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61.
62.
Phosphoserine peptides, obtained by phosphorylation of synthetic precursors with cyclic AMP-dependent protein kinase, can be efficiently separated from the corresponding non-phosphorylated peptides and from each other by ion-pair high-performance liquid chromatography. All experiments were performed under isocratic conditions on a C18 column, using phosphate buffers with pH 3.2–4.5, n-hexane sulfonic acid as counter ion, and ethanol as organic modifier. 相似文献
63.
64.
Vendela Parrow Eewa Nnberg Jari Heikkil Ulf Hammerling Sven Phlman 《Journal of cellular physiology》1992,152(3):536-544
SH-SY5Y human neuroblastoma cells can be induced to differentiate into a neuronal phenotype by treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA). In other cell systems, TPA treatment frequently leads to down-regulation of protein kinase C (PKC). However, we now report that TPA-treated and non-treated SH-SY5Y cells express PKC-alpha, but not PKC-beta and PKC-gamma, mRNA. Furthermore, only a slight down-regulation of the PKC-alpha protein could be seen during prolonged treatment with 16 nM TPA, the concentration giving optimal differentiation. In contrast, a higher concentration of TPA (1.6 microM) results in a poor neuronal differentiation and a complete down-regulation of PKC-alpha. PKC-alpha was rapidly translocated to the particulate fraction and remained membrane bound for at least 4 days during treatment with 16 nM TPA. In such cells a sustained increased level of the phosphorylated form of a 80,000 Dalton PKC-substrate was found. In addition to this sustained augmented phosphorylation, administration of fresh TPA at day 4 caused a small but reproducible further increased level of phosphorylated substrate. When the PKC activity was measured by the histone phosphorylation assay a substantial fraction of the initial enzyme activity could still be detected after 4 days of TPA treatment. Taken together, the data demonstrate that PKC remains functionally active during TPA induced differentiation of SH-SY5Y cells, which may suggest a continuous role for the enzyme during the differentiation process. 相似文献
65.
Christoph Syldatk Dirk Völkel Ulrich Bilitewski Karsten Krohn Hartmut Höke Fritz Wagner 《Biotechnology letters》1992,14(2):105-110
Summary Resting cells ofArthrobacter sp. (DSM 3745) with the ability to form L-tryptophan from D,L-5-(3-indolylmethy)hydantoin were used for the bioconversion of D,L-5-- and D,L-5--naphthylmethylhydantoin (D,L-5-- and D,L-5--NMH) to the corresponding L-amino acids. Under the optimal reaction conditions of pH 9.7 and 40°C specific productivities of 0.2 (-naphtylalanine) and 0.6 (-naphtylalanine) mM amino acid x g cell dry mass–1 x h–1 were obtained in a 0.1 M Na2CO3/NaHCO3-buffer in a strirred bioreactor. 相似文献
66.
The chemical and kinetic mechanisms of purified aspartate-beta-semialdehyde dehydrogenase from Escherichia coli have been determined. The kinetic mechanism of the enzyme, determined from initial velocity, product and dead end inhibition studies, is a random preferred order sequential mechanism. For the reaction examined in the phosphorylating direction L-aspartate-beta-semialdehyde binds preferentially to the E-NADP-Pi complex, and there is random release of the products L-beta-aspartyl phosphate and NADPH. Substrate inhibition is displayed by both Pi and NADP. Inhibition patterns versus the other substrates suggest that Pi inhibits by binding to the phosphate subsite in the NADP binding site, and the substrate inhibition by NADP results from the formation of a dead end E-beta-aspartyl phosphate-NADP complex. The chemical mechanism of the enzyme has been examined by pH profile and chemical modification studies. The proposed mechanism involves the attack of an active site cysteine sulfhydryl on the carbonyl carbon of aspartate-beta-semialdehyde, with general acid assistance by an enzyme lysine amino group. The resulting thiohemiacetal is oxidized by NADP to a thioester, with subsequent attack by the dianion of enzyme bound phosphate. The collapse of the resulting tetrahedral intermediate leads to the acyl-phosphate product and liberation of the active site cysteine. 相似文献
67.
