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:2.7.7.48 (transcriptase)
9,479 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We have studied the effect of protein phosphokinase (EC 2.7.1.37; ATP:protein phosphotransferase) and phosphoprotein phosphatase (EC 3.1.3.16; phosphoprotein phosphohydrolase) on reverse transcriptase (RNA-dependent DNA nucleotidyltransferase) activity of Rous sarcoma virus. Protein kinase from Rous sarcoma virus-transformed chick embryo fibroblasts was purified by DEAE-cellulose chromatography, Sephadex gel filtration, and isoelectric focusing. Purified reverse transcriptase from Rouse sarcoma virus was preincubated with protein kinase and ATP under conditions allowing incorporation of phosphate into substrate protein. After the preincubation, reverse transcriptase activity was assayed in the presence of poly(rA).oligo(dT) as template. A 2- to 5-fold increase of reverse transcriptase activity was found after the preincubation of reverse transcriptase with protein kinase and ATP. Incubation of reverse transcriptase with heat-treated, inactive protein kinase and ATP had no effect on transcriptase activity. When the transcriptase preparation was incubated with protein kinase and [gamma-32P]ATP and subsequently purified by chromatography on phosphocellulose and Sephadex gel filtration, significant amounts of 32P-labeled proteins were found in the fractions exhibiting reverse transcriptase activity, suggesting 32P incorporation into transcriptase or transcriptase-associated proteins. A 20-60% decrease of reverse transcriptase activity was observed after incubation of reverse transcriptase with phosphatase. The results suggest that phosphorylative modification of reverse transcriptase may be critical in the regulation of reverse transcriptase-catalyzed DNA synthesis.
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PMID:Protein kinase and its regulatory effect on reverse transcriptase activity of Rous sarcoma virus. 5 72

Poliovirus-specific RNA-dependent RNA polymerase (3Dpol) was purified to apparent homogeneity. A single polypeptide of an apparent molecular weight of 63,000 catalyzes the synthesis of dimeric and monomeric RNA products in response to the poliovirion RNA template. Analysis of purified 3Dpol by two-dimensional electrophoresis showed multiple forms of 3Dpol, suggesting posttranslational modification of the protein in virus-infected cells. The two major forms of 3Dpol appear to have approximate pI values of 7.1 and 7.4. Incubation of purified 3Dpol with calf intestinal phosphatase resulted in almost complete disappearance of the pI 7.1 form and a concomitant increase in the intensity of the pI 7.4 form of 3Dpol. Addition of 32P-labeled Pi during infection of HeLa cells with poliovirus resulted in specific labeling of 3Dpol and 3CD, a viral protein which contains the entire 3Dpol sequence. Both 3Dpol and 3CD appear to be phosphorylated at serine residues. Ribosomal salt washes prepared from both mock- and poliovirus-infected cells contain phosphatases capable of dephosphorylating quantitatively the phosphorylated form (pI 7.1) of 3Dpol.
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PMID:Multiple isoelectric forms of poliovirus RNA-dependent RNA polymerase: evidence for phosphorylation. 255 40

