Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.1.3.1 (alkaline phosphatase)
 47,916 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Improved histochemical techniques for the demonstration of NADP+-specific isocitrate dehydrogenase and malate dehydrogenase in tissue sections are described. With these techniques a semipermeable membrane is interposed between the incubating solutions and the tissue sections preventing diffusion of enzymes into the medium during incubation. In the histochemical system the NADP+-dependent enzymes catalyze the electron transfer from threo-Ds-isocitrate or L-malate into NADP+. Phenazine methosulphate and menadione serve as intermediate electron acceptors between reduced coenzyme and nitro-BT. Sodium-azide and amytal are incorporated into the incubating-medium to block electron transfer to the cytochromes. For demonstrating enzyme activities in sections containing non-specific alkaline phosphatase, a phosphatase inhibitor is added into the incubation media. Problems involved in the histochemical demonstration of both enzymes are discussed.
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PMID:Semipermeable membranes for improving the histochemical demonstration of enzyme activities in tissue sections. V. Isocitrate: NADP+ oxidoreductase (decarboxylating) and malate: NADP+ oxidoreductase (decarboxylating). 23 22

Enzyme histochemical techniques were applied to frozen sheep uteri from different stages of the oestrous cycle. The localization and activities of succinate, lactate, glucose-6-phosphate, and isocitrate (NADP+) dehydrogenases and acid and alkaline phosphatases were studied in the luminal and glandular epithelia, caruncle and myometrium. Enzyme activity in the sections was scored on a scale of 0--5. In general the enzyme activity in the uterine caruncles and epithelia was higher than in the myometrium. The myometrium did not show any alkaline phosphatase activity and isocitrate dehydrogenase (NADP+) activity was negligible. The low activities of acid phosphatase and lactate dehydrogenase and the moderate levels of glucose-6-phosphate and succinate dehydrogenases in the myometrium were constant. The caruncular tissue showed high levels of phosphatases and glucose-6-phosphate dehydrogenase, moderate levels of lactate and succinate dehydrogenases, and low levels of isocitrate dehydrogenase (NADP+) throughout the oestrous cycle. Much lower phosphatase and isocitrate dehydrogenase (NADP+) levels were found in the epithelium of deep glands compared with superficial glands. The high activity of acid and alkaline phosphatases in the luminal epithelium and the superficial glands was constant from mid-cycle to ovulation, but a significant decrease was observed immediately after ovulation. The level of dehydrogenases in epithelia was generally high and did not change during the oestrous cycle.
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PMID:Enzyme histochemistry of the sheep uterus during the oestrous cycle. 54 17

A simple and sensitive method has been described for the determination of UDPglucuronic acid pyrophosphatase activity. Pyrophosphatase-free alkaline phosphatase preparation is added to the reaction mixture in order to hydrolyze the phosphate esters (UMP and alpha-D-glucuronic acid 1-phosphate) produced by pyrophosphatase. The inorganic phosphate liberated is measured by a modification of Fiske and SubbaRow's method. The phosphatase coupled method is time saving, easy to perform and accurate. It can also be used for pyrophosphatase assays with other nucleotide substrates like UDPglucose, UDP-N-acetylglucosamine, NAD+, NADH, NADP+ and NADPH.
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PMID:UDPglucuronic acid pyrophosphatase assay with the aid of alkaline phosphatase. 84 23

p- and o-Aminomethamphetamine were synthesized as haptens to be coupled with carrier protein at the benzene ring of methamphetamine. Immunogens were prepared by the glutaraldehyde method or the MBS (N-(m-maleimidobenzoyloxy)succinimide) type cross-linking reagent method. In particular, immunization with p-aminomethamphetamine-bovine serum albumin (BSA) conjugate prepared by the glutaraldehyde method gave an anti-methamphetamine antiserum having a low cross-reactivity with methylephedrine. With the antiserum, three kinds of immunoassays for methamphetamine were established. An enzyme immunoassay (EIA) and an enzyme-linked immunosorbent assay (ELISA) were developed with alkaline phosphatase (ALP) as a label enzyme. The amount of antibody bound ALP conjugate was determined by its activity in dephosphorylating p-nitrophenyl phosphate in EIA and nicotinamide adenine dinucleotide phosphate (NADP+) in ELISA. The range of methamphetamine measurable by ELISA was 0.025-0.5 ng/well and its sensitivity was superior to that of EIA (0.3-300 ng/tube). A latex agglutination inhibition reaction test (LAIRT) was also developed for the mass screening method of urine samples. The sensitivity of this method for methamphetamine was 0.1 micrograms/ml urine.
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PMID:Immunoassay for methamphetamine with a new antibody. 218 Jul 98

