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991.
Structure of Pyridoxal Kinase from Sheep Brain and Role of the Tryptophanyl Residues 总被引:3,自引:0,他引:3
Bruno Maras Sofia Valiante Stefania Orru Maurizio Simmaco Donatella Barra Jorge E. Churchich 《Journal of Protein Chemistry》1999,18(3):259-268
The primary structure of sheep brain pyridoxal kinase has been determined by direct chemical and physical methods. The enzyme contains 312 amino acid residues with an acetylated methionine at the N-terminus, yielding a molecular mass of 34,861 Da. The functional role played by the two tryptophanyl residues in positions 52 and 244 of the polypeptide chain has been investigated by fluorescence spectroscopy. The tryptophanyl residues are not completely exposed to the rapidly relaxing solvent and they are poorly accessible to collisional quenchers. Chemical modification with NBS abolishes the catalytic activity of the kinase. The amino acid sequence of the sheep brain enzyme shows high similarity (86.2% identity) with the human pyridoxal kinase recently reported [Hanna, Turner, and Kirkness, (1997), J. Biol. Chem.
272, 10756–10760]. Comparison of the mammalian proteins with bacterial and yeast putative pyridoxal kinases retrieved from the Swiss-Prot data bank shows a low degree of overall similarity. In particular, the putative ATP-binding domain is conserved, whereas the region that appears to be crucial in the binding of the pyridoxal substrate is not. Thus, the assignment of the bacterial and yeast cDNA-deduced proteins as pyridoxal kinases should be taken with caution. 相似文献
992.
There are indications from genetic, biochemical and cell biological studies that protein kinase CK2 (formerly casein kinase II) has a variety of functions at different stages in the cell cycle. To further characterize CK2 and its potential roles during cell cycle progression, one of the objectives of this study was to systematically examine the expression of all three subunits of CK2 at different stages in the cell cycle. To achieve this objective, we examined levels of CK2, CK2 and CK2 on immunoblots as well as CK2 activity in samples prepared from: (i) elutriated populations of MANCA (Burkitt lymphoma) cells, (ii) serum-stimulated GL30-92/R (primary human fibroblasts) cells and (iii) drug-arrested chicken bursal lymphoma BK3A cells. On immunoblots, we observed a significant and co-ordinate increase in the expression of CK2 and CK2 following serum stimulation of quiescent human fibroblasts. By comparison, no major fluctuations in CK2 activity were detected during any other stages during the cell cycle. Furthermore, we did not observe any dramatic differences between the relative levels of CK2 to CK2 during different stages in the cell cycle. However, we observed a significant increase in the amount of CK2 relative to CK2 in cells arrested with nocodazole. We also examined the activity of CK2 in extracts or in immunoprecipitates prepared from drug-arrested cells. Of particular interest is the observation that the activity of CK2 is not changed in nocodazole-arrested cells. Since CK2 is maximally phosphorylated in these cells, this result suggests that the phosphorylation of CK2 by p34cdc2 does not affect the catalytic activity of CK2. However, the activity of CK2 was increased by incubation with p34cdc2 in vitro. Since this activation was independent of ATP we speculate that p34cdc2 may have an associated factor that stimulates CK2 activity. Collectively, the observations that relative levels of CK2 increase in mitotic cells, that CK2 and CK2 are phosphorylated in mitotic cells and that p34cdc2 affects CK2 activity in vitro suggest that CK2 does have regulatory functions associated with cell division. 相似文献
993.
