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)

A method is described to determine the presence and the relative amount of proteins within specific protein-DNA complexes. The system studied is the LexA repressor from Escherichia coli and its interaction with the operator of the caa gene encoding the bacterial toxin colicin A. After separation of the free and the complexed 32P-labeled DNA on a native polyacrylamide gel, the bound proteins are transferred on a polyvinylidine difluoride (PVDF) membrane after sodium dodecyl sulfate denaturation. Development of the protein on the membrane was achieved on reaction with an anti-LexA antibody and the use of a second anti-antibody crosslinked with alkaline phosphatase. The phosphatase activity is monitored using 5-bromo-4-chloro-3-indolyl phosphate as a substrate and 4-nitroblue tetrazolium salt. A quantitation by densitometry of both the stained protein bands on the PVDF membrane and the DNA on autoradiograms allowed us to assign the relative stoichiometry of the two different complexes formed between LexA and the caa operator. The method should allow unraveling of complicated band shift patterns arising from the presence of several binding sites for a same protein, as in our case, or from the presence of different proteins binding to a same DNA fragment.
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PMID:Specific protein-DNA complexes: immunodetection of the protein component after gel electrophoresis and Western blotting. 306 51

Different aspects of biliary physiology were studied in rat model of liver cirrhosis induced by CCl4/phenobarbitone. We measured bile flow, bile salt secretion, biliary secretion pressure and bile-to-plasma ratios of inert solutes under basal conditions and during infusion of taurocholate (0.4 and 0.8 mumol.min-1.100 g body wt.-1) in 11 cirrhotic and 10 control male Sprague-Dawley rats. Bile flow and biliary bile salt secretion did not differ between the two groups. Analyzing the relationship between bile salt secretion and bile flow, however, we found an increased slope (P less than 0.02) in the cirrhotic animals, suggesting a higher apparent osmotic activity of the bile salts secreted. Maximal biliary secretion pressure was maintained in cirrhotic animals (22.5 +/- 2.5 vs. 25.0 +/- 2.9 cmH2O) in the absence of exogenous bile salt. During taurocholate infusion it decreased to a lesser extent (P less than 0.001) in cirrhotic animals (13.5 +/- 3.4 vs. 19.3 +/- 3.8 cmH2O). Bile-to-plasma ratios of [3H]sucrose and [14C]ferrocyanide were markedly increased in cirrhotic rats. Biliary [14C]erythritol clearance was equal to bile flow in both groups. In the cirrhotic group, the [3H]sucrose bile/plasma ratio was positively correlated with spleen weight (r = 0.744, P less than 0.01), serum concentration of alkaline phosphatase (r = 0.583, P less than 0.05) and basal maximum biliary secretion pressure (r = 0.801, P less than 0.001). We conclude that chronic portal hypertension is associated with increased permeability of the blood/bile barrier. Nevertheless, bile flow and bile salt secretion are maintained in cirrhotic rats, giving support to the intact cell hypothesis for this important hepatocellular function.
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PMID:Canalicular bile flow and bile salt secretion are maintained in rats with liver cirrhosis. Further evidence for the intact cell hypothesis. 318 52

A new technique of quantitative histochemistry has been developed to study the cellular composition of the follicle-associated epithelium of the mouse Peyer's patch. This technique involves applying naphthol AS-BI phosphate to the surface of intact tissue where it is hydrolysed by alkaline phosphatase present in the luminal membrane of the epithelial cells. Naphthol AS-BI produced by this reaction is then coupled to Fast Red TR diazonium salt at the site of hydrolysis. M cells present in the epithelium contain little alkaline phosphatase activity and, therefore, remain white. Treatment with Alcian Blue is finally used to label goblet cells. Subsequent quantitative analysis of alkaline phosphatase-rich cells is carried out by scanning microdensitometry. Using this technique it is possible to detect two populations of alkaline phosphatase-containing cells in mice reared in a normal animal house environment. These results are discussed in relation to possible interactions taking place between enteric antigens and the gut-associated lymphoid tissue which could reduce the ability of follicle-associated enterocytes to express alkaline phosphatase.
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PMID:Automated histochemical analysis of cell populations in the intact follicle-associated epithelium of the mouse Peyer's patch. 319 21

