Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UMLS:C0272170 (SDS)
 50,377 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The Ca2(+)-and calmodulin-dependent protein kinase III, which specifically phosphorylates the eukaryotic elongation factor 2 (eEF-2), has been purified to apparent homogeneity from the post-ribosomal fraction of rabbit reticulocytes by an efficient four-step method. The method results in a more than 4000-fold purification of the enzyme. SDS-gel electrophoresis showed that the purified kinase contained only one polypeptide with the apparent molecular mass of 90 kDa. The kinase activity was associated with the 90-kDa protein as shown by analyzing the phosphorylating activity of SDS gel electrophoretically purified protein electroblotted to nitrocellulose membranes. The purified kinase was dependent on Ca2+, Mg2+ and calmodulin for activity. Kinetic analysis of the phosphorylation reaction indicates that the turnover number of the kinase was approximately 1 s-1. The Km for the two substrates ATP and eEF-2 was calculated to be approximately 100 microM and 10 microM, respectively. The activity of the kinase was competitively inhibited by cAMP. The inhibition constant Ki (0.5 mM) was found to be in the same order of magnitude as that calculated for the competitive product inhibition caused by ADP. GTP was ten-times less efficient as competitor, indicating that the kinase had a preference for adenosine nucleotides. Phosphorylation of eEF-2 did not interfere with the diphtheria-toxin-catalysed ADP-ribosylation of the factor nor did ADP-ribosylation inhibit phosphorylation.
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PMID:Kinetic characterisation of the enzymatic activity of the eEF-2-specific Ca2(+)-and calmodulin-dependent protein kinase III purified from rabbit reticulocytes. 167 64

Guanylate cyclase from rod photoreceptors of amphibian (toad, Bufo marinus, and frog, Rana catesbeiana) and bovine retinas was solubilized and purified by a single chromatography step on a GTP-agarose column. Silver staining of purified amphibian enzymes in SDS/polyacrylamide gels disclosed a doublet band (110 and 115 kDa), while the bovine enzyme appeared as a singlet band (110 kDa). The identification of these guanylate cyclases was confirmed using three chromatography systems with the purified enzymes. Specific binding to Con A-Sepharose suggested that rod guanylate cyclase is a glycoprotein. Two-dimensional gel electrophoresis of purified toad, frog, and bovine enzymes resolved two, three, and five variants, respectively, that differed in isoelectric point. Two variants of toad guanylate cyclase showed differences in various characterizations. These data suggest multiple mechanisms for regulation of guanylate cyclase activity in vertebrate rod photoreceptors.
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PMID:Polymorphism in purified guanylate cyclase from vertebrate rod photoreceptors. 167 87

We have initiated the characterization of the DNA helicases from HeLa cells, and we have observed at least 4 molecular species as judged by their different fractionation properties. One of these only, DNA helicase I, has been purified to homogeneity and characterized. Helicase activity was measured by assaying the unwinding of a radioactively labelled oligodeoxynucleotide (17 mer) annealed to M13 DNA. The apparent molecular weight of helicase I on SDS polyacrylamide gel electrophoresis is 65 kDa. Helicase I reaction requires a divalent cation for activity (Mg2+ greater than Mn2+ greater than Ca2+) and is dependent on hydrolysis of ATP or dATP. CTP, GTP, UTP, dCTP, dGTP, dTTP, ADP, AMP and non-hydrolyzable ATP analogues such as ATP gamma S are unable to sustain helicase activity. The helicase activity has an optimal pH range between pH8.0 to pH9.0, is stimulated by KCl or NaCl up to 200mM, is inhibited by potassium phosphate (100mM) and by EDTA (5mM), and is abolished by trypsin. The unwinding is also inhibited competitively by the coaddition of single stranded DNA. The purified fraction was free of DNA topoisomerase, DNA ligase and nuclease activities. The direction of unwinding reaction is 3' to 5' with respect to the strand of DNA on which the enzyme is bound. The enzyme also catalyses the ATP-dependent unwinding of a DNA:RNA hybrid consisting of a radioactively labelled single stranded oligodeoxynucleotide (18 mer) annealed on a longer RNA strand. The enzyme does not require a single stranded DNA tail on the displaced strand at the border of duplex regions; i.e. a replication fork-like structure is not required to perform DNA unwinding. The purification of the other helicases is in progress.
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PMID:A DNA helicase from human cells. 170 1

