共查询到20条相似文献,搜索用时 31 毫秒
1.
A simple and sensitive spectrophotometric method for the determination of tryptophan (TRP) is described. The method is based on the coupling reaction of tryptophan with diazotized p-phenylenediamine dihydrochloride (PPDD) in sulfuric acid medium to give the colored product having an absorption maximum at 520 nm. The coupled product was stable for 2h. Beer's law is obeyed in the tryptophan concentration range of 0.25-11 microg/ml. The method is applied for the analysis of pharmaceutical preparations of tryptophan and also in protein samples for tryptophan. Common excipients used as additives in pharmaceutical preparations do not interfere in the proposed method and the significant feature of the method is that most of the amino acids do not interfere in the determination of tryptophan. 相似文献
2.
K Gerth 《Analytical biochemistry》1985,144(2):432-435
The method described comprises the transformation of ammonium into ammonia, the rapid and gentle liberation of the ammonia followed by the measurement of the nitrogen in a Dohrmann nitrogen analyzer. Untreated biological samples (1-50 microliters) were pipetted onto magnesium oxide tablets at 130 degrees C and the ammonia liberated was transferred by a continuous stream of nitrogen carrier gas into the nitrogen analyzer. There the ammonia was determined by oxidative pyrolysis and subsequent chemiluminescence measurement of the excited NO2. The result could be read in nanograms ammonia nitrogen within 6.5 min. Apart from volatile amines, which are usually negligible in biological samples, the method was specific for ammonia because under the given conditions of volatilization the labile groups of glutamine and asparagine did not interfere. The assay was sensitive in the range of 1.5-150 nmol ammonia and suitable for the routine analysis of small samples. 相似文献
3.
Use of the avidin-biotin-peroxidase complex (ABC) method for the localization of rabbit cathepsin B in cells and tissues 总被引:2,自引:0,他引:2
Visualization of rabbit cathepsin B was achieved utilizing monospecific sheep antibodies and the avidin-biotin-peroxidase complex (ABC) method. This technique was applied to stain 1) paraffin sections of the liver, 2) fixed fibroblasts from tissue culture, and 3) fixed mesenteries. Cathepsin B was found to be localized within cells of the lining of the liver sinusoids (most probably Kupffer cells), in perinuclear granules of cultured fibroblasts, and within histiocytes of the mesentery. The results demonstrate that the method permits precise and highly sensitive localization of cathepsin B within cells and tissues. Compared to fluorescent staining of cathepsin B, the ABC method has the advantage that routine paraffin sections can be stained, and that all the orthodox histological staining procedures can still be carried out. 相似文献
4.
Richard E. Miller 《Analytical biochemistry》1976,75(1):91-99
A simple, sensitive fluorometric assay for l-glutamine is described. l-Glutamine is quantified by measuring the amount of NADPH oxidized in the presence of a glutamine-containing sample (2–20 nmol), 5 mm α-ketoglutarate, 1 mm EDTA, 63 μM NADPH, and purified Escherichia coli glutamate synthase (EC 2.6.1.53). High concentrations of l-glutamate and ammonia do not interfere. The method has been applied to quantification of l-glutamine in serum. 相似文献
5.
A highly sensitive spectrophotometric method for the selective detection of catechol compounds such as catechol siderophores (e.g., enterobactin) is described. The basis of the method involves the ability of the vicinal aromatic hydroxyl groups under acidic conditions to bring about a reduction of Fe3+ (from ferric ammonium citrate) to Fe2+. Detection of Fe2+ in the presence of Fe3+ is made with 1,10-phenanthroline under previously established conditions. The assay mixture is heated at 60 degrees C for 1 h to accelerate the development of color which is subsequently measured at 510 nm. The Beer-Lambert law is obeyed over the range of 0.16 to 60 microM 2,3-dihydroxybenzoic acid. Compared to the Arnow nitration method, the assay is more responsive, is approximately seven times more sensitive, and is effective with catechols substituted at positions 3 and 4. The method gives positive results with catechols such as DL-DOPA, L-dopamine, (+/-)-epinephrine, and DL-norepinephrine. Very rapid color development is obtained with ascorbic acid and p-diols, while m-diols are poorly detected. Low degrees of reactivity are shown by hydroxylamino and hydroxamate compounds. Phenolic, sulfydryl, indolyl, and quinonyl derivatives do not interfere with the reaction. The method has been adapted to determine catechol compounds in the culture medium of bacterial cells grown at different iron concentrations. 相似文献
6.
