In vitro synthesis of rat brain hexokinase

Hexokinase (ATP: D-hexose 6-phosphotransferase, EC 2.7.1.1) has been synthesized in the rabbit reticulocyte lysate system directed by poly(A)⁺ mRNA isolated from rat brain. Identification of the in vitro synthesis product as hexokinase was based on its immunoprecipatation with anti-hexokinase serum as well as the generation of identical peptide maps after partial cleavage of the in vitro product and authentic hexokinase with Staphylococcus aureus V8 proteinase or chymotrypsin. The in vitro product and authentic hexokinase were indistinguishable in molecular weight (SDS-gel electrophoresis); thus, despite the fact that, in situ, much of the hexokinase in brain is found in association with mitochondria, it is not synthesized in the form of a higher molecular weight precursor as is characteristic of other mitochondrial proteins. This is in accord with the view that hexokinase is best considered as a classical ‘soluble’ enzyme which is capable of exhibiting reversible association with mitochondria. The in vitro product cochromatographs (during anion-exchange HPLC) with authentic hexokinase previously shown to have a blocked (presumably acetylated) N-terminus; this procedure is capable of resolving the N-terminally blocked form of the enzyme from a partially proteolyzed form having a free N-terminal amino group. Thus the in vitro product is apparently N-acetylated by an enzyme system previously shown to be present in reticulocyte lysates. A significant fraction of the in vitro synthesized hexokinase attained a conformation characteristic of the native enzyme as judged by the observations that (1) it could be immunoprecipitated by monoclonal antibodies recognizing the native enzyme but not by antibodies recognizing denatured hexokinase, and (2) limited tryptic cleavage of the in vitro product gave fragments identical to those seen with the native enzyme and thought to reflect the organization of structural domains in that enzyme. However, based on these same criteria, the majority of the hexokinase synthesized in vitro appears to exist in a folding state that is not identical to that of either the fully denatured or native enzyme.     

用λ－ZAPⅡ作载体构建豌豆卷须cDNA文库

用改进的异硫氰酸胍－苯酚－氯仿抽提法提取了豌豆卷须总ＲＮＡ并纯化了ｍＲＮＡ,合成双链ｃＤＮＡ后加上人工接头,Ｓｅｐｈａｒｏｓｅ２Ｂ柱层析去掉小片段,最后连入载体λ－ＺＡＰⅡ并用噬菌体包装蛋白进行体外包装,经稀释测定出含有重组子的文库大小为９．２×１０~５．原位杂交筛选出了肌动蛋白阳性克隆。     

携带定居因子抗原的非毒素原性大肠杆菌 CF138 株特性的鉴定

对自腹泻病人粪便标本中分离的携带定居因子抗原的菌株（命名为ＣＦ１３８）的特性,侧重从两个方面进行了鉴定。通过电子显微镜观察,发现其菌体表面具有ＣＳ１及ＣＳ３纤毛结构,聚乙烯珠粘附试验结果证实具有粘附作用,甘露糖抗性血凝试验呈抗性凝集,与ＣＦＡ／Ⅱ抗血清作用显示强凝集阳性,属０６：Ｈ１６血清型,采用家兔肠攀试验,乳鼠ＳＴ活性测定及用ＬＴ、ＳＴ放射性ＤＮＡ探针检测等现行测定ＬＴ及ＳＴ肠毒素的方法,证实该株不产生肠毒素,结果表明该株属具有定居因子抗原的非毒素原性大肠杆菌自然诱变株。灌注该株菌体的免疫豚鼠产生了抵抗肠毒原性大肠杆菌攻击的保护力,表明该株系一潜在的抗ＥＴＥＣ感染的侯选菌苗株。     

Aluminum detoxification mechanism in Pseudomonas fluorescens is dependent on iron

Abstract Hexose phosphorylation was studied in Aspergillus nidulans wild-type and in a fructose non-utilising mutant ( frA ). The data indicate the presence of at least one hexokinase and one glucokinase in wild-type A. nidulans , while the fr A1 mutant lacks hexokinase activity. The A. nidulans gene encoding hexokinase was isolated by complementation of the fr A1 mutation. The absence of hexokinase activity in the fr A1 mutant did not interfere with glucose repression of the enzymes involved in alcohol and l-arabinose catabolism. This suggests that, unlike the situation in yeast where mutation of hexokinase PII abolishes glucose repression, the A. nidulans hexokinase might not be involved in glucose repression.     

CsRCA超表达对高温胁迫下黄瓜幼苗光合作用的影响

以CsRCA超表达黄瓜株系T1-7、T1-2和野生型‘08-1’为试材,在三叶一心时用光照培养箱模拟高温环境[40 ℃,光量子通量密度(PFD) 600 μmol·m^-2·s^-1],研究了CsRCA超表达对高温胁迫下黄瓜幼苗光合作用的调控机理.结果表明: CsRCA超表达可显著提高转基因黄瓜幼苗核酮糖-1,5-二磷酸羧化/加氧酶(Rubisco)大、小亚基的mRNA 表达量,Rubisco和Rubisco活化酶(RCA)活性亦显著高于野生型植株.高温胁迫2 h后,超表达和野生型黄瓜幼苗的光合速率(P_n)、以吸收光能为基础的光化学性能指数(PI_ABS)、Rubisco活性和RCA活性及其mRNA表达量均显著降低.经JIP-test分析发现,高温胁迫导致叶绿素荧光快速诱导动力学曲线中K点明显上升, 而捕获的激子将电子传递到电子传递链中Q_A下游的其他电子受体的概率(Ψ_o)和用于电子传递的产额(φ_E0)均显著下降,说明PSⅡ放氧复合体(OEC)和Q_A之后的电子传递链在高温下受到抑制,但是超表达植株的变化幅度要小于野生型植株.可见CsRCA超表达可以通过提高Rubisco、RCA和PSⅡ活性,缓解高温对黄瓜幼苗光合作用的影响,增强其对高温的适应性.     