Rotaviruses transcribe mRNA containing a 7mGpppGmp cap at the 5' end in vitro. Guanylyltransferase activity associated with the viral particle was detected by SDS-PAGE due to the formation of a nucleotide-enzyme complex when the virus was incubated with [alpha-32P]GTP. Using purified viral particles it was shown that only the core polypeptide VP3 exhibits the ability to form a complex with the nucleotide. The reaction is specific for GTP or dGTP when Mg2+ is used as a cofactor. The reaction also depends on the incubation temperature and the pH, as described for other guanylyltransferases. The GMP-VP3 complex transfers the GMP to pyrophosphate, synthesizing GTP or GDP, resulting in the formation of a GpppG cap. These properties of the complex allowed the core polypeptide VP3 to be identified as the rotavirus guanylyltransferase.
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PMID:Characterization of rotavirus guanylyltransferase activity associated with polypeptide VP3. 170 11

In bovine aortic smooth muscle, GTP-binding activity was equally distributed in the membrane and cytosol fractions. The most abundant GTP-binding proteins (G proteins) in each fraction were purified to near homogeneity and characterized. The most abundant G protein in the membrane fraction had a Mr value of about 22,000 (m22K G) as estimated on sodium dodecyl sulfate-polyacryl-amide gel electrophoresis (SDS-PAGE). m22K G and the human platelet smg p21, a ras p21 like G protein having the same effector domain as ras p21s, were eluted at the same retention time on C4 reversed-phase high performance liquid chromatography (HPLC). Moreover, m22K G was specifically recognized by an anti-smg p21 polyclonal antibody. m22K G was phosphorylated by cyclic AMP-dependent protein kinase with a stoichiometry of one phosphate/molecule of protein. The most abundant G protein in the cytosol fraction had a Mr value of about 21,000 (c21K G) as estimated on SDS-PAGE. c21K G was ADP-ribosylated by botulinum ADP-ribosyltransferase and about 0.4 mol of ADP-ribose was maximally incorporated into 1 mol of c21K G. c21K G and the bovine brain rhoA p21, another ras p21 like G protein, were eluted at the same retention time on C4 reversed-phase HPLC and migrated at the same position on two-dimensional gel electrophoresis. These results indicate that the major G proteins in the membrane and cytosol fractions of bovine aortic smooth muscle are smg p21 and rhoA p21, respectively. Possible roles of these G proteins in vascular smooth muscle are discussed.
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PMID:Small GTP-binding proteins in bovine aortic smooth muscle. 174 79

Platelet membrane glycoprotein IIb-IIIa has been widely studied in the last years because of its role as an activation-dependent, adhesive protein receptor. Recently we demonstrated that occupancy of glycoprotein IIb-IIIa-receptor sites by specific ligands exerts an inhibitory effect on platelet responses induced by mild stimulation, leading us to suppose that this event may interact with activation pathways. Although the mechanisms of signal transduction in human platelets are not completely elucidated, the hypothesis that GTP-binding proteins are involved is generally accepted. Our results demonstrate that platelet ConA receptors, known to be located mainly on GP IIb-IIIa, are able to bind [35S]GTP gamma S; the GTP-binding activity is specific and is due to the association with the receptors of two G-proteins, with apparent molecular masses of 25 and 21 kDa, respectively. After the purification of GP IIb-IIIa, a glycoprotein complex electrophoretically pure was obtained that was still associated with a GTP-binding activity, migrating in SDS-polyacrylamide gel electrophoresis as a narrow band of about 21 kDa.
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PMID:Platelet glycoprotein IIb-IIIa is associated with 21-kDa GTP-binding protein. 175 27