Werner Mstle Ulrich Link Wolfgang Witschel Ulf Thewalt Thomas Weber Manfred Rothe 《Biopolymers》1991,31(6):735-744
The title compound represents the smallest member of cyclic proline peptides corresponding to the general formula c(DDLL-Pro4)n with a strictly D,D,L,L double-alternating sequence of the chiral amino acid residues. The cyclopeptides with n greater than or equal to 2 could be synthesized from both DDLL-Pro4 (1) and DLLD-Pro4 (2). The cyclic monomer (n = 1) resulted only from 2, whereas not even a trace could be found by cyclization of 1. The peptide exists in a strongly strained Ci symmetrical conformation (x-ray analysis) with alternating cis and trans peptide bonds (ctct form I). The cis peptide bonds deviate from planarity (omega = 22 degrees); two of the pyrrolidine rings show a "South" conformation (phi = -94 degrees), whereas the other residues exhibit C alpha-endo puckering (phi = -124 degrees). Two of the psi angles surprisingly occur at +41 degrees (anti-cis'), the others are located in the trans' region. A quantitative ring opening occurs with trifluoroacetic acid at room temperature. In solution the existence of an isomeric ctcc sequence (form Ia) is indicated. Dreiding model studies also suggested a favorable conformation with a tctc sequence (form II). Consequently, we performed molecular mechanics calculations, based on the CHARMM force field and semiempirical quantum mechanical AM1 calculations (MOPAC program). Pronounced differences in the backbone parameters were found using these two methods. However, the theoretical studies evidenced the experimentally obtained differences in the cyclization tendencies of the linear precursors. 相似文献
68.
Transformed 3T3 cells have reduced levels and altered subcellular distribution of the major PKC substrate protein marcks 总被引:1,自引:0,他引:1
The MARCKS (myristylated alanine-rich C-kinase substrate) protein is an abundant calmodulin-binding protein that is a major and specific endogenous substrate of protein kinase C (PKC). Stimulation of cells with phorbol esters or other activators of PKC has been shown previously to result in rapid phosphorylation of MARCKS proteins and redistribution of these myristylated C-kinase substrates from membrane to cytosol. Here we show that NIH3T3 murine fibroblasts transformed by p21-HA-C-RAS or pp60-V-SRC oncoproteins have markedly reduced levels of p68-MARCKS and that most of the remaining MARCKS protein is found in the cytosol. 3T3 cells containing a nontransforming oncoprotein p26-BCL2, in contrast, exhibited normal levels and distribution of p68-MARCKS. When taken together with recent evidence that MARCKS proteins are involved in regulating organization of the membrane cytoskeleton, our findings suggest that oncoprotein-mediated alterations in MARCKS protein levels and subcellular distribution may contribute to the development or maintenance of the transformed phenotpe. 相似文献
69.
The formation of a microbial biofilm on glass surfaces arranged in lamellar piles parallel with circulating sea water (3 cm·sec–1) was studied. The increase in dry weight, protein content, nucleotide content (ATP, ADP), and diatoms was followed over a period of 62 days. Dry weight and protein were estimates of the total biofilm development, whereas the nucleotide measurements revealed the viability of the biofilm and reflected the dynamics in the community structure. 相似文献
70.
A common sequence in the inverted terminal repetitions of human and avian adenoviruses 总被引:8,自引:0,他引:8
The termini of the avian chick embryo lethal orphan (CELO) virus DNA have been sequenced. The results revealed a 63-bp-long inverted terminal repetition (ITR) which shared the sequence ATAATA with all adenovirus termini, thus far analyzed. The CELO virus ITR differed from those of the mammalian adenoviruses in two major aspects: (i) it is not a perfect duplication; (ii) it begins with a 5'-guanylic acid residue instead of the cytidylic acid normally observed in adenoviruses. 相似文献