草菇低温诱导蛋白分离纯化及其部分性质的测定

采用硫酸铵分级沉淀和制备电泳分离技术,从低温处理过的草菇菌丝中分离纯化得到三种低温诱导蛋白,分别命名为CspDZG1,CspDZG2,CspDZG3。IEF测定了等电点,分别为3．7,4．4,4．4。SDS-PAGE结果表明,CspDZG1,CspDZG2由一条多肽组成,分子量分别为56kD,54．5kD;CspDZG3由两个亚基组成,分子量分别为54．5kD,68kD。经S.aureus V8蛋白酶酶解后,测定了N端氨基酸序列。     

施氏鲟卵黄蛋白原及其相关蛋白的研究

采用凝胶柱层析、聚丙烯凝胶电泳、免疫印迹（Western blotting）和免疫扩散等方法对施氏鲟卵黄蛋白原（Vi-tellogenin,Vg）及其相关蛋白（Yolk protein,YP）进行了研究。结果表明,施氏鲟血清Vg是一种糖脂磷蛋白,其相对分子量为410kD,由分子量为205kD的两个同源亚基组成。Vg的3种相关蛋白YP1、YP2和YP3。其中YP1相对分子量为370kD,是一种糖脂磷蛋白,由相对分子量为97kD和33kD的两个亚基构成。YP2是一种相对分子量为144kD的磷脂蛋白,由相对分子量为94kD和45kD的2个亚基构成。YP3为相对分子量为66kD的磷蛋白,由相对分子量为30kD的同源亚基构成。     

日本桃叶珊瑚的冷驯化及抗寒机制研究

冷驯化能大大提高植物的抗寒性。研究测定了引进日本桃叶珊瑚(Auccuba japonica Thunb.cv.Variegata)苗木在不同越冬条件下的抗寒性指标及在低温锻炼前后的半致死温度,并通过双向电泳分离与抗寒性有关的特异蛋白。结果表明,日本桃叶珊瑚能安全地越冬(最低气温为-18℃),日本桃叶珊瑚叶片抗冻的细胞结构特征在于其叶肉细胞排列疏松,间隙大,有利于防止细胞内结冰;经过4℃冷驯化的植株低温半致死温度为-21℃,而未经冷驯化的植株低温半致死温度为-7．5℃;双向电泳分析发现至少有3个常温下存在而低温下消失的蛋白多肽,分子量分别为A15kD、B14kD、C56kD、D26kD。依据分子量和等电点初步分析,A与组蛋白H2A相似,B与组蛋白H2B相似;另B与Rubisco小亚基分子量相近,C与Rubisco大亚基分子量相近,前一种情况可能与有关抗寒基因的表达有关,后一种情况可能与低温下的光合作用减弱有关。     

牛脑Ca~(2+)/CaM依赖性蛋白激酶Ⅱ的纯化和鉴定

通过一系列层析法,首次从牛脑纯化得到胶凝电泳匀一的Ca~(2+)/CaM PKⅡ。凝胶过滤法测定全酶分子量为550kD,SDS-PAGE法测定亚基分子量为55kD,推测牛脑Ca~(2+)/CaM PK Ⅱ由十个相同的亚基组成。该酶活性绝对依赖于Ca~(2+)和CaM,以63kD PDE同工酶为底物,其AC_(50)分别为0.85μmol/L和0.18μmol/L;以酪蛋白为底物,其AC_(50)分别为0.22μmol/L和0.06μmol/L。牛脑Ca~(2+)/CaM PK Ⅱ旣能催化63kD PDE同工酶等多种蛋白或酶磷酸化,又能进行自身磷酸化。该酶催化63kD PDE同工酶最大磷酸参入量为1mol/mol亚基。磷酸化型63kD PDE同工酶的Ca~(2+)的AC_(50)高于非磷酸化型。     

白鲢鱼与黄鳝血清转铁蛋白的分离纯化及其结构和性质的比较

1.白鲢鱼与黄鳝血清转铁蛋白在分离纯化上的差异。2.用SDS-PAGE测定分子量,白鲢鱼血清转铁蛋白有两个组份,分子量分别为77kD和70kD;黄鳝血清转铁蛋白为单一组份,分子量为68.1 kD。3.白鲢鱼与黄鳝血清转铁蛋白都含糖,但都不与ConA-Sepharose柱结合。4.白鲢鱼与黄鳝血清转铁蛋白氨基酸组成的测定和比较。5.白鲢鱼与黄鳝血清转铁蛋白用胰蛋白酶在相同条件下进行酶解,白鲢鱼能得到分子量在37kD左右的二个片段,而黄鳝则几乎不能被胰蛋白酶酶解。6.白鲢鱼血清转铁蛋白在404.5nm处有一特异吸收峰,而黄鳝则在407.5nm处。     

