Amino terminal myristylation of the protein kinase p60src, a retroviral transforming protein

The transforming protein of Rous sarcoma virus, p60src, was shown to be acylated at its amino terminus with the long-chain fatty acid myristic acid by isolation of a tryptic peptide with the following structure: myristylglycylserylseryllysine. The occurrence of this unusual posttranslational modification in the cyclic adenosine monophosphate-dependent protein kinase and in several transforming protein kinases of mammalian retroviruses suggests that myristylation of the amino terminal glycyl residue may be critical for the function of certain proteins related to cell transformation and growth control.     

The 36-kilodalton substrate of pp60v-src is myristylated in a transformation-sensitive manner

A primary intracellular substrate for pp60v-src kinase in a variety of avian and mammalian cells is a protein of 34 to 39 kilodaltons (kD). After incubation of chicken embryo fibroblasts (CEF) with [3H]myristic acid for 4 hours, the 36-kD protein contained covalently bound myristic acid by several criteria: (i) the radioactively labeled material comigrated with the 36-kD protein on sodium dodecyl sulfate-polyacrylamide gels in one and two dimensions, (ii) the labeled material was insoluble in chloroform-methanol, and (iii) radioactively labeled myristate could be recovered from the purified 36-kD protein. The resistance of the acyl fatty acid moiety to hydrolysis by hydroxylamine suggested that the covalent linkage to the 36-kD protein may be through an amide linkage. The [3H]myristic-acid labeling of the 36-kD protein in Rous sarcoma virus-transformed CEF showed a reduction of up to 45 percent when compared to an identical amount of 36-kD protein derived from normal cells; this reduction was not due to general changes in myristic acid metabolism in transformed cells.     

Identification of geranylgeranyl-modified proteins in HeLa cells

Previous studies have shown that animal cells contain isoprenoid-modified proteins and that one of these proteins, lamin B, contains a thioether-linked farnesyl group that is attached to cysteine. In the present study, a novel isoprenoid-modification was identified by labeling HeLa cells with [3H]mevalonic acid and analyzing proteolytic digests of the total cell protein. Radioactive fragments were purified from these digests and treated with Raney nickel. The released, labeled material was analyzed by gas-liquid chromatography (GC) and mass spectrometry (MS). This approach revealed that an all-trans geranylgeranyl group was a major isoprenoid modification.     

Posttranslational glutamylation of alpha-tubulin

The high degree of tubulin heterogeneity in neurons is controlled mainly at the posttranslational level. Several variants of alpha-tubulin can be posttranslationally labeled after incubation of cells with [3H]acetate or [3H]glutamate. Peptides carrying the radioactive moiety were purified by high-performance liquid chromatography. Amino acid analysis, Edman degradation sequencing, and mass spectrometric analysis of these peptides led to the characterization of a posttranslational modification consisting of the successive addition of glutamyl units on the gamma-carboxyl group of a glutamate residue (Glu445). This modification, localized within a region of alpha-tubulin that is important in the interactions of tubulin with microtubule-associated proteins and calcium, could play a role in regulating microtubule dynamics.     

Interaction of Hsp 70 with newly synthesized proteins: implications for protein folding and assembly

The 70-kilodalton family of heat shock proteins (Hsp 70) has been implicated in posttranslational protein assembly and translocation. Binding of cytosolic forms of Hsp 70 (Hsp 72,73) with nascent proteins in the normal cell was investigated and found to be transient and adenosine triphosphate (ATP)-dependent. Interaction of Hsp 72,73 with newly synthesized proteins appeared to occur cotranslationally, because nascent polypeptides released prematurely from polysomes in vivo can be isolated in a complex with Hsp 72,73. Moreover, isolation of polysomes from short-term [35S]Met-labeled cells (pulsed) revealed that Hsp 72,73 associated with nascent polypeptide chains. In cells experiencing stress, newly synthesized proteins coimmunoprecipitated with Hsp 72,73; however, in contrast to normal cells, interaction with Hsp 72,73 was not transient. A model consistent with these data suggests that under normal growth conditions, cytosolic Hsp 72,73 interact transiently with nascent polypeptides to facilitate proper folding, and that metabolic stress interferes with these events.     

