溴氰菊酯对鼠脑蛋白质磷酸化作用的影响

以小鼠大脑碎片与[γ-~(32)P]ATP一起保温,观察到溴氰菊酯对蛋白1—3磷酸化的刺激作用和对4、5磷酸化的抑制作用,表明溴氰菊酯对大脑蛋白质磷酸化产生了影响。从鼠脑分离了C、D、S三个组分,分别进行的蛋白质磷酸化试验结果表明,C、D组分可能是重要的磷酸化部位。 蛋白1、2、3的磷酸化明显地受到溴氰菊酯的刺激,这三个蛋白质可能是“蛋白Ⅲb”的几种形式。溴氰菊酯对“蛋白Ⅲb”磷酸化的刺激,可能会影响神经末梢的神经激素释放,从而影响到与其相关的某些神经功能。     

溴氰菊酯对不同品系家蝇脑突触体膜蛋白磷酸化及ATP酶活性的影响

以敏感品系家蝇和溴氰菊酯抗性品系家蝇(Musca domestica L.)为材料,研究和比较了神经毒剂溴氰菊酯对其脑突触体蛋白磷酸化作用的影响。结果表明,浓度为10^-5 mol/L溴氰菊酯抑制了敏感品系家蝇脑突触体蛋白磷酸化作用,而对抗性品系家蝇脑突触体蛋白磷酸化作用无明显影响。若反应体系中加入2.5×10^-6 mol/L的cAMP显著激活了敏感品系家蝇脑突触体蛋白磷酸化水平,但是当0.6 mmol/Lca²⁺或0.6 mmol/L Ca²⁺加10^-5 mol/L钙调蛋白时明显增强了抗性品系家蝇脑突触体蛋白磷酸化水平,甚至超过了其对敏感品系的作用水平。此外,还发现不同浓度的溴氰菊酯可抑制突触膜上的Na/K-ATP酶和Ca-ATP酶活力,浓度越高抑制作用也越大,并且敏感品系家蝇对溴氰菊酯的敏感度要高于抗性品系。     

Rous sarcoma virus-induced changes in the pattern of phosphorylation of non-histone nuclear proteins

We here studied the protein kinase activity and in vitro phosphorylable sites of non-histone nuclear proteins, 0.4 M NaCl extracts (mostly chromosomal proteins) from chick embryo fibroblasts (CEF), infected or not with a Schmidt Ruppin strain subgroup A of Rous sarcoma virus (RSV).The infection and transformation of chick fibroblasts by RSV induced an increase in kinase activity and endogenous phosphorylation of non-histone chromosomal (NHC) proteins. The stimulation, by a change of medium, of the proliferation of dense cultures of normal chick fibroblasts also induced an increase in the kinase activity and endogenous phosphorylation of NHC proteins.However, two-dimensional gel electrophoresis of the ³²P-phosphorylated proteins showed that stimulation due to a change of medium and that due to the expression of transformation were very different. The stimulation by a change of medium increased to a greater or lesser extent the phosphorylation of the different NHC proteins, with no fundamental variations in the pattern of protein phosphorylation. In contrast, RSV infection induced significant changes in the pattern of protein phosphorylation. One of the most striking feature was the large increase of amount and phosphorylation of high molecular weight (HMW) proteins in particular of phosphoproteins having an evaluated molecular weight (MW) of 78 K and 82 K and pI>8.2.The percent of phosphotyrosine residues in NHC proteins was clearly increased when the proteins were extracted from transformed cells instead of normal cells. But the alkaline treatment of two-dimensional gel electrophoresis indicated that the 80 K phosphoproteins did not contain phosphotyrosine residues, and thus cannot be considered as substrates for pp60^src kinase.     

