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31.
Phorbol ester inhibits phosphatidylserine synthesis in human promyelocytic leukaemia HL60 cells. Possible involvement of free radicals and correlation with phosphorylation of nuclear protein 1b. 下载免费PDF全文
Treatment of human promyelocytic leukaemia HL60 cells in conditioned medium with 12-O-tetradecanoylphorbol 13-acetate (TPA) for 4 h resulted in 25-30% inhibition of labelling of phosphatidylserine (PS) with [U-14C]serine. PS labelling was 40% lower, and no inhibitory TPA effect was observed when the experiments were performed in fresh medium. Cycloheximide or puromycin also inhibited PS labelling by 38-44%; their inhibitory effects were non-additive with that of TPA and occurred only in conditioned medium. Catalase (CAT) and superoxide dismutase (SOD), both free-radical scavengers, and H7, a protein kinase C inhibitor, reversed to various extents the inhibitory effect of TPA on PS synthesis. On the other hand, chlorobenzoic acid, a free-radical-generating agent, also inhibited PS synthesis by 22% after 4 h treatment when conditioned medium was used. When ethanolamine was added to cells in conditioned medium to quench PS formation through the exchange of free serine with the ethanolamine moiety of phosphatidylethanolamine (PE), PS labelling was decreased by 33% and the inhibitory TPA effect was significantly decreased. On the other hand, ethanolamine had marginal quenching effect on PS labelling when added to cells in fresh medium. TPA increased the phosphorylation of various proteins in the cells, including protein lb (Mr 80,000; pI 5.5) shown to be localized mainly in the nuclear fraction. Chlorobenzoic acid selectively stimulated the phosphorylation of protein lb, whereas CAT and SOD specifically attenuated the TPA-stimulated phosphorylation of this protein. All these agents affected phosphorylation of protein lb only if conditioned medium was used. The findings suggested that net synthesis of PS through the base-exchange mechanism was stimulated in HL60 cells by cell products present in the conditioned medium. TPA inhibited this stimulated PS synthesis by a mechanism which appeared to involve active oxygen species and protein synthesis and might be related to the phosphorylation of protein lb. 相似文献
32.
Protein kinase C in fibroblasts. Characteristics of its intracellular location during growth and after exposure to phorbol esters and other mitogens 总被引:11,自引:0,他引:11
D L Halsey P R Girard J F Kuo P J Blackshear 《The Journal of biological chemistry》1987,262(5):2234-2243
Using an N-bromosuccinimide cleavage fragment of histone H1 as a relatively specific substrate for protein kinase C, we evaluated the partitioning of this kinase activity between soluble and particulate cellular fractions in 3T3-L1 fibroblasts. In confluent, serum-deprived cells, protein kinase C activity was approximately equally divided between soluble and detergent-extractable particulate fractions; both rapidly growing and transformed cells appeared to contain higher levels of particulate enzyme activity. Soluble protein kinase C activity and immunoreactivity decreased to virtually undetectable levels after exposure of the cells to phorbol 12-myristate 13-acetate (PMA), associated with a commensurate increase in particulate kinase activity and immunoreactivity. In intact cells, PMA appeared to cause a shift of immunoreactive protein kinase C from the cytosol to the perinuclear region, as assessed by immunofluorescent microscopy; however; subcellular fractionation revealed that PMA caused increases in the protein kinase C activity associated primarily with non-nuclear membranes. Exposure of the cells to sn-1,2-dioctanoylglycerol resulted in a modest and transient membrane association of protein kinase C, whereas platelet-derived growth factor, fibroblast growth factor, and bombesin caused no detectable increases in the membrane association of the kinase. Activation of protein kinase C by growth factors in fibroblasts may occur without the gross disturbances in intracellular kinase location which occur in response to phorbol esters. 相似文献
33.
