共查询到20条相似文献,搜索用时 15 毫秒
1.
R A Dodds A A Pitsillides G T Frost 《The journal of histochemistry and cytochemistry》1990,38(1):123-127
Although decarboxylases, particularly ornithine decarboxylase, are of considerable importance in cell metabolism, it has been impossible to demonstrate their activity histochemically, as this depends on trapping carbon dioxide at neutral pH values. A new reagent, lead hydroxyisobutyrate, has been shown capable of such trapping. It has been applied to the demonstration of ornithine decarboxylase activity in mouse kidney. Optimal concentrations of substrate, co-factor and trapping agent, as well as the pH optimum, have been determined for cryostat sections stabilized with a collagen polypeptide. The activity was inhibited by the specific ornithine decarboxylase inhibitor alpha-difluoromethyl ornithine. 相似文献
2.
The operational stability of an enzyme can be quantified by its half-life, or the length of time after which 50% of its original activity has degraded. Ideally, continuous methods for measuring half-lives are preferred but they can be expensive and relatively low throughput. Batch methods, while simple, cannot be used for all enzymes. For example, batch reactions can be difficult when there is a gas phase reactant or when there is significant product or substrate inhibition. Here we describe a repeated-batch method for measuring the half-life of carbonic anhydrase (CA)-based biocatalysts by automated periodic switching between a forward and reverse reaction. This method is inexpensive and can be multiplexed for high-throughput analysis of enzyme variants. Several purified CA enzymes as well as whole-cell biocatalysts with engineered CA activity were evaluated with this method. The results indicate a significant increase in operational stability is achieved upon immobilization of CA in the cellular periplasm of Escherichia coli. 相似文献
3.
Using a carbonic anhydrase assay based on membrane inlet mass spectrometry (MIMS), we have extended our earlier investigations of Photosystem II (PSII)-associated carbonic anhydrase activity in spinach PSII preparations (W. Hillier, I. McConnell, M. R. Badger, A. Boussac, V.V. Klimov G. C. Dismukes, T. Wydrzynski Biochemistry 2006, 45:2094). The relationship between the carbonic anhydrase activity and O(2) evolution has been evaluated in terms of the effects of metal ion addition, preparation type, light, and response to specific inhibitors. The results indicate that the PSII-associated carbonic anhydrase activity is variable and appears not to be associated specifically with the oxygen evolving activity nor the 33 kDa extrinsic manganese stabilising protein. 相似文献
4.
I.L. McConnell 《BBA》2007,1767(6):639-647
Using a carbonic anhydrase assay based on membrane inlet mass spectrometry (MIMS), we have extended our earlier investigations of Photosystem II (PSII)-associated carbonic anhydrase activity in spinach PSII preparations (W. Hillier, I. McConnell, M. R. Badger, A. Boussac, V.V. Klimov G. C. Dismukes, T. Wydrzynski Biochemistry 2006, 45:2094). The relationship between the carbonic anhydrase activity and O2 evolution has been evaluated in terms of the effects of metal ion addition, preparation type, light, and response to specific inhibitors. The results indicate that the PSII-associated carbonic anhydrase activity is variable and appears not to be associated specifically with the oxygen evolving activity nor the 33 kDa extrinsic manganese stabilising protein. 相似文献
5.
6.
A cytochemical method for measuring enzyme activity in individual preovulatory mouse oocytes 总被引:2,自引:0,他引:2
G G De Schepper C J Van Noorden F Koperdraad 《Journal of reproduction and fertility》1985,74(2):709-716
The activities of 6 enzymes involved in carbohydrate metabolism were determined quantitatively in preovulatory oocytes by cytochemical means per individual cell as well as biochemically in cell homogenates. Oocytes were incorporated in a polyacrylamide matrix for appropriate enzyme cytochemical staining. This incorporation preserves the morphology of the cells very well, and the enzymes keep their activity for a considerable period of time. This method could also be used to demonstrate more than one enzyme activity in the same cell. The results obtained by cytochemical means appeared to correlate very well with the biochemical data (P less than 0.005). Glucose 6-phosphate dehydrogenase, the key-enzyme in the pentose phosphate pathway, had very high activity in these preovulatory oocytes, but 6-phosphogluconate dehydrogenase activity was only about 2% of that of glucose 6-phosphate dehydrogenase. The activities of lactate dehydrogenase and to a lesser extent glucose phosphate isomerase and D-glyceraldehyde-3-phosphate dehydrogenase also appeared to be very high, while hexokinase showed a very low activity. 相似文献
7.
