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)

Propagation in vitro of rat tibial osteoblasts (ROB) is accompanied by increased expression of the early osteogenic marker alkaline phosphatase (AP) and maturation of the osteogenic phenotype. In order to establish the pattern of the integrin expressed in ROB during progression to the mature osteoblastic phenotype, we have used biosynthetic, immunoblotting and immunohistochemical assays. We immunoprecipitated from osteoblasts, expanded for 1.5- and 7.5-doubling, alpha 5 beta 1, alpha v beta 3, alpha 3 beta 1, alpha 6 beta 1 and alpha 1 beta 1 integrin heterodimers; furthermore beta 5, alpha 2 and alpha 4 chains were detected by immunoblots and indirect immunofluorescence. alpha v, alpha 1, alpha 6 subunits in most cells, and beta 3 and beta 1 subunits in a minority, were found to be associated with adhesion plaques in osteoblasts of 1.5-, 4.5- and 7.5-doubling grown in the presence of FCS, while all other subunits stained diffusely all the cells. Adhesion to fibronectin (FN), laminin (LN), collagen type I (COL I) and III(COL III) by ROB at different doubling (1.5-11) was dependent on substratum concentration, and after 2.5 h at 55 nM 60% of the cells adhered to all substrata. Arg-Gly-Asp-Ser (RGDS) containing peptides inhibited adhesion of cells differentially, according to substratum; no dependence on extent of progation in vitro was observed. In conclusion, ROB cultured in vitro for 1.5- to 11-doubling had an unchanged pattern of expression of integrin subunits, heterodimer association and cellular distribution. Adhesion specificity and affinity were also unchanged. These results suggest that the phenotypic maturation, detected as an increase in AP expression, is not accompanied by major changes in the potential for cell-matrix interactions, and does not correspond to changes in the type of integrin subunits expressed by osteoblasts.
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PMID:Osteoblastic cells from rat long bone. II: Adhesion to substrata and integrin expression in primary and propagated cultures. 904 3

Sprague-Dawley rats received deionized water (controls) during 28 days or drinking water with added D-proline, L-proline, D-aspartic acid or L-aspartic acid corresponding to a mean daily load of approximately 50 mg amino acid enantiomer kg-1 body weight. Parameters indicating the physiological status (food intake and body weight, glutamic-oxalic-transaminase, glutamic-pyruvic-transaminase, alkaline phosphatase, urea and creatinine in serum, and creatine and osmomolality of urine) were determined. After 28 days the weights of the supposed target organs of toxicity (kidney, liver, brain, thymus) were determined and organs were inspected for macroscopic and microscopic alterations. No pathological changes in the organs were observed and no signs of subacute toxicity (liver, kidney) were found. In serum, homogenates of liver, kidney and brain, and in part, in urine, the amounts of D-amino acids (D-AAs) were quantitatively determined using chiral phase capillary gas chromatography-selected ion monitoring mass spectrometry. Significant levels of certain D-AAs (Ala, Pro, Ser, Asx, Glx, Orn and Lys) were already detectable in kidney and liver homogenates and serum of controls. In brain homogenates the highest amounts among the D-AAs were found for D-Ser (up to 382 nmol g-1), moderate amounts for D-Ala, D-Asx and D-Glx, and, in a few cases, trace amounts for D-Orn and D-Lys (1-2 nmol g-1). D-Pro was not detected either in the brains of controls or in the brains of animals loaded with D-Pro. Feeding with D-Pro resulted in a 20-30 fold increased renal excretion of D-Pro at the end of the experiment. Continuous feeding with D-Asp did not increase renal excretion of this enantiomer, but in the serum, higher amounts (0.8-4.0 mumol-1) were determined in comparison to the control group (0.3-0.9 mumol-1). Feeding with D-Pro led to an increase of this enantiomer in serum (1.3-10.5 mumol-1). Feeding with D-Asp did not increase its amounts in brain homogenates (38 and 43 nmol g-1) in comparison to controls.
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PMID:Evaluation of D-amino acid levels in rat by gas chromatography-selected ion monitoring mass spectrometry: no evidence for subacute toxicity of orally fed D-proline and D-aspartic acid. 914 Jul 53