A new method for the assay of glutaminase activity: direct measurement of product formation by an ammonia electrode 总被引:1,自引:0,他引:1
Y Z Huang 《Analytical biochemistry》1974,61(2):464-470
A simple, reliable procedure is described for the quantitative assay of glutaminase reaction by measuring product formation using an ammonia electrode. The ammonia electrode is a gas-detecting electrode, sensing the level of dissolved ammonia in aqueous solutions. Ammonia concentration can be read from calibration curves after converting ammonium ion to ammonia by adding sufficient base. Sample color and turbidity do not affect measurements, and samples need not be distilled. The concentrations of the three glutaminase isoenzymes from rat tissues measured by this method are strictly comparable to those measured by other methods. 相似文献
7.
Persistent reovirus infections of murine L929 (L) fibroblast cells select mutant (LX) cells that do not support proteolytic disassembly of reovirus virions within the endocytic pathway. To better understand the function and regulation of endocytic proteases, we conducted experiments to define the block to reovirus disassembly displayed by LX cells. In contrast to parental L cells, mutant LX cells harbor defects that interfere with the maturation and activity of cathepsin B and cathepsin L but not cathepsin H. The cDNAs encoding cathepsin B and cathepsin L in L cells are identical to those in LX cells, indicating that LX cells manifest an extrinsic block to the function of these enzymes. Mixed lysates of L cells and LX cells lack activity of both cathepsin B and cathepsin L, suggesting the presence of an inhibitor of cathepsin function in LX cells. A cathepsin B-green fluorescent protein (GFP) fusion protein expressed in L cells and purified by immunoprecipitation retains cathepsin B activity, whereas cathepsin B-GFP expressed in LX cells does not. However, activity of cathepsin B-GFP expressed in LX cells can be recovered by incubating the immunoprecipitate with L cell lysate followed by immunoprecipitation, providing further evidence that LX cells express a cathepsin inhibitor. Native-gel electrophoresis and gel filtration chromatography demonstrate that, in both cell lines, the double-chain form of cathepsin B is sequestered in a large molecular weight complex that renders this form of the enzyme inactive. Alteration of this sequestration complex appears to be responsible for inhibition of cathepsin B in LX cells. These findings suggest that cathepsins can be regulated within the endocytic pathway. Moreover, this regulation influences host cell susceptibility to intracellular pathogens. 相似文献
8.
We present an improved and simple direct assay for formation of inorganic sulfide from L-cysteine in a beta-replacement reaction catalyzed by tryptophan synthase. This method provides a useful enzymatic assay for pyridoxal phosphate-dependent beta-replacement reactions in which the amino acid substrate is L-cysteine and the cosubstrate is 2-mercaptoethanol. The assay should be applicable to similar reactions with L-cysteine and other cosubstrates. The method has several advantages over other methods which have been used to assay similar beta-replacement reactions. The assay is highly reproducible and sensitive and is conveniently carried out in disposable 1.5-ml centrifuge tubes. The color remains stable for several hours. The thiol compounds L-cysteine and 2-mercaptoethanol do not interfere at the concentrations used. The method has useful applications to studies of the rates and reaction specificities of several other pyridoxal phosphate enzymes which catalyze beta-replacement reactions. We demonstrate the use of the method to study the effects of site-directed mutagenesis on the reaction specificity and mechanism of the tryptophan synthase alpha 2 beta 2 complex. 相似文献
9.
