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)

The Escherichia coli glpT gene encodes a transport protein that mediates uptake of sn-glycerol-3-phosphate. This permease is a member of a class of bacterial organophosphate permeases which transport substrates by antiport with inorganic phosphate. The glpT gene product, probably an oligomer of a single polypeptide chain, is thought to span the cytoplasmic membrane several times, as predicted by the hydropathic profile. Protein fusions, in which varying lengths of the amino-terminal end of the permease is attached to alkaline phosphatase (phoA) and to beta-galactosidase (lacZ) were constructed. On the assumption that phoA fusions only exhibit high enzymatic activity when fused to extra-cytoplasmic regions of the target protein, whereas lacZ fusions will only be active when the beta-galactosidase portion is attached to cytoplasmic domains of the target protein, the activities of the fusions were used to test a two-dimensional model for the permease. The model proposes that GlpT contains 12 transmembrane segments divided by a larger cytoplasmic region. Despite some limitation caused by hot-spot sites of transpositions, the TnphoA approach was consistent with the model. In contrast, we feel that the enzymatic activity of lacZ fusions is only a limited parameter for studying the topology of a complex membrane protein.
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PMID:The transmembrane topology of the sn-glycerol-3-phosphate permease of Escherichia coli analysed by phoA and lacZ protein fusions. 314 44

The 200-kD subunit of neurofilaments (NF-H) functions as a cross-bridge between neurofilaments and the neuronal cytoskeleton. In this study, four phosphorylated NF-H variants were identified as major constituents of axons from a single neuron type, the retinal ganglion cell, and were shown to have characteristics with different functional implications. We resolved four major Coomassie Blue-stained proteins with apparent molecular masses of 197, 200, 205, and 210 kD on high resolution one-dimensional SDS-polyacrylamide gels of mouse optic axons (optic nerve and optic tract). Proteins with the same electrophoretic mobilities were radiolabeled within retinal ganglion cells in vivo after injecting mice intravitreally with [35S]methionine or [3H]proline. Extraction of the radiolabeled protein fraction with 1% Triton X-100 distinguished four insoluble polypeptides (P197, P200, P205, P210) with expected characteristics of NF-H from two soluble neuronal polypeptides (S197, S200) with few properties of neurofilament proteins. The four Triton-insoluble polypeptides displayed greater than 90% structural homology by two-dimensional alpha-chymotryptic iodopeptide map analysis and cross-reacted with four different monoclonal and polyclonal antibodies to NF-H by immunoblot analysis. Each of these four polypeptides advanced along axons primarily in the Group V (SCa) phase of axoplasmic transport. By contrast, the two Triton-soluble polypeptides displayed only a minor degree of alpha-chymotryptic peptide homology with the Triton-insoluble NF-H forms, did not cross-react with NF-H antibodies, and moved primarily in the Group IV (SCb) wave of axoplasmic transport. The four NF-H variants were generated by phosphorylation of a single polypeptide. Each of these polypeptides incorporated 32P when retinal ganglion cells were radiolabeled in vivo with [32P]orthophosphate and each cross-reacted with monoclonal antibodies specifically directed against phosphorylated epitopes on NF-H. When dephosphorylated in vitro with alkaline phosphatase, the four variants disappeared, giving rise to a single polypeptide with the same apparent molecular mass (160 kD) as newly synthesized, unmodified NF-H. The NF-H variants distributed differently along optic axons. P197 predominated at proximal axonal levels; P200 displayed a relatively uniform distribution; and P205 and P210 became increasingly prominent at more distal axonal levels, paralleling the distribution of the stationary neurofilament network.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Multiple phosphorylated variants of the high molecular mass subunit of neurofilaments in axons of retinal cell neurons: characterization and evidence for their differential association with stationary and moving neurofilaments. 314 56

