1,25-双羟胆钙化醇及其受体含量检测的实验研究

 建立了一种改良的血清1,25-双羟胆钙化醇(1,25-Dihydroxycholecalciferol,DHCC)超微量放射受体检测(RRA)技术。完成了灵敏度、精密度、准确度、稳定性及特异性等技术指标。报告了我国健康青年血清DHCC正常值;检测了先天性佝偻病、青春期佝偻病病人及患肾性骨病奶牛等血清DHCC水平。 根据配体与受体相互结合的定量关系,建立了DHCCR(DHCC受体)检测技术。在游离与结合配基分离方面,除建立与比较了DCC(葡聚糖包埋的活性炭)及HAP(羟基磷灰石)方法外,还首次将IEF(等电聚焦电泳)应用于DHCCR分离技术。对佝偻病鸡小肠粘膜上皮细胞受体含量进行了检测并比较了鸡小肠、输卵管壳腺及肝组织DHCCR含量。     

人白血病细胞癌性促凝物(CP)的分离纯化及其特性的初步研究

用三步纯化法从人M_3型白血病细胞中分离纯化出人类肿瘤癌性促凝物(CP)。促凝活性回收率为24％,CP纯化倍数为2481倍。纯化CP在SDS-PAGE上为单一区带,其理化和酶学特性类似于动物肿瘤CP,分子量约为70 000,PI为4.8,在FVⅡ缺乏血浆中以及在含有组织因子(TF)抑制剂情况下仍能激活FX。CP促凝活性能被半胱氨酸蛋白酶抑制剂HgCl_2抑制,纯化CP能与抗动物肿瘤CP抗体形成免疫沉淀反应。     

根霉葡萄糖淀粉酶的复性复活动力学研究

本文利用荧光、紫外差光谱研究了根霉葡萄糖淀粉酶在盐酸胍变性后的复性、复活动力学。结果表明,该酶在小于4mol/L盐酸胍中变性是可逆的,其复性过程遵循一级反应方程。酶复活过程是由两个一级反应组成的复合反应,构象变化速度与复活过程中较快的反应速度相差无几,这可能是在Trp及Tyr微区的构象变化基本完成之后,酶活力恢复还没有完成造成的。     

Mutational analysis of the transin (rat stromelysin) autoinhibitor region demonstrates a role for residues surrounding the "cysteine switch"

The family of mammalian extracellular matrix metalloproteases (MMPs) are secreted by cells in an inactive (latent) proenzyme form. A highly conserved amino acid sequence, PRCGVPDV, is found near the COOH-terminal end of the pro-domain of these MMPs and believed to act as an "autoinhibitor." Recent studies (Springman, E. B., Angleton, E. L., Birkedal-Hansen, H., and Wart, H. E. V. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 364-368) indicate the Cys of this sequence ligands to the active-site zinc keeping the proenzyme in an inactive state, and mutational analysis (Sanchez-Lopez, R., Nicholson, R., Gesnel, M. C., Matrisian, L. M., and Breathnach, R. (1988) J. Biol. Chem. 263, 11892-11899) suggests that the conserved residues surrounding this Cys are required for latency. We have constructed 16 new site-directed mutations of the PRCGVPDV autoinhibitor region of the MMP transin (rat stromelysin) and tested whether these mutant enzymes are produced in a latent or activated form. We find that the conserved Arg as well as the Cys are essential for maintaining latency. The Cys cannot be replaced by other zinc-liganding amino acids, and the Arg cannot be replaced by Lys. Residues immediately surrounding the Cys are sensitive to even conservative amino acid substitutions. We show that a synthetic peptide PRCGVPDV is capable of acting as a weak inhibitor of transin and that replacement of the Cys with a Ser abolishes inhibition by the peptide. A review of the current knowledge of MMP substrate specificity in combination with these new results suggests that the PRCGVPDV sequence does not inhibit activity by mimicking the known substrates of the protease.     

Phosphorylation causes a conformational change in the carboxyl-terminal domain of the mouse RNA polymerase II largest subunit

The carboxyl-terminal domain (CTD) of the largest subunit of eukaryotic RNA polymerase II can be phosphorylated by a p34cdc2/CDC28-containing CTD kinase. Phosphorylated serine (or threonine) is located at positions 2 and 5 in the repetitive heptapeptide consensus sequence Tyr1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7. We show here that phosphorylation of the mouse CTD retards its electrophoretic mobility in sodium dodecyl sulfate-polyacrylamide gels in a way similar to that observed for the II0 form of the largest subunit of RNA polymerase II phosphorylated in vivo. At the maximum level of phosphorylation by CTD kinase in vitro, there are 15-20 phosphates evenly distributed among the 52 heptapeptide repeats that comprise the mouse CTD. Gel filtration chromatography and sucrose gradient ultracentrifugation analyses indicate that phosphorylation induces a dramatic conformational change in the CTD with the phosphorylated form adopting a far more extended structure than the unphosphorylated CTD.     