Insulin rapidly increases diacylglycerol by activating de novo phosphatidic acid synthesis

The mechanisms whereby insulin increases diacylglycerol in BC3H-1 myocytes were examined. When [3H]arachidonate labeling of phospholipids was used as an indicator of phospholipase C activation, transient increases in [3H]diacylglycerol were observed between 0.5 and 10 minutes after the onset of insulin treatment. With [3H]glycerol labeling as an indicator of de novo phospholipid synthesis, [3H]diacylglycerol was increased maximally at 1 minute and remained elevated for 20 minutes. [3H]Glycerol-labeled diacylglycerol was largely derived directly from phosphatidic acid. Insulin increased de novo phosphatidic acid synthesis within 5 to 10 seconds; within 1 minute, this synthesis was 60 times greater than that of controls. Thus, the initial increase in diacylglycerol is due to both increased hydrolysis of phospholipids and a burst of de novo phosphatidic acid synthesis. After 5 to 10 minutes, de novo phosphatidic acid synthesis continues as a major source of diacylglycerol. Both phospholipid effects of insulin seem important for generating diacylglycerol and other phospholipid-derived intracellular signaling substances.     

Activation of the cellular proto-oncogene product p21Ras by addition of a myristylation signal

The 21-kD proteins encoded by ras oncogenes (p21Ras) are modified covalently by a palmitate attached to a cysteine residue near the carboxyl terminus. Changing cysteine at position 186 to serine in oncogenic forms produces a nonpalmitylated protein that fails to associate with membranes and does not transform NIH 3T3 cells. Nonpalmitylated p21Ras derivatives were constructed that contained myristic acid at their amino termini to determine if a different form of lipid modification could restore either membrane association or transforming activity. An activated p21Ras, altered in this way, exhibited both efficient membrane association and full transforming activity. Surprisingly, myristylated forms of normal cellular Ras were also transforming. This demonstrates that Ras must bind to membranes in order to transmit a signal for transformation, but that either myristate or palmitate can perform this role. However, the normal function of cellular Ras is diverted to transformation by myristate and therefore must be regulated ordinarily by some unique property of palmitate that myristate does not mimic. Myristylation thus represents a novel mechanism by which Ras can become transforming.     

滇南风吹楠种子油脂的提取及脂肪酸成分分析

[目的]提取滇南风吹楠（Horsfieldia tetratepala）种子油脂,并对其脂肪酸成分进行分析。[方法]采用索氏提取法提取滇南风吹楠种仁的油脂,进行甲酯化处理后用气相色谱-质谱（GC/MS）联用仪检测其脂肪酸组成及含量。[结果]滇南风吹楠种仁的平均含油率为54.73%。GC-MS分析表明,滇南风吹楠种子油脂中含有16种脂肪酸,其中月桂酸的含量较高,为48.66%,肉豆蔻酸的含量次之,为43.29%。[结论]该研究为滇南风吹楠的保护及利用提供了依据。     

呋喃唑酮代谢物人工抗原的合成及半抗原偶联比的测定

[目的]合成呋喃唑酮代谢物人工抗原,并测定该半抗原偶联比。[方法]通过混合酸酐法将羧基化改造的小分子抗原AOZ与HSA偶联后,运用紫外光谱分析判断合成的结果,并计算小分子抗原与蛋白的偶联比率。[结果]该试验成功合成了呋喃唑酮代谢物AOZ,对其进行羧基化改造后与载体蛋白HSA成功偶联,偶联比经测定为4∶1。[结论]利用紫外光谱分析测定小分子抗原偶联比率的方法简便,可靠性强。     

酵母提取物诱导重组大肠杆菌合成HrpNEcc蛋白的研究

 【目的】HrpNEcc蛋白是一种起细胞信号作用的蛋白激发子,通过激活植物遗传系统的多基因表达调控,诱导植物的广谱抗病性、驱虫性和抗逆性,促进植物生长发育,因此在农业生产中具有重要意义。在hrpNEcc重组大肠杆菌高密度发酵廉价诱导剂的筛选工作中,偶然发现不添加任何外源诱导剂的TB培养基对照处理也有HrpNEcc蛋白合成。本研究旨在找出引起对照处理中HrpNEcc蛋白合成的诱导因子,并研究诱导因子的来源和含量对HrpNEcc蛋白合成的影响。【方法】摇瓶发酵培养重组大肠杆菌,离心收集菌体,用考马斯亮蓝染色法测定菌体总蛋白,SDS-PAGE检测目的蛋白条带,并结合Bandscan软件计算出HrpNEcc蛋白含量。【结果】在不添加任何外源诱导剂的情况下,用TB培养基发酵生产重组大肠杆菌E. coli BL21(DE3)/pET30a(+)hrpNEcc,HrpNEcc蛋白最高产量可达301.45 mg?L-1,比在IPTG诱导下,用LB培养基发酵生产的HrpNEcc蛋白产量提高72.43%。进一步研究证实,TB培养基中的酵母提取物（yeast extract）含有某些诱导因子能够诱导hrpNEcc基因表达合成HrpNEcc蛋白,并且hrpNEcc的表达水平随酵母提取物来源和浓度的不同而存在较大差异。【结论】在不添加任何外源诱导剂的情况下,高含量的酵母提取物诱导重组大肠杆菌hrpNEcc表达合成HrpNEcc蛋白,但其诱导机制还有待进一步研究。     

