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)

The localization of alkaline phosphatases in dentinogenically active rat incisor odontoblasts was studied by means of subcellular fractionation and electron microscopical histochemistry. Subcellular fractionation revealed the predominant phosphatase activity to be present in the microsome fraction and to a lesser extent in the mitochondrial fraction. Adenosine triphosphate degrading enzyme activity was determined in the presence or absence of (+/-)-6(m-bromophenyl)-5, 6-dihydroimidazo(le) (2,1-b) thiazole oxalate (R 8231). Before the histochemical study, the effects on phosphatase activities by aldehyde fixation were studied by biochemical assay. A method of fixation for optimal preservation of phosphatase activity is presented. Phosphatase electron microscopic histochemistry was performed by using ATP as a substrate and with or without addition of the inhibitor R 82319 Precipitates were seen in the membranes of vesicles present in the odontoblast process and the Golgi region. When there were signs of insufficient fixation, precipitates were also seen in the outer membranes of mitochondria. No phosphatase activity was seen in the cell membrane. ATP degrading enzyme activities mediated by nonspecific alkaline phosphatase (APase) and Ca2+ -adenosine triphosphatase thus have the same morphological localization. This close association is consistent with earlier biochemical studies.
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PMID:Ultrastructural localization of alkaline phosphatases in rat incisor odontoblasts. 2 17

Previous studies in vivo and with isolated perfused rat livers have suggested that the deleterious effect of ethanol on hepatic pyridoxal 5'-phosphate metabolism is mediated by acetaldehyde. Inasmuch as acetaldehyde has no effect on the synthesis of pyridoxal phosphate, it has also been postulated that acetaldehyde accelerates pyridoxal phosphate degradation by displacing this coenzyme from binding proteins, which protect it against hydrolysis. To test these hypotheses, studies have been performed with isolated rat hepatocytes, subcellular fractions of rat liver, and human erythrocytes. Ethanol oxidation lowered the pyridoxal phosphate content of isolated liver cells when acetaldehyde oxidation was inhibited by either disulfiram or prior treatment of rats with cyanamide. Additions of 7.5 mM acetaldehyde alone at 40-min intervals to cell suspensions decreased hepatic pyridoxal phosphate content only slightly because acetaldehyde was rapidly metabolized. However, when acetaldehyde oxidation and reduction were inhibited by cyanamide treatment and by 4-methyl-pyrazole and isobutyramide, respectively, a 40% decrease in hepatic pyridoxal phosphate content was observed in 80 min of incubation. In equilibrium dialysis experiments, acetaldehyde, 7.5 and 15 mM, displaced protein-bound pyridoxal phosphate in undialyzed hepatic cytosol and in hemolysate supernate containing added pyridoxal phosphate. In the presence of alkaline phosphatase, acetaldehyde accelerated the degradation of pyridoxal phosphate in dialyzed hemolysate supernate and hepatic cytosol with added pyridoxal phosphate. Acetaldehyde also inhibits tyrosine aminotransferase. The kinetics of inhibition were mixed competitive-noncompetitive with respect to pyridoxal phosphate. These observations support the hypothesis that the deleterious effect of ethanol oxidation on pyridoxal phosphate metabolism is mediated at least in part by acetaldehyde which displaces this coenzyme from protein binding, thereby enhancing its degradation.
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PMID:The role of acetaldehyde in mediating the deleterious effect of ethanol on pyridoxal 5'-phosphate metabolism. 2 31