Influenza B/LEE/40, B/Rome/1/67, B/Hong Kong/8/73, and B/Victoria/98926/70 viruses have a similar polypeptide composition as analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis. These viruses are composed of six or seven polypeptides, depending on whether one or two high-molecular-weight polypeptides are resolved, ranging in molecular weights from 27,000 to 90,400. Three of these polypeptides, namely the heavy and light hemagglutinin chains and the neuraminidase, have attached carbohydrate. Highly purified influenza B/LEE/40 and B/Rome/1/67 virus preparations have RNA-dependent RNA polymerase activity equivalent to the incorporation of 100 and 30 pmol, respectively, of (3)H-UMP per mg of virus protein per h at 37 C, which is demonstrated only in detergent-treated virus suspensions. However, no RNA-dependent DNA polymerase enzyme activity was detected in the two viruses although virus suspensions were "activated" by heat, alpha-chymotrypsin, and detergents. Other enzymatic activities were associated with purified preparations of influenza B virus and were attributed to minor contamination of virus with host cell enzymes. Thus, nucleoside and deoxynucleoside phosphohydrolase enzymes were active in the absence of detergents and catalyzed the release of 1,200 and 1,800 nmol of P(i) per mg of virus protein in 30 min at 37 C from ATP and dATP substrates. Thin-layer chromatography indicated that the products of the phosphohydrolase enzymes of influenza B/LEE/40 were mainly nucleoside diphosphate and monophosphate. The latter enzymes were tightly bound to influenza B/LEE/40 virus and could not be removed completely by repeated centrifugation, including centrifugation of the virus to equilibrium in density gradients of 25 to 40% (wt/vol) cesium chloride. A low degree of RNase (approximately 0.01 mug% contamination) and phosphatase (10-30 nmol of P(i) released per mg of virus protein per 30 min) activity was detected in some, but not all, influenza B/LEE/40 virus preparations.
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PMID:Polypeptide composition of Influenza B viruses and enzymes associated with the purified virus particles. 435 55

We have isolated the murine cDNA homologue of the human protein tyrosine phosphatase PTP-PEST (MPTP-PEST) from an 18.5-day mouse embryonic kidney library. The cDNA isolated has a single open reading frame predicting a protein of 775 amino acids. When expressed in vitro as a glutathione S-transferase fusion protein, the catalytic domain (residues 1-453) shows intrinsic phosphatase activity. Reverse transcriptase PCR and Northern-blot analysis show that MPTP-PEST mRNA is expressed throughout murine development. Indirect immunofluorescence in COS-1 cells against a heterologous epitope tag attached to the N-terminus of MPTP-PEST, together with cellular fractionation and Western-blot experiments from different murine cell lines, indicate that MPTP-PEST is a free cytosolic protein of 112 kDa. Finally, sequence analysis indicates that the C-terminal portion of the protein contains four regions rich in proline, glutamate, serine and threonine, otherwise known as PEST sequences. These are characteristic of proteins that display very short intracellular half-lives. Despite the presence of these motifs, pulse-chase labelling experiments demonstrate that MPTP-PEST has a half-life of more than 4 h.
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PMID:Murine protein tyrosine phosphatase-PEST, a stable cytosolic protein tyrosine phosphatase. 777 23

We analyzed complexed and free prostate-specific antigen (PSA), the free/total PSA and complexed/free PSA ratios, acid phosphatase, and prostatic phosphatase in serum from 36 patients with prostatic carcinoma and from 48 non-neoplastic control patients (20 with prostatitis and 28 with benign prostatic hyperplasia). Receiver-operating characteristic plots showed that serum PSA was the most efficient variable, singly used, in discriminating neoplastic from non-neoplastic patients. At a cut-off value of 10.0 ng/ml, serum PSA had a diagnostic sensitivity of 87% and a diagnostic specificity of 83%. In particular, three patients with prostatic carcinoma and twenty non-neoplastic controls had serum PSA levels of between 4 and 10 ng/ml. The subsequent analysis of the serum free/total PSA ratio, in this subgroup, using a cut-off level of 15%, allowed us to classify correctly all prostatic cancer cases and 18/20 non-neoplastic diseases. We next analyzed PSA mRNA in circulating cells using an improved reverse-transcriptase polymerase chain reaction dot blot procedure, from six patients with prostatic carcinoma with distant metastases, and in seventeen with localized cancer. The analysis had a high sensitivity (up to dilutions 1:10(6) of total RNA from prostatic cancer cells vs total RNA from normal blood cells). The analysis revealed circulating micrometastatic cells in 3/6 (50%) cases of metastatic cancer and in 4/17 cases of localized cancer. To conclude, serum total PSA combined with the free/total PSA ratio is a very efficient algorithm in discriminating neoplastic from non-neoplastic prostatic diseases, while other mRNA species must be analyzed, in addition to PSA mRNA, in circulating cells to increase the efficiency in detecting metastatic prostatic cancer.
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PMID:Prostate-specific antigen (protein and mRNA) analysis in the differential diagnosis and staging of prostate cancer. 935 30