A reappraisal of the histochemical method for carbonic anhydrase 总被引:1,自引:0,他引:1
8.
We have developed a method of measuring the activity and characteristics of carbonic anhydrase (CA) using the disappearance of 18O from CO2 in 1 ml of gas contained in a glass chamber as it exchanges with H2O in 0.01 ml 0.25 M NaHCO3 solution in a thin (25 micron) porous membrane. Serial gas samples (approximately 0.02 ml) are analyzed in a mass spectrometer to obtain the rate of disappearance of the label. The enzyme activity can be measured inside intact cell or particle membranes. As little as 10(-15) mol of high-activity type CA can be detected at 25 degrees C, and the activity of 200 times this amount can be measured. The uncatalyzed hydration reaction velocity constant was 0.056 +/- 0.004 s-1, in agreement with published data. 相似文献
9.
10.
Histochemical demonstration of carbonic anhydrase activity 总被引:14,自引:4,他引:10
Holger P. J. Hansson 《Histochemistry and cell biology》1967,11(2):112-128
Summary Freeze-dried frozen sections are floated on the surface of the freshly prepared incubation mixture (CoSO4 1.75 × 10–3
M, H2SO4 5.3 × 10–2
M, NaHCO3 1.57 × 10–2
M and KH2PO4 1.17 to 11.7 × 10–3
M; demonstration of weak activity requires high phosphate). A compound containing cobalt and phosphorous precipitates at carbonic anhydrase sites and is converted to CoS. Adequate staining requires only 2–10 minutes of incubation. Actazolamide inhibits the staining reaction in specific concentrations. Actazolamidein vivo, 20 mg/kgi.v. to mice 30 minutes before sacrifice also inhibited the staining. The proportion phosphorous in the specific precipitate increases with KH2PO4 of the medium (shown by the addition of60Co and32P). An explanation of the reaction mechanism is given, based on the catalyzed loss of CO2 in the surface layer. The inclusion of phosphate in the medium makes this modification ofHäusler's method so sensitive that it shows carbonic anhydrase activity in for instance stratum spinosum of the skin.This investigation was supported by grants from the Medical Faculty, University of Uppsala and from the U.S. National Institutes of Health (Grant NB 3060 to E.Bárány). 相似文献
11.
A quantitative cytochemical method for the demonstration of phosphofructokinase has been successfully applied to a range of plant tissues. The findings indicate that this enzyme system may be assayed as an indicator of glycolytic activity in plant cells, and furthermore tha the very high endogenous phosphoenolpyruvate concentrations may not be rate limiting in vivo. 相似文献
12.
Carbonic anhydrase activity of intact carbonic anhydrase II-deficient human erythrocytes 总被引:1,自引:0,他引:1
Dodgson S. J.; Forster R. E. nd; Sly W. S.; Tashian R. E. 《Journal of applied physiology》1988,65(4):1472-1480
Intact erythrocytes from subjects with deficiency of blood carbonic anhydrase (CA) II and from normal subjects were assayed for enzyme activity by use of an 18O exchange technique in a solution containing 25 mM (CO2 + NaHCO3) plus 125 mM NaCl. At 25 degrees C and pH 7.4, the catalyzed reaction velocity was 0.32 +/- 0.04 M/s for the CA II-deficient and 1.60 +/- 0.12 M/s for the normal cells, a ratio of 1:5. Under the same conditions at 37 degrees C the relative difference between the CA II-deficient and normal cells was much less: the velocity for the CA II-deficient cells was 0.84 +/- 0.07 M/s and for the normal cells 1.60 +/- 0.32 M/s, a ratio of 1:1.9. Results were comparable for the hemolysates with the NaHCO3 reduced to 85 mM (the corresponding intracellular concentration): at 25 degrees C CA II-deficient cells had a velocity of 0.36 +/- 0.01 M/s compared with 1.12 +/- 0.04 M/s for the normal cells, a ratio of 1:3.1. At 37 degrees C again the relative difference between hemolysates from CA II normal and deficient cells was much less: the CA II-deficient cells had a reaction velocity of 1.17 +/- 0.22 M/s vs. 2.60 +/- 0.36 M/s for the normal cells, a ratio of 1:2.2. The greater fractional reduction of enzyme velocity of CA II-deficient cells at 25 degrees C compared with 37 degrees C appears to be explained by a greater chloride inhibition of the presumed CA I at the lower temperature.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
13.
