peroxisomal disorders. 相似文献
17.
A new rapid, sensitive and convenient procedure is presented allowing determination of dUTPase activity. With [5-(3)H]dUTP used as the substrate, dUTPase, converts it to the corresponding monophosphate and is coupled with thymidylate synthase-catalyzed reaction, resulting in tritium release from [5-(3)H]dUMP. Following charcoal absorption of the labeled nuleotides, radioactivity of tritiated water is determined. The new assay was tested to show comparable results with a previously described assay, based on measuring dUTPase-catalyzed [5-(3)H]dUMP production. 相似文献
18.
19.
S Lemaire M Dumont F Jolicoeur B Belleau 《Canadian journal of physiology and pharmacology》1986,64(6):707-711
14-beta-Methyl-8-oxacyclorphan (BC-3016) was tested for its ability to depress the electrically evoked contractions of the guinea pig ileum (GPI) and of the mouse vas deferens (MVD) and to compete with the binding of prototype ligands selective for kappa-, mu-, or delta-opioid receptors in membrane preparations of rat brain and guinea pig cerebellum. BC-3016 was a very potent agonist in the GPI and MVD preparations, with ID50 of 0.7 and 31 nM, respectively. The activity of levorphanol, a standard alkaloid related to BC-3016, was much lower in both assays with ID50 values of 44 and 86 nM, respectively. Conversely, the activity of BC-3016 was quite comparable to that of dynorphin-A(1-13) in both preparations. In the GPI assay, a putative kappa-receptor antagonist, MR-2266, was 6.6 and 5.5 times more potent than naloxone in blocking the activity of BC-3016 and dynorphin-A(1-13), respectively. BC-3016 was also very potent in displacing bound [3H]ethylketocyclazocine ([3H]EKC) to membrane preparations of the guinea pig cerebellum, a brain component containing predominantly kappa-opioid receptors (Ki of 0.58 nM). Its potency in the displacement of the bound mu-ligand, 3H-labelled (D-Ala2,MePhe4,Gly-OH5)-enkephalin ([3H]DAGO), to rat brain homogenates was somewhat lower (Ki of 0.8 nM) but still high when compared with its ability to displace the delta-ligand, 3H-labelled (D-Ser2, Thr6)-Leu-enkephalin ([3H]DSLET) to rat brain homogenates (Ki of 4.45 nM). The affinity of BC-3016 for the opioid receptor was 2.1-fold higher than that of U-50488H, a selective kappa-opioid ligand.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
20.
Rate constants for binding of five inhibitors of human immunodeficiency virus (HIV) protease were determined by stopped-flow spectrofluorometry. The two isomers of quinoline-2-carbonyl-Asn-Phe psi-[CH(OH)CH2N]Pro-O-t-Bu (R diastereomer = 1R; S diastereomer = 1S) quenched the protein fluorescence of HIV protease and thus provided a spectrofluorometric method to determine their binding rate constants. The dissociation rate constants for acetyl-Thr-Ile-Leu psi(CH2NH)Leu-Gln-Arg-NH2 (2), (carbobenzyloxy)-Phe psi[CH(OH)CH2N]Pro-O-t-Bu (3), and pepstatin were determined by trapping free enzyme with 1R as 2, 3, and pepstatin dissociated from the respective enzyme.inhibitor complex. Association rate constants of 1R, 2, and pepstatin were calculated from the time-dependent inhibition of protease-catalyzed hydrolysis of the fluorescent substrate (2-aminobenzoyl)-Thr-Ile-Nle-Phe(NO2)-Gln-Arg-NH2 (4). The kinetic data for binding of 1S to the protease fit a two-step mechanism. Kd values for these inhibitors were calculated from the rate constants for binding and were similar to the respective steady-state Ki values. 相似文献