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1.
Catechol-O-methyl transferase (COMT) activity can be monitored continuously using a coupled enzyme assay in which the inhibitory product S-adenosylhomocysteine (SAH) is converted to S-inosylhomocysteine (SIH). A simple spectrophotometric assay for COMT is described based on the difference in the ultraviolet absorption spectra between SAH and SIH.  相似文献   

2.
A rapid spectrophotometric assay for catechol-O-methyltransferase   总被引:1,自引:0,他引:1  
A new assay technique for catechol-O-methyltransferase is described. 3,4-Dihydroxyacetophenone is used as the substrate for the assay and the products, 3-hydroxy-4-methoxyacetophenone and 4-hydroxy-3-methoxyacetophenone are detected spectrophotometrically at 344 nm in borate buffer, pH 10.0. This spectrophotometric procedure is simple, rapid, and inexpensive while retaining reasonably high sensitivity and precision.  相似文献   

3.
A rapid assay for measuring the activities of catechol-O-methyltransferase (COMT) is described. The method is based on high-performance liquid chromatography (HPLC)-fluorescence detection, and includes on-line extraction of catecholamines with a precolumn, separation of norepinephrine (NE) and normetanephrine (NMN) on an ODS column, electrochemical oxidation, and post-column fluorogenic derivatization using ethylenediamine. The method took less than 25 min for one sample, which is half that of the previous method and the sensitivity was similar. The intra-day assay precisions were 0.52-1.6%, and the inter-day assay precisions were 3.6-5.8% for rat liver and cerebral cortex (n = 5). The method is suitable for the rapid measurement of COMT activities of many biological samples.  相似文献   

4.
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Summary A sensitive radioenzymatic assay of catechol-O-methyltransferase (COMT) in hair root cells is presented. Only five hair roots with intact bulb and sheath are needed for one assay. By pulling 15–20 hairs, 3–4 parallel assays can be performed. As in erythrocytes the COMT activity in hair root cells is constant for each individual. Nevertheless, there is no high correlation between the enzyme activities in erythrocyte and in hair root cells (r=0.26, 0.1>P>0.05, N=46).The determination of COMT in hair root cells offers a further application of this source in genetic research, as in the study of a correlation between COMT activity and various endogenous psychiatric disorders.Part of the thesis of T. Strohmeyer, Faculty of Medicine, University of HamburgDedicated to Prof. H. Holzer on the occasion of his 60th birthday  相似文献   

6.
Membrane proteins constitute 20-30% of all proteins encoded by the genome of various organisms. While large amounts of purified proteins are required for pharmaceutical and crystallization attempts, there is an unmet need for the development of novel heterologous membrane protein overexpression systems. Specifically, we tested the application of Brevibacillus choshinensis cells for the biosynthesis of human membrane bound catechol-O-methyltransferase (hMBCOMT). In terms of the upstream stage moderate to high expression was obtained for complex media formulation with a value near 45 nmol/h/mg for hMBCOMT specific activity achieved at 20 h culture with 37 °C and 250 rpm. Subsequently, the efficiency for reconstitution of hMBCOMT is markedly null in the presence of ionic detergents, such as sodium dodecyl sulphate (SDS). In general, for non-ionic and zwiterionic detergents, until a detergent critic micellar concentration (CMC) of 1.0 mM, hMBCOMT shows more biological activity at lower detergent concentrations while for detergent CMC higher than 1 mM, higher detergent concentrations seem to be ideal for hMBCOMT solubilization. Indeed, from the detergents tested, the non-ionic digitonin at 0.5% (w/v) appears to be the most suitable for hMBCOMT solubilization.  相似文献   

7.
8.
A luminol-dependent chemiluminescence assay for the assessment of the phagocytosis of erythrocytes sensitized with anti-D IgG immunoglobulin by mononuclear leukocytes is described. The mononuclear leukocytes were obtained by apheresis enriched by centrifugation through a density gradient and stored in liquid nitrogen before use. The total reaction mixture, consisting of mononuclear leukocytes-luminol-erythrocytes (either anti-D IgG sensitized or unsensitized controls) was 500 μl, light detection was by an LKB 1251 luminometer. Peak luminescence was seen between 35–45 minutes, the reaction being exhausted by 120 minutes. Determination of the reproducibility of the assay gave intra- and inter-assay coefficients of variation of 5% and 13% respectively. We found the chemiluminescent response to be affected by the number of erythrocytes used in the assay and by the composition of the medium in which the cells were resuspended, particularly the pH at the initiation of the assay. We also compared the chemiluminescence assay to a microscopic phagocytic assay and found the results virtually identical. However, the former chemiluminescence assay was much easier to perform, marginally more sensitive, less laborious and eliminated any possibility of subjective error.  相似文献   