A phosphatase, hydrolyzing pyridoxal-5-phosphate (P5P), a physiologically active component of the vitamin B6 complex and an essential co-enzyme in the synthesis of neurotransmitters, has been localized cytochemically in the perikarya of neurons in the peripheral, autonomic and central nervous systems of the rat. Neurons in dorsal root ganglia, sympathetic ganglia and ventral horn of spinal cord were studied by light and electron microscopy, while Purkinje cells, neurons in the dentate nucleus of the cerebellum, thalamus, and hypothalamus were studied by light microscopy only. Optimal conditions for demonstrating this activity in aldehyde-fixed tissue were determined with dorsal root ganglia. At the optimal pH of 5.0, neurons in these ganglia and in all other neurons studied show pyridoxal-5-phosphatase (P5Pase) activity in GERL. Small neurons in dorsal root ganglia also display enzyme activity in the endoplasmic reticulum (ER); activities in GERL and ER are also appreciably high at neutral pH. Small and large neurons in these ganglia, and neurons of sympathetic ganglia, show variable P5Pase activity in the Golgi apparatus. These localizations differ from the usual sites of both acid phosphatase and alkaline phosphatase activities. The P5Pase activity, demonstrated cytochemically, is a new acid hydrolase activity in GERL.
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PMID:Pyridoxal phosphatase: cytochemical localization in GERL and other organelles of rat neurons. 3 96

Using freeze-dried or sections from fresh-frozen or aldehyde-fixed material nitro BT (NBT), tetranito BT (TNBT), distyryl nitro BT (DS-NBT), thiocarbamyl nitro BT (TC-NBT) or benzothiazolylstyrylphthalhydrazidyl tetrazolium chloride (BSPT) were tested as auxiliary reagents for the localization of glycosidases, phosphatases and non-specific esterases with indoxyl substrates in rat tissues. By means of NBT or TNBT as a tetrazolium salt acid beta-D-galactosidase, beta-D-N-acetylglucosaminidase, acid phosphatase, neuraminidase and non-specific esterase can only be localized at the cellular level; a more precise localization is possible for lactase-beta-D-glucosidase in the intestinal brush border, and the best results are obtained in the demonstration of alkaline phosphatase; among all methods described previously the tetrazolium procedure with TNBT is the method of choice for the light microscopic localization of this enzyme. Reverse data are observed with BSPT as a tetrazolium salt; then, all acid and neutral hydrolases can be exactly localized in lysosomes, secretion granules, cytoplasm and/or microvilli of many cells and tissues provided BSPT-formazan is stabilized by osmification. Furthermore, this procedure enables the reliable ultracytochemical demonstration of these enzymes. However, in the case of alkaline phosphatase only sites with high enzyme activity reveal a positive reaction. -DS- and TC-NBT are inferior to NBT, TNBT or BSPT.
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PMID:[Tetrazolium methods for the histochemical investigation of hydrolases (author's transl)]. 10 69

The activity of a pyrophosphate-splitting tissue factor in jaws, teeth and intestinal mucosa has been studied by means of histochemistry. Freeze-cut, unfixed sections of whole animals were incubated in a buffered medium (pH 8.6) containing inorganic pyrophosphate (PP) Pb2+, Mg2+, and Zn2+ at various concentrations. The effects of compound R 8231, heat, aldehyde fixation, and demineralization with EDTA were also investigated. In sections showing optimal staining, deposition of incubation products was found in the stratum intermedium and the subodontoblastic cells of the developing tooth, in the osteoblastic layers, and at the surface of the intestinal mucosa. The hard tissues were also stained except in the demineralized sections. Treatment with heat or compound R 8231 resulted in loss of originally observed soft tissue staining while short-time demineralization with EDTA enhanced the staining reaction. It is argued that a nonspecific deposition of the capturing ion, Pb2+, can hardly explain the observed soft tissue staining. The results point to the presence of a PP-splitting enzyme, and it is suggested that the enzyme exhibits features of an alkaline phosphatase with PP-phospholhydrolytic properties rather than of an inorganic pyrophosphatase.
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PMID:Histochemical localization of alkaline pyrophosphate- phosphohydrolase in tooth-forming cells of rat. 17 19