Differences in the cyclic AMP-dependent phosphorylation of plasma membrane proteins of differentiated and undifferentiated L6 myogenic cells

Differences in the cyclic AMP-dependent plasma membrane phosphorylation system of undifferentiated and differentiated L6 myogenic cells have been detected. Endogenous plasma membrane protein phosphorylation in undifferentiated L6 myoblasts was stimulated more than three fold by 5 x 10(-5) M cyclic AMP, whereas no statistically significant cyclic AMP-dependent phosphorylation of endogenous plasma membrane proteins was observed in differentiated L6 cells. In undifferentiated cells cyclic AMP promoted the phosphorylation of several proteins, the most prominent of which had a molecular weight of 110,000. In differentiated cells cyclic AMP did not selectively promote the phosphorylation of specific plasma membrane proteins. Both differentiated and undifferentiated L6 cells, however, contain a cyclic AMP-dependent protein kinase capable of catalyzing the phosphorylation of exogenous substrates, such as histone f2b. Therefore, the data show that differentiation in L6 cells is associated with a selective change in the activity of a plasma membrane cyclic AMP-dependent protein kinase which employs endogenous membrane proteins as substrate.     

小菜蛾溴氰菊酯敏感和抗性品系蛋白质表达谱的比较分析

杀虫剂抗性是害虫防治中的一个主要挑战。为有效治理抗性, 我们必须了解杀虫剂诱导的害虫生理生化变化。目前害虫抗药性的一些机理已经清楚, 但更多的相关机制还有待探究。本研究通过蛋白质组学方法检测了小菜蛾Plutella xylostella溴氰菊酯敏感和抗性品系间蛋白质组的表达差异。结果显示： SDS-PAGE胶上有大约300个蛋白差异点, 其中23个蛋白点具2.5倍以上的表达差异, 通过MALDI-TOF-MS, 我们成功鉴定出8个蛋白, 其中包括化感蛋白CSP2、 铜锌超氧化物歧化酶和peroxiredoxin样蛋白。通过实时定量PCR（real-time quantitative PCR, qPCR）分析了其中5个蛋白的mRNA 表达水平, 结果表明mRNA 表达水平不能真实反映蛋白的表达水平。免疫印迹验证了双向电泳中SOD1的表达差异。本研究有力地证明溴氰菊酯诱导小菜蛾成虫蛋白质组表达变化, 这为进一步筛选抗性靶标提供很大的帮助。     

Changes in the pattern of protein phosphorylation during meiosis reinitiation in starfish oocytes

Endogenous protein phosphorylation in oocytes of Marthasterias glacialis was examined by incubating living oocytes with [³²P]phosphate and cortical and endoplasmic fractions with [γ-³²P]ATP. Individual phosphorylated proteins were detected by autoradiography after bidimensional and monodimensional electrophoresis, using SDS-polyacrylamide gradient gel slabs. An increased phosphorylation of several protein species was observed as early as 5 min following in vivo hormonal stimulation by 1-methyladenine. No dephosphorylation nor any change in protein staining of the gels was observed. In vitro phosphorylation patterns were consistent with those observed in vivo. They did not change upon in vitro 1-methyladenine addition and remained unaffected when the incubations were carried out in the presence of cAMP or beef heart protein kinase inhibitor. Cortical phosphorylation was inhibited by calcium ions. The results suggest that the hormone promotes alterations in the availability of phosphorylation sites in some proteins already present in the control oocytes which, as well as the corresponding activated cAMP-independent protein kinases, may play a significant role during formation of the maturation promoting factor.     

Differences in the Cyclic AMP-Dependent Phosphorylation of Plasma Membrane Proteins of Differentiated and Undifferentiated L6 Myogenic Cells

Differences in the cyclic AMP-dependent plasma membrane phosphorylation system of undifferentiated and differentiated L₆ myogenic cells have been detected. Endogenous plasma membrane protein phosphorylation in undifferentiated L₆ myoblasts was stimulated more than three fold by 5 × 10⁻⁵ M cyclic AMP, whereas no statistically significant cyclic AMP-dependent phosphorylation of endogenous plasma membrane proteins was observed in differentiated L₆ cells. In undifferentiated cells cyclic AMP promoted the phosphorylation of several proteins, the most prominent of which had a molecular weight of 110,000. In differentiated cells cyclic AMP did not selectively promote the phosphorylation of specific plasma membrane proteins. Both differentiated and undifferentiated L₆ cells, however, contain a cyclic AMP-dependent protein kinase capable of catalyzing the phosphorylation of exogenous substrates, such as histone f₂b. Therefore, the data show that differentiation in L₆ cells is associated with a selective change in the activity of a plasma membrane cyclic AMP-dependent protein kinase which employs endogenous membrane proteins as substrate.     

