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: UNIPROT:Q07644 (polypeptide)
72,197 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Thrombomodulin (TM) is an anionic (pI approximately 4) protein cofactor that promotes thrombin (THR) cleavage of protein C to generate activated protein C (APC), a potent anticoagulant. We find that the cationic platelet alpha-granule protein platelet factor 4 (PF4) stimulates 4-25-fold the cofactor activity of rabbit TM and two differentially glycanated versions of an extracellular domain human TM polypeptide in which the glycosaminoglycan (GAG) is either present (GAG+ TM) or absent (GAG- TM) with an ED50 of 3.3-10 micrograms/ml. No such stimulation occurs in response to beta-thromboglobulin or thrombospondin, or when protein C lacking its gamma-carboxyglutamic acid (Gla) domain is the substrate. Heparin and chondroitin sulfates A and E reverse PF4 stimulation. PF4 minimally affects the Kd for THR but decreases 30-fold (from 8.3 to 0.3 microM) the Km for protein C of APC generation by GAG+ TM. PF4 also strikingly transforms the [Ca2+] dependence profile of rabbit and GAG+ TM to resemble that of GAG- TM. A potential explanation for this is that PF4, like Ca2+, induces heparin-reversible alterations in native (but not Gla-domainless) protein C conformation as assessed by autofluorescence emission analysis. We conclude that PF4 stimulates TM APC generation by interacting electrostatically with both the TM GAG and the protein C Gla domain to enhance markedly the affinity of the THR.TM complex for protein C. By this mechanism, PF4 may play a previously unsuspected role in the physiologic regulation of clotting.
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PMID:Platelet factor 4 stimulates thrombomodulin protein C-activating cofactor activity. A structure-function analysis. 752 87

In this study we report that a synthetic peptide of the effector domain of rab3A (rab3AL(33-48)) stimulates both amylase secretion and inositol 1,4,5-trisphosphate (IP3)-accumulation in digitonin-permeabilized pancreatic acini in an analogous way to cholecystokinin-octapeptide (CCK8). Maximum CCK8-induced IP3-accumulation was observed at five seconds after addition of CCK8 to the acini. Maximum rab3AL(33-48)-induced IP3-production occurred 15 to 30 seconds after addition of rab3AL(33-48); then the acinar IP3 content declined towards the basal level. Heparin, an inhibitor of IP3 binding to its receptor, inhibited both rab3AL(33-48)- and CCK8-stimulated amylase secretion without affecting the response to vasoactive intestinal polypeptide. rab3AL(33-48) had no effect in intact acini, indicating that the site of action of rab3AL(33-48) is intracellular. We conclude that rab-like small molecular weight GTP-binding proteins regulate phospholipase C activity and thereby amylase secretion from inside of the cell.
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PMID:A synthetic peptide of the effector domain of rab3A stimulates inositol 1,4,5-trisphosphate production in digitonin-permeabilized pancreatic acini. 768 59

Transglutaminase, a zero length cross-linker that catalyzes the formation of isopeptide bonds between proximal Gln and Lys side chains, was used as a structural and conformational probe of the hexadecameric phosphorylase kinase molecule (alpha beta gamma delta)4. Brief cross-linking of nonactivated kinase caused formation of alpha-beta dimers, with no cross-linking involving the gamma- and delta-subunits. When the kinase was first activated by autophosphorylation, significant amounts of alpha-alpha dimers were also observed in addition to the alpha-beta, demonstrating the occurrence of a conformational change in the alpha-subunits concomitant with activation. Both dimers resulted from intramolecular cross-linking. Because the COOH-terminal regions of the alpha-subunits are at the lobe tips of this bilobal kinase (Wilkinson D. A. Marion, T. N., Tillman, D. M., Norcum, M. T., Hainfeld, J. F., Seyer, J. M., and Carlson, G. M. (1994) J. Mol. Biol. 235, 974-982), the formation of zero length cross-linked alpha-alpha dimers indicates that the polypeptide backbones of these subunits must stretch from the lobe tips to a more central location where they abut each other. Excess putrescine, as the amine substrate in place of endogenous Lys, was incorporated by transglutaminase predominately into the alpha-subunits of the kinase, with only slight modification of the beta- and gamma-subunits. Exogenous calmodulin (delta'), an activator of the kinase with a binding site on the alpha-subunits (James, P., Cohen, P., and Carafoli, E. (1991) J. Biol. Chem. 266, 7087-7091), was a potent inhibitor of cross-linking. It also inhibited incorporation of putrescine into the alpha-subunits but stimulated incorporation into the beta- and gamma-subunits. Heparin, another activator of the kinase, had the same effects as exogenous calmodulin on cross-linking and putrescine incorporation, suggesting a commonality in the mechanism through which these two effectors activate the holoenzyme, including promoting a conformational change that increases the surface accessibility of target Gln residues on the catalytic gamma-subunit.
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PMID:Zero length conformation-dependent cross-linking of phosphorylase kinase subunits by transglutaminase. 796 56