植物光合机构对光环境的适应机制: 状态转换

在自然条件下,植物接受的照光量经常变化,而植物在进化过程中已形成了相应的适应机制,用以维持光环境变化过程中2个光反应之间光能转换的能量平衡.植物的调控系统不但能通过调控叶片和叶绿体的运动以及光合色素的积累调节光的吸收,还可以通过光系统的状态转换灵活地调节捕光色素蛋白复合体吸收的能量分配.特别是在低光强下,植物通过可对电子传递链的氧化还原状态做出响应的激酶和磷酸酶调控光系统Ⅱ捕光色素蛋白复合体(LHCⅡ)的可逆磷酸化,从而调节激发能在PSⅠ与PSⅡ之间的分配.植物的状态转换机制是植物适应光质等光环境变化的重要机制.本文综述了植物状态转换机制的研究进展,阐述了LHCⅡ的磷酸化及其在PSⅠ与PSⅡ两个光系统间的移动及其状态转换在植物适应光环境变化中的生理意义,并展望了今后的主要研究方向.     

基于己糖激酶与葡萄糖-6-磷酸脱氢酶共表达的辅酶NADPH高效再生

【目的】构建己糖激酶与葡萄糖-6-磷酸脱氢酶的大肠杆菌共表达体系,以葡萄糖为底物实现辅酶NADPH的高效再生。【方法】通过分子生物学方法,克隆己糖激酶HKgs、HKpp基因,并于Escherichia coli BL21(DE3)中表达,再将己糖激酶HKgs、HKpp分别与葡萄糖-6-磷酸脱氢酶Gpd PP共表达,实现NADPH的原位再生。比较两个共表达工程菌的辅酶再生效果,并针对催化活力较高的工程菌BL21(HKgs+Gpd PP)进行表达条件优化。【结果】NADPH再生活力达到856 U/L。该辅酶再生体系与醇脱氢酶Adh R联合催化,使不对称还原4-氯乙酰乙酸乙酯的催化活力提高至原始值的2.5倍。【结论】通过己糖激酶与葡萄糖-6-磷酸脱氢酶在大肠杆菌中的共表达,构建了一个新的NADPH高效再生体系,并用于醇脱氢酶催化的不对称还原反应。     

Non-PTS uptake and subsequent metabolism of glucose in Pediococcus halophilus as demonstrated with a double mutant defective in phosphoenolpyruvate:mannose phosphotransferase system and in phosphofructokinase

Pediococcus halophilus possesses phosphoenolpyruvate:mannose phosphotransferase system (man:PTS) as a main glucose transporter. A man:PTS defective (man:PTS^d) strain X-160 could, however, utilize glucose. A possible glucose-transport mechanism other than PTS was studied with the strain X-160 and its derivative, man:PTS^d phosphofructokinase defective (PFK^–) strain M-13. Glucose uptake by X-160 at pH 5.5 was inhibited by any of carbonylcyanide m-chlorophenylhydrazone, nigericin, N,N-dicyclohexylcarbodiimide, or iodoacetic acid. The double mutant M-13 could still transport glucose and accumulated intracellularly a large amount of hexose-phosphates (ca. 8 mM glucose 6-phosphate and ca. 2 mM fructose 6-phosphate). Protonophores also inhibited the glucose transport at pH 5.5, as determined by the amounts of accumulated hexose-phosphates (< 4 mM). These showed involvement of proton motive force (P) in the non-PTS glucose transport. It was concluded that the non-PTS glucose transporter operated in concert with hexokinase or glucokinase for the metabolism of glucose in the man:PTS^d strain.Abbreviations BM basal medium - BM-G basal medium containing glucose - CM complex medium - man:PTS phosphoenolpyruvate:mannose phosphotransferase system - CCCP carbonylcyanide m-chlorophenylhydrazone - DCCD N,N-dicyclohexyl carbodiimide - P proton motive force - pH transmembrane pH gradient - transmembrane electrical potential difference - MNNG N-methyl-N-nitro-N-nitrosoguanidine - PIPES piperazine-N,N-bis(-ethanesulfonic acid) - MES 4-morpholineethanesulfonic acid - G-6-P glucose 6-phosphate - F-6-P fructose 6-phosphate - FDP fructose 1,6-bisphosphate - EMP Embden-Meyerhof-Parnas pathway - PFK phosphofructokinase - GK glucokinase - HK hexokinase - IAA iodoacetic acid - II^man enzyme II component of man:PTS