Stage-specific changes in protein phosphorylation during preimplantation development in the mouse

To gain insight into the role of protein phosphorylation during early mammalian development, seven mouse preimplantation stages were metabolically labeled with radioactive orthophosphate and the radiolabeled proteins identified using gel electrophoresis and autoradiography. The results obtained indicate that there are marked differences in protein phosphorylation patterns between the zygote and two-cell stage and between the morula and blastocyst stage. In addition, there is a compaction-specific change in the phosphorylation profile of three components of Mr 37,000. This compaction-specific change takes place during compaction in the eight-cell embryo; thus, it is the first biochemical change specifically correlated to this important event of early development.     

溴氰菊酯胁迫下小菜蛾幼虫体内差异表达蛋白的分析

小菜蛾Plutella xylostella L.是世界性十字花科蔬菜的主要害虫, 已对多种杀虫剂产生抗性, 其中以对拟除虫菊酯类杀虫剂的抗性发展最快。溴氰菊酯是拟除虫菊酯杀虫剂中杀虫毒力最强的品种。我们前期的研究发现, 小菜蛾溴氰菊酯敏感品系（DS）和抗性品系（DR）成虫期的蛋白质双向电泳(2-DE)图谱存在显著差异。本研究通过双向电泳技术从小菜蛾4龄幼虫中分离出89个有明显差异的蛋白点, 从中选出30个进行串联质谱(MALDI-TOF-MS)实验, 并利用蛋白质数据库检索这些在抗性品系中表达而在敏感品系中不表达或者不同品系中差异表达的蛋白质的归属、 性质和功能, 最终成功鉴定出10个蛋白。对其中的3个基因进行了荧光定量PCR验证, 发现这些蛋白质在mRNA水平的表达与在蛋白水平的表达是一致的。这些在溴氰菊酯胁迫下差异表达的蛋白为研究溴氰菊酯的作用靶标和作用机理, 以及筛选与其抗性相关的蛋白质提供了依据。     

Deltamethrin induced changes in choline transport and phosphorylation activities in synaptosomes from the optic lobe of squid, Loligo pealei

1. Deltamethrin, a powerful synthetic pyrethroid causes a significant change in choline transport in freshly prepared synaptosomes from squid optic lobes. 2. At resting state (nondepolarized) such an effect manifested as a reduction of 14C-choline uptake in a short term (1 min) uptake experiment. 3. At depolarized state, or under conditions where synaptosomes are subjected to osmotic, aging and other stress conditions, deltamethrin caused stimulation of 14C-choline uptake, resulting in elevation of the levels of total radiocarbons in synaptosomes. 4. Such changes are accompanied with changes in overall phosphorylation activities in synaptosomes.     

Protein tyrosine phosphorylation in sperm during gamete interaction in the mouse: the influence of glucose

A key intracellular event during capacitation is protein tyrosine phosphorylation, but its involvement during sperm interaction with the oocyte has not been investigated. Glucose is necessary to achieve fertilization and thus may have an influence on sperm protein tyrosine phosphorylation. The objectives of this study were to 1) visualize protein tyrosine phosphorylation patterns in sperm during capacitation and interaction with the oocyte and 2) determine the influence of glucose. Protein tyrosine phosphorylation was investigated by Western analysis and immunofluorescence. Protein tyrosine phosphorylation was increased during capacitation, and immunofluorescence revealed that zona binding and gamete fusion were correlated with an increase in tyrosine phosphorylation of proteins in the midpiece. During capacitation, the absence of glucose led to a delay in the appearance of protein tyrosine phosphorylation. Following binding to the zona pellucida and the oolemma, tyrosine phosphorylation in the flagellum was also delayed in the absence of glucose and resulted in a significant inhibition of the midpiece phosphorylation. The correlation between successful gamete fusion and the tyrosine phosphorylation of midpiece proteins suggests that the effect of glucose on sperm-oocyte interaction is mediated through regulation of protein tyrosine phosphorylation in a specific area of the fertilizing sperm.     