Borrelia burgdorferi adhere to mammalian cells in vitro but neither the ligand(s) nor the receptor(s) has (have) been clearly established. Using an in vitro attachment-inhibition assay, a B. burgdorferi attachment mechanism has been identified. Heparin, heparan sulfate, and dermatan sulfate reduced the attachment of virulent B. burgdorferi strain 297 to HeLa cells by approximately 60%. In addition, virulent, but not avirulent, B. burgdorferi strains B31, N40, and HB19 demonstrated heparin attachment-inhibition. Attachment to Chinese hamster ovary cells deficient in heparan sulfate proteoglycans was reduced by 68% compared to attachment to wild-type cells and was identical to attachment at maximum heparin inhibition to the wild-type cells. Pretreatment of HeLa cell monolayers with heparitinase, heparinase, and chondroitinase ABC, but not with chondroitinase AC, reduced borrelial attachment by approximately 50%. A moderately high affinity, low copy number, promiscuous B. burgdorferi glycosaminoglycan receptor was demonstrated by equilibrium binding studies. A 39-kD polypeptide, purified by heparin affinity chromatography from Triton X-100 extracts derived from virulent borrelia, was a candidate for this receptor. These studies indicate that one mode of B. burgdorferi attachment to eukaryotic cells is mediated by a borrelial glycosaminoglycan receptor attaching to surface-exposed proteoglycans on mammalian cells.
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PMID:Borrelia burgdorferi bind to epithelial cell proteoglycans. 811 83

Tissue factor pathway inhibitor (TFPI) is a multivalent Kunitz-type protease inhibitor, which inhibits factor Xa directly and in a factor Xa dependent manner inhibits the factor VIIa/tissue factor catalytic complex. Altered forms of recombinant TFPI (rTFPI) were tested for their ability to inhibit human factor Xa and bovine gamma-carboxyglutamate (Gla)-domainless factor Xa in the presence and absence of calcium ions, heparin, phospholipids, and factor Va. Sequential deletions of the positively charged C-terminus of TFPI produces proteins that have decreasing inhibitory activity against factor Xa as well as decreasing affinity for heparin-agarose. Deletion of the C-terminus distal to Leu181, which eliminates the third Kunitz-type domain, results in the loss of heparin-agarose binding at physiological ionic strength. Furthermore, the entire C-terminal polypeptide, including at least a portion of the third Kunitz-type domain, appears to be involved in heparin binding. Residues 230-241 probably form an alpha helix in which Lys231 and Arg237 within the Kunitz domain and Lys240 and Lys241 could provide a positively charged surface epitope capable of binding heparin. Heparin and Ca2+ together, but not individually, enhance the rate of factor Xa inhibition by full-length TFPI. The effect of heparin is concentration dependent and biphasic (maximal between 0.1 and 1.0 unit/ml) suggesting that the acceleration of factor Xa inhibition occurs at least in part through a 'template' mechanism.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Structural requirements for tissue factor pathway inhibitor interactions with factor Xa and heparin. 829 16