Cathepsin E is an intracellular aspartic proteinase of the pepsin family predominantly expressed in cells of the immune system and believed to contribute to homeostasis by participating in host defense mechanisms. Studies on its enzymatic properties, however, have been limited by a lack of sensitive and selective substrates. For a better understanding of the importance of this enzyme in vivo, we designed and synthesized a highly sensitive peptide substrate for cathepsin E based on the sequence of the specific cleavage site of alpha2-macroglobulin. The substrate constructed, MOCAc-Gly-Ser-Pro-Ala-Phe-Leu-Ala-Lys(Dnp)-D-Arg-NH2 [where MOCAc is (7-methoxycoumarin-4-yl)acetyl and Dnp is dinitrophenyl], derived from the cleavage site sequence of human alpha2-macroglobulin, was the most sensitive and selective for cathepsin E, with k(cat)/K(m) values of 8-11 microM(-1) s(-1), whereas it was resistant to hydrolysis by the analogous aspartic proteinases cathepsin D and pepsin, as well as the lysosomal cysteine proteinases cathepsins B, L, and H. The assay allows the detection of a few fmol of cathepsin E, even in the presence of plasma and cell lysate, and gives accurate results over a wide enzyme concentration range. This substrate might represent a useful tool for monitoring and accurately quantifying cathepsin E, even in crude enzyme preparations. 相似文献
10.
Futoshi Aranishi Kenji Hara Kiyoshi Osatomi Tadashi Ishihara 《Comparative biochemistry and physiology. Part B, Biochemistry & molecular biology》1996,114(4):371-376
Cathepsin B was purified from the crude extract of carp (Cyprinus carpio) hepato-pancreas by the method involving ammonium sulfate fractionation and five sequential chromatographies monitored the activity with Z-Arg-Arg-MCA as a substrate, and the specific activity increased about 11,400 fold with a 2% recovery. Although the homogeneity of the purified cathepsin B was established on Native-PAGE, it migrated as two bands of 29,000 and 25,000 molecular weights by the single and heavy chains on SDS-PAGE, respectively. The monospecific antibody against the homogeneous cathepsin B was purified by the affinity chromatography on cathepsin B-Sepharose 4B, and did not immunologically react with rat cathepsin B, carp cathepsins H and L but only with carp cathepsin B by immunoelectrophoretic blot analysis. As the result of the tissue and liver distributions of cathepsin B, the remarkable immunological reactivities in the extracts of spleen, kidney and hepato-pancreas in carp and those of pacific cod, yellow fin tuna, skip jack tuna and common mackerel in pisces were detected with the anti-carp hepato-pancreas cathepsin B at molecular weight of nearby 29,000 or 25,000. 相似文献
11.
A procedure has been developed for the measurement of lysosmal peptidase activity that is based upon highly sensitive fluorometric amino acid analysis. The fluorochrome results from the reaction of phthalaldehyde and mercaptoethanol with amino acids or similar compounds at pH 9.5. The fluorometric method is 10–100 times more sensitive than the colorimetric ninhydrin procedure for the measurement of lysosomal peptidases. The method lends itself to the measurement of any peptidase or protease that yields free amino groups in the product. The method has been applied to the measurement of cathepsin A and lysosomal dipeptidase activity. 相似文献
12.
Kumar GD Chavarria GE Charlton-Sevcik AK Yoo GK Song J Strecker TE Siim BG Chaplin DJ Trawick ML Pinney KG 《Bioorganic & medicinal chemistry letters》2010,20(22):6610-6615
A series of thiosemicarbazone analogs based on the benzophenone, thiophene, pyridine, and fluorene molecular frameworks has been prepared by chemical synthesis and evaluated as small-molecule inhibitors of the cysteine proteases cathepsin L and cathepsin B. The two most potent inhibitors of cathepsin L in this series (IC(50)<135 nM) are brominated-benzophenone thiosemicarbazone analogs that are further functionalized with a phenolic moiety (2 and 6). In addition, a bromo-benzophenone thiosemicarbazone acetyl derivative (3) is also strongly inhibitory against cathepsin L (IC(50)=150.8 nM). Bromine substitution in the thiophene series results in compounds that demonstrate only moderate inhibition of cathepsin L. The two most active analogs in the benzophenone thiosemicarbazone series are highly selective for their inhibition of cathepsin L versus cathepsin B. 相似文献
13.