The purpose of this study was to try to differentiate histochemically between the various enzymes which may catalyze the hydrolysis of ATP in developing rat dental tissues. Freeze cut and freeze dried sections of molar and incisor teeth were incubated in lead capture-based media at pH 5.0, 7.2 or 9.4 with one of the following substrates: beta-glycerophosphate, AMP, ADP, ATP, AMP-PNP and tetrasodium pyrophosphate. To establish the enzymatic nature of the hydrolysis parallel sections were incubated after prior fixation in either formaldehyde or glutaraldehyde. By comparing the enzymatic stainings obtained with the various substrates and at the different pH:s, it was concluded that ATP can be visibly hydrolyzed in rat dental tissues by alkaline phosphatase (stratum intermedium, apical part of maturation ameloblasts, basal part of all ameloblasts, odontoblasts and subodontoblastic layer), specific ATPase (apical and basal parts of secretory ameloblasts) and ATP pyrophosphatase and/or adenylate cyclase (stratum intermedium, odontoblasts). Acid phosphatase, specific ADPase, 5'-nucleotidase, inorganic pyrophosphatase, 3':5'-cyclic-AMP-phosphodiesterase and adenylate kinase on the other hand, seem not to be engaged in the ATP hydrolysis to such a degree as to complicate the interpretation of the histochemical staining. The alkaline phosphatase part of the ATP hydrolysis appeared to be rather insensitive to aldehyde fixation, while the hydrolysis effected by specific ATPase and ATP pyrophosphatase and/or adenylate cyclase was extinguished after fixation with formaldehyde for 4 h or glutaraldehyde for 10 min.
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PMID:Adenosine triphosphate hydrolysis in rat dental tissues. A histochemical study to differentiate the enzymes involved. 18 60

Using fresh frozen, freeze-dried or cryostate sections from aldehyde fixed rat tissues 13 diazonium salts were tested as simultaneous coupling reagents for the localization of acid, neutral and alkaline hydrolases with azo indoxyl methods. Hexazotized new fuchsine and/or Fast blue B are the diazonium salts of choice for the demonstration of acid beta-galactosidase, neuraminidase, beta-N-acetylglucosaminidase, acid phosphatase, and non-specific esterase followed by hexazotized p-rosaniline. Fast blue VB, BB and RR and Fast violet B are recommended for the investigation of alkaline phosphatase and lactase, Fast garnet GBC for acid beta-galactosidase, glucosaminidase and lactase. Fast red B, RC, RL and TR and Fast black K can only be employed for lactase studies. The exact concentration of the coupling reagent depends on the activity of the enzyme and the organ imvestigated. On the average 0.01-0.02 ml unstable diazonium salt/ml and 0.3--1 microgram stable diazonium salt/ml are sufficient for the correct localization of these hydrolases. Freeze-dried cryostat sections yield the best results in the demonstration of lactase and alkaline phosphatase independent on the coupling reagent used. Sections from formaldehyde or glutaraldehyde fixed organs are superior for the localization of the other hydrolases; an exception is the investigation of acid beta-galactosidase and glucosaminidase with Fast garnet GBC. Then, excellent results are obtained also with freeze-dried material. Fresh frozen sections are suitable for the localization of lactase with hexazotized new fuchsine or p-rosaniline and of alkaline phosphatase with Fast blue VB and BB or violet B. The total activity of acid, neutral and alkaline hydrolases can be investigated using semipermeable membranes in combination with all unstable and stable diazonium salts of choice. Reliable osmification of the azoindoxyl dye is only possible if hexazotized p-rosaniline is employed for coupling; without further posttreatment all azoindoxyl dyes are extracted by ethanol, isopropanol or xylol. 7 incubation media are given for the demonstration of hydrolases with azoindoxyl methods at the level of light microscopy for routine studies and typical examples for the application of these methods are presented. A modified procedure is described for the freeze-drying of cryostat sections with the Edwards-Pearse tissue dryer EPD3.
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PMID:[Azoindoxyl methods for the investigation of hydrolases. IV. Suitability of various diazonium salts (author's transl)]. 36 63

