Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: 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)

We describe a microassay for measuring galactose (Gal) and galactose 1-phosphate (Gal-1-P) in dried blood spots. After a coupled enzyme reaction involving galactose dehydrogenase (GADH, EC 1.1.1.48) and alkaline phosphatase (AP, EC 3.1.3.1) in a microplate well, NADH fluorescence is measured by a highly sensitive fluorometric microplate reader, capable of rapid measurement of fluorescence (2 min per 96 samples). Within- and between-run CVs for measurements of Gal at 90 mg/L with Gal-1-P at 130 mg/L were both less than 5% (n = 8), and analytical recoveries for Gal at 90 mg/L and Gal-1-P at 130 mg/L were 98% and 92%, respectively. Five hundred dried blood-spot samples can be assayed within 2 h, with full calculation of results by an on-line microcomputer. This rapid and reliable assay system is very useful for the routine screening of newborns for galactosemia.
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PMID:Microassay for screening newborns for galactosemia with use of a fluorometric microplate reader. 277 26

In cultured normal rat liver epithelial cells, the specific activity and/or isozyme expression of NADH-diaphorase (NADH-D), pyruvate kinase (PK), glucose-6 phosphate dehydrogenase (G6PD), gamma-glutamyl transpeptidase (GGT), and alkaline phosphatase (AP) were markedly dependent on the growth state of the cultures. Proliferating, preconfluent cells had higher specific activities of PK, NADH-D, and G6PD but lower activities of GGT and AP than did the more stationary confluent cells. Addition of epidermal growth factor [EGF] to the media of proliferating cells enhanced the specific activities of PK, NADH-D, G6PD, GGT, and lactate dehydrogenase (LDH) of these cells, but the specific activity of AP was markedly depressed. The increase in activity of PK and GGT by EGF appeared to involve new protein synthesis, whereas the effect of EGF on AP appeared to involve the EGF-directed suppression of the synthesis of a form of AP that is produced exclusively by cells in confluent cultures. Furthermore, the preconfluent cells were more responsive to the action of EGF on AP than were confluent cells, i.e., the EGF-mediated decrease in AP activity was seen at lower concentration in preconfluent than in confluent cells. Paradoxically, confluent cells exhibited a two-to threefold higher capacity to bind [125 I]EGF because of an increase in surface receptor number. The results of this study indicate that enzymatic or other biochemical studies performed on cultured cells must take into account the growth-state of the cultures. EGF can modulate enzyme activity in growing and nongrowing cells; one effect of EGF is to maintain higher activity of glycolytic enzymes, suggesting that EGF or EGF-like factors may contribute to the high rate of glycolysis in certain neoplasms.
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PMID:The effects of epidermal growth factor and the state of confluence on enzymatic activities of cultured rat liver epithelial cells. 286 16

NADH-cytochrome b5 reductase is the predominant NADH-diaphorase found in the human neutrophil (Blood 62:152, 1983). Although this reductase segregates with the light membranes of nitrogen-cavitated neutrophils separated on Percoll gradients (which include the plasma membrane markers alkaline phosphatase and NADPH-oxidase), it is approximately 95% excluded from plasma membrane-enriched phagocytic vacuoles. The reductase constitutes approximately 5% of the light membrane fraction FAD-flavoprotein (14.8 +/- 5.5 pmol/mg protein) and was found in equimolar concentration with a high potential b cytochrome also present in this light membrane fraction and tentatively identified as cytochrome b5. Isolation of the reductase from human neutrophils was accomplished by Triton X-114 solubilization of the light Percoll gradient membranes, followed by temperature-dependent phase separation and then affinity chromatography on AMP-Sepharose. The active preparation contained 1.3 mol FAD/mol protein, migrated on sodium dodecyl sulfate-polyacrylamide gels as a single band corresponding to an apparent mol wt of 45,000 daltons, exhibited a pl of 5.7 on chromatofocusing and was obtained in greater than 70% yield, with an overall purification of almost 900-fold. The purified enzyme was characterized by a high specificity for NADH as electron donor (Km = 6.4 mumol/L v Km greater than 1.6 mmol/L for NADPH) and exhibited a maximal turnover of ca. 30,000 min-1 at 22 degrees C with either ferricyanide or cytochrome b5 (Km = 10 nmol/L) as electron acceptor. Although the physical characterization and biochemical properties described here demonstrate that this neutrophil NADH b5 reductase is similar to the corresponding liver and erythrocyte enzymes, its unique function in the neutrophil has yet to be determined.
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PMID:Purification and characterization of the human neutrophil NADH-cytochrome b5 reductase. 299 39