Troponin I: Inhibitor or facilitator 总被引:1,自引:0,他引:1
Perry SV 《Molecular and cellular biochemistry》1999,190(1-2):9-32
TN-I occurs as a homologous group of proteins which form part of the regulatory system of vertebrate and invertebrate striated muscle. These proteins are present in vertebrate muscle as isoforms, Mr 21000-24000, that are specific for the muscle type and under individual genetic control. TN-I occupies a central position in the chain of events starting with the binding of calcium to troponin C and ending with activation of the Ca2+ stimulated MgATPase of the actomyosin filament in muscle. The ability of TN-I to inhibit the MgATPase of actomyosin in a manner that is accentuated by tropomyosin is fundamental to its role but the molecular mechanism involved is not yet completely understood. For the actomyosin ATPase to be regulated the interaction of TN-I with actin, TN-C and TN-T must undergo changes as the calcium concentration in the muscle cell rises, which result in the loss of its inhibitory activity. A variety of techniques have enabled the sites of interaction to be defined in terms of regions of the polypeptide chain that must be intact to preserve the biological properties of TN-I. There is also evidence for conformational changes that occur when the complex with TN-C binds calcium. Nevertheless a detailed high resolution structure of the troponin complex and its relation to actin/tropomyosin is not yet available. TN-I induces changes in those proteins with which it interacts, that are essential for their function. In the special case of cardiac TN-I its effect on the calcium binding properties of TN-C is modulated by phosphorylation. It has yet to be determined whether TN-I acts directly as an inhibitor or indirectly by interacting with associated proteins to facilitate their role in the regulatory system. 相似文献
994.
EF-1a binds aminoacyl-tRNA to the ribosome with the hydrolysis of GTP; the complex facilitates the exchange of GDP for GTP to initiate another round of elongation. To examine the subunit structure of EF-1 and phosphorylation by protein kinase CKII, recombinant , , and subunits from rabbit were expressed in E. coli and the subunits were reconstituted into partial and complete complexes and analyzed by gel filtration. To determine the availability of the and subunits for phosphorylation by CKII, the subunits and the reconstituted complexes were examined as substrates for CKII. Formation of the nucleotide exchange complex increased the rate of phosphorylation of the subunit and reduced the Km, while addition of to or the complex inhibited phosphorylation by CKII. However, a had little effect on phosphorylation of . Thus, the and subunits in EF-1 were differentially phosphorylated by CKII, in that phosphorylation of was altered by association with other subunits, while the site on was always available for phosphorylation by CKII. From the availability of the subunits for phosphorylation by CKII and the composition of the reconstituted partial and complete complexes, a model for the subunit structure of EF-1 consisting of (22)2 is proposed and discussed. 相似文献
995.
Lee Gap Ryol Kim Se Nyun Noguchi Kohei Park Sang Dai Hong Seung Hwan Cho-Chung Yoon S. 《Molecular and cellular biochemistry》1999,195(1-2):77-86
Expression of the RI regulatory subunit of protein kinase A type I is increased in human cancer cell lines, in primary tumors, in cells after transformation, and in cells upon stimulation of growth. Ala99 (the pseudophosphorylation site) of human RI was replaced with Ser (RI-p) for the structure-function analysis of RI. MCF-7 hormone- dependent breast cancer cells were transfected with an expression vector for the wild-type RI or mutant RI-p. Overexpression of RI-P resulted in suppression of protein kinase A type II, the isozyme of type I kinase, production of kinase exhibiting reduced cAMP activation, and inhibition of cell growth showing an increase in G0/G1 phase of the cell cycle and apoptosis. The wild-type RI overexpression had no effect on protein kinase A isozyme distribution or cell growth. Overexpression of protein kinase A type II regulatory subunit, RII, suppressed RI and protein kinase A type I and inhibited cell growth. These results show that the growth of hormone-dependent breast cancer cells is dependent on the functional protein kinase A type I. 相似文献
996.