An ELISA method for the quantitation in vitro of HCV serine proteinase activity was developed. A peptide substrate, Ac-Gly-Glu-Ala-Gly-Asp-Asp-Ile-Val-Pro-Cys-Ser-Met-Ser-Tyr-Thr-Trp-Thr-L ys (biotin) -OH (Sub-1), was hydrolyzed by a recombinant NS3 proteinase fused with maltose binding protein (MBP-NS3) into a product with a free amino moiety at the N-terminus. The product was immobilized, and the amino moiety was analyzed by digoxigenin labeling followed by immunological reaction with anti-digoxigenin-alkaline phosphatase conjugate and then the colorimeteric reaction. This method is suited for the high throughput screening of inhibitors, and the screening can be accelerated by automatic operation.
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PMID:An enzyme-linked immunosorbent assay for detecting proteolytic activity of hepatitis C virus proteinase. 917 84

Amino acid exchanges in the Alcaligenes eutrophus nickel permease (HoxN) were constructed by site-directed mutagenesis, and their effects on nickel ion uptake were investigated. Mutant hoxN alleles were expressed in Escherichia coli, and activity of the altered permeases was examined via a recently described physiological assay (Wolfram, L., Friedrich, B., and Eitinger, T. (1995) J. Bacteriol. 177, 1840-1843). Replacement of Cys-37, Cys-256, or Cys-318 by alanine did not severely affect nickel ion uptake. This activity of a C331A mutant was diminished by 60%, and a similar phenotype was obtained with a cysteine-less mutant harboring four Cys to Ala exchanges. Alterations in a histidine-containing sequence motif (His-62, Asp-67, His-68), which is conserved in microbial nickel transport proteins, strongly affected or completely abolished transport activity in the E. coli system. The analysis of HoxN alkaline phosphatase fusion proteins implied that His-62, Asp-67, and His-68 exchanges did not interfere with overall membrane topology or stability of the nickel permease. These mutations were reintroduced into the A. eutrophus wild-type strain. Analyses of the resulting HoxN mutants indicated that exchanges in the histidine motif led to a clearly decreased affinity of the permease for nickel ion.
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PMID:A Ni2+ binding motif is the basis of high affinity transport of the Alcaligenes eutrophus nickel permease. 920 33

An Escherichia coli expression system that exploits the bacterial alkaline phosphatase (PhoA) signal sequence to translocate recombinant human epidermal growth factor (hEGF) to the periplasm was used to evaluate how changes in the composition and sequence of amino acids near the PhoA-hEGF junction influence the periplasmic accumulation of recombinant protein. A series of chimeric structural genes was generated by in vitro replacement of hEGF sequence with analogous segments from the EGF-like domain of human heregulin (HRG), significantly altering the electrostatic character of the amino-terminal region of the mature protein. Quantitation of HRG/EGF protein in E. coli periplasmic extracts, by RP-HPLC, showed a fourfold decrease after one of two acidic residues located in the amino-terminal region of the mature hEGF, near the PhoA junction, was replaced. An additional threefold decrease was observed when the second acidic residue was replaced with a positively charged lysine. Further extension of the amino-terminal HRG sequence, beyond the first six residues, resulted in net neutralization of a more distant EGF acidic residue with no additional effect on protein yield. The importance of having a negatively charged group in the amino-terminal region of the mature protein was confirmed when insertion of an aspartic acid near the amino-terminus of two poorly expressed hybrid protein sequences resulted in a five- to eightfold increase in their recovery from the periplasm. This study demonstrates the importance of having negatively charged residues near the fusion junction of recombinant proteins expressed in E. coli using the PhoA signal sequence for protein export.
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PMID:Amino-terminal charge affects the periplasmic accumulation of recombinant heregulin/EGF hybrids exported using the Escherichia coli alkaline phosphatase signal sequence. 926 80

