北京棒杆菌天冬氨酸激酶突变体P184Q的酶学性质表征

通过分析谷氨酸棒杆菌天冬氨酸激酶(AK)的结构,筛选可能影响别构抑制剂结合的Pro184位点,对其进行饱和定点突变,成功筛选出突变菌株P184Q.酶学性质研究表明:突变体P184Q的V_(max)比野生型(WT)提高了3倍;n=1.39,低于WT的值(2.6),正协同性下降,同时Km值减小,对底物的亲和力增大;P184Q最适pH=6.5,最适反应温度为25℃,半衰期为2.8h;P184Q对金属离子和有机溶剂均表现出良好的抗性,解除抑制剂苏氨酸和甲硫氨酸、赖氨酸和甲硫氨酸、苏氨酸和赖氨酸、赖氨酸对酶活力的抑制作用.     

Kinetic parameters of oscillating reaction of amino acid- BrO3^——Mn^2＋-H2SO4-acetone system

In the previous papers[1—3], the oscillating reactionusing amino acids as organic substrates was studied. Inorder to obtain information about the kinetic parameters ofoscillating reaction of amino acids in amino acid- BrO3 - ?Mn2 -H2SO4-acetone sy…     

薯干粉柠檬酸发酵中氨基酸的消长

本文分析研究了薯干粉黑曲霉深层柠檬酸发酵过程中胞内外氨基酸消长规律。在发酵前期，随菌体生长胞内氨基酸增加，胞外氨基酸减少，变化较大的有Ａｓｐ，Ｇｌｕ，Ｔｈｒ等。在产酸期，胞内外氨基酸含量变化较小。添加Ｔｈｒ或Ｍｅｔ，可以促进菌体生长和产酸。并分析了添加氨基酸后菌体蛋白、胞外蛋白及游离氨基酸的氨基酸组成     

鹿血抗氧化活性肽分子量测定及其氨基酸组成分析

分离提纯了鹿血酶解产物中的抗氧化活性肽,并用高效液相色谱(HPLC)体积排阻法(SEC)测定分子量,分析其氨基酸组成.经SephadexG-25和DEAE-52柱层析纯化,得到了带有较弱负电荷的抗氧化活性肽.HPLC分析结果表明,该抗氧化活性肽为低分子量肽类物质,且是以相对分子量为876,463,146Da为主的小肽混合物.氨基酸组成分析表明,该肽所含17种氨基酸中以亮氨酸、赖氨酸和丙氨酸的质量分数较高,分别为17.2%、12.3%和12.1%,人体必需氨基酸(缬氨酸、异亮氨酸、亮氨酸、苯丙氨酸、蛋氨酸、苏氨酸、赖氨酸)的含量为52.7%.     

计算机辅助木糖还原酶定点突变的生物信息学分析

木糖还原酶（xylose reductase,XR）是木糖代谢生成乙醇途径中一个重要的酶,目前利用纤维素生成酒精的关键问题之一：木糖代谢过程中XR和木糖醇脱氢酶（xylitol dehydrogenase,XDH）的氧化还原不平衡。本研究借助生物信息学手段（酶三维结构建模、酶和辅酶分子对接）,充分分析数据库资源,找到了一些可能影响XR酶活性或辅酶依赖性的关键氨基酸。毕赤氏酵母XR与NADP之间有Lys21（K）、Val222（V）、Glu223（E）、Phe236（F）和Thr273（T）;毕赤氏酵母XR与NAD之间有Val222（V）、Glu223（E）、Phe236（F）、Glu237（E）和Thr273（T）;热带假丝酵母XR与NADP之间有Asn278（N）和Arg282（R）。对比两种辅酶与毕赤氏酵母XR形成氢键的氨基酸,如果使毕赤氏酵母XR只与辅酶NAD结合,则可以将Lys21替换成其它的氨基酸,因Lys21在所有XR序列中完全保守,需要进行氨基酸替代模拟计算预实验,在确保酶三维结构不变及NAD可以结合XR的前提条件下替代Lys21;如果使毕赤氏酵母XR只与辅酶NADP结合,则可以将Glu237（不完全保守）替换成其它的氨基酸。另外,还可以根据需要将这些形成氢键的氨基酸进行组合替代。要改变热带假丝酵母XR的NADP依赖性,可以替代Asn278（N）和/或Arg282（R）（不完全保守）。本研究为进一步酶的理性设计（提高活性及改变辅酶依赖性）并在分子水平上对木糖还原酶进行改造打下了基础。     