Induction of axonal neuritogenesis in NB2a/d1 cells was associated with an increased content of neurofilament proteins (NFPs) by immunoblot analysis. The major NFP subunits in differentiated [NB2a(+)] cells included microheterogenous forms with apparent molecular weights of 200-190 kDa (NFP-H), 143-142 kDa (NFP-M) and 70 kDa (NFP-L) on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Only NFP-L was detected in cytoskeletal preparations of undifferentiated [NB2a(-)] cells. All three NFPs of NB2a(+) cells incorporated 32P-orthophosphate in intact cells. A 160/155 kDa NFP-H immunoreactive polypeptide in NB2a(-) and NB2a(+) cells represented a relatively unmodified form of the 200 kDa NFP-H, since dephosphorylation of the 200 kDa NFP-H in vitro with alkaline phosphatase generated the 160/155 kDa forms. Triton-extracted NB2a(+) cells displayed NFP-H immunoreactivity in neurites and occasionally in perikaryal regions at the base of neurites. NFP-M was present throughout the neurites and somata of NB2a(+) cells, and was regularly detected in portions of perikarya in NB2a(-) cells. NFP-L immunoreactivity was distributed throughout the Triton-insoluble cytoskeleton of NB2a(-) and NB2a(+) cells. Immunocytochemical analyses revealed that extensively phosphorylated forms of NFP-H were largely restricted to the neurites of NB2a(+) cells, and less modified forms predominated throughout both perikarya and neurites of NB2a(-) and NB2a(+) cells.
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PMID:Neurofilament triplet proteins of NB2a/d1 neuroblastoma: posttranslational modification and incorporation into the cytoskeleton during differentiation. 314 7

The excretory/secretory antigens released during in vitro culture of infective third-stage Heligmosomoides polygyrus larvae were analyzed by enzyme-linked immunosorbent assay and immunoblotting using sera from repeatedly infected mice. During the first 8-10 hr of culture at 37 C, freshly exsheathed larvae released only one antigen that cosedimented with trypsin (24 kDa) upon ultracentrifugation and was composed of a single 23-kDa polypeptide chain. After 10 hr of culture, the larvae released additional antigens identified by bands equivalent to polypeptides of approximately 18, 25, 26, 32, 58, and 76 kDa on nonreduced Western blots. The release of these molecules was maintained for up to 60 hr. Their staining intensity on blots was in the order 23 much greater than 25 greater than 76 greater than 18 greater than or equal to 58 greater than or equal to 32 greater than or equal to 26 kDa. Velocity sedimentation analysis showed that the 76-kDa component exists as a monomeric 76-kDa "native" antigen. The 32-, 58-, and 76-kDa antigens were specifically adsorbed by concanavalin A (Con A)-Sepharose and the 76-kDa molecule was detected on blots incubated with alkaline phosphatase-conjugated Con A, indicating the presence of mannose-like residues on these molecules. The 18-, 23-, 25-, and 26-kDa antigens did not bind to Con A-Sepharose. Hyperimmune antisera raised against lyophilized larvae had negligible antibody activity against the larval ES antigens, suggesting that the ES antigens are released soon after synthesis rather than being stored in significant quantities within the larvae.
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PMID:Heligmosomoides polygyrus: excretory/secretory antigens released in vitro by exsheathed third-stage larvae. 319 58

Uricase is a peroxisomal liver enzyme that catalyzes the oxidation of uric acid to allantoin during purine catabolism. It is present in vertebrates in most species of fish, amphibians, and mammals but its enzymatic activity is absent in hominoids. We have used Western blot analysis in a comparative study to establish a homology among uricases from different species of vertebrates. Using antibodies against denatured rat liver uricase, we have been able to detect for the first time cross-reactivity with the uricase of species ranging in the evolutionary scale from fish to primates (macaque). Our results suggest that these uricases have a common evolutionary origin. Our conclusion is also supported by the fact that uricase from different species exhibits identical tissue, subcellular localization, and similarity of molecular weights. This study was extended to include human liver samples. Using the same approach but with a more sensitive detection system (alkaline phosphatase instead of peroxidase), we did not detect polypeptide species related to rat uricase in human fetal or adult liver samples, which indicates that during hominoid evolution, the mutational event responsible for the loss of uricase activity in humans precluded formation of a translatable uricase mRNA.
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PMID:Uricase protein sequences: conserved during vertebrate evolution but absent in humans. 319 41