牛蛙乳酸脱氢酶同工酶的分离纯化及其性质研究

牛蛙心脏中乳酸脱氢酶在聚丙烯酰胺凝胶电泳上显示3种同工酶区带,分别命名为LDH1、LDH2、LDH3,其中LDH1的活力占绝对优势.采用HiTrap^TM Blue HP 亲和层析和DEAE-Sephadex A离子交换层析对牛蛙骨骼肌中的LDH3进行了分离纯化.纯化的LDH3比活力为295 U/mg,Km NADH=0.028,Km丙酮酸=1.242,在SDS-PAGE上显示两条带,提示该同工酶是由两种亚基组成的,亚基的分子量分别为35.3 kD和37.6 kD.     

新城疫病毒F蛋白中两段七肽重复序列的克隆和表达

从新城疫病毒 (NDV)中国强毒株F4 8E9和弱毒株长春株F蛋白的cDNA中亚克隆出两段七肽重复序列(HeptadRepeatRegion ,HR1,HR2 ) ,将HR1和HR2分别插入表达载体pGEX 6p 1,在大肠杆菌BL2 1(DE3 )中表达 ,将与载体中的GST(GlutathioneS Trasferase)融合表达的可溶性融合蛋白用GST亲和层析柱纯化。纯化的融合蛋白用蛋白酶酶切后 ,先用GST亲和层析柱除去GST ,再加热进一步纯化。纯化的HR1和HR2质谱分析其分子量 ,结果表明 ,强株的HR1和HR2的分子量分别为 7 10 3kD和 6 3 0 1kD ,弱株的HR1和HR2的分子量分别为 7 10 7kD和6 3 0 9kD ,强弱株HR1和HR2的分子量都基本一致。本工作为研究HR1、HR2的结构以及它们在NDV与宿主细胞融合中的作用奠定了基础。     

超滤分离和鉴定三种香菇多糖

用热水从香菇子实体中浸提出香菇多糖,采用两种超滤陶瓷膜将粗多糖分级成三部分Le1,Le2和Le3。所有的这三种多糖都由两组分所组成,采用凝胶过滤色谱测定了多糖分子量,13CNMR和IR光谱测定显示多糖Le1为含α糖甙键的多糖,多糖Le3为含β糖甙键的多糖。采用气相色谱法测定了三种多糖的单糖组成,结果显示三种多糖都由葡糖糖,阿拉伯糖,木糖,甘露糖和半乳糖组成,Le1,Le2和Le3中阿拉伯糖、木糖、甘露糖、半乳糖、葡萄糖的摩尔比分别为0.15∶0.52∶1.00∶1.20∶7.20、0.21∶0.68∶1.00∶1.02∶11.56、0.29∶0.42∶1.00∶0.85∶16.20。三种多糖Le1,Le2和Le3的平均分子量分别为4.02×104、2.16×105和8.93×105。     

N2和NH4^＋培养下粪产碱菌固氮酶钼铁蛋白的合成及其特性

应用柱层析和制备电泳分别将N_2和 NH_4~ 培养的粪产碱菌固氮酶钼铁蛋白(Af 1和Af 1)分离并纯化,两者理化性质十分相似。分子量分别为226 kD和 222 kD;α亚基和β亚基分子量分别为57 kD和60 kD;氨基酸种类相同,总残基数分别为1790和1750;每分子Af 1和Af 1均含有2个原子Mo和32个原子 Fe;金属原子簇的氧化还原当量数为6。Af 1的比活性为 1477 nmolC_2H_4 mg~(-1)protein min~(-1),Af 1无活性;分子结构两者有差异。     

黑皮扁豆凝集素提纯及部分性质研究

采用NaCl抽提、硫酸铵分部沉淀和2步离子交换法从黑皮扁豆中纯化得到一种甘露糖专一凝集素(DPL)。在非变性电泳中纯化的DPL只有一条蛋白带,在还原及非还原条件下的SDS-PAGE中呈现两条蛋白带,其分子量分别为36．8kD和39．8kD;利用FPLC经Sephacryl S-300凝胶过滤测得其表观分子量为155kD,表明DPL由2个α亚基和2个β亚基组成,无二硫键。DPL的等电点为pH6．18,含1．6％糖,为酸性糖蛋白。DPL的热稳定性和pH稳定性,与已报道的甘露糖/葡萄糖专一的豆科凝集素相似。     

