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91.
92.
Activation of mitogen-activated protein kinase and its activator by ras in intact cells and in a cell-free system. 总被引:10,自引:0,他引:10
S Hattori M Fukuda T Yamashita S Nakamura Y Gotoh E Nishida 《The Journal of biological chemistry》1992,267(28):20346-20351
Mitogen-activated protein (MAP) kinase is a serine/threonine kinase whose function is thought to be essential for the transduction of mitogenic signals. MAP kinase is activated by phosphorylation induced by a variety of extracellular stimuli, and its direct upstream activator has been identified. Using amphibian and mammalian systems, we show here that ras can activate MAP kinase and its activator. Injection of v-Ha-ras p21 into Xenopus immature oocytes activated both MAP kinase and maturation-promoting factor (MPF) activities. The activation of MAP kinase preceded that of MPF, demonstrating that ras activates MAP kinase in an MPF-independent pathway. Moreover, we found that the MAP kinase activator is also activated in ras-injected oocytes. Activation of MAP kinase and its activator occurred also when the v-Ki-ras gene was conditionally induced in rat fibroblastic 3Y1 cells. Furthermore, we observed that ras activated MAP kinase and its activator in a cell-free system prepared from Xenopus oocytes. Using an antibody against the Xenopus 45-kDa MAP kinase activator, we demonstrated that the 45-kDa activator molecule was activated by ras. These findings suggest that the MAP kinase activator/MAP kinase system may be the downstream components of ras signal transduction pathways. 相似文献
93.
Poly-N-acetyllactosamine extension has been found in O-glycans in addition to N-glycans and glycosphingolipids. Attempts were made in HL-60 and K562 cells to determine the amount of poly-N-acetyllactosaminyl O-glycans in the major sialoglycoprotein, leukosialin. Leukosialin was immunoprecipitated from [3H]glucosamine-labeled HL-60 and K562 cells. Glycopeptides were prepared by Pronase digestion, and O-glycan-containing glycopeptides were isolated by affinity chromatography using Jacalin-agarose. The glycopeptides bound to Jacalin-agarose and those unbound were treated with alkaline borohydride, and the released O-glycans were fractionated by Bio-Gel P-4 filtration. Sequential glycosidase digestion of the O-glycans, with or without pretreatment by fucosidase or neuraminidase, revealed the following conclusions. 1) Leukosialin from HL-60 cells contains about 1-2 poly-N-acetyllactosaminyl O-glycan chains/molecule. 2) About 50% of these poly-N-acetyllactosaminyl O-glycans contain sialyl Le(x) termini, NeuNAc alpha 2-->3Gal beta 1-->4 (Fuc alpha 1-->3)GlcNAc beta 1-->R. The amount of sialyl Le(x) structure in leukosialin is roughly equivalent to that on cell surfaces of HL-60 cells. 3) Leukosialin from K562 cells, on the other hand, contains no detectable amount of poly-N-acetyllactosaminyl O-glycans. 4) The presence of poly-N-acetyllactosamine in O-glycans is dependent on the core 2 beta 1,6-N-acetylglucosaminyl transferase. 5) Jacalin-agarose binds to sialylated small oligosaccharides such as NeuNAc alpha 2-->3Gal beta 1-->3(NeuNAc alpha 2-->6) GalNAc but not the hexasaccharide NeuNAc alpha 2-->3Gal beta 1-->3(NeuNAc alpha 2-->3Gal beta 1-->4GlcNAc beta 1-->6) GalNAc. These results indicate that the formation of polylactosaminyl O-glycans and sialyl Le(x) structure in O-glycans is dependent on the core 2 formation. 相似文献
94.
95.