9.
Allantoin is separated by thin-layer chromatography (TLC) and sprayed with an acidic solution ofp-dimethylaminobenzaldehyde. The yellow color produced is read in a densitometer and compared with that of a standard. The lower limit of quantitation is 0.1 μg per spot (0.5 mg/100 ml). The method can be utilized for the estimation of allantoin in serum, lymph, and urine.  相似文献   

10.
Abstract Polysialic acids occur as capsular polysaccharides of several pathogenic bacteria. An understanding of how polysialyltransferase functions in the synthesis of polysialic acid will require enzyme purification and characterization in concert with genetic analysis. A rapid filter assay has been developed for bacterial polysialyltransferase suitable for enzyme purification. The filter assay and the currently used paper chromatography methods are equivalent in parallel experiments. The Escherichia coli K92 polysialyltransferase exhibited the same pH and temperature optima, Mg2+ dependence and acceptor specificity in both assays. [14C]Sialic acid bound in filter assays correlates with polymer formed by gel filtration. Specificity may be increased by the addition of exogenous acceptors.  相似文献   

11.
A sensitive assay for glutamyltransferase   总被引:4,自引:0,他引:4  
J Thorndike  L Reif-Lehrer 《Enzyme》1971,12(2):235-241
  相似文献   

12.
A glass micro-chamber which allows polarographic assay in a volume of 180 microliter is described. The conical shape of this chamber allows efficient mixing with a Teflon magnetic flea. This chamber facilitates the study of the small quantities of mitochondria obtained from human tissue biopsies or animal sources. The polarographic assay of mouse liver mitochondria is described.  相似文献   

13.
An assay for biotinidase using biocytin, the natural substrate, is described. The fluorometric procedure uses 1,2-diacetylbenzene which reacts selectively with lysine allowing its direct determination in mixture with biocytin. We have examined the applicability of the assay using human serum biotinidase.  相似文献   

14.
Research on the mechanism of action of cellulases has been hampered by the lack of a rapid, continuous, or kinetic assay. A linked assay system that uses glucose oxidase and horseradish peroxidase has been coupled with β-glucosidase to yield an assay system that can be used for kinetic assays for cellobiase-producing enzymes as well as a measure of cellobiose degradation by β-glucosidases. This assay shows a 20-fold increase in sensitivity over the traditional reducing sugar assay.  相似文献   

15.
16.
Summary A modification is described of the capillary assay for chemotaxis. It employs a 96-well dilution plate and its cover. Capillary tubes are inserted through the cover and are supported by small rubber collars. The method is faster and less tedious and gives more precise results than earlier methods.  相似文献   

17.
A new radioisotopic assay for polyamine oxidase with N1-acetylspermine as substrate is presented. A modified method for the chemical synthesis of radioactive N1-acetylspermine, which gave a good yield, is also described. The reaction mixture, containing N1-[14C]acetylspermine and tissue homogenate, was incubated for the enzyme reaction and applied to a minicolumn of Amberlite CG-50. The reaction product 3-[14C]-acetamidopropanal did not adsorb to the column, but passed through it; thus the eluate could be directly subjected to liquid scintillation counting. The blank levels were low and relatively constant even with crude tissue homogenates. The detection limit obtained was 0.05 nmol per tube. This method is simple, highly sensitive, and highly specific.  相似文献   

18.
19.
A titrimetric method for the assay of glycogen phosphorylase is presented in which a direct and continuous course of reaction is obtained over a wide range of enzyme concentrations (7.2–378.3 μg/ml). The method resulted in rates which were in agreement with those obtained using the inorganic phosphate method, and the expected value of the equilibrium concentration ratio of inorganic phosphate to glucose-1-phosphate was obtained. The method can be extended to higher concentrations, and it can be used to measure the rate in either direction. The Km and Vmax values of each substrate, glucose-1-phosphate and inorganic phosphate, were determined.  相似文献   

20.
N-myristoylation is the irreversible attachment of a C(14) fatty acid, myristic acid, to the N-terminal glycine of a protein via formation of an amide bond. This modification is catalyzed by myristoyl-coenzyme A (CoA):protein N-myristoyltransferase (NMT), an enzyme ubiquitous in eukaryotes that is up-regulated in several cancers. Here we report a sensitive fluorescence-based assay to study the enzymatic activity of human NMT1 and NMT2 based on detection of CoA by 7-diethylamino-3-(4-maleimido-phenyl)-4-methylcoumarin. We also describe expression and characterization of NMT1 and NMT2 and assay validation with small molecule inhibitors. This assay should be broadly applicable to NMTs from a range of organisms.  相似文献   

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