粘虫不同发育期体内储存蛋白的检测及苦皮藤素Ⅴ对其含量的影响

采用PAGE和SDS-PAGE以及Western blot 的方法,分析了粘虫Mythimna separata幼虫、蛹及成虫体内的储存蛋白。结果表明,粘虫体内存在两种储存蛋白,其中一种为SP-1,即幼虫特异性储存蛋白,从6龄粘虫幼虫的2日龄开始出现在血淋巴中,到末日龄时达到峰值,停止取食后从血淋巴中消失;另一种为SP-3,在化蛹时开始出现在脂肪体中,一直到成虫期仍可持续表达,因此属于持续性储存蛋白。SP-1为分子量约94 kD和100 kD的2种亚基组成的蛋白质,而SP-3为分子量约94 kD的1种亚基组成的蛋白质。SP-1含8.16%的芳香类氨基酸,3.06%的甲硫氨酸。经苦皮藤素Ⅴ亚致死剂量处理5龄粘虫幼虫后的6龄2、3、4日龄粘虫幼虫体内储存蛋白的含量明显低于对照组,对5日龄后粘虫处理组和对照组体内储存蛋白的含量及雌性成虫产卵量没有明显影响。     

粘虫不同发育期体内储存蛋白的检测及苦皮藤素Ⅴ对其含量的影响

采用PAGE和SDS-PAGE以及Western blot 的方法,分析了粘虫Mythimna separata幼虫、蛹及成虫体内的储存蛋白。结果表明,粘虫体内存在两种储存蛋白,其中一种为SP-1,即幼虫特异性储存蛋白,从6龄粘虫幼虫的2日龄开始出现在血淋巴中,到末日龄时达到峰值,停止取食后从血淋巴中消失;另一种为SP-3,在化蛹时开始出现在脂肪体中,一直到成虫期仍可持续表达,因此属于持续性储存蛋白。SP-1为分子量约94 kD和100 kD的2种亚基组成的蛋白质,而SP-3为分子量约94 kD的1种亚基组成的蛋白质。SP-1含8.16%的芳香类氨基酸,3.06%的甲硫氨酸。经苦皮藤素Ⅴ亚致死剂量处理5龄粘虫幼虫后的6龄2、3、4日龄粘虫幼虫体内储存蛋白的含量明显低于对照组,对5日龄后粘虫处理组和对照组体内储存蛋白的含量及雌性成虫产卵量没有明显影响。     

Isolation and characteristics of dihydropyridine calcium antagonist receptor from rabbit skeletal muscles

Up to 80% of the dihydropyridine receptor is solubilized from transverse tubules of rabbit skeletal muscle by 3-[(3-cholamidopropyl)-dimethylammonium]-2-oxy-1-propane sulfonate (CHAPSO). The DHP receptor is an oligomeric complex made up of two subunits with molecular masses of 160 and 53 kD as shown by DHP-Sepharose affinity chromatography and SDS gel electrophoresis of specifically eluted proteins. The reduction of disulfide bridges of the 160 kD subunit is accompanied by a decrease in its apparent molecular mass up to 125 kD. A method is proposed for preparative isolation of the DHP receptor which is based on ion-exchange chromatography and WGA-Sepharose chromatography. Individual subunits of DHP receptor were isolated by Sepharose 4B gel filtration in SDS; their amino acid composition was determined. Both the 160 and 53 kD subunits are N-glycosylated, and the oligosaccharide portions make up to 7.5% and 6.6%, respectively.     

Purification and Properties of Exopolyphosphatase from the Cytosol of Saccharomyces cerevisiae Not Encoded by the PPX1 Gene

A novel exopolyphosphatase has been isolated from the cytosol of Saccharomyces cerevisiae grown to the stationary phase after its transfer from phosphate-deficient to complete medium. The PPX1 gene responsible for 40-kD exopolyphosphatase of the cytosol does not encode it. Specific activity of the preparation is 150 U/mg, purification degree is 319, and the yield is 16.9%. The minimal molecular mass of the active but unstable enzyme complex is approximately 125 kD. A stable enzyme complex with a molecular mass of approximately 500 kD is composed of two polypeptides of approximately 32 and 35 kD and apparently polyphosphates (polyP). Unlike the enzyme encoded by PPX1, the high-molecular-mass exopolyphosphatase is slightly active with polyP3, not inhibited by antibodies suppressing the activity of 40-kD exopolyphosphatase, inhibited by EDTA, and stimulated by divalent cations to a lesser extent. The high-molecular-mass exopolyphosphatase hydrolyzes polyP with an average chain length of 208 to 15 phosphate residues to the same extent, but is inactive with ATP, PPi, and p-nitrophenyl phosphate. The activity with polyP3 is 13% of that with polyP208. The Km values for polyP208, polyP15, and polyP3 hydrolysis are 3.5, 75, and 1100 microM, respectively. The enzyme is most active at pH approximately 7. Co2+ at the optimal concentration of 0.1 mM stimulates the activity 6-fold, while Mg2+ at the optimal concentration of 1 mM enhances it 2-fold. The enzyme under study is similar in some properties to an exopolyphosphatase purified earlier from yeast vacuoles.     