A sensitive immunochemical method was developed for the detection of glycosphingolipids on thin-layer chromatograms. The procedure involves oxidation of diol groups of glycosphingolipids with sodium periodate, derivatization of the formed aldehyde groups with digoxigenin-hydrazide, and reaction of the bound digoxigenin with an alkaline phosphatase-labeled polyclonal anti-digoxigenin antibody. The latter is detected by an insoluble indigo-like dye as a result of dephosphorylation of 5-bromo-4-chloro-3-indolyl phosphate. The detectability of all glycosphingolipid species was improved over that of the orcinol and resorcinol staining methods. Two nanograms of the standard gangliosides GM1, GD1A, and GT1 was detected, whereas the detection limit for short-chain neutral glycosphingolipids was in the range of 20-50 ng. Long-chain glycosphingolipids were detectable with a particularly high sensitivity. Selective staining of the gangliosides could be achieved by the use of low periodate concentrations.
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PMID:Immunochemical detection of glycosphingolipids on thin-layer chromatograms. 169 50

Osteopenia is a recognized complication of diabetes mellitus in humans and experimental animals. We recently found that tetracyclines prevent osteopenia in the streptozotocin-induced diabetic rat and that this effect was associated with a restoration of defective osteoblast morphology (Golub et al., 1990). The present study extends these initial ultrastructural observations by assessing osteoblast function in the untreated and tetracycline-treated diabetic rats. After a 3-week protocol, non-diabetic control and diabetic rats, including those orally administered a tetracycline, minocycline (MC), or a non-antimicrobial tetracycline analog (CMT), were perfusion-fixed with an aldehyde mixture; the humeri were dissected and processed for ultracytochemical localization of alkaline phosphatase (ALPase) and Ca-ATPase activities. Some rats from each experimental group received an intravenous injection of 3H-proline as a radioprecursor of procollagen, and the humeri were processed for light microscopic autoradiography. In addition, the osteoid volume in each experimental group was quantitatively examined by morphometric analysis of electron micrographs. During the diabetic state, active cuboidal osteoblasts in the endosteum of control rats were replaced by flattened bone-lining cells that contained few cytoplasmic organelles for protein synthesis (Golgi-RER system), and active transport (mitochondria). Treating diabetic rats with MC, and even more so with CMT, appeared to "restore" osteoblast structure. During diabetes, bone-lining cells incorporated little 3H-proline or secreted little labeled protein and produced only a very thin osteoid layer. Tetracycline administration to the diabetics increased both the incorporation of 3H-proline by osteoblasts and their secretion of labeled protein toward the osteoid matrix, in a pattern similar to that seen in the non-diabetic controls.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Tetracycline administration restores osteoblast structure and function during experimental diabetes. 183 18

We used in situ nucleic acid hybridization cytochemistry to examine cell types and subcellular sites expressing albumin (alb) or pro alpha 2 collagen (col) mRNA in livers from normal and analbuminemic rodents. Biotinylated cDNA or RNA probes were applied to aldehyde-fixed, non-frozen sections and the resulting DNA-RNA or RNA-RNA hybrids were subsequently visualized by enzymatic detection of either peroxidase or alkaline phosphatase conjugated to anti-biotin IgG or streptavidin. In normal rat liver, alb mRNA was expressed in all hepatocytes and was localized to discrete subcellular structures distributed as aggregates in the cytoplasm and in specific structures encircling the nucleus; these subcellular structures most likely represent the endoplasmic reticulum and nuclear envelope. In mouse liver, pro alpha 2 col mRNA was identified in a subpopulation of sinusoidal lining cells which have the morphological appearance of lipocytes. In liver from analbuminemic rats, a small number of hepatocytes, distributed throughout the hepatic lobule, expressed alb mRNA at high levels; the subcellular distribution of this alb mRNA was essentially identical to that observed in normal rat hepatocytes. Since non-radioactive in situ hybridization detected mRNA within the boundaries of individual cells and showed its precise subcellular location under conditions in which there was excellent preservation of tissue morphology, this procedure should be useful for a wide variety of histopathologic studies.
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PMID:Albumin and collagen mRNA expression in normal and analbuminemic rodent liver: analysis by in situ hybridization using biotinylated probes. 240 55


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