Interactions of protein kinase CK2 subunits 总被引:3,自引:0,他引:3
Korn I Gutkind S Srinivasan N Blundell TL Allende CC Allende JE 《Molecular and cellular biochemistry》1999,191(1-2):75-83
Several approaches have been used to study the interactions of the subunits of protein kinase CK2. The inactive mutant of CK2 that has Asp 156 mutated to Ala (CK2A156) is able to bind the CK2 subunit and to compete effectively in this binding with wild-type subunits and . The interaction between CK2A156 and CK2 was also demonstrated by transfection of epitope-tagged cDNA constructs into COS-7 cells. Immunoprecipitation of epitope-tagged CK2A156 coprecipitated the subunit and vice-versa. The assay of the CK2 activity of the extracts obtained from cells transiently transfected with these different subunits yielded some surprising results: The CK2 specific phosphorylating activity of these cells transfected with the inactive CK2A156 was considerably higher than the control cells transfected with vectors alone. Assays of the immunoprecipitated CK2A156 expressed in these cells, however, demonstrated that the mutant was indeed inactive. It can be concluded that transfection of the inactive CK2A156 affects the endogenous activity of CK2. Transfection experiments with CK2 and subunits and CK2A156 were also used to confirm the interaction of CK2 with the general CDK inhibitor p21WAF1/CIP1 co-transfected into these cells. Finally a search in the SwissProt databank for proteins with properties similar to those derived from the amino acid composition of CK2 indicated that CK2 is related to protein phosphatase 2A and to other phosphatases as well as to a subunit of some ion-transport ATPases. 相似文献
997.
Amyloid -peptide (A), the main constituent of senile plaques in Alzheimer's disease (AD) brain, is hypothesized to be a key factor in the neurodegeneration seen in AD. Recently it has been shown by us and others that the neurotoxicity of A occurs in conjunction with free radical oxidative stress associated with the peptide. A(1–40) and several other fragments of the A sequence are associated with free radicals in solution that are detectable using electron paramagnetic resonance spectroscopy. These free radicals were shown to attack brain cell membranes, initiate lipid peroxidation, increase Ca2+ influx and damage membrane and cytosolic proteins. In AD brain obtained under rapid autopsy protocol, the activity of the oxidatively-sensitive enzyme creatine kinase was shown to be significantly reduced. We reasoned that A-associated free radical-induced modification of creatine kinase activity and other markers of cellular damage might be modulated by free radical scavengers. Accordingly, this study demonstrates that vitamin E can modulate A(25–35)-induced oxidative damage to creatine kinase and cellular proteins in cultured embryonic hippocampal neurons. These results, consistent with the hypothesis of free radical-mediated A toxicity in AD, are discussed with deference to potential free radical scavengers as therapeutic agents for slowing the progression of AD. 相似文献
998.
999.
内源性CO对内皮素促大鼠主动脉平滑肌细胞增殖及MAPK活性的影响 总被引:1,自引:0,他引:1
血红素加氧酶(heme oxygenase,HO)是血红素分解代谢过程中的限速酶,它能使细胞内的血红素降解成胆绿素和一氧化碳(carbonmonoxide,CO),近来资料表明内源性一氧化碳对生理和病理状态下的血管张力有重要的调节作用,目前尚不不禁内源性HO/CO刘否参与平滑肌细胞增殖过程的调节,本实验在体内培养的大鼠主动脉平滑肌细胞模型上,用血色素加氧酶抑制剂卟啉锌-9(zinc protopo 相似文献
1000.
Yu BZ Zheng J Yu AM Shi XY Liu Y Wu DD Fu W Yang J 《Cell biochemistry and function》2004,22(5):291-298
The mechanism of development of mouse fertilized eggs from the one-cell stage to the two-cell stage remains unclear to date. In the present study, we have evaluated protein kinase C (PKC) and M-phase promoting factor (MPF) kinase activity in fertilized mouse eggs treated with a PKC modulator. PKC and MPF activity have similar activity. The two subunits of MPF, p34(cdc2) and cyclin B, were shown to be included in the substrates phosphorylated by PKC in fertilized mouse eggs, while PKC modulator affected the electrophoretic mobility shift of cdc2 and cdc25C by dephosphorylation and phosphorylation. These results clearly indicate that PKC may affect the progression of the cell cycle through post-translational modification of MPF activity. 相似文献