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)

Rat liver alkaline phosphatase (ALP) was markedly induced by treatment of rats by bile-duct ligation and colchicine injection. Taking this advantage for enrichment of ALP mRNA, we constructed a lambda gt11 liver cDNA library using polyadenylated RNA prepared from the treated rat liver, and isolated an ALP cDNA clone. The 2165 bp cDNA contained an open reading frame that encodes a 524-amino-acid-residue polypeptide with a predicted molecular mass of 57737 Da. The precursor protein contained a presumed signal peptide of 17 amino acid residues followed by 28 amino acid residues identical with the N-terminal sequence determined from the purified rat liver ALP. It was also confirmed that amino acid sequences of two CNBr-cleavage peptides obtained from liver ALP were contained within the cDNA-encoded protein. Five possible N-linked glycosylation sites were found in the molecule and a highly hydrophobic amino acid sequence at the C-terminus. The deduced polypeptide of rat liver ALP showed 88% homology to that of the human liver-type enzyme in osteosarcoma cells. RNA blot hybridization analysis identified a single species of ALP mRNA with 2.7 kb in both the control and the treated rat livers. An approx. 20-fold increase of the mRNA was detected in the treated liver at 12 h after the onset of stimulation, compared with that in the control liver.
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PMID:Primary structure of rat liver alkaline phosphatase deduced from its cDNA. 289 32

Native, cell-surface insulin receptor consists of two glycoprotein subunit types with apparent masses of about 125,000 daltons (alpha subunit) and 90,000 daltons (beta subunit). The alpha and beta subunits are derived from a single polypeptide precursor by one or more proteolytic cleavages. The predominant subunit configuration in the native insulin receptor is a disulfide-linked heterotetrameric structure containing two alpha and two beta subunits. The alpha and beta insulin-receptor subunits seem to have distinct functions such that alpha appears to bind hormone whereas beta appears to possess intrinsic tyrosine kinase activity. In detergent extracts, insulin activates receptor autophosphorylation of tyrosine residues on its beta subunit, whereas in the presence of reductant, the alpha subunit is also phosphorylated. Other physiologically relevant substrates of the insulin receptor tyrosine kinase in target cells, if any, have not yet been identified. In intact cells, insulin activates serine/threonine phosphorylation of insulin receptor beta subunit as well as tyrosine phosphorylation. The biological role of the receptor-associated tyrosine kinase is not known. Tyrosine phosphorylation, catalyzed by either autophosphorylation or purified src kinase, of insulin receptor beta subunit in vitro activates the receptor kinase activity, whereas dephosphorylation with alkaline phosphatase deactivates the receptor kinase. The insulin receptor kinase is regulated by beta-adrenergic agonists and other agents that elevate cAMP in adipocytes, presumably via the cAMP-dependent protein kinase. Such agents decrease receptor affinity for insulin and partially uncouple receptor tyrosine kinase activity from activation by insulin. These effects appear to contribute to the biological antagonism between insulin and beta-agonists. The insulin receptor kinase is also inhibited in intact cells by phorbol esters that mediate serine/threonine phosphorylation of the insulin receptor, presumably via the Ca++-phospholipid-dependent protein kinase. These data suggest the hypothesis that a complex network of tyrosine and serine/threonine phosphorylations on the insulin receptor modulate its binding and kinase activities in an antagonistic manner.
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PMID:The nature and regulation of the insulin receptor: structure and function. 298 34

Prolyl hydroxylase was purified from human placentae, specific antiserum against it was prepared, and a new radioimmunoassay system employing 125I-labelled enzyme preparation was established. The molecular weight of the placental enzyme was shown to be 320,000 by gel filtration. SDS-polyacrylamide gel electrophoresis showed two bands of unequal intensity having molecular weights of 60,000 and 130,000. Their amino acid compositions were identical to each other, suggesting the polypeptide with a molecular weight of 130,000 might be a dimer of the polypeptide with a molecular weight of 60,000. The new radioimmunoassay established had a sensitivity of the order of 10 ng/ml, indicating it was more sensitive than previous radioimmunoassay employing 3H-labelling method. Clinical studies on patients with liver diseases disclosed that the concentrations of serum immunoreactive prolyl hydroxylase were elevated both in cases of hepatocellular damage and in cases of cholestasis. In cases of hepatocellular damage the enzyme behaved like cytoplasmic enzymes such as glutamic oxaloacetic transaminase, glutamic pyruvic transaminase and lactic dehydrogenase, but in cases of cholestasis it resembled biliary enzymes such as alkaline phosphatase and gamma-glutamyl transpeptidase. This result might be associated with the peculiar location of the enzyme within the cell, in the membrane of rough endoplasmic reticulum.
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PMID:Human prolyl hydroxylase. Purification, radioimmunoassay and clinical studies in liver diseases. 299 Oct 66