New lanthanide methods for the histochemical detection of non-specific alkaline phosphatase in the light microscope are described and compared with already existing techniques for the light microscopical demonstration of this enzyme. To avoid formation of insoluble lanthanide hydroxide at alkaline pH citrate complexes with the capture ions cerium, lanthanum and didymium were used. A molar ratio of 11 mM citrate/14 mM capture reagent is proposed. For preincubated sections, pretreatment in chloroform-acetone and fixation in glutaraldehyde, for non-preincubated sections fixation in glutaraldehyde yielded the best results. 4-Methylumbelliferyl and 5-Br-4-Cl-3-indoxyl phosphate were found to be the most suitable substrates. For routine purposes 4-nitrophenyl, 1-naphthyl, 2-naphthyl and 2-glycerophosphate were also sufficient; naphthol AS phosphates were inferior but still suitable. After incubation for 5-60 min at 37 degrees C lanthanide phosphate was converted into lead phosphate which was visualized as lead sulfide. At pH 9.2-9.5 enzyme activity was demonstrated at many sites such as intestinal, uterine, placental, renal and epididymal microvillous zones, plasma membranes of arterial, sinus and capillary endothelial cells, vaginal and urethral epithelium, smooth muscle cells, myoepithelial cells as well as excretory duct cells of salivary and lacrimal glands and in secretory granules of laryngeal glands. In comparison with Gomori's calcium, Mayahara's lead, Burstone's and Pearse's azo-coupling, McGadey's tetrazolium salt and Gossrau's azoindoxyl coupling technique the lanthanide methods detected alkaline phosphatase activities at identical or additional sites depending on the respective procedure.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Light-microscopic histochemistry of non-specific alkaline phosphatase using lanthanide-citrate complexes. 323 44

We have isolated from the high salt wash of rabbit reticulocyte ribosomes two forms of the polypeptide chain initiation factor 2 (eIF-2) which differ with respect to their beta-subunit, GDP content, and sensitivity to Mg2+ in ternary (eIF-2 X GTP X Met-tRNAf) and binary (eIF-2 X GDP) complex formation. The form of eIF-2 eluting first from a cation exchange (Mono S, Pharmacia) column has a beta-subunit of lower molecular weight (eIF-2(beta L] and a more acidic pI value than the form eluting at a higher salt concentration (eIF-2(beta H]. These two forms of eIF-2 beta-polypeptides are also detected in reticulocyte lysates when the proteins are resolved by two-dimensional isoelectric focusing-dodecyl sulfate polyacrylamide gel electrophoresis followed by immunoblotting. The peptide mapping of the isolated beta-subunits after limited proteolysis by papain, pancreatic protease, alpha-chymotrypsin, or Staphylococcus aureus V8 protease further demonstrates that the two forms of beta-subunits are not the product of a non-specific proteolytic action that occurred during the purification procedure, but rather reflects the existence in vivo of both forms of eIF-2. The GDP content of eIF-2(beta L) and eIF-2(beta H) is approximately 0.85 and 0.22 mol of GDP/mol of eIF-2, respectively. The KD for GDP of eIF-2(beta L) was lower (2.2 X 10(-9) M) than that of eIF-2(beta H) (6.0 X 10(-8) M). In the presence of 1 mM Mg2+, the activities of eIF-2(beta L) and eIF-2(beta H) in forming a binary and a ternary complex are inhibited 90 and 25%, respectively. The extent of Mg2+ inhibition and its reversal by the guanine nucleotide exchange factor is directly proportional to the amount of GDP bound to eIF-2. No inhibition by Mg2+ is observed when eIF-2-bound GDP is removed by alkaline phosphatase. In the presence of the guanine nucleotide exchange factor, both forms of eIF-2 are equally active in ternary complex formation, and the complex formed is quantitatively transferred to 40 S ribosomal subunits.
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PMID:The isolation and characterization from rabbit reticulocytes of two forms of eukaryotic initiation factor 2 having different beta-polypeptides. 330 29