M P Neeper L M Kuo M C Kiefer R J Robb 《Journal of immunology (Baltimore, Md. : 1950)》1987,138(10):3532-3538
High affinity receptors for interleukin 2 (IL 2) contain the Tac protein as one ligand-binding subunit. Localization of the IL 2-binding site on this molecule, as well as localization of the complementary site on IL 2, should provide insight into the design of IL 2 analogs. In this report, we examine the ability of normal and modified Tac protein to bind IL 2 and several antibodies that recognize the native Tac molecule. Using a transient L cell expression system, we have determined that transfection with cDNA-missing Tac exon 4 resulted in expression of spliced protein that had no measurable binding to IL 2 or the monoclonal anti-receptor antibodies, anti-Tac, and 7G7/B6. This protein was detected, however, by rabbit polyclonal antibodies prepared against synthetic Tac peptides. Thus, one or more amino acids encoded by exon 4 is important, either for direct ligand contact or for the proper folding of critical segments of the Tac molecule. In addition, insertion of stop codons at a unique restriction enzyme site near the beginning of exon 5 resulted in cellular secretion of truncated Tac molecules that were capable of binding IL 2, anti-Tac, and 7G7/B6. Amino acids encoded by exons 5 to 8 thus play no critical role in IL 2 binding. The ligand association demonstrated for truncated Tac protein produced by exons 2 to 4 should guide attempts to define the IL 2-binding segment of the Tac molecule. 相似文献
34.
Lawrence G. Palmer 《The Journal of membrane biology》1987,96(2):97-106
Epithelial Na channels are apparently pore-forming membrane proteins which conduct Na much better than any other biologically abundant ion. The conductance to Na can be 100 to 1000 times higher than that to K. The only other ions that can readily get through this channel are protons and Li. Small organic cations cannot pass through the channel, and water may also be impermeant. The selectivity properties of epithelial Na channels appear to be determined by at least three factors: A high field-strength anionic site, most likely a carboxyl residue of glutamic or aspartic acid residues on the channel protein, probably accounts for the high conductance through these channels of Na and Li and to the low conductance of K, Rb and Cs. A restriction in the size of the pore at its narrowest point probably accounts for the low conductance of organic cations as well as the possible exclusion of water molecules. The outer mouth of the channel appears to be negatively charged and may control access to the region of highest selectivity and may serve as a preliminary selectivity filter, attracting cations over anions. These conclusions are illustrated by the cartoon of the channel in Fig. 3. This picture is obviously both fanciful and simplified, but its general points will hopefully be testable. It leaves open a number of important questions, including: does amiloride block the channel by binding within the outer mouth? what does the inner mouth of the channel look like, and does this part of the channel contribute to selectivity? and what, if any, are the interactions between the features of the channel that impart selectivity and those that control the regulation of the channel by hormonal and other factors? 相似文献
35.
Freeze-Thawing of Aquaspirillum magnetotacticum Cells Selectively Releases Periplasmic Proteins 总被引:1,自引:1,他引:0 下载免费PDF全文
Lawrence C. Paoletti Kevin A. Short Nancy Blakemore Richard P. Blakemore 《Applied microbiology》1987,53(10):2590-2592
Cells of the gram-negative bacterium Aquaspirillum magnetotacticum, when suspended in buffer and freeze-thawed, produced pinkish orange supernatant fluid. The fluid contained ≤2.0% of total extractable outer membrane component 2-keto-3-deoxyoctonate or of the cytoplasmic membrane marker succinic dehydrogenase. Electrophoretic banding patterns and difference spectra of proteins and hemoproteins released by freeze-thawing cells were distinct from those of membrane-associated substances and similar to those of periplasmic substances obtained by applying conventional fractionation methods to this organism. 相似文献
36.
Molecular Evolution and Nucleotide Sequences of the Maize Plastid Genes for the α Subunit of Cf1 (atpA) and the Proteolipid Subunit of Cf0 (atpH) 下载免费PDF全文
The nucleotide sequences of the maize plastid genes for the alpha subunit of CF1 (atpA) and the proteolipid subunit of CF0 (atpH) are presented. The evolution of these genes among higher plants is characterized by a transition mutation bias of about 2:1 and by rates of synonymous and nonsynonymous substitution which are much lower than similar rates for genes from other sources. This is consistent with the notion that the plastid genome is evolving conservatively in primary sequence. Yet, the mode and tempo of sequence evolution of these and other plastid-encoded coupling factor genes are not the same. In particular, higher rates of nonsynonymous substitution in atpE (the gene for the epsilon subunit of CF1) and higher rates of synonymous substitution in atpH in the dicot vs. monocot lineages of higher plants indicate that these sequences are likely subject to different evolutionary constraints in these two lineages. The 5'- and 3'-transcribed flanking regions of atpA and atpH from maize, wheat and tobacco are conserved in size, but contain few putative regulatory elements which are conserved either in their spatial arrangement or sequence complexity. However, these regions likely contain variable numbers of "species-specific" regulatory elements. The present studies thus suggest that the plastid genome is not a passive participant in an evolutionary process governed by a more rapidly changing, readily adaptive, nuclear compartment, but that novel strategies for the coordinate expression of genes in the plastid genome may arise through rapid evolution of the flanking sequences of these genes. 相似文献
37.