萝卜中具溶菌酶活性组分的分子结构分析

[目的]分析萝卜具有溶菌酶活性组分CBPs的分子结构,以期为其作用机制和在萝卜中的生理功能提供资料。[方法]利用亲和层析法及CM-纤维素离子交换柱层析分离纯化CBPs,测定其氨基酸组成、糖基和溶菌酶活性中心残基。[结果]从萝卜中得到了2个具溶菌酶活性且无糖基的组分:CBP1和CBP2,它们间氨基酸组成差异不大;Asp/Glu和His专一性化学修饰剂单独作用后,CBP1和CBP2相对溶菌酶活力均大幅度降低,预先加入竞争性抑制剂则下降的幅度减小。[结论]萝卜中有2个具溶菌酶活性的非糖蛋白组分,其溶菌酶活性中心氨基酸残基均可能含有Asp/Glu和His。     

α?芋螺毒素LvIA第11位氨基酸的新突变体合成及其靶点活性

为了进一步探究第11位氨基酸的性质对LvIA靶点结合活性的影响，设计了LvIA的2个新型突变体[D11R]LvIA和[D11H]LvIA，即用2个碱性氨基酸?精氨酸（R）和组氨酸（H）分别替换原来的酸性氨基酸D。先人工合成了这2个新突变体的线性肽，然后采用2步氧化法进行折叠，以获得在第1位和第3位半胱氨酸（Cys 1～3）、第2位和第4位半胱氨酸（Cys 2～4）之间定点连接形成二硫键。经高效液相色谱分离纯化和质谱鉴定，合成了含有Cys(1～3, 2～4)二硫键连接方式的多肽，其分子质量正确，纯度在95%以上。利用双电极电压钳电生理学技术对这2种突变体与α3β2 nAChR的结合活性进行了检测。结果发现，当该位点的氨基酸性质由酸性转换为碱性后，对LvIA的活性影响巨大，直接导致对α3β2 nAChR的阻断活性丧失。[D11R]LvIA和[D11H]LvIA的活性与野生型LvIA相比分别降低了574.38%和408.62%。由此表明，第11位氨基酸的酸碱性对LvIA的活性至关重要。     

An unusual form of lipid linkage to the CD45 peptide

Some protein kinases and phosphatases are myristoylated on their amino terminus, which perhaps contributes to subcellular localization or regulation. Glycoprotein CD45, a hematopoietic tyrosine phosphatase, was examined for fatty acid content. The CD45 protein incorporated [3H]myristate, but little [3H]palmitate. The label was not metabolized and reincorporated into amino acids or saccharides, as revealed by peptide maps of CD45 labeled with [3H]myristate, 14C-labeled amino acids, [35S]methionine, or 125I, and glycosidase treatments, respectively. The myristate label was resistant to mild alkaline methanolysis and was found in fatty acid and sphingosine, indicating an unusual form of lipid attachment to CD45.     

两歧双歧杆菌PRL2010细胞壁蛋白预测和功能分析

[目的]运用生物信息学方法预测两歧双歧杆菌PRL2010锚定于细胞壁的蛋白和功能分析,为后续研究该菌与宿主相互作用的分子机制奠定基础。[方法]用Phobius和Signal P 4.0软件对两歧双歧杆菌PRL2010全基因组编码蛋白中细胞壁蛋白进行预测,同时采用COG(Cluster of Orthologous Groups of proteins)功能数据库对预测的细胞壁蛋白进行功能注释和聚类分析。[结果]两歧双歧杆菌PRL2010基因组1 706个编码蛋白中,28个蛋白含有细胞壁的锚定结构域。细胞壁蛋白功能分析结果显示,28个细胞壁蛋白中,21个蛋白没有功能注释,7个蛋白有功能注释,其中1个蛋白(exo-alpha-sialidase)参与碳水化合物代谢与转运,2个蛋白(bacteriophage endolysin和cell wall-associated protein)参与细胞壁和细胞膜的生物合成,2个蛋白(hypothetical protein BBPR_0440和Subtilisin family peptidase)参与蛋白质翻译后修饰,1个蛋白(beta-n-acetylhexosaminidase Nag Z)参与细胞内运输、分泌和囊泡运输,1个蛋白(metallophosphoesterase)功能只是预测的。[结论]两歧双歧杆菌PRL2010的大多数细胞壁蛋白功能未知,还需要在以后的研究中进一步分析和注释。     

Identification and isolation of a variant surface glycoprotein from Trypanosoma vivax