1. We have used the patch-clamp technique to study modulation of the inwardly rectifying K+ current (IK(IR)) in cultured bovine pulmonary artery endothelial cells (CPAE cells). In whole-cell mode, IK(IR) was defined as the Ba(2+)-sensitive current. In single channel recordings, we observed a strongly inwardly rectifying and K(+)-selective channel with a conductance of 31 +/- 3 pS. 2. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis and functional data suggest that the endothelial IRK is most probably Kir2.1. 3. Intracellular ATP is required to prevent run-down of IRK in whole-cell mode. Single channel activity disappeared in inside-out patches exposed to ATP-free solution and in cell-attached patches on cells exposed to metabolic inhibition (KCN, 2-deoxyglucose). 4. The non-hydrolysable ATP analogues, ATP gamma S and adenylyl imidodiphosphate (AMP-PNP), did not prevent run-down. Run-down did not occur in the presence of okadaic acid, a phosphatase inhibitor, but was enhanced in the presence of protamine, an activator of phosphatase 2A (PP2A). 5. GTP gamma S and AlF4- inhibited IRK, also in the presence of ATP. GTP beta S antagonized the GTP gamma S effect. Pretreatment of the cells with PTX did not affect the GTP gamma S-induced inhibition. Okadaic acid, however, slowed this inhibition. 6. Neither activation of protein kinase A (PKA) nor activation of protein kinase C (PKC) affected IRK. Additionally, neither cytochalasin B nor a high concentration of intracellular Ca2+ affected the time course of IRK run-down. 7. We conclude that run-down of IRK is probably due to dephosphorylation by PP2A. Activation of a PTX-insensitive G protein inhibits this current by a mechanism that is neither mediated via the PKA and PKC pathways nor by intracellular Ca2+, but supposedly by a G protein-dependent activation of a phosphatase.
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PMID:Modulation of inwardly rectifying potassium channels in cultured bovine pulmonary artery endothelial cells. 940 63

All RNA viruses known to date encode an RNA-dependent RNA polymerase (RdRp) that is required for replication of the viral genome. We have expressed and purified the turnip yellow mosaic virus (TYMV) RdRp in insect cells using a recombinant baculovirus, either in its native form, or fused to an hexa-histidine tag. Phosphorylation of the protein was demonstrated by labelling experiments in vivo, as well as phosphatase treatment of the purified protein in vitro. Phospho amino acid analysis and immunoblotting experiments identified serine and threonine residues as being the subject of phosphorylation. Peptide mass mapping using MS analysis of a protein digest revealed that phosphorylation sites are localized within a putative PEST sequence [a sequence rich in proline (P), glutamic acid (E), serine (S) and threonine (T) residues] in the N-terminal region of the protein. Using monoclonal antibodies specific for ubiquitin conjugates, we were able to demonstrate that the TYMV RdRp is conjugated to ubiquitin molecules when expressed in insect cells. These observations suggest that the TYMV RdRp may be processed selectively by the ubiquitin/proteasome degradation system upon phosphorylation of the PEST sequence.
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PMID:Evidence for phosphorylation and ubiquitinylation of the turnip yellow mosaic virus RNA-dependent RNA polymerase domain expressed in a baculovirus-insect cell system. 1088 Mar 40