The pathophysiological changes that occur in mice experimentally infected with Schistosomatium douthitti were studied. Male ICR mice, 6-8 weeks in age, were exposed to 100 cercariae of S. douthitti from infected snails (Lymnaea catascopium) and sacrificed weekly for a total of 13 weeks. Liver homogenates, serum samples, and histological sections of liver tissue were examined. Results showed that body weights of animals with prepatent infections were higher than those of corresponding controls. After patency, which occurred at 5 weeks, body weights were lower and liver weights were higher resulting in significantly increased liver weight/body weight ratios. Hematocrit values declined progressively in patent infections. Total cholesterol in liver was generally higher in the parasitized groups reaching significance during patency. Values rose with age in both control and parasitized groups, but sooner in the latter. Free cholesterol was increased in the liver of animals with patent infections. Total lipid content of the liver was reduced in the infected animals throughout the study. Both liver glycogen and serum glucose levels in the infected animals rose over the control values. The activity of alkaline phosphatase (E.C.3.1.3.1) was elevated in liver tissue of infected mice. Glutamic-pyruvic transaminase (E.C.2.6.1.2) activity was higher in serum but lower in the livers of animals harboring patent infections. Total bile salt concentration in parasitized animals did not differ appreciably from control values; however, gallbladders were enlarged five times in the infected animals. Histologically, liver sections from infected mice showed granulomas in various stages of formation and degeneration. Granulomas contained from 1 to 40 schistosome eggs. After 6 weeks of infection, granulomas were characterized by many neutrophils and monocytes. Few lymphocytes and eosinophils were present. As the granulomas developed, fibroblasts and connective tissue became more prominent. Glycogen deposits were observed surrounding granulomas and were increased in older infections. Adult worms contained abundant amounts of glycogen and cholesterol in their parenchymal tissues.
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PMID:Schistosomatium douthitti: biochemical and morphological effects of an experimental infection in mice. 335 Jan 1

The T3-binding activity of salt-extractable nuclear proteins from rat liver was affected when ATP (2-10 mM; pH 8.0) was added concomitantly with T3 in the incubation medium. Scatchard analysis revealed that the equilibrium association constant was significantly reduced [5 mM ATP, 0.3 +/- 0.1 (+/- SE) 10(10) M-1; control, 1.1 +/- 0.15 X 10(10) M-1], but the maximum binding capacity remained unchanged. Similar values of inhibition were obtained when unbound receptors were preincubated with ATP. ATP achieved its maximal effect after 45 min of incubation at 30 C. Dilution experiments indicated that the effect of ATP was reversible. The inhibiting potency of nucleoside triphosphates at pH 8.0 was in the following order: ATP = CTP greater than GTP, whereas UTP had no effect. Nonhydrolyzable analogs of ATP were also inhibitory, and HPLC fractionation showed an approximately 98% recovery of ATP after incubation with nuclear extract. The adenine ring with at least two phosphates was essential, since ADP was as potent as ATP, whereas AMP had no effect. When the pH of the incubation medium was lowered to 7.3, the T3-binding activity was inhibited by ATP in the 0.1-1 mM range. Magnesium (3 mM) greatly increases the ATP effect at pH 7.3, but not at pH 8. The T3-binding activity was also drastically reduced when calf intestine alkaline phosphatase was added concomitantly in the incubation medium. Eight micrograms per ml enzyme were necessary to inhibit the T3 specific binding by 50% (30 C for 45 min). Scatchard analysis showed that the receptor affinity for T3 was decreased (control, 1.1 +/- 0.02 x 10(10) M-1; alkaline phosphatase, 0.41 +/- 0.03 x 10(10) M-1; n = 6), whereas the maximum binding capacity remained unchanged. Incubations performed with increasing concentrations of beta-mercaphoethanol (2.5, 5, 10, and 25 mM) revealed that the phosphatase inhibitory effect is thiol dependent. The inhibition was maximal at 2.5 mM and progressively decreased at 5 and 10 mM. No inhibition occurred at 25 mM. When a saturating concentration of T3 was employed, the specific binding was decreased at low thiol concentrations. These observations show that the nuclear T3 receptors may be modulated by ATP/ADP and phosphorylation/dephosphorylation processes. It is proposed that in vitro dephosphorylation leads to rapid oxydation of sulfhydryl groups which are essential for optimum T3 binding.
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PMID:Effects of adenosine triphosphate and alkaline phosphatase on solubilized 3,5,3'-triiodothyronine-binding activity. 340 84

