A new method for the assay of glutaminase activity: direct measurement of product formation by an ammonia electrode

A simple, reliable procedure is described for the quantitative assay of glutaminase reaction by measuring product formation using an ammonia electrode. The ammonia electrode is a gas-detecting electrode, sensing the level of dissolved ammonia in aqueous solutions. Ammonia concentration can be read from calibration curves after converting ammonium ion to ammonia by adding sufficient base. Sample color and turbidity do not affect measurements, and samples need not be distilled. The concentrations of the three glutaminase isoenzymes from rat tissues measured by this method are strictly comparable to those measured by other methods.     

Site-directed mutagenesis of the ionizable groups in the active site of Zymomonas mobilis pyruvate decarboxylase: effect on activity and pH dependence.

Pyruvate decarboxylase (PDC, EC 4.1.1.1) is a thiamin diphosphate-dependent enzyme about which there is a large body of structural and functional information. The active site contains several absolutely conserved ionizable groups and all of these appear to be important, as judged by the fact that mutation diminishes or abolishes catalytic activity. Previously we have shown [Schenk, G., Leeper, F.J., England, R., Nixon, P.F. & Duggleby, R.G. (1997) Eur. J. Biochem. 248, 63-71] that the activity is pH-dependent due to changes in kcat/Km while kcat itself is unaffected by pH. The effect on kcat/Km is determined by a group with a pKa of 6.45; the identity of this group has not been determined, although H113 is a possible candidate. Here we mutate five crucial residues in the active site with ionizable side-chains (D27, E50, H113, H114 and E473) in turn, to residues that are nonionizable or should have a substantially altered pKa. Each protein was purified and characterized kinetically. Unexpectedly, the pH-dependence of kcat/Km is largely unaffected in all mutants, ruling out the possibility that any of these five residues is responsible for the observed pKa of 6.45. We conjecture that the kcat/Km profile reflects the protonation of an alcoholate anion intermediate of the catalytic cycle.     

Cleavage of territrems by alkaline hydrogen peroxide: Isolation and characterization of the aromatic moiety of territrems

The chemical reaction of cleavaging territrem B to give 3,4,5-trimethoxy benzoic acid by alkaline hydrogen peroxide was investigated. The method was applied for confirmation of the chemical structure of the aromatic moiety of territrem A, A’, B, and B’. The physicochemical properties of the aromatic cleavage product of territrem Aindicated the structure as 3,4-methylendioxy, 5-methoxy benzoic acid (or 4-methoxy, 6-carboxy, 1, 3-benzodioxole). The experiment also gave the evidences that territrem A and A’, on the other hand territrem B and B’ have the identical aromatic moieties on their structures.     

Learning enhancement and behavioral arousal induced by yohimbine

Learning of a food motivated delayed reinforcement autoshaping task was investigated in rats treated with water vehicle or the prototypical anxiogenic agent and alpha 2-adrenergic antagonist yohimbine (0.5 or 1.5 mg/kg, i.p. 30 min before behavioral testing). Unconditioned exploratory rearing activity was monitored concomitantly with acquisition of a lever touch response. The low dose of yohimbine enhanced learning, but it also increased unconditioned behavioral arousal. The high dose retarded acquisition, but when it was withdrawn the animals learned but exploratory activity increased beyond control levels prior to acquisition. Learning thus appeared to be related to the behavioral arousal produced by yohimbine, suggesting that learning enhancement by anxiogenic substances is not due to a direct effect on processes intrinsic to information storage and retrieval; rather, anxiogenic substances may be important modulators of vigilance and performance variables.     

The effect of light and phytochrome on 1-aminocyclopropane-1-carboxylic Acid metabolism in etiolated wheat seedling leaves

While light-grown wheat leaves produced ethylene at a low rate of <0.1 nanomoles per gram per hour and contained 1-aminocyclopropane-1-carboxylic acid (ACC) at low levels of <2.5 nanomoles per gram, etiolated wheat leaves produced ethylene at a rate of 2 nanomoles per gram per hour and accumulated concentrations of ACC at levels of 40 nanomoles per gram. Upon illumination of 8-day-old etiolated wheat seedlings with white light, the ethylene production rate increased initially, due to the activation of ethylene-forming activity, but subsequently declined to a low level (0.1 nanomoles per gram per hour) at the end of the 6-hour illumination. This light-induced decline in ethylene production rate resulted from a decline (more than 35 nanomoles per gram) in ACC level, which was accompanied by a corresponding increase in 1-(malonylamino)cyclopropane-1-carboxylic acid content. These data indicate that illumination promoted ACC malonylation, resulting in reduced ACC level and consequently reduced ethylene production. However, light did not cause any significant increase in the extractable ACC-malonyltransferase activity. The effect of continuous white light on promotion of ACC malonylation was also observed in intermittent white light or red light. A far-red light treatment following red light partially reversed the red light effect, indicating that phytochrome participates in the promotion of ACC malonylation.     