The insulin receptor is an integral membrane glycoprotein (Mr approximately 300,000) composed of two alpha-subunits (Mr approximately 130,000) and two beta-subunits (Mr approximately 95,000) linked by disulphide bonds. This oligomeric structure divides the receptor into two functional domains such that alpha-subunits bind insulin and beta-subunits possess tyrosine kinase activity. The amino acid sequence deduced from cDNA of the single polypeptide chain precursor of human placental insulin receptor revealed that alpha- and beta-subunits consist of 735 and 620 residues, respectively. The alpha-subunit is hydrophilic, disulphide-bonded, glycosylated and probably extracellular. The beta-subunit consists of a short extracellular region which links the alpha-subunit through disulphide bridges, a hydrophobic transmembrane region and a longer cytoplasmic region which is structurally homologous with other tyrosine kinases like the src oncogene product and EGF receptor kinases. The cellular function of insulin receptors is dual: transmembrane signalling and endocytosis of hormone. The binding of insulin to its receptor on the cell membrane induces transfer of signal from extracellular to cytoplasmic receptor domains leading to activation of cell metabolism and growth. In addition, hormone-receptor complexes are internalized leading to intracellular proteolysis of insulin, whereas receptors are recycled to the membrane. These phenomena are kinetically well-characterized, but their molecular mechanisms remain obscure. Insulin receptor in different tissues and animal species are homologous in their structure and function, but show also significant differences regarding size of alpha-subunits, binding kinetics, insulin specificity and receptor-mediated degradation. We suggest that this heterogeneity of receptors may be linked to the diversity in insulin effects on metabolism and growth in various cell types. The purified insulin receptor phosphorylates its own beta-subunit and exogenous protein and peptide substrates on tyrosine residues, a reaction which is insulin-sensitive, Mn2+-dependent and specific for ATP. Tyrosine phosphorylation of the beta-subunit activates receptor kinase activity, and dephosphorylation with alkaline phosphatase deactivates the kinase. In intact cells or impure receptor preparations, a serine kinase is also activated by insulin. The cellular role of two kinase activities associated with the insulin receptor is not known, but we propose that the tyrosine- and serine-specific kinases mediate insulin actions on metabolism and growth either through dual-signalling or sequential pathways.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Protein kinase activity of the insulin receptor. 301 97

Proteolytic fragments of simian virus 40 tumor (T) antigen and T antigen that was dephosphorylated with alkaline phosphatase bound between 1.5 to 2 times more origin-containing simian virus 40 DNA than did intact T antigen in DNA saturation experiments. Kinetic experiments showed that these treatments also enhanced the rate at which T antigen bound to the DNA. The enhanced binding of T-antigen fragments correlated with the generation of DNA-binding fragments that lacked the NH2-terminal region. Dephosphorylation of T antigen in vitro resulted in the removal of phosphate groups from the NH2-terminal region as well as from the COOH-terminal region. To test the effects of dephosphorylation on the size of the protein, immunoaffinity-purified T antigen was subjected to sedimentation with and without prior treatment with alkaline phosphatase. Most of the purified protein sedimented as a monomer and no significant effect was observed after dephosphorylation, indicating that the enhanced DNA-binding activity was probably not due to the uncovering of additional binding sites buried specifically in oligomerized T antigen. Taken together, these results indicate that in vivo phosphorylation of the NH2-terminal region (residues 106 to 124) decreases the binding of the protein to the DNA origin. The effect is reversed by in vitro dephosphorylation or by proteolysis which removes the highly phosphorylated NH2-terminal arm of the polypeptide. We suggest that phosphorylation inactivates one of two distinct DNA-binding activities on the polypeptide chain perhaps corresponding to two separate regions in T antigen.
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PMID:Phosphorylation downregulates the DNA-binding activity of simian virus 40 T antigen. 302 78