Transport of organic cations by a renal epithelial cell line (OK)

The goal of this study was to determine the mechanisms involved in the transport of the organic cation, tetraethylammonium (TEA), across the apical membrane of OK cells. [14C]TEA accumulated in OK cell monolayers reaching equilibrium in 2 h. The uptake of [14C]TEA at equilibrium was dependent upon temperature and was inhibited by sodium azide and by various organic cations, including N1-methylnicotinamide (NMN), mepiperphenidol, and cimetidine but not by the organic anion, p-aminohippuric acid. The initial uptake of [14C]TEA was characterized by a saturable process. The mean +/- S.D. Km was 27.8 +/- 2.6 microM and the Vmax was 414 +/- 26.5 pmol/mg protein/min. Both an accelerated efflux and influx of [14C]TEA in the presence of a trans-gradient of unlabeled TEA and NMN was observed, whereas a deaccelerated influx and efflux was observed in the presence of a trans-gradient of mepiperphenidol. The mechanism of interaction between NMN and TEA was examined. NMN significantly increased the apparent Km (mean +/- S.D.) of TEA to 82.8 +/- 16.4 microM (p less than 0.001), whereas the Vmax (mean +/- S.D.) was only slightly affected (478 +/- 72 pmol/mg protein/min) suggesting a competitive inhibition. The stimulatory effect of trans-gradients of NMN on TEA transport was due to an increase in the Vmax of TEA suggesting that NMN trans-stimulates TEA transport by increasing the turnover rate of the exchanger. In the presence of an inwardly directed proton gradient, the efflux at 30 s of [14C]TEA from the OK cell monolayers was significantly accelerated (p less than 0.05). Studies with the pH-sensitive fluorescent probe, 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein, suggested that TEA could drive the countertransport of protons. In apical membrane vesicles prepared from OK cells, the uptake of [3H]NMN exhibited an apparent "overshoot phenomenon" in the presence of an initial outwardly directed proton gradient. Protons competitively inhibited TEA uptake suggesting that the proton/organic cation and the organic cation/organic cation self exchange mechanism are the same mechanism. This is the first report describing both TEA self-exchange and proton/TEA exchange in the apical membrane of a continuous cell line. OK cells are an excellent model for the study of organic cation transport across the apical membrane.     

Dissociative inhibition of dimeric enzymes. Kinetic characterization of the inhibition of HIV-1 protease by its COOH-terminal tetrapeptide

Human immunodeficiency virus 1 (HIV-1) protease is an aspartyl protease composed of two identical protomers linked by a four-stranded antiparallel beta-sheet consisting of the NH2- and COOH-terminal segments (Weber, I.T. (1990) J. Biol. Chem. 265, 10492-10496). Kinetic analysis of the HIV-1 protease-catalyzed hydrolysis of a fluorogenic substrate demonstrates that the enzyme is an obligatory dimer. At pH = 5.0, 0.1 M sodium acetate, 1 M NaCl, 1 mM EDTA buffer, 37 degrees C, the equilibrium dissociation constant, Kd = 3.6 +/- 1.9 nM. We found that the tetrapeptide Ac-Thr-Leu-Asn-Phe-COOH, corresponding to the COOH-terminal segment of the enzyme, is an excellent inhibitor of the enzyme. Kinetic analysis shows that the inhibitor binds to the inactive protomers and prevents their association into the active dimer (dissociative inhibition). The dissociative nature of this inhibition is consistent with the results obtained from sedimentation equilibrium experiments in which the apparent molecular weight of the enzyme was observed to be 20,800 +/- 1,500 and 12,100 +/- 300, in the absence and presence of the COOH-terminal tetrapeptide, respectively. The dissociation constant of the protomer-inhibitor complex is Ki = 45.1 +/- 1.8 microM. This is the first kinetic analysis and direct experimental demonstration of noncovalent dissociative inhibition.     

Lateral diffusion of membrane-spanning and glycosylphosphatidylinositol-linked proteins: toward establishing rules governing the lateral mobility of membrane proteins.

In the plasma membrane of animal cells, many membrane-spanning proteins exhibit lower lateral mobilities than glycosylphosphatidylinositol (GPI)-linked proteins. To determine if the GPI linkage was a major determinant of the high lateral mobility of these proteins, we measured the lateral diffusion of chimeric membrane proteins composed of normally transmembrane proteins that were converted to GPI-linked proteins, or GPI-linked proteins that were converted to membrane-spanning proteins. These studies indicate that GPI linkage contributes only marginally (approximately twofold) to the higher mobility of several GPI-linked proteins. The major determinant of the high mobility of these proteins resides instead in the extracellular domain. We propose that lack of interaction of the extracellular domain of this protein class with other cell surface components allows diffusion that is constrained only by the diffusion of the membrane anchor. In contrast, cell surface interactions of the ectodomain of membrane-spanning proteins exemplified by the vesicular stomatitis virus G glycoprotein reduces their lateral diffusion coefficients by nearly 10-fold with respect to many GPI-linked proteins.