Glial fibrillary acidic protein (GFAP) is soluble in low ionic strength solutions but shows a strong tendency toward assembly with increasing ionic strength as revealed by electron microscopy and turbidity measurements. Increasing K+, Na+, and Li+ concentrations cause an increase followed by a decrease in GFAP turbidity with a maximum at 200 mM, but their effects are much weaker than effects of divalent cations at the same ionic strength. Ca2+, Mg2+, Mn2+, and Ba2+ promote assembly at millimolar concentrations, and 10 microM Cu2+ causes rapid aggregation. The critical concentration for GFAP assembly was 0.08 +/- 0.04 mg/mL in 2 mM Tris-HCl, 60 mM KCl, and 1 mM CaCl2, pH 6.8. The Mr 38,000 rod domain of GFAP obtained by limited chymotryptic digestion is more soluble in 100 mM imidazole hydrochloride buffer, pH 6.8, than the intact molecule, and removal of the end pieces greatly reduces the ability of GFAP to form filaments. BNPS-skatole (2-[(2-nitrophenyl)sulfenyl]-3-methyl-3-bromoindolenine) treatment releases a Mr 30,000 N-terminus and a Mr 20,000 C-terminus. The Mr 30,000 polypeptide shows a higher affinity than the Mr 20,000 fragment for intact GFAP. Arginine and lysine at low concentrations slightly accelerate GFAP assembly, but above 100 mM both amino acids inhibit assembly. ATP, GTP, CTP, and UTP do not show significant effects on GFAP assembly. Dephosphorylation by alkaline phosphatase slightly reduces the assembly ability of GFAP, but phosphatase-treated GFAP still is assembly competent.
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PMID:Factors modulating filament formation by bovine glial fibrillary acidic protein, the intermediate filament component of astroglial cells. 319 99

To examine the role of lipid metabolism in the growth and function of osteoblast-like cells, we studied ROS 17/2.8 osteosarcoma cells and primary cultures of rat calvarial osteoblasts during growth in a serum-free medium supplemented by purified human lipoproteins or by liposomes. Increase in ROS cell number was measured in sparse (1-5 X 10(3)/cm2) cultures over 6-8 days. Liposomes (0-300 micrograms/ml) and high (HDL), low (LDL), and very low density (VLDL) lipoprotein fractions (0-300 micrograms apoprotein) markedly stimulated cell growth. Cells plated at 5 X 10(3)/cm2 achieved growth rates in the presence of LDL or HDL comparable to 10% fetal bovine serum. Serum-free culture with exogenous lipid maintained the response of cell cyclic AMP accumulation to parathyroid hormone. Cyclic AMP response to parathyroid hormone was enhanced by glucocorticosteroid, and was attenuated by 1,25-dihydroxyvitamin D (1,25(OH)2D) with an EC50 (10(-10) M) comparable to that previously observed in serum-cultured cells (J. Biol. Chem. 258:736, 1985). 1,25(OH)2D also increased the alkaline phosphatase activity in ROS cells cultured in lipid-supplemented serum-free culture. Lipoproteins or liposomes also markedly enhanced the proliferative response of sparse cultures of normal rat osteoblasts to polypeptide mitogens.
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PMID:Growth of rat osteoblast-like cells in a lipid-enriched culture medium and regulation of function by parathyroid hormone and 1,25-dihydroxyvitamin D. 322 57