Immunostaining of normal human fibroblasts with a monoclonal antibody (MAb) (V22AC12) revealed typical cytoplasmic arrays of vimentin filaments in both mitotic and interphase cells. In human A8387 fibrosarcoma cells and SV40-virus-transformed human fibroblasts, the same antibody showed positivity only in mitotic cells and in interphase cells only after treatment of the fixed cells with alkaline phosphatase. Upon immunoblotting with the MAb, an Mr 57,000 vimentin polypeptide was seen in normal fibroblasts. In fibrosarcoma cells the same polypeptide was revealed by this antibody only after treatment with alkaline phosphatase. The Mr 57,000 vimentin polypeptide was a major cytoskeletal protein in both fibroblasts and fibrosarcoma cells. Inclusion of Ca2+ into the cytoskeleton extraction medium brought about a somewhat increased degradation of vimentin in fibroblasts. In fibrosarcoma cells, such treatment caused a quantitative disappearance of the Mr 57,000 protein with a concomitant appearance of 3 distinct, low-molecular-weight degradation products in the detergent-soluble fraction. Another Ca2+-induced change in the polypeptide profile of fibrosarcoma cells was the disappearance of the Mr 240,000 non-erythroid alpha-spectrin and the concomitant appearance of a prominent Mr 140,000 degradation product. Inclusion of proteolysis inhibitors in the Ca2+-supplemented extraction medium inhibited degradation of both vimentin and alpha-spectrin polypeptides. The results suggest differences in the composition of the cytoskeletons of normal fibroblasts and fibrosarcoma cells, manifested in the differential Ca2+-susceptibility of vimentin and non-erythroid alpha-spectrin. Results with MAb V22AC12 suggest that differential phosphorylation of vimentin could account for at least part of this difference.
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PMID:Differential immunoreactivity and Ca2+-dependent degradation of vimentin in human fibroblasts and fibrosarcoma cells. 304 35

The presence of many types of polypeptide growth factors in the mineralized extracellular matrix of bone is now well established. These factors are generally referred to as bone-derived growth factors (BDGFs), and are similar, or possibly identical, to the following species; platelet-derived growth factor (PDGF); acidic and basic forms of fibroblast growth factor (aFGF, bFGF); transforming growth factor beta (TGF-beta); and insulin-like growth factor 1 (IGF-1). Several osteoinductive factors, such as bone morphogenetic protein (BMP) and osteogenin, a skeletal growth factor (SGF), and osteoblast-derived BDGFs, have also been identified. Complete description of the biological functions of these BDGFs which are relevant to bone will ultimately require specific bioassays involving specific cell types in vitro, as well as in vivo animal implant models. Studies with primary rat osteoblast-like cells exposed either to mixed BDGFs, pure TGF-beta, or heparin-purified PDGF, aFGF, or bFGF from bovine bone have shown a general dose-dependent mitogenic effect. Phenotypic changes which accompany the BDGF-induced wave of proliferation include: decreased osteocalcin secretion and a reduction in 1,25-(OH)2 vitamin D3-stimulated osteocalcin synthesis; reduced alkaline phosphatase specific activity; decreased cyclic AMP responsiveness to parathyroid hormone (PTH); and increased collagen synthesis. Bone exhibits the most complex spectrum of growth factor activities of any tissue yet described. In bovine bone powder free of blood and cartilage contamination, the volume concentration of mitogens is up to 20 times greater than that in serum. Bone cells and other indigenous cell types must be considered as possible sources of the BDGFs, in addition to sequestration from blood. Mechanisms for the unmasking or release of BDGFs from the mineralized matrix that result in local action on osteoblasts, endothelial cells, and other target cells are undoubtedly important for the development and maintenance of bone tissue.
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PMID:Polypeptide growth factors in bone matrix. 306 10