Steroidogenic acute regulatory protein (StAR) plays a critical role in steroid hormone synthesis. StAR is thought to increase the delivery of cholesterol to the inner mitochondrial membrane where P450scc resides. Tropic hormones acting through the intermediacy of cAMP rapidly increase pregnenolone synthesis, and this rapid steroidogenic response is believed to be due to StAR's action. The StAR protein contains two consensus sequences for phosphorylation catalyzed by protein kinase A that are conserved across all species in which the amino acid sequence of the StAR protein has been determined. We demonstrated that human StAR expressed in COS-1 cells exists in at least four species detectable by two-dimensional gel electrophoresis followed by Western blotting. The two more acidic species disappeared after treatment of the cell extracts with alkaline phosphatase. 32P was incorporated into StAR protein immunoprecipitated from COS-1 cell extracts, and a 10-min treatment with 8-bromo-cAMP increased 32P incorporation into the StAR preprotein. StAR protein generated by in vitro transcription/translation was phosphorylated by the protein kinase A catalytic subunit in the presence of [gamma-32P]ATP. Mutation of potential sites for protein kinase A-mediated phosphorylation at serine 57 and serine 195 to alanines, individually, reduced 32P incorporation from labeled ATP into StAR preprotein produced by in vitro transcription/translation when incubated with protein kinase A catalytic subunit. 32P labeling of StAR protein expressed in COS-1 cells was also reduced when serine 57 or serine 195 were mutated to alanines. A double mutant in which both serine 57 and serine 195 were changed to alanines displayed markedly reduced 32P incorporation. To determine the functional significance of StAR phosphorylation, we tested the steroidogenic activity of the wild-type StAR and mutated StAR proteins in COS-1 cells expressing the human cholesterol side chain cleavage enzyme system. Mutation of the conserved protein kinase A phosphorylation site at serine 57 had no effect on pregnenolone synthesis. However, mutation of the serine residue at 195 resulted in an approximately 50% reduction in pregnenolone production. The S195A mutant construct did not yield the more acidic species of StAR detected in two-dimensional Western blots, indicating that the mutation affected the ability of the protein to be post-translationally modified. Mutation of the corresponding serine residues in murine StAR (Ser56 and Ser194) to alanines yielded results that were similar to those obtained with human StAR; the S56A mutant displayed a modest reduction in steroidogenic activity, whereas the S194A mutant had approximately 40% of the activity of murine wild-type StAR. In contrast to the human S195A mutation, conversion of serine 195 to an aspartic acid residue had no effect on steroidogenic activity, consistent with the idea that a negative charge at this site modulates StAR function. Our observations suggest that phosphorylation of serine 194/195 increases the biological activity of StAR and that this post- or co-translational event accounts, in part, for the immediate effects of cAMP on steroid production.
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PMID:Phosphorylation of steroidogenic acute regulatory protein (StAR) modulates its steroidogenic activity. 940 83

CrossLaps peptide [Glu-Lys-Ala-His-Asp-Gly-Gly-Arg], a part of the C-telopeptide of the alpha 1-chain of type I collagen of bone, is a recently developed biochemical marker of bone turnover. In this study, the clinical utility of measurement of urinary CrossLaps was investigated in eleven premenopausal women who received a gonadotropin-releasing hormone (GnRH) agonist for 6 months for treatment of adenomyosis (n = 1) or leiomyomas (n = 10). Along with urinary CrossLaps, the levels of various biochemical markers, and serum estradiol, calcitonin and intact parathyroid hormone (i-PTH) were measured, and lumbar spine bone mineral density (BMD) was also monitored before, during, and at the end of the course of GnRH agonist therapy. Apart from CrossLaps, markers of bone resorption tested were urinary pyridinoline, deoxypyridinoline and hydroxyproline. Markers of bone formation tested were serum osteocalcin and bone-specific alkaline phosphatase (B-ALP). Serum estradiol levels decreased to undetectable levels at 2 months of GnRH agonist therapy. The values for all biochemical markers increased significantly throughout the therapy. The degree of an increase in CrossLaps levels was greater than that in all other markers. Mean lumbar spine (L2-L4) BMD was decreased by 7.2% at 6 months of treatment. The percent change in BMD at 6 months of treatment correlated inversely with the percent change in CrossLaps levels from the baseline to 1, 2, and 5 months of treatment. These results indicate that measurement of urinary CrossLaps might be a useful tool to predict the risk of bone loss caused by hypoestrogenism including GnRH agonist therapy.
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PMID:Clinical usefulness of urinary CrossLaps as a sensitive marker of bone metabolism. 944 79