To investigate if G-protein-receptor interactions can be characterized using sucrose density gradients (SDG) we have determined the experimental conditions for muscarinic acetylcholine receptor (mAChR) solubilization and analysis on SDG. Solubilization of 65-80% of [3H]QNB bound mAChR was accomplished with 1% of detergent. Analysis of solubilized receptors on SDG containing 0.4 M KCl and 0.1% detergent demonstrated that the physical properties of the receptor-detergent complexes are influenced by the solubilizing detergent as well as detergents included in the SDG. Neither GTP gamma S nor NaF and AlCl3 altered the sedimentation properties of mAChR, suggesting that the solubilized mAChR is no longer associated with G-protein under these conditions. Receptors bound to [3H]oxotremorine and [3H]QNB had similar sedimentation properties, suggesting that, once solubilized, mAChRs do not remain associated with G-proteins. Covalent labeling with [3H]PrBCM followed by solubilization and analysis on SDS-gel electrophoresis demonstrated the presence of intact receptor molecule. These observations suggest that the changes in the sedimentation properties of detergent-receptor complexes are independent of G-protein interactions and are influenced by the nature of the detergent associated with the mAChR during analysis.
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PMID:Solubilization and sedimentation analysis of muscarinic acetylcholine receptors. 175 82

Two forms of E. coli initiation factor IF2, IF2 alpha and IF2 beta, have been known for several years. Both forms are products of the gene infB with translational initiation at codon 1 (AUG) and codon 158 (GUG) in the same reading frame. In this work we demonstrate that IF2 beta exists in two forms, IF2 beta and IF2 beta' with initiation codons 158 (GUG) and 165 (AUG) and molecular masses of 79.7 kDa and 78.8 kDa respectively. We have recently described a fast purification method for IF2 alpha, using an FPLC procedure consisting of ion-exchange liquid chromatography on Q Sepharose HP, Mono Q and Mono S. After the Mono Q step, an apparently homogeneous IF2 beta was observed when analyzed by SDS-PAGE. However the chromatography on Mono S results in the elution of two peaks containing IF2 beta. The N-terminal amino acid sequence of the two proteins identified the first peak to be IF2 beta and the second as a protein which we term IF2 beta' starting seven residues downstream at the AUG codon 165. The activity in vitro of the two purified forms of IF2 beta was tested by measuring the stimulation of binding of the initiator fMet-tRNA(fMet) to 70S ribosomes in the presence of GTP and poly(A,U,G) as messenger-RNA. In this assay no difference in activity is detected.
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PMID:Tandem translation of E. coli initiation factor IF2 beta: purification and characterization in vitro of two active forms. 176 5

A GTP-binding protein with an apparent molecular weight of 25 kDa was detected in hepatocyte extracts using SDS-PAGE and [alpha-32P]GTP. p21ras proteins could only be detected by immunological analysis. The amounts of p21ras proteins present in isolated hepatocytes and in a highly purified preparation of liver plasma membrane vesicles were 0.3 and 4 ng p21ras protein/micrograms membrane protein, respectively. In comparison with the total cell extract, the degree of enrichment of plasma membrane vesicles with p21ras was similar to that of 5'-nucleotidase. The p21ras proteins were tightly associated with the membrane. Treatment of [3H]choline-labelled plasma membranes with an excess concentration of the anti-p21ras antibody Y13-259 failed to inhibit either basal or guanosine 5'-[gamma-thio]triphosphate (GTP[S])-stimulated [3H]choline release. It is concluded that in hepatocytes (a) the majority of p21ras is bound to the plasma membrane and (b) p21ras is not directly involved in the activation by GTP[S] of phospholipase D.
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PMID:Low molecular weight GTP-binding proteins in hepatocytes and an assessment of the role of p21ras proteins in the activation of phospholipase D. 177 29

Polyclonal antibodies to the alpha subunits of G0 type G proteins (G0 alpha) were coupled to agarose gel and used to isolate G0 alpha from solubilized membranes of various bovine tissues. The cholate extract of membranes was applied to the anti-G0 alpha-agarose gel column. The column was washed extensively, then bound proteins were eluted at a neutral pH using a commercial ActiSep Elution Medium. The proteins in the eluate displayed a single band of 39 kDa on SDS-polyacrylamide gel electrophoresis. They bound to GTP gamma S and were ADP-ribosylated by pertussis toxin. The yield of the immunoreactive G0 alpha from the extract was about 40%. Isoelectric focusing, immunoassay and peptide mapping analysis of the G0 alpha-like proteins purified from the heart and adrenal medulla indicated that these proteins were very similar to the alpha subunit of a minor subtype of G0 in the brain which was previously referred to as G0 * alpha.
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PMID:Immuno-affinity purification and characterization of the alpha subunits of G0 type G proteins from various bovine tissues. 177 78


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