Hendrikus J. Laanbroek Marie-Jos Br-Gilissen Hans L. Hoogveld 《Applied microbiology》2002,68(3):1454-1457
Ammonia-starved cells of Nitrosomonas europaea are able to preserve a high level of ammonia-oxidizing activity in the absence of ammonium. However, when the nitrite-oxidizing cells that form part of the natural nitrifying community do not keep pace with the ammonia-oxidizing cells, nitrite accumulates and may subsequently inhibit ammonia oxidation. The maintenance of a high ammonia-oxidizing capacity during starvation is then nullified. In this study we demonstrated that cells of N. europaea starved for ammonia were not sensitive to nitrite, either when they were starved in the presence of nitrite or when nitrite was supplied simultaneously with fresh ammonium. In the latter case, the initial ammonia-oxidizing activity of starved cells was stimulated at least fivefold. 相似文献
14.
I Assfalg-Machleidt G Rothe S Klingel R Banati W F Mangel G Valet W Machleidt 《Biological chemistry Hoppe-Seyler》1992,373(7):433-440
The dipeptidyl rhodamine diamide substrates (Z-Phe-Arg)2-R110 and (Z-Arg-Arg)2-R110 are 820- and 360-fold more selective for cathepsin L than for cathepsin B allowing a sensitive determination of cathepsin L activity in the presence of high activity of cathepsin B. The results obtained with cell lysates suggest that the cysteine proteinase activity of vital macrophages detected by flow cytometry with these substrates is mainly due to cathepsin L. 相似文献
15.
R Ando T Sakaki Y Morinaka C Takahashi Y Tamao N Yoshii S Katayama K Saito H Tokuyama M Isaka E Nakamura 《Bioorganic & medicinal chemistry》1999,7(4):571-579
By focusing on the amphiphilic properties of cyclopropenone (e.g. a good electrophile and a precursor for a stable 2pi-aromatic hydroxycyclopropenium cation), a new class of cysteine proteinase inhibitors containing a cyclopropenone moiety was designed. For the purpose of the present research, we needed to devise a new method to introduce a peptide-related moiety as a substituent on the cyclopropenone residue. We investigated the reaction of metalated cyclopropenone acetal derivatives (2, R2 = metal) with N-protected alpha-aminoaldehydes 4 to obtain the adduct 5, and succeeded in the preparation of highly potentiated cysteine proteinase inhibitors 8 after several steps transformations. They showed strong inhibitory activities only to cysteine proteinases such as calpain, papain, cathepsin B, and cathepsin L and not to serine (e.g. thrombin and cathepsin G) and aspartic proteinases (e.g. cathepsin D). Kinetic studies indicated that they are competitive inhibitors, and by the examinations of their inhibitory mechanism it became clear that they are reversible inhibitors. 相似文献
16.
Mouse testicular and sperm-associated cathepsin activities were compared for optimal conditions of assay, molecular sieving properties, inhibition data, and electrophoretic mobility. An extract of testes had several isoenzymes of cathepsin among which cathepsin B1 may be a major form while a sperm-associated component may be a minor form. The sperm-associated component was highly sensitive to pepstatin and may be cathepsin D. 相似文献
17.