不同品系小菜蛾成虫脑突触体 蛋白质磷酸化的研究

对小菜蛾Plutella xylostella(L.)敏感品系、抗溴氰菊酯品系、抗杀虫双品系和抗杀螟丹品系的成虫脑突触体蛋白质磷酸化进行了研究比较。结果表明：蛋白质磷酸化在各个品系中的表现是不一样的。cAMP和钙加钙调蛋白对不同品系小菜蛾脑蛋白质磷酸化都有不同程度的刺激作用;3种杀虫剂均对各品系小菜蛾的磷酸化反应有影响,杀虫双、杀螟丹表现为抑制,溴氰菊酯表现为加强。这种影响在敏感品系中表现得比抗性品系中要强烈。     

The identification of Dictyostelium phosphoproteins altered in response to the activation of RasG

Dictyostelium RasG has been implicated in the regulation of a variety of cellular processes, including the initiation of development, cell movement, and cytokinesis, but the molecular components of the signaling pathways involved are largely unknown. We used a tetracycline-regulated protein expression system to study the effect of activated RasG, RasG(G12T), expression on the phosphorylation state of Dictyostelium proteins. Over 70 vegetative phosphoprotein components were resolved by two-dimensional (2-D) immunoblot analysis and of these 16 phosphothreonine and three phosphotyrosine protein components were found to reproducibly change upon RasG(G12T) expression. Thirteen of these were recovered from 2-D gels and identified by mass spectrometry of in-gel tryptic digestions. The proteins identified include the signaling proteins RasGEF-R and protein kinase B, the adhesion protein DdCAD-1, the cytoskeletal protein actin, the mitochondrial division protein FtsZA, and proteins involved in translation and metabolism. In addition to the direct demonstration of the phosphorylation of putative downstream targets of RasG activation, these findings reveal previously undetected phosphorylation of several proteins.     

Protein tyrosine phosphorylation during prolonged in vitro incubation of ejaculated bovine spermatozoa is regulated by the oxidative state of the medium

Protein tyrosine phosphorylation plays a regulatory role in a multitude of physiological processes in sperm. Changes in protein tyrosine phosphorylation, viability, and motility were studied as a function of extended incubation of bovine sperm in vitro at ambient temperature (18-20 degrees C). Fresh ejaculates were incubated after dilution for 8 days. On Days 0, 2, 5, and 8, an aliquot of sperm was incubated with or without theophylline at 37 degrees C for 30 min prior to assessing sperm viability, motility, and tyrosine phosphorylation of soluble and whole-cell proteins. There was a time-dependent decline in sperm motility, which was to some extent reversed by incubation with theophylline. The sum of the phosphotyrosine signal from two soluble proteins (M(r) 67 000 and 36 000) declined with incubation time in both theophylline-treated and untreated sperm. There were major differences in the pattern of tyrosine phosphorylation during incubation between ejaculates from different bulls. Tyrosine phosphorylation of a number of proteins from whole-cell extracts increased in a time-dependent manner during in vitro incubation and was unaffected by the presence of theophylline in the medium. The oxygenation state of the incubation medium had profound effects on sperm motility, viability, and tyrosine phosphorylation of proteins from whole-cell extracts. Sperm motility and viability declined more rapidly under aerobic compared with anaerobic conditions. Tyrosine phosphorylation of proteins from whole-cell extracts increased considerably during anaerobic incubation, while there was no significant change during aerobic incubation. This increase in phosphorylation due to anaerobic incubation was reversed when sperm were transferred from an anaerobic to an aerobic environment, indicating that the oxygenation state of the medium regulates both protein tyrosine kinases and phosphatases. In addition, sperm incubated under aerobic conditions for 5 days retained the ability to phosphorylate proteins when transferred to an anaerobic environment. The increase in protein tyrosine phosphorylation during in vitro incubation took place in a medium that did not contain capacitating substances such as heparin, sodium bicarbonate, or BSA. It transpired over a time scale of days and was not augmented by an increase in intracellular cAMP concentration through phosphodiesterase inhibition. Protein tyrosine phosphorylation during extended in vitro incubation at ambient temperature was significantly inhibited by the presence of oxygen in the medium.     

