Functional groups at the catalytic site of F1 adenosine triphosphatase

The protection of F1 ATPase by inorganic phosphate, ADP, ATP, and magnesium ion against inactivation by 1-fluoro-2,4-dinitrobenzene, 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole, and 1-(ethoxycarbonyl)-2-ethoxy-1,2-dihydroquinoline, respectively, has been investigated. Dissociation equilibrium constants and rate constants for the labeling reactions have been deduced from a quantitative treatment of the kinetic data. Comparison of these dissociation constants with each other and with the corresponding literature values indicates that the essential Tyr, Arg, Lys, and Glu or Asp residues are indeed located at the catalytic site of the enzyme. Examination of the rate constants for the labeling reactions in the presence of excess inorganic phosphate, ADP, ATP, or magnesium ion, respectively, suggests that the essential phenol and amino groups are located nearer to the bound inorganic phosphate or the gamma-phosphate group than to the alpha- or beta-phosphate group of the bound ATP, that the essential guanidinium group is located nearer to the alpha- or beta-phosphate group than to the gamma-phosphate group of the bound ATP or the bound inorganic phosphate, and that the essential carboxylate group is located slightly farther away but complexed with magnesium ion which it shares with the bound inorganic phosphate. A mechanism consistent with these topographical relationships is proposed for the catalytic hydrolysis and synthesis of ATP.     

Characterization of human glucocerebrosidase from different mutant alleles.

Human cDNA was mutagenized to duplicate six naturally occurring mutations in the gene for glucocere-brosidase. The mutant genes were expressed in NIH 3T3 cells. The abnormal human enzymes were purified by immunoaffinity chromatography and characterized. The Asn370----Ser mutant protein differed from normal enzyme in its inhibition by both conduritol B epoxide and glucosphingosine demonstrating that the 370 mutant enzyme has an abnormal catalytic site. In addition, the 370 mutant enzyme is less activated by saposin C, but more stimulated by phosphatidylserine than the wild type enzyme. The Arg463----Cys mutant protein was normal with respect to conduritol B epoxide and glucosphingosine inhibition, but was less activated by both saposin C and phosphatidylserine. The Arg120----Gln mutant protein was catalytically inactive. The Leu444----Pro, the pseudopattern, and the Pro415----Arg mutants appear to have reduced amounts of enzyme protein in cells. The studies demonstrated that mutations in the gene for glucocerebrosidase have different effects on the catalytic activity and stability of the enzyme.     

The biology of beta-adrenergic receptors: analysis in human epidermoid carcinoma A431 cells.

1. G-protein-linked transmembrane signaling has emerged as a major pathway for information transduction across the cell membrane. 2. In addition to photopigments that propagate the signal from light, cell-surface receptors for hormones, neurotransmitters, and autacoids propagate signals from ligand binding to membrane-bound effector units via G-proteins. 3. Biochemical and molecular features of one prominent member of these receptors, the beta-adrenergic receptor, will be highlighted in the present article. 4. The role of the human epidermoid carcinoma A431 cells as a model for the study of the structure and biology of beta-adrenergic receptors will be emphasized. 5. A model for receptor regulation, gleaned from recent advances in the biochemistry, cell and molecular biology of beta-adrenergic receptors, is discussed.     

固氮蓝藻“红圈病”的研究——Ⅰ.“红圈病”在大面积培养蓝藻中的危害以及防范措施

对蓝藻“红圈病”的病症、发病条件、传播程度方式、危害进行了研究。结果表明,发病条件与蓝藻最佳生长条件基本一致;病原通过水体和带病藻体传播,对藻的产量和质量均造成严重危害。同时探讨了综合防治措施。     

基因定点诱变删除及天然α型心钠素的分泌型表达

用基因定点诱变技术,删除了pO_1α ANF表达质粒中的33对碱基,使人α型心钠素结构基因直接与大肠杆菌分泌型表达质粒pIN-Ⅲ-OmPA中的信号肽酶切位点编码区相连,构成天然人α型心钠素的表达质粒pANF,在IPTG诱导下表达28肽的天然人α型心钠素。纯化后的表达产物具有天然心钠素的放免活性和很强的舒张血管的生物活性。     

The disulfide bridge pattern of snake venom disintegrins, flavoridin and echistatin.

Flavoridin and echistatin, isolated from the venom of Trimeresurus flavoviridis and Echis carinatus, respectively, belong to the disintegrin family of integrin beta 1 and beta 3 inhibitors of low molecular weight RGD-containing, cysteine-rich peptides. Since disulfide bonds are critical for expression of biological activity, we sought to determine their location in these two proteins. In flavoridin, direct evidence for the existence of linkage between Cys4-Cys19 and between Cys45 and Cys64 was obtained by analysis of proteolytic products, and indirect evidence suggests links between Cys6-Cys14 and Cys13-Cys36. In echistatin, links between Cys8-Cys37 and Cys20-Cys39 were identified by direct chemical analysis.     

Quantitation of model digestive mixtures by 13C NMR.

13C nuclear magnetic resonance (NMR) spectra were obtained at 50.3 and 100.5 MHz for methanolic and aqueous mixtures of sodium taurocholate, 1-monocapryloyl-rac-glycerol, and caprylic acid. Distortionless Enhancement by Polarization Transfer (DEPT) was used to improve spectral sensitivity and resolution, and to generate calibration curves for quantitative determinations of each lipid in methanol. Alternatively, the heights for nonoverlapping peaks in a 13C NMR spectrum acquired with inverse-gated decoupling provide reliable quantitative estimates for each component of the mixture, particularly when the data are obtained in methanol. These experiments also demonstrate the feasibility of detailed NMR structural investigations in model systems for glyceride digestion.