珍珠薏米乳酸发酵饮料的研究

研究了以珍珠和薏米为主要原料制作乳酸发酵饮料的工艺。通过正交试验,确定最佳调配组合为:蔗糖10%,香精0.1%,薏米发酵液与珍珠水解液的体积比为2∶1。对饮料的可溶性固形物、酸度、还原糖、金属元素、氨基酸含量等指标进行了测定。结果表明,还原糖质量浓度为11.9 mg.mL-1;可溶性固形物为4.6%;pH值为4.97(16.5℃时);Zn、Mg、Fe、Mn、Na、Ca、K的质量浓度分别为0.1672,5.14340,.23260,.2933,241.76,136.093,4.87μg.mL-1;Cd、Pb、Cr、Cu和Co等均未测出;且含有Asp、Thr、Ser、Glu、Gly、Ala、Val、MetI、le、Leu、Tyr、Phe、Lys、His、Arg和Pro等16种氨基酸。     

不同季节中兰州鲇肌肉水解氨基酸

目的研究兰州鲇(Silurus lanzhouensis)肌肉中常规营养成分和不同季节中肌肉水解氨基酸的组成和含量。方法采用氨基酸自动分析仪测定肌肉中水解氨基酸。结果兰州鲇肌肉中含有18种氨基酸,组成和含量顺序未呈现出季节性变化特点;蛋氨酸、异亮氨酸、亮氨酸、天冬氨酸、丙氨酸这5种氨基酸的含量及氨基酸总量,均随季节的推移呈上升趋势,但在3个季节中仅有秋季显著高于春季(P<0.05);苏氨酸、丝氨酸和谷氨酸等9种氨基酸的含量和必需氨基酸总量等虽有季节性上升趋势,但在3个季节间均无显著差异(P>0.05);胱氨酸、组氨酸和精氨酸等4种氨基酸含量未呈现出季节性变化。结论兰州鲇是一种蛋白质含量较高的经济鱼类,肌肉中水解氨基酸的种类、含量顺序及多数氨基酸的含量在季节变化中有较大的保守性。     

四种植物蛋白的成分与营养学特性分析

分析了4种植物蛋白的氨基酸组成特征和营养学特性。玉米蛋白粉、大豆粉、棉籽蛋白粉、花生饼粉蛋白质质量分数分别为61.60%,38.18%,51.49%,43.15%。大豆粉氨基酸组成比较均衡;棉籽蛋白粉、花生饼粉中多种必需氨基酸组成与大豆蛋白接近,但蛋氨酸含量较低;玉米蛋白粉富含亮氨酸、异亮氨酸、缬氨酸等支链氨基酸,但其中赖氨酸、色氨酸含量明显低于其他3种植物蛋白。建议根据蛋白资源的氨基酸特色组成,合理搭配互补,以提高谷物蛋白质营养价值。     

鸭的羽毛组成成分比较分析

用氨基酸分析仪测定北京鸭、金定鸭及其杂种大土北鸭羽毛的氨基酸组成,发现不同种的鸭毛所含的氨基酸种类相同,但相应的各种氨基酸的含量却有差别。大土北鸭羽毛的异亮氨酸、甲硫氨酸和脯氨酸的含量接近于北京鸭;半胱氨酸、谷氨酸(包括谷氨酰胺)、甘氨酸、亮氨酸、苯丙氨酸和酪氨酸的含量接近于金定鸭;天冬氨酸(包括天冬酰胺)的含量略低于两亲本;赖氨酸的含量显著低于两亲本;丙氨酸、精氨酸、组氨酸、苏氨酸和缬氨酸的含量高于两亲本;丝氨酸的含量则与两亲本相近。经光谱分析表明,三种鸭毛所含金属元素的种类亦相同,且铜含量相差无几;北京鸭羽毛的锌、铁含量明显高于金定鸭;大土北鸭羽毛的锌含量接近于金定鸭,而铁含量则接近于北京鸭。     

The endophilin-CIN85-Cbl complex mediates ligand-dependent downregulation of c-Met

Ligand-dependent downregulation of tyrosine kinase receptors is a critical step for modulating their activity. Upon ligand binding, hepatocyte growth factor (HGF) receptor (Met) is polyubiquitinated and degraded; however, the mechanisms underlying HGF receptor endocytosis are not yet known. Here we demonstrate that a complex involving endophilins, CIN85 and Cbl controls this process. Endophilins are regulatory components of clathrin-coated vesicle formation. Through their acyl-transferase activity they are thought to modify the membrane phospholipids and induce negative curvature and invagination of the plasma membrane during the early steps of endocytosis. Furthermore, by means of their Src-homology 3 domains, endophilins are able to bind CIN85, a recently identified protein that interacts with the Cbl proto-oncogene. Cbl, in turn, binds and ubiquitinates activated HGF receptor, and by recruiting the endophilin-CIN85 complex, it regulates receptor internalization. Inhibition of complex formation is sufficient to block HGF receptor internalization and to enhance HGF-induced signal transduction and biological responses. These data provide further evidence of a relationship between receptor-mediated signalling and endocytosis, and disclose a novel functional role for Cbl in HGF receptor signalling.     