Subcutaneous adipose tissue was obtained from fetuses removed from pregnant obese (Ossabaw) and lean (crossbred) sows at three stages of gestation (70, 90, and 110 days). Histochemical analysis for nucleo-side phosphatase (NPase), alkaline phosphatase (APase), and NADH tetrazoleum reductase (NADH-TR) was conducted on fresh-frozen cryostat sections. Age- associated changes in NPase and NADH-TR reactions in the arteriolar system were correlated with the morphological development of the medial layer of arterioles and arteries. For instance, a strong NPase reaction in small arterioles was associated temporally with the assumption of a normal smooth muscle cell morphology and arrangement in the medial layer. In the youngest fetuses, strong NADH-TR reactions were only evident in small and presumptive arterioles and venules (associated with fat cells). Little NADH-TR reactivity was evident in larger arterioles and venules in 70-day tissue. Arteries and large arterioles were distinguished from veins and venules (strong reactions vs. weak reactions) with NADH-TR and NPase reactions in the oldest fetuses. In the younger fetuses, the NPase distinction (arterioles vs. veinules) was obvious before NADH-TR distinction. Small adipocyte-associated vessels were APase positive in the youngest fetuses, but APase reactivity was limited to short segments of vessel between arterioles and capillaries in the oldest fetuses. With the following exceptions, all the above observations were independent of fetal strain. In obese fetuses (110 day) small venules and small arterioles were equally reactive for NPase activity. Capillaries in obese fetuses (110 day) were NADH-TR reactive, whereas no activity was evident in capillaries from lean fetuses (110 day).(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Differentiation of blood vessels in the adipose tissue of lean and obese fetal pigs, studied by differential enzyme histochemistry. 299 39

We have developed a method for isolation of plasma membranes from rabbit endometrium, with high yield and purification. Endometrial homogenates are precipitated with calcium chloride and the resulting supernatant is fractionated by centrifugation in a self-forming gradient of 20% Percoll. Before fractionation, the intact luminal epithelial surface was labelled with 125I-labelled soyabean agglutinin. Between buoyant densities of 1.015 and 1.017 g/ml, a discrete peak of surface label was obtained, which coincided with activities for 5'-nucleotidase and alkaline phosphatase, enzyme markers for the plasma membrane. This peak was well separated from the majority of cellular protein, and from marker enzyme activities for mitochondria and microsomes (NADH cytochrome C reductase) and lysosomes (acid phosphatase). Electron microscopy of the purified membranes showed membrane sheets and vesicles free from other cellular organelles. Analysis of detergent-soluble membrane proteins, fractionated by concanavalin A-affinity chromatography, revealed differences in the protein pattern of membranes from uteri of rabbits receptive (Day 6 of pregnancy) and non-receptive (Day 3) for implantation. The method will be useful for generation of immunological and affinity probes for surface antigens involved in ovoimplantation.
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PMID:Purification of rabbit endometrial plasma membranes from receptive and non-receptive uteri. 299 83

We describe the ultrastructure of various types of gastric carcinoma cells as well as their histochemical properties as visualized at the electron-microscope level. The histochemical properties of tumour cells were compared with those of homologous normal epithelial cells. The localization and activity of ATPase, IDPase, acidic phosphatase and alkaline phosphatase as well as of the oxidoreductases (cytochrome oxidase, succinate dehydrogenase and NADH-dehydrogenase) were studied. Our findings demonstrated that, in tumour cells, a complicated process of structural-functional restructuring takes place. It seems that a number of ultracytochemical properties may be preserved or may disappear altogether; also, such properties may become enhanced or weaker. This heterogeneity of the histochemical properties of tumour cells is discussed with regard to the role of the stem (polypotent) cell in the process of the histogenesis (cytogenesis) of human gastric carcinomas.
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PMID:Ultrastructural ultracytochemical investigation of human gastric carcinomas. 301 11

The antitumor antibiotic mitomycin C is shown to form a covalent complex with calf thymus DNA under anaerobic conditions in the presence of either NADPH cytochrome c reductase/NADPH, xanthine oxidase/NADH, or the chemical reducing system H2/PtO2. Digestion of the complex with DNase I/snake venom diesterase/alkaline phosphatase yields a single mitomycin deoxyguanosine adduct as the major DNA alkylation product, identified as N2-(2'' beta,7''-diaminomitosen-1'' alpha-yl) 2'-deoxyguanosine (Structure 2). Two minor adducts, 2-5% each of the total adduct pool, are isolated and identified as the 1'' beta stereoisomer of 2 (Structure 3), and 10''-decarbamoyl-2 (Structure 7). The same results were obtained with M13 DNA and poly(dG-dC).poly(dG-dC); however, in the latter case, a minor adduct apparently possessing two deoxyguanosine and one mitomycin unit is isolated. Digestion of the covalent mitomycin-calf thymus DNA complex with nuclease P1 yields four dinucleotide adducts, all of which consist of 2 linked at its 3' end to each of the four possible 5' nucleotides (A, T, G, and C). Upon treatment of each dinucleotide adduct with snake venom diesterase/alkaline phosphatase, 2 is released along with the corresponding free nucleoside. In apparent conflict with the present results, previous reports from another laboratory have indicated that modification of calf thymus DNA by mitomycin C under conditions identical to those described here result in the isolation of three mitomycin C mononucleotide adducts possessing linkages of the drug to N2 and O6 of guanine and N6 of adenine. Evidence is shown suggesting that the latter adducts are actually three of the above four dinucleotide derivatives of 2 obtained independently by us and, thus, all of them in fact possess an identical N2-mitosenylguanine adduct moiety. Model-building studies indicate an excellent fit of the guanine N2-linked drug molecule inside the minor groove of B-DNA with no appreciable distortion of the DNA structure.
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PMID:Reaction of DNA with chemically or enzymatically activated mitomycin C: isolation and structure of the major covalent adduct. 301 44

