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:2.7.10.1 (ERK)
95,504 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The components required for specific transcription of ribosomal RNA were isolated from logarithmically growing Acanthamoeba castellanii. The transcription initiation factor fraction, TIF, and RNA polymerase I were extracted from whole cells at 0.35 M KCl. The extract was fractionated with polyethylenimine, then chromatographed on phosphocellulose (P11) which resulted in the separation of TIF from RNA polymerase I. The fractions containing TIF were further chromatographed on DEAE cellulose (DE52), Heparin Affigel, and Matrex green agarose, followed by sedimentation through glycerol gradients. TIF was purified approximately 17,000-fold, and shown to have a native molecular weight of 289 kD, and to bind specifically to rRNA promoter sequences by DNase I footprinting. The addition of homogeneous RNA polymerase I to this complex permitted the initiation of specific transcription in vitro. The phosphocellulose fractions containing RNA polymerase I were chromatographed on DEAE cellulose, Heparin-Sepharose, DEAE-Sephadex, and sedimented through sucrose gradients. Polymerase I was purified to apparent homogeneity with a yield of 8.1% and a specific activity of 315. It contained one fewer subunit than previously reported. DNase I protection experiments demonstrated that in both partially purified and homogeneous fractions, RNA polymerase I was capable of stable binding to the TIF-rDNA complex, and correctly initiating transcription on rDNA templates.
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PMID:Purification of components required for accurate transcription of ribosomal RNA from Acanthamoeba castellanii. 162 Jun 19

Inositol phospholipid metabolism during mitogen-induced Schwann cell proliferation has been examined. Addition of axolemma- and myelin-enriched membrane fractions (AXL and MYE, respectively) to cultured Schwann cells stimulated 32P incorporation into phosphatidylinositol 4-monophosphate [PtdIns(4)P] and phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2]. During the first 5 min of incubation with the mitogens, the amount of 32P incorporated into PtdIns(4)P and PtdIns(4,5)P2 was four- to fivefold above control values. The phosphorylation of the inositol phospholipids was dependent on the concentration of membrane mitogens and was maximal within 1 h. Schwann cells that were prelabeled with [3H]glycerol and then stimulated with AXL and MYE displayed a 30-70% increase in the amounts of [3H]PtdIns(4)P and [3H]PtdIns(4,5)P2 and a 60-80% increase in the amount of [3H]phosphatidic acid. A concomitant 20% decrease in the content of [3H]PtdIns was observed after stimulation. These results suggest that the increased metabolism of PtdIns, PtdIns(4)P, and PtdIns(4,5)P2 may be one of the initial molecular events in the transduction of the mitogenic signal across the Schwann cell plasma membrane.
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PMID:Schwann cell proliferation is accompanied by enhanced inositol phospholipid metabolism. 282 49

The mammalian cytosolic thymidine kinase is one of a number of enzymes involved in DNA replication whose activities increase dramatically during S phase of the cell cycle. As a first step in defining the mechanisms that control the S phase induction of thymidine kinase activity, we have purified the human enzyme from HeLa cells and raised a specific immune serum against the purified protein. The enzyme was isolated from cells arrested in S phase by treatment with methotrexate and purified to near homogeneity by ion-exchange and affinity chromatography. Stabilization of the purified enzyme was achieved by the addition of digitonin. An electrophoretic Rm of 0.2 in nondenaturing gels characterizes the purified enzyme activity as cytosolic thymidine kinase. The enzyme has a Stoke's radius of 40 A determined by gel filtration and a sedimentation coefficient of 5.5 S determined by glycerol gradient sedimentation. Based on these hydrodynamic values, a native molecular weight of 96,000 was calculated for the purified enzyme. When electrophoresed in denaturing sodium dodecyl sulfate-polyacrylamide gels under reducing conditions, the most purified enzyme fraction was found to contain one predominant polypeptide of Mr = 24,000. Several lines of evidence indicate that this polypeptide is responsible for thymidine kinase enzymatic activity. 1) The Mr = 24,000 polypeptide co-migrates with thymidine kinase activity in electrophoretic and sedimentation analyses. 2) A subunit Mr = 25,504 is predicted by the nucleotide sequence of a recently isolated cDNA clone that encodes HeLa thymidine kinase. 3) Mouse LTK- cells transformed with this clone express a cytosolic thymidine kinase activity, as well as a novel Mr = 24,000 polypeptide detectable with immune serum raised against the purified human enzyme.
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PMID:Human cytosolic thymidine kinase. Purification and physical characterization of the enzyme from HeLa cells. 333 3

C-type particles produced by a tissue culture-adapted BALB/c myeloma were characterized. It was determined that although the particles were morphologically and antigenically similar to murine leukemia and sarcoma virus, the size of their RNA was different, they lacked RNA-dependent DNA polymerase, they were unstable in NET buffer, sucrose and citrate but were stable in glycerol and Earle balanced salt solution, and they behaved differently from oncornaviruses when treated with ether and detergent.
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PMID:Characterization of C-type particles produced by a tissue culture-adapted murine myeloma. 412 86

