Three residues involved in binding and catalysis in the carbamyl phosphate binding site of Escherichia coli aspartate transcarbamylase

Site-directed mutagenesis was used to create four mutant versions of Escherichia coli aspartate transcarbamylase at three positions in the catalytic chain of the enzyme. The location of all the amino acid substitutions was near the carbamyl phosphate binding site as previously determined by X-ray crystallography. Arg-54, which interacts with both the anhydride oxygen and a phosphate oxygen of carbamyl phosphate, was replaced by alanine. This mutant enzyme was approximately 17,000-fold less active than the wild type, although the binding of substrates and substrate analogues was not altered substantially. Arg-105, which interacts with both the carbonyl oxygen and a phosphate oxygen of carbamyl phosphate, was replaced by alanine. This mutant enzyme exhibited an approximate 1000-fold loss of activity, while the activity of catalytic subunit isolated from this mutant enzyme was reduced by 170-fold compared to the wild-type catalytic subunit. The KD of carbamyl phosphate and the inhibition constants for acetyl phosphate and N-(phosphono-acetyl)-L-aspartate (PALA) were increased substantially by this amino acid substitution. Furthermore, this loss in substrate and substrate analogue binding can be correlated with the large increases in the aspartate and carbamyl phosphate concentrations at half of the maximum observed specific activity, [S]0.5. Gln-137, which interacts with the amino group of carbamyl phosphate, was replaced by both asparagine and alanine. The asparagine mutant exhibited only a small reduction in activity while the alanine mutant was approximately 50-fold less active than the wild type. The catalytic subunits of both these mutant enzymes were substantially more active than the corresponding holoenzymes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Molecular cloning and expression of a proteinase gene from Lactococcus lactis subsp. cremoris H2 and construction of a new lactococcal vector pFX1

The 6.5 kb HindIII DNA fragment of the Lactococcus lactis subsp. cremoris H2 plasmid pDI21 was cloned into Escherichia coli POP13 with NM1149, and also directly into Lactococcus lactis subsp. lactis 4125 using a newly-constructed broad host-range vector pFX1. Proteinase was experessed in both transformed organisms. The proteinase resembles a PI type since it preferentially degraded -casein. The restriction map of the 6.5 kb proteinase gene fragment has minor differences from those of published plamid proteinase genes. High-efficiency electroporation with pFX1 provides a direct approach for gene cloning in lactococci.Abbreviations cfu colony forming units - HEPES N-[2-hydroxyethyl]piperazine-N-[2-ethanesulphonic acid] Dedicated to Prof. Dr. G. Drews on the occasion of his 65th birthday     

大鼠缰核与下丘脑外侧区在调节心血管活动方面的机能联系

电刺激大鼠下丘脑外侧区（LH）,动脉压明显升高,心率加快,在刺激电极同侧缰核（Hb)内微量注射盐酸利多卡因、电刺激LH引起的升压反应可被阻断38.9%,心率增快反应可被阻断44.4%,双侧Hb内微量注射盐酸利多卡因,电刺激LH引起的升压反应可被阻断40.7%,心率增快反应可被阻断41.2% ,单侧或双侧Hb内微量注射人工脑脊液均不能阻断电刺激LH引起的心血管反应。电刺激大鼠Hb,动脉压明显升高,心率无明显改变,在刺激电极同侧LH内微量注射盐酸利多卡因,电刺激Hb引起的升压反应可被阻断63.2%,双侧LH内微量注射盐酸利多卡因,电刺激Hb引起的升压反应可被阻断62.6%,单侧或双侧LH内微量注射人工脑脊液均不能阻断电刺激Hb引起的心血管反应。本实验提示Hb与LH在调节心血管活动方面有协同作用。     

血栓素合成酶抑制剂的研究现状和展望

血栓素合成酶抑制剂(TxSI)是一类新型抗血小板药,其显著特点是阻断花生四烯酸(AA)代谢过程中诱聚性及血管收缩性产物血栓素A_2(TxA_2)的形成,而不抑制甚至增加抗聚性及血管扩张性前列环素(PGI_2)的生成。这类药物比环氧酶抑制剂阿司匹林等经典抗血小板药具有更高的特异性作用,可望于心血管疾病的临床应用。     

The effect of carbamylcholine on CFU-s differentiation

The proportion of spleen colony-forming units (CFU-s) killed by hydroxyurea was greatly increased after bone marrow cells (BMCs) from LACA mice were exposed to carbamylcholine (Cach; 1 X 10(-13) to 1 X 10(-9) in vitro and there was a marked change in the proportion of spleen colony types. Following treatment with Cach, granulocytic and mixed erythroid-type colonies increased from 20 to 26.3% and 16.1 to 29.6% in 9-day colonies and from 8.3 to 28.2% and 21.7 to 39.4% in 13-day colonies, respectively. Single cell suspensions of spleen colonies were made for granulocyte-macrophage progenitor (CFU-gm) and late erythroid progenitor (CFU-e) assays. The number of CFU-gm from Cach-treated BMC was about twice that from control BMC for both day 9 and day 13 groups; the number of CFU-e decreased relatively. The results suggest that cholinergic receptors on CFU-s may increase the tendency to differentiate into the granulocytic/monocytic line.     

Secondary structure prediction for the spectrin 106-amino acid segment, and a proposed model for tertiary structure

A collective secondary structure prediction for the human erythrocyte spectrin 106-residue repeat segment is developed, based on the sequences of nine segments that have been reported in the literature, utilizing a consensus of several secondary structure prediction methods for locating turn regions. The analysis predicts a five-fold structure, with three alpha-helices and two beta-strand regions, and differs from previous models on the lengths of the helices and the existence of beta-strand structure. We also demonstrate that this structural motif can be folded into tertiary structures that satisfy the experimental spectrin data and several general principles of protein organization.     

环境因子对两种海洋双壳类幼体趋性行为中的调节作用

本文研究了温度、盐度和重金属等几种因子对缢蛏和长牡蛎幼体趋地性和趋光性行为的影响。结果表明:1)降低温度能增强缢蛏幼体的正趋地性,而长牡蛎幼体却由正趋地性转变为负趋地性,同时两种幼体的正趋光性受到抑制作用;升高温度对缢蛏幼体趋光性的影响不明显,但能加强牡蛎幼体的正趋光性。2)盐度提高时缢蛏幼体的正趋光性趋于增强,正趋地性趋于减弱。3)重金属Cu和Zn在各自的亚致死浓度下具有提高缢蛏幼体正趋地性和降低正趋光性的作用,而牡蛎幼体的正趋地性却趋于减弱,正趋光性趋于增强。本文据上述幼体的行为变化,还讨论了这几种因子对幼体垂直分布的调节作用。     

草蛉科纳草蛉属研究（Ⅱ）—六新种记述（脉翅目：草蛉科）

本文是纳草蛉属Navasius研究续报(研究Ⅰ见“动物分类学报”第15卷第3期)。报道了纳草蛉属6新种:跃纳草蛉N.igneus sp.nov.,冠纳草蛉N.lophophorus sp.nov.,黑角纳草蛉N.nigricornutus sp.nov.显沟纳草蛉N.phantosulcus sp.nov.康纳草蛉N.sanus sp.nov.及三齿纳草蛉N.tridentatus sp.nov.。