高效液相色谱法分析宫颈癌及宫颈上皮内瘤变患者的血浆氨基酸变化

 利用高效液相色谱（HPLC）代谢组学技术检测宫颈癌、宫颈上皮内瘤变及健康人血浆游离氯基酸含量,探讨宫颈癌及宫颈上皮内瘤变患者血浆游离氯基酸含量的改变及其临床意义。收集新疆医科大学第一附属医院病理确诊的26 例宫颈上皮内瘤变（CIN）患者、22 例宫颈癌（CSSC）患者及35 例健康人血液标本,应用偏最小二乘判别分析法（PLS-DA）对HPLC 谱数据进行模式识别分析,将其结果与健康人血浆游离氯基酸进行比较。结果表明,与健康人相比,CIN 和宫颈癌患者血浆存在明显的氨基酸代谢异常。天冬氨酸、谷氨酸、天冬酰胺、丝氨酸、甘氨酸、组氨酸、牛磺酸、丙氨酸、脯氨酸、酪氨酸、缬氨酸、蛋氨酸、异亮氨酸、亮氨酸、赖氨酸、苯丙氨酸含量自健康人、宫颈上皮内瘤变到宫颈癌呈下降趋势,且以宫颈癌血浆氨基酸含量下降最为显著,差异具有统计学意义（P<0.05）;而精氨酸和苏氨酸在宫颈上皮内瘤变血浆中较健康人明显增高,差异具有统计学意义（P<0.05）。由此得出,肿瘤细胞可能选择性地从血浆中摄取特定氨基酸来满足自身不同阶段的生长需求。     

Adaptive eradication of methionine and cysteine from cyanobacterial light-harvesting proteins

Sulphur is unique among the main elements of living cells in that it is covalently bound to biopolymers but does not occur in the biopolymer backbone. Indeed, most of the bacterial sulphur content resides in the methionine and cysteine side-chains of proteins. The growth yield of an organism under conditions of sulphur limitation could therefore be greatly enhanced by mutations that substitute Met and Cys in the organism's proteins for sulphur-free amino acids. Because the saving in sulphur would increase with such accumulating mutations, Met and Cys changes could be progressively selected. Abundant proteins should be the prime targets of such a selection. A few published observations give credence to this scenario. Sulphate permease, which is abundantly produced by sulphur-starved Salmonella typhimurium, lacks Met and Cys residues. Also, two species of marine purple bacteria synthesize more protein than can be expected from a limited sulphate supply. We now report that the cyanobacterium Calothrix sp. PCC 7601 (referred to here as Calothrix) encodes sulphur-depleted versions of its most abundant proteins--phycocyanin and its auxiliary polypeptides--which it specifically expresses under conditions of sulphur limitation. Although these proteins do not take part in the fixation of sulphur, their elevated synthesis affects the sulphur budget of cyanobacterial cells. Direct evidence is thus provided that the structure of macromolecules can be subject to metabolic optimization.     

Ancestral lysozymes reconstructed, neutrality tested, and thermostability linked to hydrocarbon packing

The controversy surrounding the idea that neutral mutations dominate protein evolution is attributable in part to the inadequacy of the tools available to evolutionary investigators. With a few exceptions, most investigations into the force driving protein evolution have relied on indirect criteria for distinguishing neutral and non-neutral variants. To investigate a particular pathway of molecular evolution, we have reconstructed by site-directed mutagenesis likely ancestral variants of the lysozymes of modern game birds (order Galliformes), tested their activity and thermostability and determined their three-dimensional structure. We focused on amino acids at three positions that are occupied in all known game birds either by the triplet Thr 40, Ile 55, Ser 91, or by the triplet Ser 40, Val 55, Thr 91. We have synthesized proteins representing intermediates along the possible three-step evolutionary pathways between these triplets. Although all of these are active and stable, none of these intermediates is found in known lysozymes. A comparison of the structures and thermostabilities of the variants reveals a linear correlation between the side-chain volume of the triplet and the thermostability of the protein. Each pathway connecting the two extant triplet sequences includes a variant with a thermostability outside the range of the extant proteins. This observation is consistent with a non-neutral evolutionary pathway. The existence of variants that are more stable than the extant proteins suggests that selection for maximum thermostability may not have been an important factor in the evolution of this enzyme.     