To determine the effects of differences in glycosylation on the structure and functional properties of recombinant human antithrombin (rHAT), we have characterized the properties of the recombinant protein overexpressed by baby hamster kidney cells. Three forms of rHAT, I-III, were isolated which differed in affinity for heparin. Form I had the lowest affinity and contained a high proportion of highly branched complex carbohydrate. Form II had higher affinity and contained both complex and high mannose-type chains. Form III had the highest affinity and was similar to form II in the type of carbohydrate present, but had a lower level of glycosylation, consistent with the absence of carbohydrate at one of the four glycosylation sites. 1H NMR spectra of plasma HAT and rHAT forms I-III suggested very similar protein structures for all forms. Heparin pentasaccharide produced almost identical NMR perturbation difference spectra. The only functional difference found was in the rates of inactivation of factor Xa. Forms II and III gave second order rate constants similar to that of plasma HAT, whereas form I gave a biphasic inhibition, with the first phase having a rate about four times that of the other forms. We conclude that carbohydrate heterogeneity does not alter the structure of the HAT polypeptide or the heparin-induced conformational change, but does affect the heparin affinity and can alter the rate of proteinase inhibition.
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PMID:Heterogeneity of recombinant human antithrombin III expressed in baby hamster kidney cells. Effect of glycosylation differences on heparin binding and structure. 834 38

Protein phosphatase 2A2 is inactivated by phosphorylation following incubation with purified preparations of an autophosphorylation-activated protein kinase (Hong Guo and Zahi Damuni (1992) Proc. Natl. Acad. Sci. U.S.A. 90, 2500-2504). This protein kinase was purified about 250,000-fold from extracts of bovine kidney to apparent homogeneity. The purified preparations exhibited a single polypeptide of apparent M(r) approximately 36,000. Up to 1 mol of phosphoryl groups was incorporated per mol of the purified kinase following incubation with ATP. This autophosphorylation reaction (t1/2 approximately 0.5-1 min) was accompanied by a approximately 10-fold activation of the kinase. Autophosphorylation and activation were reversed by protein phosphatase 2A2 or the catalytic subunit of protein phosphatase 1. Phosphoamino acid analysis indicated that the kinase underwent autophosphorylation on threonines. The rate of autophosphorylation was independent of the concentration of the enzyme and a slope of 0.97 (gamma = 0.998) was obtained by van't Hoff's plot indicating that autoposphorylation was intramolecular. Relative to myelin basic protein, the enzyme exhibited about 8, 62, 130, 33, 5, and < 0.1% activity with histones H1, H2A, H2B, H3, and H4 and with glycogen synthase alpha, respectively. Heparin inhibited the activity of the enzyme half-maximally at about 20 micrograms/ml. The results indicate that this autophosphorylation-activated kinase is a new protein kinase.
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PMID:Purification and characterization of an autophosphorylation-activated protein serine threonine kinase that phosphorylates and inactivates protein phosphatase 2A. 838 87

Proliferation and extracellular matrix (ECM) overproduction by glomerular mesangial cells characterizes many types of glomerulonephritis and often precedes the development of glomerulosclerosis. Heparin is a potent inhibitor of mesangial cell growth in vitro. We examined whether standard heparin can inhibit mesangial cell proliferation in vivo in the mesangioproliferative anti-Thy 1.1 nephritis. Untreated control rats were compared to rats infused with heparin either early (day -2 to 1) or late (day 2 to 5) after induction of anti-Thy 1.1 nephritis. The results show that heparin treatment significantly reduced mesangial cell proliferation regardless of when it was initiated. Heparin (either early or late treatment) also reduced mesangial basic fibroblast growth factor (bFGF) expression and platelet-derived growth factor (PDGF) receptor up-regulation as reflected by immunostaining, whereas PDGF B-chain expression was reduced only by late heparin treatment. Furthermore, heparin treatment markedly inhibited the mesangial matrix expansion for a variety of ECM proteins, including laminin, type I and IV collagen, fibronectin and entactin. Heparin did not affect the initial mesangiolysis, glomerular macrophage influx, deposition of anti-Thy 1.1 IgG or fibrinogen, or the glomerular platelet influx. These results suggest that heparin, via its antiproliferative rather than anticoagulant effect, can inhibit mesangial cell proliferation, overexpression of polypeptide growth factors, and ECM protein overproduction in vivo. The beneficial effect of heparin can be demonstrated even if treatment is initiated after the development of nephritis. By virtue of these properties, heparin may be an effective agent in the treatment of human mesangioproliferative disease and in the prevention of glomerulosclerosis.
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PMID:Heparin suppresses mesangial cell proliferation and matrix expansion in experimental mesangioproliferative glomerulonephritis. 844 Dec 32