We have developed a novel method for quantitating protein phosphorylation by a variety of protein kinases. It can be used with purified kinases and their substrates in vitro or in combination with cell extracts. The method is based on the knowledge that protein kinase C (PKC) adds three phosphates to each molecule of its preferred substrate, myelin basic protein (MBP). A time course is performed in which a kinase is allowed to phosphorylate its preferred substrate or the protein under investigation in the presence of [gamma-32P]ATP. At the same time PKC is allowed to fully phosphorylate MBP. After resolving the products by SDS-PAGE, electrophoretic transfer, and determining the degree of incorporation of 32P by phosphorImager analysis, the data are converted to moles phosphate/mole protein by normalization with phosphorylated MBP. The method is both sensitive and relatively rapid and all the steps are commonly available in the biochemistry laboratory. We have used this method to confirm and extend information on the relationship of MEK1 and MAPK/Erk2 in rat lung fibroblasts exposed to V(2)O(5). A 4-h exposure to V(2)O(5) results in partial phosphorylation of MAPK/Erk2 such that 25% of the potential phosphorylation sites are occupied. We also demonstrate that despite multiple potential phosphorylation sites, recombinant human AP endonuclease is weakly phosphorylated in vitro (4% at best) by PKC, cGMP-dependent protein kinase, casein kinase II, and casein kinase I and not at all phosphorylated by MAPK. Furthermore we are unable to demonstrate phosphorylation in cell extracts from HeLa cells, mouse fibroblasts after oxidative damage with H(2)O(2) or alkylation damage with methylmethane sulfonate, or rat lung fibroblasts after oxidative damage with V(2)O(5). 相似文献
14.
15.
The salt bridge probe cyanogen (ethanedinitrile, C2N2; N?C–-C?N) inhibits the bovine carbonic anhydrase (EC 4.2.1.1.) hydrolase activity toward various types of esters without significant effect on its hydrolyase activity. Two sets of pyridine derivatives that were isosteric substrates for the two activities were differentially affected. Acetazolamide and salamide are reversible inhibitors of the enzyme; only salamide affords protection of the hydrolase activity against the action of C2N2. Since each is known to bind in different positions within the active site, the selective effect of salamide may arise from its position covering one CO2 site as well as a site important for hydrolase activity. The C2N2 concentration dependence of the time course of hydrolase inhibition is consistent with the existence of a high C2N2 affinity site with slow covalent change and a second site with lower C2N2 affinity, but higher rate of covalent modification of the enzyme. © 1993 John Wiley & Sons, Inc. 相似文献
16.
Summary A simple method for separation of carbonic anhydrase activity into components by electrophoresis on cellulose acetate strips is described. With this method, using barbiturate buffer systems at various pH values, two main components of CAH in rat erythrocytes, and the splitting of each of these into two minor components were revealed. Two components were also observed in the CAH activity in kidney and lens homogenates, and one component in brain homogenate. A modification of Häusler's histochemical method for CAH was adapted for visualization of the electrophoretically separated bands. This rendered the evalution of the results easier than with the quantitative measurements alone. The quantitative measurement of CAH activity in electrophoretic strips corresponded with the degree of staining by the histochemical method. This among other facts supports the view of the specificity of the histochemical method used. Some examples of the histochemical staining pattern of the CAH activity in rat tissues are given. 相似文献
17.