Administration of the antiestrogen ethamoxytriphetol (MER-25) during baboon gestation results in a marked decline in placental progesterone production. Since this effect in primates may be modulated via an estrogen receptor, the present study investigated the possible existence of an estrogen receptor in human placenta. Villous tissue of human, term placentas was homogenized in 0.01M Tris-HCl, ethylenediaminetetraacetic acid, dithiothreitol, glycerol buffer. Cytosol was incubated with 10(-8)M [3H] 17 beta-estradiol (E2) in the presence or absence of 10(-6)M diethylstilbestrol (DES). A single peak of [3H]E2 binding occurred in the 5.2 S region after glycerol density gradient centrifugation, which was competed for by DES, E2, and enclomiphene. Scatchard analysis demonstrated E2 binding, which was saturable, of high affinity (Kd = 1.90 X 10(11)M) and of low capacity (N = 0.13 X 10(-14) moles/mg cytosolic protein). Competition for [3H]E2 binding was DES greater than E2 greater than estrone greater than MER-25 greater than enclomiphene, whereas androgens, progestins, and corticosteroids were ineffective. The results fulfill the criteria for a specific estrogen receptor. The influence of antiestrogen and, possibly, estrogen upon placental function in baboons may be modulated by an estrogen receptor.
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PMID:A specific cytosolic estrogen receptor in human term placenta. 714 4

This research characterizes the effects of selected physiological stressors such as work and various environmental heat loads in rhesus monkeys. Non-human primates (N = 6) were behaviorally conditioned to exercise in a wheel ergometer at approximately 3 METs (1 MET = 3.5 ml O2 uptake.kg-1.min-1). On separate days, each monkey attempted six work/rest cycles (10 min work: 1 min rest) at 15, 25, and 35 degrees C (Tdb), vapor pressure < 10 mm Hg. Core temperature (Tco), body weight (BW) and blood samples were taken immediately before and after exercise. Excessively high heat storage rates dictated that the 35 degrees C trial be limited to three work/rest bouts. The change in Tco during exercise was significantly greater in the 35 degrees C trial than during the 15 and 25 degrees C work bouts. Sweat rate, as determined by the change in BW over time, was also significantly greater during the 35 degrees C trial as compared to the 15 and 25 degrees C trials. Glucose levels (mean +/- SE) declined significantly during exercise from 4.35 +/- 0.1 and 4.58 +/- 0.4 mmol to surprisingly low levels of 1.67 +/- 0.2 and 1.76 +/- 0.2 mmol in the 15 and 25 degrees C trials, respectively. Increases in blood lactate, glycerol, and triglycerides were observed independent of environmental temperature. Free fatty acids increased during exercise in the 15 and 25 degrees C trials but declined slightly during the shorter 35 degrees C trial. The findings of this study indicate that the increased heat storage observed at higher environmental heat loads appears to substantially limit the amount of work these primates can perform, possibly the result of a limited sweat production capacity. Additionally, plasma glucose following exercise decreased to levels not typically seen in humans. The general metabolic profile in these primates was otherwise similar to that observed in humans at this work level.
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PMID:Physiological responses of rhesus monkeys to exercise at varied temperatures. 772 77

Tumor necrosis factor-alpha (TNF) induces clustering of theca-interstitial cells (TIC) isolated from immature, hypophysectomized rats, while inhibiting luteinizing hormone (LH)-stimulated androstenedione in vitro. Stimulators of PKC, 1-oleoyl-2-acetyl-sn-glycerol (OAG, 50 and 100 microM) and phorbol-12-myristate-13-acetate (PMA, 50 nM), caused TIC clustering by 6 days in vitro. Clustering induced by these compounds resembled that induced by TNF. The protein kinase inhibitor, staurosporine at 1 and 10 nM, impaired TNF-induced TIC clustering for 6 days, as did the protein kinase inhibitor, 1-(5-isoquinolinesulfonyl)-2-methylpiperizine dihydrochloride (H-7); conversely, the protein kinase inhibitor, chelerythrine chloride (0.1, 1.0 or 10 microM), did not attenuate TNF-directed clustering. The protein kinase inhibitors did not reverse the suppression of LH-stimulated androstenedione by TNF. Inhibitors of the EGF receptor PTK, A23 (10, 50, or 100 microM) and A46 (0.1, 1.0, 10, or 50 microM), impaired TNF-induced TIC clustering, while TNF suppression of LH-directed androstenedione was unaffected. EGF-induced TIC clustering was also impaired by A46, while A23 was less effective. Both A23 and A46 blocked EGF attenuation of LH-directed androstenedione after 4 days. When challenged with TNF (1 ng/ml) or PMA (50 nM), PKC activity increased in TIC. A23 (50 microM) and A46 (10 microM) each alone blocked the TNF-associated increase in PKC activity; however, PKC activity attributable to PMA was unaffected by A46. Together, these results suggest that TNF-induced TIC clustering involves activation of PTK which directs subsequent increases in PKC activity; however, mechanisms by which TNF inhibits LH-stimulated steroidogenesis remains elusive.
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PMID:Involvement of protein kinase C and protein tyrosine kinase pathways in tumor necrosis factor-alpha-induced clustering of ovarian theca-interstitial cells. 814 4

