An isothermal titration calorimetric method to determine the kinetic parameters of enzyme catalytic reaction by employing the product inhibition as probe.

An isothermal titration calorimetric (ITC) method was developed to measure the kinetic parameters of ribonuclease A catalytic hydrolysis of cytidine 2',3'-cyclic monophosphate. Employing the inhibition of product as a probe, the K(m), K(i), k(c), and DeltaH(m) can be determined by two simple calorimetric measurements. First, the substrate was titrated into the cell containing high concentration of enzyme. The molar reaction heat was calculated from the titration peak area divided by substrate moles per titration, and the initial catalytic reaction rate in the presence of various concentrations of product can be calculated from the peak height and the molar reaction heat. From Michaelis-Menten function in the presence of inhibitors, the relationship between K(m) and K(i) can be obtained. Then, the dissociation constant, which is equal to K(i), was measured by a regular ITC experiment. Thus, K(m) and k(c) can be calculated. The method developed here can be applied in other enzyme catalytic systems with inhibitive products.     

Selected contribution: effects of ischemia-reperfusion on vascular contractility and alpha(1)-adrenergic-receptor signaling in the rat tail artery.

To determine the effects of ischemia-reperfusion (I/R) on alpha(1)-adrenergic-receptor (alpha(1)-AR) functions, alpha(1)-AR-mediated contraction, inositol phosphate (IP) accumulation, and alpha(1)-AR-G protein coupling were examined in the tail arteries of anesthetized rats after 60 min of ischemia and 60 min of reperfusion. The contractile response to norepinephrine (NE) was significantly increased after I/R, whereas the contractile response to KCl remained unchanged. This was accompanied by a 69% increase in NE-stimulated IP accumulation. Furthermore, receptor-stimulated coupling of alpha(1a)-AR to G alpha(q/11) proteins was increased, whereas the coupling of alpha(1b)-AR or alpha(1d)-AR to their G proteins was not altered by I/R. These changes in vascular alpha(1)-AR function occurred without concurrent alteration in expression levels of membrane alpha(1)-AR subtypes or in the associated G proteins. These data demonstrate that I/R increases alpha(1a)-AR-G(q/11) protein coupling and alpha(1)-AR-stimulated IP accumulation in the tail artery. The alterations in alpha(1)-AR signaling are associated with and may underlie the enhanced contractile response of the tail artery to adrenergic stimulation after I/R.     

BJ-HCC-20, a potential novel cancer-testis antigen.

In an effort to identify novel Cancer-Testis genes, we analyzed the sequence in the q26-28 region of human X chromosome by several on-line tools. The candidate sequences were then confirmed by experiments. We have obtained a novel Cancer-Testis gene, BJ-HCC-20. In vivo, it was found to have two isoforms. In samples of liver, colon, gastric and lung cancer tested, the expression frequency of BJ-HCC-20 is 25%, 17%, 21% and 15%, respectively. Full-length cDNAs of both BJ-HCC-20 isoforms were isolated and their gene structures and promoter regions were characterized. BJ-HCC-20 might have implications in theoretical and practical tumor biology.     

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.     

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.     

心不甘中甾体皂甙元的分离和结构鉴定(2)

自心不甘(Tupistra aurantiaca Wall et Backer)根的醋酸乙酯萃取物经硅胶柱层析分离除可得到1β、2β、3β、4β、5β、7α-hexahydroxyspirost-25(27)-en-6-one外,还得到7个游离的甾体皂甙元A—G,其中A及B分别为3-epiruscogenin及3-epi-neoruscogenin,F为△~(25(27))-pentrogenin(6)、C、D和E系新化合物,经IR、MS、~1H NMR及~(13)C NMR谱鉴定分别推定为ranmogenin A(3)、B(4)和C(5)(兰茂甙元甲、乙和丙)。     

心不甘中甾体皂甙元的分离和结构鉴定(3)

本文报告心不甘(Tupistra aurantiaca Wall et Backer)根的粗甙经盐酸水解后的总甙元中分离的甙元E和F的结构鉴定。甙元F经鉴定为△~(25(27))-pentrogenin(2)。甙元E(ranmo-genin D)为新化合物,其结构经IR、MS、~1H NMR和~(13)C NMR谱分析推定如(1)所示。     

大鼠发育过程中肝、肺r-GT酶活性和定位

用生物化学和组织化学方法研究正常发育中大鼠肝、肺r-GT活性和定位。结果表明:肝r-GT活性自胚龄17天开始升高,21天达高峰,出生第一天明显降低,第六天降至接近成年低水平。在胚胎期肝r-GT主要位于肝细胞内,出生后则主要位于胆小管。该结果提示胚胎期肝r-GT主要参与肝细胞膜上氨基酸的转运。出生后可能主要参与解毒功能,大鼠肺r-GT活性随发育逐步升高,主要分布于肺支气管上皮细胞。提示肺r-GT可能参与解毒功能。