N-Arylazido-beta-alanyl-NAD+ [N3'-O-(3-[N-(4-azido-2-nitrophenyl)amino]propionyl)NAD+] has been prepared by alkaline phosphatase treatment of arylazido-beta-alanyl-NADP+ [N3'-O-(3-[N-(4-azido-2-nitrophenyl)amino]propionyl)NADP+]. This NAD+ analogue was found to be a potent competitive inhibitor (Ki = 1.45 microM) with respect to NADH for the purified bovine heart mitochondrial NADH dehydrogenase (EC 1.6.99.3). The enzyme was irreversibly inhibited as well as covalently labeled by this analogue upon photoirradiation. A stoichiometry of 1.15 mol of N-arylazido-beta-alanyl-NAD+ bound/mol of enzyme, at 100% inactivation, was determined from incorporation studies using tritium-labeled analogue. Among the three subunits, 0.85 mol of the analogue was bound to the Mr = 51,000 subunit, and each of the two smaller subunits contained 0.15 mol of the analogue when the dehydrogenase was completely inhibited upon photolysis. Both the irreversible inactivation and the covalent incorporation could be prevented by the presence of NADH during photolysis. These results indicate that N-arylazido-beta-alanyl-NAD+ is an active-site-directed photoaffinity label for the mitochondrial NADH dehydrogenase, and are further evidence that the Mr = 51,000 subunit contains the NADH binding site. Previous studies using A-arylazido-beta-alanyl-NAD+ [A3'-O-(3-[N-(4-azido-2-nitrophenyl)amino]propionyl)NAD+] demonstrated that the NADH binding site is on the Mr = 51,000 subunit [Chen, S., & Guillory, R. J. (1981) J. Biol. Chem. 256, 8318-8323]. Results are also presented to show that N-arylazido-beta-alanyl-NAD+ binds the dehydrogenase in a more effective manner than A-arylazido-beta-alanyl-NAD+.
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PMID:N-arylazido-beta-alanyl-NAD+, a new NAD+ photoaffinity analogue. Synthesis and labeling of mitochondrial NADH dehydrogenase. 234 Feb 77

This communication presents the results obtained in tubular aggregates of 24 enzyme histochemical techniques for demonstrating activity of oxidoreductases, transferases, hydrolases and isomerases. The activity characteristics of the tubular aggregates in m. gluteus medius of 18 patients with diseases of the neuromuscular system were almost identical. A high activity of the mitochondrial enzymes, NADPH: tetrazolium oxidoreductase, NADH:tetrazolium oxidoreductase and cytochrome c oxidase, could be shown in the pathological structures, whereas the activity of the mitochondrial enzymes, glycerol-3-phosphate:menadione oxidoreductase, succinate:PMS oxidoreductase, malate:NAD+ oxidoreductase and isocitrate:NAD+ oxidoreductase, and the partial mitochondrial enzymes, malate:NADP+ oxidoreductase and isocitrate:NADP+ oxidoreductase, was very slight or even absent. There was a moderate to strong activity of the glycolytic enzymes lactate:NAD+ oxidoreductase, glyceraldehyde-3-phosphate:NAD+ oxidoreductase, phosphofructokinase, phosphoglucomutase and glucose phosphate isomerase. In contrast, the activity of alpha-glucan phosphorylase was slight. The activity of phosphogluconate:NADP+ oxidoreductase, glucose-6-phosphate:NADP+ oxidoreductase and 5'-nucleotidase was slight, whereas there was no activity of myosin ATPase and mitochondrial ATPase, acid phosphatase or alkaline phosphatase. The high activity of AMP-deaminase was very striking. The activity of peroxidase was moderate. Results obtained with adsorption studies point to adsorption of some of the enzymes studied to the tubular aggregates in vivo and this phenomenon very probably determined the histochemical characteristics of these structures.
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PMID:Histochemical features of tubular aggregates in diseased human skeletal muscle fibres. 317 98