Within the uterine glands, the following enzymes were demonstrated by histochemical methods after 30, 58, 80, 100, and 110 d of pregnancy, respectively: beta-N-acetyl-hexosaminidase, beta-galactosidase, beta-glucuronidase, alpha-mannosidase, acid phosphatase, alkaline phosphatase, esterases, cytochrome oxidase, 5-nucleotidase, leucine aminopeptidase, adenosine triphosphatase, diaphorases (NADH, NADPH), glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, succinate dehydrogenase, isocitrate dehydrogenase (NAD, NADP), beta-hydroxybutyrate dehydrogenase, glycero-3-phosphate dehydrogenase, NAD-glycero-3-phosphate dehydrogenase, glutamate dehydrogenase (NAD, NADP), lactate dehydrogenase. The results show that the activities of G-6-PDH, 6-PGDH, and cytochrome oxidase increase within secreting cells during the 2nd half of pregnancy. The activities of the other enzymes remained almost unchanged during the period of investigation. The description of our results distinguishes between gland neck, middle, and distal part of the secretory unit, respectively. In general, the enzyme activities are similar within the middle and distal gland segments, but lower in the epithelia of the neck region. The activity of dehydrogenases was medium to intensive within the middle and distal gland segments, but only low to medium within the neck portion. Of the hydrolases, the acid phosphatase, ATPase, leucine aminopeptidase, and beta-galactosidase demonstrated an intensive activity within activity secreting cells. The enzyme activities of the gland epithelia are compared with these of the uterine surface epithelia and the histochemical results are discussed in context with their significance in histiotrophic nutrition.
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PMID:[Enzyme histochemistry of the pig placenta. III. Histotopics of enzymes in the uterine epithelium]. 309 49

Incubation of rabbit liver microsomes with alkaline phosphatase resulted in a marked decrease of NADPH-dependent monooxygenase activities. This decrease was found to be correlated with the decrease of NADPH-cytochrome c reductase activity catalyzed by NADPH-cytochrome P-450 reductase. Neither the content of cytochrome P-450, as determined from its CO difference spectrum, nor the peroxide-supported demethylase activity catalyzed by cytochrome P-450 alone was affected by the phosphatase treatment. NADH-cytochrome b5 reductase and cytochrome b5 were not affected by the phosphatase either. NADPH-cytochrome P-450 reductase purified from rabbit liver microsomes lost its NADPH-dependent cytochrome c reductase activity upon incubation with phosphatase in a way similar to that of microsome-bound reductase. Flavin analysis showed that the phosphatase treatment caused a decrease of FMN with concomitant appearance of riboflavin. Alkaline phosphatase, therefore, inactivates the reductase by attacking its FMN, and the inactivation of the reductase, in turn, leads to a decrease of the microsomal monooxygenase activities.
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PMID:The effects of phosphatase on the components of the cytochrome P-450-dependent microsomal monooxygenase. 310 84

Wistar rats (110-125 g) were irradiated with a single dose of 500 R. Histochemical studies were done concerning the glycoproteins (GP) of sublingual glands, gastric, small intestine and colon mucosa, and some intestinal enzymes: acid and alkaline phosphatase (ACP, ALP) leucineaminopeptidase (LAP), Mg-dependent ATP-ase, NADH-diaphorase, lactic dehydrogenase (LDH). After irradiation all these reactions were diminished, with a maximal effect between 3-5 days. This impairment is in accord with the maximal lethality in this interval after such a degree of irradiation that produced the gastrointestinal syndrome. Cocarboxylase, a radioprotector, improved these changes regarding the structures of the small intestine and also the GP of sublingual glands, stomach, small intestine and colon, demonstrating there its efficiency.
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PMID:Histochemical changes in the digestive tract in irradiated rats. 311 66


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