The diurnal variations in mRNA quantities of lipogenic enzymes (acetyl-CoA carboxylase, fatty acid synthase, malic enzyme and glucose-6-phosphate dehydrogenase) in rat livers were detected. When the rats began feeding actively after lights out at 1900 h, the mRNA quantities were high from 0500 h to 0900 h in the morning. The variation in fatty acid synthase mRNA quantities was the most dramatic. However, no measurable variation in any enzyme levels including fatty acid synthase was detected. It may be because the half-lives of the enzymes are too long to be effected by the mRNAs which were high for several hours. 相似文献
96.
Mechanisms of somatic embryogenesis in cell cultures: Physiology,biochemistry, and molecular biology
A. Komamine R. Kawahara M. Matsumoto S. Sunabori T. Toya A. Fujiwara M. Tsukahara J. Smith M. Ito H. Fukuda K. Nomura T. Fujimura 《In vitro cellular & developmental biology. Plant》1992,28(1):11-14
Summary One of the most characteristic cell functions in plants is totipotency. Somatic embryogenesis can be regarded as a model system
for the investigation of mechanisms of totipotency, because a high frequency and synchronous embryogenic system from single
somatic cells has been established in carrot suspension cultures. Four phases are recognized in this process, and several
molecular markers, viz. polypeptides, mRNAs, antigens against monoclonal antibodies, can be detected during the expression
of totipotency, but they disappear during its loss. Four organ-specific genes have been isolated from hypocotyls and roots
by differential screening. They were expressed preferentially after the globular-heart stages of embryogenesis, and were strongly
suppressed by auxin. A CEM 1 gene was isolated by differential screening of embryogenic cell clusters. This gene was expressed
strongly and transiently during the proglobular and globular stages. The sequence of CEM 1 was found to encode a polypeptide
showing high homology to the elongation factor isolated from eucaryotic cells. Thus good progress is being made in understanding
the basic mechanisms of somatic embryogenesis.
Presented in the Session-in-Depth Developmental Biology of Embryogenesis at the 1991 World Congress on Cell and Tissue Culture,
Anaheim, California, June 16–20, 1991. 相似文献
97.
Lactate dehydrogenase (LDH) [EC 1.1.1.27] in a crude extract (40-80% ammonium sulfate fraction) of bovine brain was adsorbed on an immobilized colchicine column and specifically eluted by addition of 1 mM NADH. The purity and subunit composition of the pooled LDH were estimated by two-dimensional gel electrophoresis. With an increase of NaCl concentration from 0 to 2.0 M, ligand saturation of LDH on immobilized colchicine increased from 6.8 to 14%, whereas that on immobilized Cibacron blue F3GA decreased from 2.1 to 0%. In the presence of high NaCl concentration, immobilized colchicine enabled both large- and small-scale purification of LDH by affinity chromatography and resulted in a yield of 117 mg from 1 kg of bovine brain in the presence of 2.5 M NaCl or higher recoveries of 54-96% from various tissues of one rat in the presence of 1.0 M NaCl. These results indicate that immobilized colchicine is an excellent adsorbent for the isolation and purification of LDH by affinity chromatography and has a high LDH-adsorbing capacity dependent upon a high NaCl concentration. Kinetic studies revealed that colchicine apparently competed with cofactor NAD for the active site of LDH and the Ki values of colchicine decreased with an increase of NaCl concentration. The chemical specificity of the colchicine-binding site of LDH was studied by the use of colchicine analogues and it is concluded that both the tropolone moiety (C-ring) and the amido bond in a side chain of colchicine structure are essential to the colchicine-LDH interaction. 相似文献
98.