38.
Sequence analysis of alpha 1(VI) and alpha 2(VI) chains of human type VI collagen reveals internal triplication of globular domains similar to the A domains of von Willebrand factor and two alpha 2(VI) chain variants that differ in the carboxy terminus. 总被引:13,自引:2,他引:11 下载免费PDF全文
M L Chu T C Pan D Conway H J Kuo R W Glanville R Timpl K Mann R Deutzmann 《The EMBO journal》1989,8(7):1939-1946
Amino acid sequences of human collagen alpha 1(VI) and alpha 2(VI) chains were completed by cDNA sequencing and Edman degradation demonstrating that the mature polypeptides contain 1009 and 998 amino acid residues respectively. In addition, they contain small signal peptide sequences. Both chains show 31% identity in the N-terminal (approximately 235 residues) and C-terminal (approximately 430 residues) globular domains which are connected by a triple helical segment (335-336 residues). Internal alignment of the globular sequences indicates a repetitive 200-residue structure (15-23% identity) occurring three times (N1, C1, C2) in each chain. These repeating subdomains are connected to each other and to the triple helix by short (15-30 residues) cysteine-rich segments. The globular domains possess several N-glycosylation sites but no cell-binding RGD sequences, which are exclusively found in the triple helical segment. Sequencing of alpha 2(VI) cDNA clones revealed two variant chains with a distinct C2 subdomain and 3' non-coding region. The repetitive segments C1, C2 and, to a lesser extent, N1 show significant identity (15-18%) to the collagen-binding A domains of von Willebrand factor (vWF) and they are also similar to some integrin receptors, complement components and a cartilage matrix protein. Since the globular domains of collagen VI come into close contact with triple helical segments during the formation of tissue microfibrils it suggests that the globular domains bind to collagenous structures in a manner similar to the binding of vWF to collagen I. 相似文献
39.
40.
Plasminogen activator inhibitor 1 (PAI-1) inhibits both tissue-type plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA) and, therefore, is an important regulator of plasminogen activation. We have developed eucaryotic and procaryotic expression systems for PAI-1 and characterized the recombinant glycosylated and non-glycosylated products, together with a non-recombinant natural control, produced in the histosarcoma cell line HT 1080. For eucaryotic expression, the PAI-1 cDNA was stably transfected into chinese hamster ovary cells (CHO cells), while procaryotic expression in Escherichia coli was examined after inserting the DNA sequence encoding the mature PAI-1 protein into an inducible expression vector. Recombinant PAI-1 from CHO cells was purified approximately 50-fold in two steps and was indistinguishable from natural PAI-1. Between 3% and 4% of total cellular protein in the procaryotic expression system consisted of PAI-1, from which it was purified approximately 30-fold, with yields of between 15% and 20%. This PAI-1 formed 1:1 complexes with uPA and also with the single- and two-chain forms of tPA. Kinetic analysis demonstrated that the procaryote-produced PAI-1 had an inhibitory activity towards all three forms of PA that resembled that of natural PAI-1 with association rate constants of approximately 10(7) M-1 s-1. In contrast to PAI-1 from eucaryotic cells, the PAI-1 from E. coli had an inherent activity equal to that of guanidine/HCl-activated natural PAI-1. The activity could not be increased by treatment with denaturants suggesting that the latent form of PAI-1 was absent. However, at 37 degrees C the procaryote-produced PAI-1 lost activity at the same rate as natural PAI-1, with approximately 50% of the activity remaining after 3 h. This activity could be partially restored by treatment with 4 M guanidine/HCl. E. coli-derived PAI-1, added to human plasma and fractionated by Sephacryl S-200 chromatography, eluted in two peaks that were similar to those obtained with guanidine-activated PAI-1 from eucaryotic cells, suggesting that it bound to the PAI-1-binding protein (vitronectin). 相似文献