A DNA fragment from Bacillus natto IFO3936 has been cloned which enhances the production of both extracellular alkaline and neutral proteases in Bacillus subtilis. The DNA sequence analysis around the gene responsible for the hyperproduction, prtR, revealed one open reading frame (comprising 60 amino acid residues) which was bounded by potential transcriptional and translational regulatory signals in its preceding and following regions. This open reading frame was not homologous to the published sequences of the structural genes of the two proteases. The calculated molecular weight (7,109) of the polypeptide predicted from the DNA sequence is much smaller than those of the two proteases, indicating that the gene product is distinct from those enzymes. In-frame fusion between the N-terminal region of the coding sequence and the lacZ gene of Escherichia coli demonstrated that the coding region was indeed translated in vivo. By deletion analysis it was suggested that prtR was the structural gene for the 60-amino-acid polypeptide. Cells carrying a prtR plasmid secreted both proteases 40 to 400 times more than the cells carrying the vector alone. Furthermore, it was found that prtR also enhanced the production of levansucrase by 1 or 2 orders of magnitude. There was no difference, however, in the amount of the other extracellular enzymes such as alpha-amylase, RNase, and alkaline phosphatase. These results indicate that prtR is specific for the hyperproduction of the proteases and levansucrase.
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PMID:Molecular cloning and nucleotide sequence of a DNA fragment from Bacillus natto that enhances production of extracellular proteases and levansucrase in Bacillus subtilis. 308 53

Monoclonal antibodies to squid neurofilament (aNFP) and intermediate filament (aIFA) proteins were used as probes for the biochemical and immunocytochemical analyses of neurofilament structure and distribution in the squid giant axon and stellate ganglion. On Western blots the aNFP antibody stained exclusively the 220 kDa and high-molecular-weight (HMW) components of neurofilaments in the giant axon, whereas the aIFA antibody primarily labeled the 60 kDa protein in the giant axon and the 60 and 65 kDa proteins in the stellate ganglion. Dephosphorylation of axoplasmic proteins by alkaline phosphatase resulted in a decrease in the molecular weights of both the 220 kDa and HMW neurofilament proteins and a concomitant loss of reactivity with the aNFP antibody on Western blots. This indicated that the aNFP antibody is specific for a phosphorylated epitope in the neurofilament. Increased dephosphorylation of the 220 kDa protein led to an enhanced immunostaining of the resultant 190 kDa polypeptide by the aIFA antibody, suggesting that the phosphorylated epitope may mask the conserved epitope recognized by aIFA. Light and electron microscopic immunocytochemical studies show intense labeling by the aNFP antibody in the giant axon. In contrast, the aIFA antibody labeled the glial cells around the giant axon intensely, while labeling of the giant axon itself was considerably less than that with the aNFP antibody. Since the 60 kDa protein in axoplasm is intensely stained by the aIFA antibody on Western blots, the relatively low amounts of labeling seen on semithin and thin sections of the giant axon by this antibody may be due to the masking of the 60 kDa protein by in situ fixed axoplasmic proteins. However, the aIFA antibody intensely labeled glial cells within the stellate ganglion and "islands" of filaments and nuclear membranes within ganglion cells. No reactivity for either antibody was seen in synapses. The aNFP antibody specifically labeled "beadlike" portions and cross-bridges on the axonal neurofilaments, suggesting that these components consist of the 220 kDa and HMW proteins. In contrast, the aIFA antibody labeled relatively smooth filaments in ganglion and glial cells. These data suggest that the 65 kDa protein represents the squid glial filament protein and that the 60 kDa protein found in axoplasm represents the low-molecular-weight subunit in the axonal neurofilament. The latter appears to be formed and/or organized in "islands" of filaments within ganglion cells.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Biochemical and immunocytochemical characterization and distribution of phosphorylated and nonphosphorylated subunits of neurofilaments in squid giant axon and stellate ganglion. 311 21

The Bacillus subtilis alpha-amylase structural gene (amyE) lacking its own signal peptide coding sequence was joined to the end of the Escherichia coli alkaline phosphatase (phoA) signal peptide coding sequence by using the technique of oligonucleotide-directed site-specific deletion. On induction of the phoA promoter, the B. subtilis alpha-amylase was expressed and almost all the activity was found in the periplasmic space of E. coli. The sequence of the five amino-terminal amino acids of the secreted polypeptide was Glu-Thr-Ala-Asn-Lys-, and thus the fused protein was correctly processed by the E. coli signal peptidase at the end of the phoA signal peptide.
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PMID:Secretion of Bacillus subtilis alpha-amylase in the periplasmic space of Escherichia coli. 311 69


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