Fas transduces apoptotic signals upon cross-linking with the Fas ligand (FasL), which is experimentally replaced by agonistic anti-Fas monoclonal antibodies (mAb). Of eight human malignant hematopoietic cell lines (HL-60, KG-1, THP-1, K562, U937, Jurkat, IM-9, RPMI-8226) examined by flow cytometric analysis, all, except K562, were found to be positive for surface Fas antigen. However, despite surface Fas expression, the agonistic anti-Fas mAb (7C11) induced apoptosis in only three of seven Fas-expressing cell lines (KG-1, Jurkat and IM-9). This Fas-resistance did not correlated with high levels of mRNA either for DcR3, a decoy receptor for FasL, or for FAP-1, a Fas-associated phosphatase that can block the apoptotic function of Fas. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis did not show consistent differences in the expression of Bcl-2 and Bax between Fas-sensitive and Fas-resistant cell lines examined. These findings indicated that the presence or absence of mRNA expression of DcR3, FAP-1, Bcl-2 and Bax did not always correlate with relative sensitivity to Fas-mediated apoptosis. Treatment of cells with cycloheximide converted the phenotype of resistant cell lines from Fas-resistant to Fas-sensitive, and enhanced the sensitivity of Fas-sensitive cell lines. These results suggest that the Fas-resistance is dependent on the presence of labile proteins that determine resistance to Fas-mediated apoptosis and the apoptotic machinery is already in place in Fas-resistant cell lines.
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PMID:Fas-mediated apoptosis and expression of related genes in human malignant hematopoietic cells. 1119 Feb 79

Adrenergic regulation of the pineal enzyme serotonin N-acetyltransferase [arylalkylamine N-acetyltransferase (AA-NAT); EC 2.3.1.87] accounts for the circadian rhythm in melatonin formation. In the present study, the role of protein phosphatases in the adrenergic regulation of rat pineal AA-NAT was investigated using specific inhibitors. In cultured pineals, the serine/threonine phosphatase type 1 and type 2A inhibitors okadaic acid and calyculin A significantly decreased adrenergically or cAMP-induced AA-NAT activity, whereas the serine/threonine phosphatase type 2B inhibitor cypermethrin and tyrosine phosphatase inhibitor dephostatin were ineffective. Reverse transcriptase-polymerase chain reaction (RT-PCR) data indicate that okadaic acid exerts its effect on cAMP-dependent AA-NAT induction by downregulating the amount of AA-NAT transcript. The 'third' messengers, inducible cAMP early repressor (ICER) and Fos-related antigene-2 (Fra-2), are believed to play a negative role in pineal AA-NAT transcription. Okadaic acid increased the cAMP responsiveness of neither ICER mRNA nor Fra-2 mRNA. Therefore, the regulatory role of pineal serine/threonine phosphatases in adrenergically stimulated AA-NAT expression probably does not depend on ICER or Fra-2.
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PMID:Serine/threonine phosphatase inhibitors decrease adrenergic arylalkylamine n-acetyltransferase induction in the rat pineal gland. 1144 72

Coronavirus genome replication and transcription take place at cytoplasmic membranes and involve coordinated processes of both continuous and discontinuous RNA synthesis that are mediated by the viral replicase, a huge protein complex encoded by the 20-kb replicase gene. The replicase complex is believed to be comprised of up to 16 viral subunits and a number of cellular proteins. Besides RNA-dependent RNA polymerase, RNA helicase, and protease activities, which are common to RNA viruses, the coronavirus replicase was recently predicted to employ a variety of RNA processing enzymes that are not (or extremely rarely) found in other RNA viruses and include putative sequence-specific endoribonuclease, 3'-to-5' exoribonuclease, 2'-O-ribose methyltransferase, ADP ribose 1"-phosphatase and, in a subset of group 2 coronaviruses, cyclic phosphodiesterase activities. This chapter reviews (1) the organization of the coronavirus replicase gene, (2) the proteolytic processing of the replicase by viral proteases, (3) the available functional and structural information on individual subunits of the replicase, such as proteases, RNA helicase, and the RNA-dependent RNA polymerase, and (4) the subcellular localization of coronavirus proteins involved in RNA synthesis. Although many molecular details of the coronavirus life cycle remain to be investigated, the available information suggests that these viruses and their distant nidovirus relatives employ a unique collection of enzymatic activities and other protein functions to synthesize a set of 5'-leader-containing subgenomic mRNAs and to replicate the largest RNA virus genomes currently known.
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PMID:The coronavirus replicase. 1560 9


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