We have demonstrated that the purified guanine nine nucleotide exchange factor (GEF) may be isolated as a complex with NADPH. Complete inhibition of the GEF-catalyzed exchange of eukaryotic initiation factor 2-bound GDP for GTP was observed in the presence of either 0.5-0.75 mM NAD+ or NADP+. Incubation of GEF with ATP results in the phosphorylation of its Mr 82,000 polypeptide. This phosphorylation is strongly inhibited by heparin but is not affected by heme or H8 (N-[2-(methylamino)ethyl]-5-isoquinolinesulfonamide dihydrochloride), an inhibitor of cAMP- and cGMP-dependent protein kinases and protein kinase C. The purification of GEF was modified to eliminate any contaminating kinase activity and the isolated protein appears to be homogeneous as judged by NaDodSO4/polyacrylamide gel electrophoresis and silver staining. The Mr 82,000 subunit of GEF is phosphorylated only upon addition of ATP and casein kinase II. The extent of phosphorylation is approximately equal to 0.55 mol of phosphate per mol of GEF, and this results in a 2.3-fold increase in the guanine nucleotide exchange activity. Following treatment of the phosphorylated GEF with alkaline phosphatase, the activity of the protein is reduced by a factor of 5. Rephosphorylation of GEF increases its specific activity to that of the phosphorylated protein. The results of this study suggest that phosphorylation/dephosphorylation of GEF plays a role in regulating polypeptide chain initiation.
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PMID:Phosphorylation of the guanine nucleotide exchange factor from rabbit reticulocytes regulates its activity in polypeptide chain initiation. 342 26

A case of adenocarcinoma of Bartholin's glands in a middle aged female is presented. This well known, but rather uncommon malignancy is reported since it shows an unusual structure consisting of a combination of two different histological entities, namely an adeno-cystic component and adenopapillary component. An interval of 21 years elapsed between the appearance of the primary lesion and the recurrence which showed extensive haematogenous and lymphogenous metastases and proved fatal within two years of the recurrence. The study includes an enzyme histochemical evaluation and a fine needle cytological examination. The former revealed a high degree of activity of the NADP+ regenerating enzymes of the pentose shunt as well as a definite, but variable, activity of many lysosomal enzymes, mainly non specific esterases, acid phosphatase, aryl sulphatase and beta-glucuronidase. In contrast to other adenocarcinomas arising in the female genital tract, especially those of endometrium and ovary, the tumour cells showed only a slight activity of alkaline phosphatase, As is the case in many other adenocarcinomas, the activity of lactate dehydrogenase was strongly evident.
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PMID:Histochemical and cytochemical studies of metastasizing carcinoma of Bartholin's gland. 645 95

A kinetic study is made of a system consisting of a specific enzymic cycling assay coupled to an enzymic reaction. A kinetic analysis of this system is presented, and the accumulation of chromophore involved in the cycle is seen to be parabolic, i.e. the rate of the reaction increases continuously with constant acceleration. The system is illustrated by the measurement of alkaline phosphatase activity using beta-NADP+ as substrate. The enzymes alcohol dehydrogenase and diaphorase are used to cycle beta-NAD+ in the presence of ethanol and p-Iodonitrotetrazolium Violet. During each turn of the cycle, one molecule of the tetrazolium salt is reduced to an intensely coloured formazan. A simple procedure for evaluating the kinetic parameters involved in the system and for optimizing this cycling assay is described. The method is applicable to the measurement of any enzyme, and its amplification capacity as well as the simplicity of determining kinetic parameters enable it to be employed in enzyme immunoassays to increase the magnitude of the measured response.
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PMID:Kinetic study of an enzymic cycling system coupled to an enzymic step: determination of alkaline phosphatase activity. 761 54

The chemiluminescent reaction of lucigenin with various reducing sugars and reducing compounds has been studied. It was found that dihydroxyacetone gave the most intense chemiluminescence (CL). We have developed highly sensitive chemiluminescent methods for alkaline phosphatase (ALP) based on the production of dihydroxyacetone using NADP+ or glycerol-3-phosphate as substrate. The detection limits for ALP using each substrate were 1.25 x 10(-19) mol/assay and 2.5 x 10(-19) mol/assay, and the coefficient of variation (n = 7) was 2.8% and 3.7%, respectively. We have also applied the method using NADP+ as substrate in enzyme immunoassays (EIA) for cholecystokinin (CCK) and human chorionic gonadotropin (hCG). CCK-8 (octapeptide sulphated form of a carboxy terminal fragment of CCK) concentrations released from alimentary canal of rat were assayed using the chemiluminescent EIA (CLEIA) and a fluorimetric EIA (ALP label). The correlation between CCK-8 values obtained by these methods was y = 1.04x + 18.21, r = 0.946, n = 28. hCG values in serum and in urine were measured. The correlation between hCG values in serum samples obtained using the CLEIA and a time-resolved fluoroimmunoassay (TR-FIA), and in urine samples obtained using the CLEIA and the fluorimetric EIA using ALP were satisfactory. The correlations were y = 1.00x - 0.04, r = 0.997 (n = 51) and y = 1.00x - 0.03, r = 0.999 (n = 10), respectively.
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PMID:A new highly sensitive chemiluminescent assay of alkaline phosphatase using lucigenin and its application to enzyme immunoassay. 776 11


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