The concentrations of cathepsins B and H in various tissues and peripheral blood cells of rats were determined by means of sensitive immunoassays. The minimum detectable amounts of cathepsins B and H were 30 pg and 20 pg/assay, respectively, and the presence of endogenous thiol proteinase inhibitors did not interfere with the immunoassays. Cathepsin B was found at high levels in the kidney, vagina, spleen, and adrenal gland, and cathepsin H at high levels in the kidney, vagina, liver, lung, and spleen. Low levels of cathepsins B and H were present in the heart, skeletal muscle, and testis. The ratios of cathepsins B and H in various organs were different: the brain and adrenal gland contained much higher levels of cathepsin B than of cathepsin H, whereas the lung and liver contained higher levels of cathepsin H than of cathepsin B. In several organs such as the kidney, spleen, liver, and lungs, the level of cathepsins B plus H was much higher than that of thiol proteinase inhibitors (TPI-alpha + TPI-beta), whereas in tissues containing large amounts of TPI-alpha, such as the skin, esophagus and stomach, the level of inhibitors was higher than that of cathepsins B plus H. Of the peripheral blood cells tested, macrophages had the highest contents of cathepsins B and H, and so their level of cathepsins B plus H was much higher than that of TPI-alpha plus TPI-beta, whereas lymphocytes and neutrophils contained comparable amounts of proteinases and inhibitors.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
18.
F Aranishi K Hara T Ishihara 《Comparative biochemistry and physiology. B, Comparative biochemistry》1992,102(3):499-505
1. Cathepsin H was purified about 5400-fold from hepatopancreas of carp (Cyprinus carpio) by the method involving ammonium sulfate fractionation, and chromatography on S-Sepharose, DEAE-Sephacel, Ultrogel AcA54, Concanavalin A-Sepharose 4B and GPC on Protein-Pak 125. 2. The purified cathepsin H gave a single protein band on analytical-PAGE, but migrated as two bands of 27,000 and 23,000 mol. wt on SDS-PAGE. 3. Cathepsin H had a pH and temperature optimum of 6.5 and 45 degrees C using Arg-MCA as a substrate, respectively, and was activated by sulfhydryl compounds and inhibited by cysteine protease inhibitors and metal compounds having high reactivities at cysteine residue. 4. The carp hepatopancreas cathepsin H immunoreacted with the monospecific antibody against rat liver cathepsin H, and did not react with the antibodies against carp hepatopancreas cathepsins B and L by the method of immunoelectrophoretic blotting. 相似文献
19.
An improved method for determining the activity of benzoyl-arginine p-nitroanilide-hydrolyzing enzyme in crude tissue extracts 总被引:1,自引:0,他引:1
The method described in this communication is sensitive and allows the direct determination in crude tissue extracts of the enzyme that hydrolyzes α-N-benzoyl-dl-arginine p-nitroanilde (Bz-Arg-NPhNO2). It may be useful in determining cathepsin B activity in crude enzyme preparations when the reaction mixture is incubated with an inhibitor of trypsinlike enzymes which are also capable of hydrolyzing Bz-Arg-NPhNO2. 相似文献
20.
Application of the most sensitive fluorescent label 2-aminobenzoic acid (anthranilic acid, AA) for characterization of carbohydrates from the glycoproteins ( approximately 15 pmol) separated by polyacrylamide gel electrophoresis is described. AA label is used for the determination of both monosaccharide composition and oligosaccharide map. For the monosaccharide determination, bands containing the glycoprotein of interest are excised from the polyvinylidene fluoride (PVDF) membrane blots, hydrolyzed in 20% trifluoroacetic acid, derivatized, and analyzed by C-18 reversed-phase high-performance liquid chromatography. For the oligosaccharide mapping, bands were digested with peptide N-glycosidase F (PNGase F) in order to release the N-linked oligosaccharides, derivatized, and analyzed by normal-phase anion-exchange chromatography. For convenience, the PNGase F digestion was performed in 1:100 diluted ammonium hydroxide overnight. The oligosaccharide yield from ammonium hydroxide-PNGase F digestion was better or equal to all the other reported procedures, and the presumed "oligosaccharide-amine" product formed in the reaction mixture did not interfere with labeling of the oligosaccharides under the conditions used for derivatization. Sequencing of oligosaccharides can be performed using the same mapping method following treatment with an array of glycosidases. In addition, the mapping method is useful for determining the relative and simultaneous distribution of sialic acid and fucose. 相似文献