We report the molecular cloning and characterization of a 49-kDa form of casein kinase I from rat testis. A cDNA clone encoding the enzyme, designated casein kinase I delta, contained an open reading frame of 1284 nucleotides that predicts a polypeptide of 428 amino acids with a M(r) of 49,121. The predicted amino acid sequence shares 76% identity with casein kinase I alpha, a 37-kDa form recently cloned from bovine brain (Rowles, J., Slaughter, C., Moomaw, C., Hsu, J., and Cobb, M. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 9548-9552), and 65% identity with HRR25, a 57-kDa form of casein kinase I from yeast shown to be involved in DNA repair (Hoekstra, M. F., Liskay, R. M., Ou, A. C., DeMaggio, A. J., Burbee, D. G., and Heffron, F. (1991) Science 253, 1031-1034). Northern analysis of rat or rabbit RNA revealed three hybridizing species of 3.5-4.1, 2.2, and 1.9 kilobase pairs (kb). The largest message was detected in all tissues examined, whereas the 1.9- and 2.2-kb species were found predominantly in testis. A probe corresponding to the 3'-untranslated region of the casein kinase I delta cDNA hybridized only to the 1.9-kb transcript. Expression of the casein kinase I delta cDNA in Escherichia coli resulted in active enzyme that phosphorylated casein, phosvitin, and the peptide substrate DDDDVASLPGLRRR. Enzyme activity was associated with a predominant polypeptide of 55-kDa, although COOH-terminal degradation products of 50 and 42 kDa were also present in partially purified enzyme. Recombinant casein kinase I delta was inhibited by the specific casein kinase I inhibitor, CKI-7, half-maximally at 12 microM. Heparin inhibited recombinant casein kinase I delta when phosvitin was the substrate, with half-maximal inhibition at 11.5 micrograms/ml. However, if the peptide substrate was used, heparin activated recombinant casein kinase I delta 4-5-fold, with half-maximal activation at 9.5 micrograms/ml. A truncated form of casein kinase I delta, lacking the COOH-terminal 111 amino acids, was no longer activated by heparin. Casein kinase I delta therefore represents a separate member of the casein kinase I family distinguished by its larger size and unique kinetic behavior with respect to heparin.
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PMID:Molecular cloning, expression, and characterization of a 49-kilodalton casein kinase I isoform from rat testis. 845 11

Two phosphatidylinositol 4-kinase isozymes, type 3 and type 2, have been separated on hydroxylapatite after solubilizing bovine brain microsomes with Triton X-114. Employing a newly developed renaturation procedure following SDS-PAGE, we demonstrate that a 200 kDa polypeptide carries the enzyme activity of this type 3 isoform. Chromatography on hydroxylapatite, Heparin-Sepharose, Superdex 200 and finally SDS-PAGE results in an approximately 30,000-fold purification. Tryptic peptides generated from the 200 kDa polypeptide after SDS-PAGE have been sequenced and the obtained data have been used for constructing and synthesizing degenerated oligonucleotides. Polymerase chain reaction as well as screening of cDNA libraries allowed several clones to be isolated from which a 4.7 kb contiguous sequence can be built up. The open reading frame covers 4.4 kb with a 0.3 kb untranslated 3' end which yields a deduced amino acid sequence of 1,467 amino acids. The C-terminal part of ca. 300 amino acids represents the catalytic domain. Sequence alignment of this domain with the mammalian counterpart, the human type 2 phosphatidylinositol 4-kinase, the yeast kinases STT4 and PIK1, as well as with the catalytic domains of bovine, human, mouse and yeast phosphatidylinositol 3-kinases reveals a high degree of identity: 26 of these approximately 300 amino acids are invariable in all of these eight catalytic domains. Five motifs indicate nuclear localization and DNA binding properties of the enzyme. Two leucine zipper motifs (amino acids 358-386, 862-882) are detectable. Furthermore, a helix loop helix motif (amino acids 716-729) as well as two nuclear localization signals (amino acids 838-854, 345-349) indicate the presence of the type 3 isoform in the nucleus.
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PMID:Identification of a 200 kDa polypeptide as type 3 phosphatidylinositol 4-kinase from bovine brain by partial protein and cDNA sequencing. 860 4


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