Substrates for protein kinase C in a cell free preparation of rat aorta smooth muscles

Protein phosphorylation has been studied in a cell free system of rat aorta smooth muscles. Addition of Ca2+ caused phosphorylation of several proteins. The addition of phosphatidylserine or calmodulin together with Ca2+ further increased the phosphorylation of proteins with apparent molecular weights of 20 and 92.5 kilodaltons. The activators of protein kinase C, 12-0-tetradecanoylphorbol-13-acetate and 1,2-diolein, increased phosphorylation of the protein bands of similar molecular weight to those increased by phosphatidylserine in the presence of Ca2+, whereas the biologically inactive phorbol ester, 4 alpha-phorbol-12,13 didecanoate (4 alpha PDD) failed to change the pattern of protein phosphorylation. These results show that proteins present in smooth muscle of rat aorta with molecular weights of 20 and 92.5 kilodaltons are substrates for protein kinase C.     

Endogenous phosphorylation of proteins and phosphatidylinositol in the plasma membranes of a human astrocytoma

Incubation of plasma membrane preparations from several tissues with [gamma-32P]ATP resulted in the phosphorylation of phosphatidylinositol as well as of proteins. The presence of an active phosphatidylinositol kinase in these membranes was indicated by equal or greater incorporation of 32P into phosphatidylinositol phosphate than into proteins. Phosphorylation of endogenous protein and lipid substrates by protein and phosphatidylinositol kinases in the plasma membranes of a human astrocytoma was investigated in detail. Maximal protein phosphorylation required the presence of Nonidet-P40 and phosphatase inhibitors (vanadate or fluoride). The rate of protein phosphorylation was greater with Mg2+ than with Mn2+, and phosphoserine accounted for 60% of the radioactivity incorporated into proteins. In the presence of Mn2+, phosphorylation of tyrosine was increased and was equal to that of serine phosphorylation (40%). With one exception, the overall pattern of phosphorylated proteins was similar with either Mg2+ or Mn2+. Maximal phosphatidylinositol phosphorylation of the astrocytoma plasma membranes also required detergent and phosphatase inhibitors. However, the enzymatic characteristics of lipid phosphorylation differed from those of protein phosphorylation with respect to divalent cation activation, ATP dependence, and sensitivity to inhibition by p-chloromercuriphenyl sulfonate, quercetin, and nucleoside derivatives. These results suggest that phosphorylation of plasma membrane proteins and phosphatidylinositol is catalyzed by different enzymes. The fact that membrane preparations exhibited phosphatidylinositol kinase activity almost 100,000 times greater than that exhibited by the purified tyrosine kinase of ros gene would exclude this and similar oncogene proteins from making a significant contribution to the overall phosphatidylinositol phosphorylation of cell membranes.     

Topographic regulation of phosphorylation in giant neurons of the squid, Loligo pealei: role of phosphatases

In previous studies of phosphorylation in squid stellate ganglion neurons, we demonstrated that a specific multimeric phosphorylation complex characterized each cellular compartment. Although the endogenous protein profile of cell body extracts (giant fiber lobe, GFL), as determined by Coomassie staining, was similar to that of axoplasm from the giant axon, in this study we show that the protein phosphorylation profiles are qualitatively different. Whereas many axoplasm proteins were phosphorylated, including most cytoskeletal proteins, virtually all phosphorylation in perikarya was confined to low molecular weight compounds (<6 kDa). Because phosphorylation of exogenous substrates, histone and casein, was equally active in extracts from both compartments, failure to detect endogenous protein phosphorylation in cell bodies was attributed to the presence of more active phosphatases. To further explore the role of phosphatases in these neurons, we studied phosphorylation in the presence of serine/threonine and protein tyrosine phosphatase (PTP) inhibitors. We found that phosphorylation of axonal cytoskeletal proteins was modulated by okadaic acid-sensitive ser/thr phosphatases, whereas cell body phosphorylation was more sensitive to an inhibitor of protein tyrosine phosphatases, such as vanadate. Inhibition of PTPs by vanadate stimulated endogenous phosphorylation of GFL proteins, including cytoskeletal proteins. Protein tyrosine kinase activity was equally stimulated by vanadate in cell body and axonal whole homogenates and Triton X-100 free soluble extracts, but only the Triton X soluble fraction (membrane bound proteins) of the GFL exhibited significant activation in the presence of vanadate, suggesting higher PTP activities in this fraction than in the axon. The data are consistent with the hypothesis that neuronal protein phosphorylation in axons and cell bodies is modulated by different phosphatases associated with compartment-specific multimeric complexes.