The ras, is a G-like protein that controls the mitogen-activated protein kinase (MAPK) pathway involved in control and differentiation of cell growth. MAPK is a key component of its signaling pathway and the aberrant activation may play an important role in the transformation process. To better understand roles of ras in the activation of MAPKs, we have established ras transformed NIH3T3 fibroblast cell line, and analyzed the MAPK module. The ras transformed cells formed numerous spikes at the edges of cells and showed loss of contact inhibition. The levels of ERK1/2 MAPKs as revealed by Western blot analysis were not significantly different between ras transformed and non-transformed cells. However, phosphorylation of ERK MAPKs and the level of MEK were significantly increased although the heavily expressed level of Raf-1, an upstream component of MAPK pathway was unchanged in ras transformed NIH3T3 cells. The sedimentation profile of the MAPK module kinases in a glycerol gradient showed the presence of a rather homogeneous species of multimeric forms of ERK1/2 and MEK as indicated by the narrow distribution peak areas. The broad sedimentation profile of the Raf-1 in a glycerol gradient may suggest possible heterologous protein complexes but the identification of interacting molecules still remains to be identified in order to understand the organization of the MAPK signal transduction pathway.
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PMID:Molecular assembly of mitogen-activated protein kinase module in ras-transformed NIH3T3 cell line. 1104 42

Chlorpyrifos oxon (CPO) activates extracellular signal-regulated kinase (ERK 44/42) in Chinese hamster ovary (CHOK1) cells but the mechanism is not defined. This study tests the hypothesis that diacylglycerol (DAG) is the secondary messenger responsible for CPO-induced ERK 44/42 activation. It is known that DAG is sequentially hydrolyzed by DAG lipase and monoacylglycerol (MAG) lipase, both of which are organophosphate sensitive. Inhibition of these enzymes might therefore lead to the accumulation of DAG and MAG, of which only DAG is a secondary messenger. The experiments show that treatment of CHOK1 cells with CPO significantly inhibits DAG/MAG lipase activity and elevates cellular DAG levels. Pretreatment of CHOK1 cells with CPO or a carbamate known to be a DAG lipase inhibitor, followed by treatment with a cell-permeable DAG (1,2-dihexanoyl-sn-glycerol), results in synergistic activation of ERK 44/42. CPO-potentiated DAG-induced ERK 44/42 activation is both time and concentration dependent. This activation is blocked by inhibitors of protein kinase C and mitogen-activated protein kinase kinase, suggesting that these enzymes are important in CPO/DAG cellular signaling. Activation by a stable DAG analogue (phorbol ester) was not altered by CPO, suggesting that DAG metabolism is the probable target for CPO-potentiated DAG-induced ERK 44/42 activation. These observations support the hypothesis that CPO potentiates DAG signaling in CHOK1 cells by inhibiting a CPO-sensitive DAG lipase, thereby providing a potential mechanism of toxicity not associated with acetylcholinesterase inhibition.
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PMID:Chlorpyrifos oxon potentiates diacylglycerol-induced extracellular signal-regulated kinase (ERK 44/42) activation, possibly by diacylglycerol lipase inhibition. 1178 Oct 77

Collagens have recently been identified as ligands for discoidin domain receptors (DDR1 and DDR2), generating an interest in studying the properties of binding of DDR to its ligand. We are interested in the interaction of DDR2 with collagen I because of its potential role in liver fibrosis. Our in vitro binding assay utilizes DDR2-Fc fusion proteins, which can be clustered (multimerized) by use of antibodies to form DDR2 complexes. Binding of DDR2 complexes to collagen I coated on plastic plates was established by a microplate-based assay using Eu(3+)-labeled proteins and time-resolved fluorometry. Clustering of the DDR2-Fc with antibody was found to be requisite for binding to collagen in vitro. Using atomic force microscopy (AFM) in an aqueous environment, we characterized the surface topographies of DDR2 complexes and collagen I, and investigated binding of this receptor-ligand pair. We were able to image and identify binding of DDR2 complexes onto individual molecules of triple-helical collagen and provide insight into the number and locations of binding sites on collagen I. In most cases, a single receptor complex bound to a single collagen molecule and there were preferred DDR2 binding sites on the collagen I triple helix. These data were validated by rotary-replication transmission electron microscopy (TEM) of glycerol-sprayed samples.
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PMID:Binding of discoidin domain receptor 2 to collagen I: an atomic force microscopy investigation. 1222 Jan 73


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