云南山楂茎尖的离体培养及其无性系快速繁殖

用云南山楂(Crataegus scabrifolia(Franch.)Rehd.)成年树茎尖和实生芽两种不同发育阶段的材料为外殖体,诱导它们休眠芽萌动,丛生芽条并诱导芽条生根。实验结果如下:1.以成年态的云南山楂侧芽为外植体,培养在附加IAA 0.1—0.5mg/l+6-BA 1-2mg/l的MS培养基上可诱导芽的萌发;将芽继代培养在附加0.5—1mg/l 6-BA的SH或MS培养基上,40天后芽数增殖4—6倍;将芽条截下置于1/2MS培养基上,附加不同浓度的IAA或IBA,可得到50—80％的生根率。2.以实生芽为外殖体,在相同条件下,则20天后芽数增殖便可获4—6倍;98％以上生根。结果表明:云南山楂的幼年态要比成年态易脱分化和再分化。     

大陆漂移与白蚁的系统发育

白蚁Termite是属于古老而又原始的一类昆虫。从许多生活习性和形态特征证明,等翅目Isoptera——白蚁与蜚蠊目Blattariae昆虫的亲缘关系十分接近,特别与隐尾蠊科Cryptocercidae的某些类群有着更为密切的关系。它们同以木材纤维为食,并且都是借助于体内共生的细菌和原生动物消化木纤维。当地球上高大乔木出现之后,就已经为这类蛀木昆虫的生存提供必要的生活条件。石炭纪Carboniferous是蜚蠊目昆虫最为繁盛的地质时期(林启彬,1978),为此,白蚁在地质历史上有可能与蜚蠊同时期,或接近     

Protein kinase C-dependent and -independent pathways of proto-oncogene induction in human astrocytoma cells

We compared the abilities of the muscarinic agonist carbachol, epidermal growth factor (EGF), and phorbol 12-myristate 13-acetate (PMA) to induce proto-oncogene mRNA accumulation and other cellular responses in normal and protein kinase C-deficient 1321-N1 human astrocytoma cells. PMA, carbachol, and EGF all stimulated rapid accumulation of mRNA for the proto-oncogenes c-fos and c-myc in the normal cells; in the protein kinase C-deficient cells, carbachol and EGF, but not PMA, retained this effect, which was not mimicked by the calcium ionophore A23187. Both carbachol and PMA activated protein kinase C in these cells, as evidenced by the stimulated phosphorylation of an acidic Mr 80,000 protein kinase C substrate protein with phosphoamino acid and peptide map identity. This response was mimicked by several other neurotransmitters in these cells, including epinephrine, histamine, oxotremorine, and serotonin, and was abolished in cells made protein kinase C-deficient by preincubation with high concentrations of PMA. Both PMA and carbachol promoted the phosphorylation of the ribosomal protein S6 and activated an S6 protein kinase in the normal but not in the protein kinase C-deficient cells. EGF, in contrast, did not appear to activate protein kinase C, but promoted the phosphorylation of S6 and activation of the S6 kinase in both normal and protein kinase C-deficient cells. We conclude that, in 1321-N1 cells, induction of c-fos and c-myc mRNA can occur through a protein kinase C-dependent pathway and one or more independent pathways, exemplified by the responses to carbachol and EGF in the protein kinase C-deficient cells.     

Purification and characterization of proteolytic fragments of lipocortin I that inhibit phospholipase A2

Human lipocortin I is a 38.5-kDa phospholipase A2 inhibitor that has been produced in Escherichia coli in large quantities by recombinant DNA technology (Wallner, B.P., Mattaliano, R.J., Hession, C., Cate, R. L., Tizard, R., Sinclair, L.K., Foeller, C., Chow, E.P., Browning, J.L., Ramachandran, K.L., and Pepinsky, R.B. (1986) Nature 320, 77-80). To localize the region within the protein responsible for its inhibitory activity, we generated a series of fragments of the recombinant product by limited proteolysis with elastase and characterized their structure by sequencing and peptide mapping. Five active fragments have been analyzed in detail. The smallest is an 18-kDa fragment derived from the amino-terminal half of lipocortin. Three of the larger fragments contain this region. The fifth fragment is missing 83 amino acids from the amino terminus. A region common to all the active fragments (amino acid residues 97-178) is 70% homologous with the corresponding region from a second member of the lipocortin family which recently was cloned (Huang, K-S., Wallner, B.P., Mattaliano, R.J., Tizard, R., Burne, C., Frey, A., Hession, C., McGray, P., Sinclair, L.K., Chow, E.P., Browning, J.L., Ramachandran, K.L., Tang, J., Smart, J.E., and Pepinsky, R.B. (1986) Cell 46, 191-199) and thus presumably is important for activity. In addition to inhibitory fragments, we have isolated a 3-kDa proteolytic fragment from the amino terminus of lipocortin I that contains the known phosphorylation site for protein-tyrosine kinases. Because of sequence homology of the 3-kDa fragment with biologically active synthetic peptides from pp60v-src and middle T antigen, its release by proteases may represent an important part of the activity of lipocortin.