The protozoan Trypanosoma vivax is one of the most important agents of African trypanosomiasis, a disease that hinders the productive use of livestock in one-third of the African continent. Trypanosoma vivax is also present in the Caribbean and in South America, posing a threat to the livestock industries of the tropical and subtropical world. Much less is known of the biology of this trypanosome than of the better studied T. brucei and T. congolense. One of the variant surface glycoproteins (VSGs) of a West African stock of T. vivax was identified, purified, and partially characterized by the use of a combination of highly resolving techniques to maximize information from the relatively small amount of parasite material available. The molecular weight of the isolated protein (46,000) is smaller than that of VSGs from other species. As with T. brucei VSGs the protein from T. vivax is complexed with sugars and incorporates 3H when living trypanosomes are incubated with [3H]myristic acid, but the T. vivax molecule is more hydrophobic than the T. brucei molecule. The small size of the T. vivax VSG may have a bearing on the functional and evolutionary relationships of variant antigens in trypanosomes.     

Protein synthesis upon acute nutrient restriction relies on proteasome function

The mechanisms that protect mammalian cells against amino acid deprivation are only partially understood. We found that during an acute decrease in external amino acid supply, before up-regulation of the autophagosomal-lysosomal pathway, efficient translation was ensured by proteasomal protein degradation. Amino acids for the synthesis of new proteins were supplied by the degradation of preexisting proteins, whereas nascent and newly formed polypeptides remained largely protected from proteolysis. Proteasome inhibition during nutrient deprivation caused rapid amino acid depletion and marked impairment of translation. Thus, the proteasome plays a crucial role in cell survival after acute disruption of amino acid supply.     

犬血清IgG的纯化、抗体制备及其鉴定

[目的]探讨犬血清IgG的纯化方法。[方法]采用硫酸铵盐析法结合SephadexG-200凝胶柱层析提取纯化犬血清IgG,利用SDS-PAGE电泳和免疫印迹法鉴定所得产品的纯度和免疫活性。[结果]获得的纯化犬血清IgG的SDS-PAGE图谱中存在2条蛋白条带,表明获得了高纯度的犬血清IgG;重链分子量为58kD,轻链分子量为27kD,IgG分子量为170kD。特异性检测发现犬血清IgG重链和轻链都能与鼠抗犬血清IgG抗体结合,表明纯化犬血清IgG具有免疫原性和反应原性。[结论]硫酸铵盐析法结合SephadexG-200凝胶柱层析可获得纯度高、活性好的纯化犬血清IgG,为犬血清IgG相关的免疫学试验提供了高质量的免疫材料。     

亚硝化单胞菌is79a3 Sec途径分泌系统及底物蛋白分析

[目的]分析亚硝化单胞菌is79a3 Sec途径分泌装置及底物蛋白。[方法]从NCBI中选取亚硝化单胞菌is79a3菌株基因组注释的蛋白质氨基酸序列,采用Blast程序搜寻基因组中编码Sec途径分泌装置的蛋白,同时采用Signal P 4.0、Lipo P、TMHMM 2.0软件分析该菌株基因组中Sec途径的底物蛋白,同时采用COG功能数据库进行功能分析。[结果]通过分析发现亚硝化单胞菌is79a3菌株编码Sec途径分泌装置有关的蛋白15个,包含Sec途径分泌系统中12个转运蛋白编码基因,1个信号肽识别蛋白编码基因和2个信号肽酶蛋白编码基因;Sec途径底物蛋白分析结果显示366个含有Sec途径Spase I类肽酶识别的信号肽,36个蛋白只含有Spase II类肽酶识别的信号肽。402个Sec途径底物蛋白功能分析结果显示:有272个蛋白功能不清楚,130个蛋白有具体的功能注释。[结论]亚硝化单胞菌is79a3 Sec途径分泌装置完整,该菌株Sec途径底物蛋白大多数没有功能注释和功能未知,在有功能注释的蛋白中,主要参与细胞壁/细胞膜的生物合成、蛋白质翻译后修饰/蛋白更替、无机离子转运代谢、能源产生与转换。     

黄羽扇豆翻译延伸因子2的序列分析

 利用Sanger双脱氧链终止法对黄羽扇豆（Lupinusluteus）翻译延伸因子2（EF2）的全长cDNA克隆进行了序列分析。该cDNA长度为2794bp,其中包括5’末端的80bp非翻译区,3’末端185bp非翻译区和2529bp长编码序列,3’末端为一18bp poly（A）尾。与其它GTP结合蛋白比较其编译的843aa（氨基酸）序列,发现它们具有显著的同源性;黄羽扇豆EF2与甜菜EF2的氨基酸序列90％相同。其差异主要存在于序列的中部;而与肽基tRNA和核糖体作用部位、与GTP结合部位和GTP酶活性有关的部位具有很高的保守性。黄羽扇豆EF2第700个氨基酸残基组氨酸是白喉毒素的ADP-核糖基化部位。