Inhibitors of protein kinase C

Protein kinase catalyzes the transfer of the γ-phosphoryl group from ATP to the hydroxyl groups o fprotein side chains, which plays critical roles in signal transduction pathways by transmitting extracellular signals across the plasma membrane and nuclear membrane to the destination sites in the cytoplasm and the nucleus. Protein kinase C (PKC) is a superfamily of phospholipid-dependent Ser/Thr kinase. There are at least 12 isozymes in PKC family.They are distributed in different tissues and play different roles in physiological processes. On account of their concern with a variety of pathophysiologic states, such as cancer,inflammatory conditions, autoimmune disorder, and cardiac diseases, the inhibitors, which can inhibit the activity of PKC and the interaction of cytokine with receptor, and interfere signal transduction pathway, may be candidates of therapeutic drugs. Therefore, intense efforts have been made to develop specific protein kinase inhibitors as biological tools and therapeutic agents. This article reviews the recent development of some of PKC inhibitors based on their interaction with different conserved domains and different inhibition mechanisms.     

C-terminal domain of Chk1 regulates its subcellular location and kinase activity for DNA repair

Effector kinase Chk1 is an evolutionarily conserved protein kinase. It is a key mediator linking the mechanisms that monitor DNA integrity to components of the cell cycle engine. In this study, recombinant vectors pEGFP-C1-Chk1/C 288/C 334/C 368 were constructed and transfected into HeLa cells to study the effect of the Chk1 regulatory domain on the regulation of subcellular Chk1 location in response to DNA damage. We found that DNA damage-induced nuclear accumulation is regulated by 34 amino acids (334–368) in the C-terminal regulatory domain. Recombinant vectors pXJ41-Chk1/C 288/C 334/C 368 were co-transfected with reporter plasmid pEGFP-N2 into HeLa cells to study the repair abilities of the different human Chk1 truncation mutants. In addition, recombinant vectors were transfected into HeLa cells to study the effects of the different truncation mutants on the cell cycle. Furthermore, to study the kinase activity of the different truncation mutants, Ser216 phosphorylation of Cdc25C was studied by Western blot analysis. We found that the enzymatic activity of C 368, missing the 108 C-terminal amino acids (368–476), was higher than that of full-length Chk1, and C 368 delayed the cell cycle progression. The enzymatic activity of C 334, missing the 142 C-terminal amino acids (334–476), was equivalent to that of full-length Chk1. C 288, missing the 188 C-terminal amino acids (288–476), had almost no enzymatic activity, suggesting that the regulatory domain contains both inhibitory and regulatory elements. This study provides useful information for further research on Chk1 function.     

甘蔗UGPase cDNA的克隆及序列分析

以甘蔗(FN95-1702)为材料,通过RT-PCR,首次克隆得到了甘蔗UGPase cDNA片段.该片段长1 495 bp,其中包含完整的ORF为1 431 bp,共编码476个氨基酸,并含有5个重要的Lys残基位点,分别为Lys257、Lys321、 Lys367、 Lys408、 Lys409,它们对维持UGPase活性及与底物结合方面发挥着重要作用.     

Maize yellow stripe1 encodes a membrane protein directly involved in Fe(III) uptake

Frequently, crop plants do not take up adequate amounts of iron from the soil, leading to chlorosis, poor yield and decreased nutritional quality. Extremely limited soil bioavailability of iron has led plants to evolve two distinct uptake strategies: chelation, which is used by the world's principal grain crops; and reduction, which is used by other plant groups. The chelation strategy involves extrusion of low-molecular-mass secondary amino acids (mugineic acids) known as 'phytosiderophores' which chelate sparingly soluble iron. The Fe(III)-phytosiderophore complex is then taken up by an unknown transporter at the root surface. The maize yellow stripe1 (ys1) mutant is deficient in Fe(III)-phytosiderophore uptake, therefore YS1 has been suggested to be the Fe(III)-phytosiderophore transporter. Here we show that ys1 is a membrane protein that mediates iron uptake. Expression of YS1 in a yeast iron uptake mutant restores growth specifically on Fe(III)-phytosiderophore media. Under iron-deficient conditions, ys1 messenger RNA levels increase in both roots and shoots. Cloning of ys1 is an important step in understanding iron uptake in grasses, and has implications for mechanisms controlling iron homeostasis in all plants.