Carbonic anhydrase III from rabbit muscle, a newly discovered major isoenzyme of carbonic anhydrase, has been found to be also a p-nitrophenyl phosphatase, an activity which is not associated with carbonic anhydrases I and II. The p-nitrophenyl phosphatase activity has been shown to chromatograph with the CO2 hydratase activity; both activities are associated with each of its sulfhydryl oxidation subforms; and both activities follow the same pattern of pH stability. This phosphomonoesterase activity of carbonic anhydrase III has an acidic pH optimum (<5.3); its true substrate appears to be the phosphomonoanion with a Km of 2.8 mm. It is competitively inhibited by the typical acid phosphatase inhibitors phosphate (Ki = 1.22 × 10?3M), arsenate (Ki = 1.17 × 10?3M), and molybdate (Ki = 1.34 × 10?7M), with these inhibitors having no effect on the CO2 hydratase or the p-nitrophenyl acetate esterase activities of carbonic anhydrase III. The p-nitrophenyl acetate esterase activity of carbonic anhydrase III, on the other hand, has the sigmoidal pH profile with an inflection at neutral pH, typical of carbonic anhydrases for all of their substrates, and is inhibitable by acetazolamide (a highly specific carbonic anhydrase inhibitor) to the same degree as the CO2 hydratase activity. The acid phosphatase-like activity of carbonic anhydrase III is slightly inhibited by acetazolamide at acidic pH, and inhibited to nearly the same degree at neutral pH. These data are taken to suggest that the phosphatase activity follows a mechanism different from that of the CO2 hydratase and p-nitrophenyl acetate esterase activities and that there is some overlap of the binding sites. 相似文献
18.
The human erythrocyte membrane is an efficient enhancer of both high (CA II) and low (CA I) activity isozymes of red blood cell carbonic anhydrase. The presence of membrane increased CO2 hydration catalyzed by bovine CA II 1.6-fold, human CA II 3.5-fold, and human CA I 1.6-fold. With the high activity CA isozymes, maximal stimulation was observed in the presence of 1-3 micrograms membrane protein/ml. The Vmax for bovine CA II (4 nM) rose from 0.302 to 0.839 mM/s, while that for human CA II (6 nM) increased from 0.113 to 0.414 mM/s in the absence and presence of membrane, respectively. The apparent Km for CO2 increased from 13.2 to 51.2 mM for bovine CA II, and from 6.5 to 38.5 mM for human CA II. Mixtures of membrane plus enzyme, upon centrifugation through linear sucrose density gradients, displayed enhanced Ca activity only in membrane-containing gradient fractions, verifying the stimulatory ability of membranes on enzyme activity and indicating tight and stable complex formation. Membrane enhancement of CA activity appears to be a general phenomenon in that mouse hepatocyte membranes also stimulated CA activity, although less efficiently than erythrocyte membranes. Of the many soluble putative effectors assayed, only imidazole enhanced CA II activity to an extent comparable with erythrocyte membranes; imidazole did not, however, stimulate the activity of human CA I. The data are consistent with a model of CA II activation by membrane association that may effect a distortion of the enzyme conformation in such a way as to facilitate intra- and/or intermolecular proton transfer between membrane-bound and enzyme-bound proton shuttling residues (perhaps the imidazole moiety of histidine) and the Zn-bound hydroxide at the catalytic site of the enzyme. 相似文献
19.
Carbonic anhydrase II (CAII) binds to and regulates transport by the NHE1 isoform of the mammalian Na(+)/H(+) exchanger. We localized and characterized the CAII binding region on the C-terminal tail of the Na(+)/H(+) exchanger. CAII did not bind to acidic sequences in NHE1 that were similar to the CAII binding site of bicarbonate transporters. Instead, by expressing a variety of fusion proteins of the C-terminal region of the Na(+)/H(+) exchanger, we demonstrated that CAII binds to the penultimate group of 13 amino acids of the cytoplasmic tail. Within this region, site-specific mutagenesis demonstrated that amino acids S796 and D797 form part of a novel CAII binding site. Phosphorylation of the C-terminal 26 amino acids by heart cell extracts did not alter CAII binding to this region, but phosphorylation greatly increased CAII binding to a protein containing the C-terminal 182 amino acids of NHE1. This suggested that an upstream region of the cytoplasmic tail acts as an inhibitor of CAII binding to the penultimate group of 13 amino acids. The results demonstrate that a novel phosphorylation-regulated CAII binding site exists in distal amino acids of the NHE1 tail. 相似文献