The 2- and 8-azido trimer 5'-triphosphate photoprobes of 2-5A have been enzymatically synthesized from [gamma-32P]2-azidoATP and [alpha-32P]8-azidoATP by 2-5A synthetase from rabbit reticulocyte lysates. Identification and structural determination of the 2- and 8-azido adenylate trimer 5'-triphosphates were accomplished by enzymatic hydrolyses with T2 RNase, snake venom phosphodiesterase, and bacterial alkaline phosphatase. Hydrolysis products were identified by HPLC and PEI-cellulose TLC analyses. The 8-azido photoprobe of 2-5A displaces p3A4[32P]pCp from RNase L with affinity equivalent to p3A3 (IC50 = 2 X 10(-9) M in radiobinding assays). The 8-azido photoprobe also activates RNase L to hydrolyze poly(U) [32P]pCp 50% at 7 X 10(-9) M in core-cellulose assays. The 2- and 8-azido photoprobes and authentic p3A3 activate RNase L to cleave 28S and 18S rRNA to specific cleavage products at 10(-9) M in rRNA cleavage assays. The nucleotide binding site(s) of RNase L and/or other 2-5A binding proteins in extracts of interferon-treated L929 cells were investigated by photoaffinity labeling. Dramatically different photolabeling patterns were observed with the 2- and 8-azido photoprobes. The [gamma-32P]2-azido adenylate trimer 5'-triphosphate photolabels only one polypeptide with a molecular weight of 185,000 as determined by SDS gel electrophoresis, whereas the [alpha-32P]8-azido adenylate trimer 5'-triphosphate covalently photolabels six polypeptides with molecular weights of 46,000, 63,000, 80,000, 89,000, 109,000, and 158,000. Evidence that the photolabeling by 2- and 8-azido 2-5A photoprobes was highly specific for the p3A3 allosteric binding site was obtained as follows.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:2- and 8-azido photoaffinity probes. 1. Enzymatic synthesis, characterization, and biological properties of 2- and 8-azido photoprobes of 2-5A and photolabeling of 2-5A binding proteins. 324 13

CellCAM 105 is an integral membrane glycoprotein, with apparent Mr 105,000, which has been purified from rat liver plasma membranes. It consists of two structurally similar, highly glycosylated polypeptide chains and is involved in cell-cell adhesion of adult rat hepatocytes in vitro. In this communication we report on the distribution and cell surface location of cellCAM 105 in rat tissues, obtained by using highly sensitive immunodetection systems based on complex formation between biotinylated antibodies, biotinylated peroxidase and avidin, or on antibodies coupled to alkaline phosphatase. CellCAM was found in many organs and organ systems, including liver, kidney, blood, blood vessels, glands, respiratory system, and gastrointestinal tract. It was mainly localized to epithelial structures but showed a varying cell surface distribution. In some cell types it was predominantly localized to cell-cell contact areas. In other cell types the highest concentrations were seen in brush-border areas containing densely packed microvilli. In addition to epithelial structures, cellCAM 105 was found in rat platelets, where it became strongly expressed on the cell surfaces after activation with ADP or collagen, suggesting that it might be involved in platelet adhesion and/or aggregation mechanisms. Granulocytes also contained cellCAM 105. By SDS-PAGE/immunoblotting, significant differences were found in the apparent Mr values of cellCAM 105 in different tissues. The collected data suggest that cellCAM 105 participates in several different cell surface membrane interactions, of which the common denominator might be membrane-membrane binding.
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PMID:Immunohistochemical localization of cellCAM 105 in rat tissues: appearance in epithelia, platelets, and granulocytes. 329 Mar 31

Over 3 1/2 years, 54 previously untreated hypopituitary children were enrolled in three studies; 48 had idiopathic pituitary dwarfism. Three preparations of somatrem were used (SI, SII, Somatonorm). Growth velocities on all three preparations were similar, though SI and SII tended to produce slightly higher growth velocities. Serum somatomedin and alkaline phosphatase levels increased during treatment. Antibodies to Escherichia coli polypeptide (ECP) increased to a maximum Z score of about 0.7 with SI, but this was considerably lower with SII and below 0.1 with Somatonorm. Anti-hGH antibodies had the highest titre in children treated with SI and these antibodies also had quite high binding capacities. Anti-hGH antibody formation was negligible during Somatonorm treatment.
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PMID:Treatment of pituitary dwarfism with biosynthetic growth hormone. 329 34


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