A Retinal Calmodulin-Dependent Kinase: Calcium/ Calmodulin-Stimulated and -Inhibited States

Abstract: A calcium/calmodulin-dependent protein kinase was isolated from retina. The retinal enzyme is composed exclusively of 50-kilodalton (kD) subunits and has a molecular mass of approximately 275 kD, in contrast to forebrain calmodulin kinase II, which is composed of 50-kD and 60-kD subunits in a 3:1 ratio and has a molecular mass of approximately 520 kD. Similar substrate specificities, kinetic properties, capacity to bind calmodulin, and immunoreactivity suggest that the retinal kinase is an isoenzyme of forebrain calmodulin kinase II. Both kinases autophosphorylate in an intramolecular manner; however, auto-phosphorylation has different effects on the activities of the two enzymes. Autophosphorylation of retinal calmodulin kinase converts the enzyme from a calcium/calmodulin-dependent to a calcium/calmodulin-inhibited kinase, with high activity in the absence of calcium, whereas autophosphorylation of the forebrain kinase results in a less active, calcium/calmodulin-independent enzyme. These properties of calmodulin kinase may play an important role in retinal function.     

人蛋白二硫键异构酶的纯化及其抗血清制备

人蛋白二硫键异构酶的纯化及其抗血清制备吴秉毅,冯桂湘,吕静,张帆,冯永清,王福琴（第一军医大学珠江医院广州５１０２８０）关键词蛋白二硫键异构酶,分离与纯化,抗血清制备二硫键在维持蛋白质的空间结构、生物学活性中起重要作用。二硫键的形成是蛋白质翻译后修饰...     

小刺猴头菌丝体多糖的结构研究

对小刺猴头过滤掉发酵液的发酵菌丝体,水提和碱提后获得的均一组分多糖HMP-w1.1和HMP-a1.1进行结构性质的研究。结果表明:HMP-w1.1是分子量为36.3 kD的α型吡喃糖,单糖组成为甘露糖(Man),葡萄糖(Glc),半乳糖(Gal),岩藻糖(Fuc);HMP-a1.1是分子量为42.8 kD的β型吡喃糖,单糖组成为甘露糖(Man),半乳糖醛酸(GalUA),葡萄糖(Glc),半乳糖(Gal),岩藻糖(Fuc)。综合高碘酸氧化和Smith降解的试验结果,推断HMP-w1.1的糖苷键构型可能为1→、1→4、1→4,6、1→6、1→2、1→2,6;HMP-a1.1的糖苷键构型可能为1→6、1→2、1→2,6。     

Identification,purification, and characterization of pathogenesis-related proteins from virus-infected Samsun NN tobacco leaves

Ten pH-3 soluble, low-molecular-weight pathogenesis-related proteins (PRs) were found to accumulate in leaves of tobacco cv. Samsun NN reacting hypersensitively to tobacco mosaic virus. Besides the previously characterized PRs 1a, 1b, 1c and 2, these proteins were provisionally designated N, O, P, Q, R, and S in order of decreasing electrophoretic mobility in native polyacrylamide gels. Two-dimensional gel electrophoresis indicated that the PRs consist of single polypeptides, except for R, which is composed of two components with slightly different molecular weights. Estimated molecular weights in SDS-containing gels were: PRs 1a and 1b 17 kD, 1c 16.5 kD, 2 31 kD, N 33 kD, O 35 kD, P 27 kD, Q 28 kD, R 13 and 15 kD, and S 25 kD. However, based on their elution from gel filtration columns and relative moblities in native gels of different acrylamide concentrations, P and Q appeared to have molecular weights similar to those of the PR 1 group. Upon chromatofocusing no additional components were resolved. The PRs were eluted between pH 7 and 4; except for R, their pIs, as judged from isoelectric focusing, appeared to lie in the range from pH 4 to 5.2. In the presence of 6 M urea PR 1a was split into two components, one of which was strongly retarded on gels, as were P and Q. None of the PRs was detected when gels were stained for glycoproteins.By combinations of gel filtration, DEAE-cellulose chromatography, and chromatofocusing, PRs 1a, 1b, 1c, 2 and N were purified, their amino acid compositions determined, and antisera raised against each of these components. By Western blotting, antisera against either PR 1a, 1b, or 1c reacted with each of the components of the PR 1 group, as well as with PR S. Similarly, the antisera against either PR 2 or N reacted with both 2 and N, as well as with O and R. On the basis of major similarities in molecular weight characteristics, amino acid compositions, and serological relationships, it is proposed to classify tobacco PRs into five groups: 1: PRs 1a, 1b, and 1c; 2: 2a (formerly 2), 2b (N), and 2c (O); 3: 3a (P), and 3b (Q); 4: 4a and 4b (the two components of R); and 5: PR 5 (S).