One point mutation which converts glycine-317 to aspartate of tissue-nonspecific alkaline phosphatase (TNSALP) was reported to be associated with lethal hypophosphatasia (Greenberg, C. R., et al. Genomics 17, 215-217, 1993). In order to define the molecular defect of TNSALP underlying the pathogenesis of hypophosphatasia, we have examined the biosynthesis of TNSALP with a Gly317-->Asp substitution. When expressed in COS-1 cells, the mutant did not exhibit alkaline phosphatase activity at all, indicating that the replacement of glycine-317 with aspartate abolishes the catalytic activity of TNSALP. Pulse-chase experiments showed that the newly synthesized mutant failed to acquire Endo H-resistance and to reach the cell surface. Interestingly, this TNSALP mutant was found to form a disulfide-bonded high-molecular-mass aggregate and was rapidly degraded within the cell, though the mutant protein was modified by glycosylphosphatidylinositol (GPI). Lactacystin, an inhibitor of the proteasome, obstructed the degradation of the mutant protein, suggesting the involvement of proteasome as a part of quality control of TNSALP.
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PMID:Intracellular retention and degradation of tissue-nonspecific alkaline phosphatase with a Gly317-->Asp substitution associated with lethal hypophosphatasia. 961 60

Detection of recurring three-dimensional side-chain patterns is a potential means of inferring protein function. This paper presents a new method for detecting such patterns and discusses various implications. The method allows detection of side-chain patterns without any prior knowledge of function, requiring only protein structure data and associated multiple sequence alignments. A recursive, depth-first search algorithm finds all possible groups of identical amino acids common to two protein structures independent of sequence order. The search is highly constrained by distance constraints, and by ignoring amino acids unlikely to be involved in protein function. A weighted root-mean-square deviation (RMSD) between equivalenced groups of amino acids is used as a measure of similarity. The statistical significance of any RMSD is assigned by reference to a distribution fitted to simulated data. Searches with the Ser/His/Asp catalytic triad, a His/His porphyrin binding pattern, and the zinc-finger Cys/Cys/His/His pattern are performed to test the method on known examples. An all-against-all comparison of representatives from the structural classification of proteins (SCOP) is performed, revealing several new examples of evolutionary convergence to common patterns of side-chains within different tertiary folds and in different orders along the sequence. These include a di-zinc binding Asp/Asp/His/His/Ser pattern common to alkaline phosphatase/bacterial aminopeptidase, and an Asp/Glu/His/His/Asn/Asn pattern common to the active sites of DNase I and endocellulase E1. Implications for protein evolution, function prediction and the rational design of functional regulators are discussed.
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PMID:Detection of protein three-dimensional side-chain patterns: new examples of convergent evolution. 964 96

A partial structure of many glycoproteins, a glycosylated asparagine carrying a complex type undecasaccharide N-glycan (Neu5Ac(alpha 2-6)Gal(beta 1-4)GlcNAc(beta 1-2)Man alpha 1-3) [Neu5Ac(alpha 2-6)Gal(beta 1-4)GlcNAc(beta 1-2)Man(alpha 1-6)]Man(beta 1-4) GlcNAc(beta 1-4)GlcNAc-Asn) was obtained by total synthesis. As a starting material served a chemically synthesized diantennary heptasaccharide azide which was deprotected in a three-step sequence in high yield. The reduction of the anomeric azide was accomplished with propanedithiol in methanol-ethyldiisopropylamine. Coupling of the glycosyl amine to an activated aspartic acid gave the benzyl protected asparagine conjugate. After removal of the six benzyl functions the resulting free heptasaccharide asparagine was elongated enzymatically in the oligosaccharide part. The use of beta-1,4-galactosyltransferase and alpha-2,6-sialytransferase in the presence of alkaline phosphatase allowed the efficient transfer of four sugar units to the acceptor resulting in a full length N-glycan, a sialyated diantennary undecasaccharide-asparagine of the complex type.
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PMID:Chemoenzymatic synthesis of a sialylated diantennary N-glycan linked to asparagine. 964 61


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