Alkaline phosphatase has been studied in hydrophobic interaction chromatography (HIC), using a bonded C1-ether phase on a silica gel support, together with an aqueous salt gradient. Its behavior under various gradient elution conditions has demonstrated good chromatographic performance and retention of enzymatic activity under aqueous conditions. It has now been studied using linear photodiode array (LDA) spectroscopy in combination with low-angle laser light scattering (LALLS) in gradient elution HIC. HIC-LALLS permitted the use of routine salt gradients for collection of molecular weight information, despite small changes in the baseline, via computerized baseline subtraction. Size-exclusion chromatography (SEC)-LALLS measurements, under various isocratic conditions, meant to mimic HIC elution, have indicated the presence of monomer/dimer, dimer/trimer, or mainly trimer. aggregates of alkaline phosphatase can also be detected under salt gradient HIC conditions, but at lower levels relative to the monomer. This paper also describes the behavior of alkaline phosphatase when detected using LDA under various chromatographic, temperature, and concentration (injected) conditions. The results indicate a facile equilibrium of at least two monomeric forms of alkaline phosphatase of the same molecular weight, which change relative populations as a function of operational conditions. Most interesting is the suggestion that alkaline phosphatase undergoes rapid conformational interconversions on the chromatographic detection time scales, and that these interconverting conformations, concentration dependent, produce a novel dual wavelength ratioing, viz., a pseudo-Gaussian peak mimicking the chromatographic elution profile at either wavelength. The reasons for these observations and their possible use in future high-performance liquid chromatographic biopolymer studies are discussed and described.
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PMID:Conformational studies of bovine alkaline phosphatase in hydrophobic interaction and size-exclusion chromatography with linear diode array and low-angle laser light scattering detection. 341 21

The purpose of this investigation was to determine whether biologically available organic phosphates other than beta-glycerophosphate were capable of inducing mineralization of bone in vitro. The chick periosteal osteogenesis model was used to demonstrate that endogenously available organic phosphates, fructose 1,6-diphosphate (F1,6-D) and phosphoethanolamine (PEA) induce calcium accumulation in bone formed in vitro. Alkaline phosphatase activity was inhibited in a dose-dependent manner by PEA, and the sodium salt of F1,6-D. There was an inverse correlation between alkaline phosphatase activity and organic phosphate-mediated mineralization. The data demonstrate that certain biologically available organic phosphates can induce mineralization and modulate bone metabolism in vitro.
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PMID:Phosphoethanolamine- and fructose 1,6-diphosphate-induced calcium uptake in bone formed in vitro. 350 31

A method is described for increasing the response of enzyme immunoassays employing alkaline phosphatase as the label initiating 2 sequential catalytic reactions. First, NADP is dephosphorylated to produce NAD, which catalytically activates a specific redox-cycle involving the enzymes alcohol dehydrogenase and diaphorase. During each turn of the cycle 1 molecule of a tetrazolium salt is reduced to an intensely coloured formazan. The method is capable of detecting as little as 0.01 amol alkaline phosphatase, and when applied to an immunoassay for TSH a sensitivity (zero + 2.5 standard deviations) of 0.0013 mIU/l was obtained.
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PMID:Enzyme amplification for immunoassays. Detection limit of one hundredth of an attomole. 351 23


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