The effect of lipid-protein interaction on the photodynamics of bacteriorhodopsin (bR) was investigated by using partially delipidated purple membrane (pm). When pm was incubated with a mild detergent, Tween 20, the two major lipid components of pm, phospholipids and glycolipids, were released in different ways: the amount of phospholipids released was proportional to the logarithm of the incubation time; the release of glycolipids became noticeable after the release of approximately 2 phospholipids/bR, but soon leveled off at approximately 50% of the initial content. It was found that the thermal decay of the photocycle intermediate N560 was inhibited by the removal of less than 2 phospholipids per bR. This inhibition was partly explained by an increase in the local pH near the membrane surface. More significant changes in the bR photoreactions were observed when greater than 2 phospholipids/bR were removed: (1) the extent of light adaptation became much smaller, and this reduction correlated with the release of glycolipids; (2) N560 became difficult to detect; (3) the M412 intermediate, which is characterized by a pH-insensitive lifetime, was replaced by a long-lived M-like photoproduct with a pH-sensitive lifetime. The heavy delipidation apparently altered the mechanism by which the deprotonated Schiff base receives a proton. An important conformational change in the protein moiety is suggested to take place during the M412 state, this conformational change being inhibited in the rigid lipid environment. 相似文献
99.
Effect of a light-induced pH gradient on purple-to-blue and purple-to-red transitions of bacteriorhodopsin 总被引:1,自引:0,他引:1
Bacteriorhodopsin-containing vesicles that were able to alkalize the extravesicular medium by greater than 1.5 pH units under illumination, i.e., inside-out vesicles, were reconstituted by reverse-phase evaporation with Halobacterium halobium polar lipids or exogenous phospholipids. Acid titration of a dark-adapted sample was accompanied by a color change from purple to blue (pKa = 2.5-4.5 in 0.15 M K2SO4), and alkali titration resulted in the formation of a red species absorbing maximally at 480 nm (pKa = 7 to greater than 9), the pKa values and the extents of these color changes being dependent on the nature of lipid. When a vesicle suspension at neutral or weakly acidic pH was irradiated by continuous light so that a large pH gradient was generated across the membrane, either a purple-to-blue or a purple-to-red transition took place. The light-induced purple-to-red transition was significant in an unbuffered vesicle suspension and correlated with the pH change in the extravesicular medium. The result suggests that the purple-to-red transition is driven from the extravesicular side, i.e., from the C-terminal membrane surface. In the presence of buffer molecules outside, the dominant color change induced in the light was the purple-to-blue transition, which seemed to be due to a large decrease in the intravesicular pH. But an apparently inconsistent result was obtained when the extravesicular medium was acidified by a HCl pulse, which was accompanied by a rapid color change to blue. We arrived at the following explanation: The two bR isomers, one containing all-trans-retinal and the other 13-cis-retinal, respond differently to pH changes in the extravesicular and the intravesicular medium. In this relation, full light adaptation was not achieved when the light-induced purple-to-blue transition was significant; i.e., only the 13-cis isomer is likely to respond to a pH change at the N-terminal membrane surface. 相似文献
100.
Human polymorphonuclear leukocytes (PMN) produced considerable amounts of platelet-activating factor (PAF) when exposed to various concentrations of lyso-PAF, especially in the absence of albumin. The amount of produced PAF in the presence of 5 microM lyso-PAF (without albumin) was 1.1 pmol/10 min per 2.5 X 10(6) cells, which was close to the level in the case of opsonized zymosan stimulation. We found that the activity of neither acetyltransferase nor acetylhydrolase was affected markedly by the treatment of cells with lyso-PAF, suggesting that the increased availability of lyso-PAF could be responsible for the induction of PAF synthesis. We also found that PAF synthesis was induced not only by lyso-PAF but also by ether-containing ethanolamine lysophospholipids, 1-alkenyl(alkyl)-sn-glycero-3-phosphoethanolamine (GPE). The addition of 1-alkenyl(alkyl)-GPE caused the degradation of pre-existing 1-alkyl-2-arachidonoyl-sn-glycero-3-phosphocholine (GPC) and an increased level of lyso-PAF, followed by the formation of PAF. By contrast, 1-acyl-GPC and 1-acyl-GPE failed to induce PAF production. These results suggest a possible key role of the availability of lyso-PAF in triggering the biosynthesis of PAF in human PMN. 相似文献