转化淋巴细胞胰岛素受体酪氨酸蛋白激酶活性及细胞内源性底物的研究

本文对增殖期的淋巴细胞胰岛素依赖性酪氨酸蛋白激酶活性及内源性废物进行了分析研究。在纯化的健康人淋巴细胞中加入适量的植物血凝素(PHA),经过72h培养即成为转化淋巴细胞(增殖期淋巴细胞)。应用~(32)P参入实验,证实转化淋巴细胞胰岛素受体具有胰岛素依赖性的酪氨酸蛋白激酶活性,与未转化的对照组相比其活性增加约9倍。Scatchard分析表明转化后淋巴细胞膜表面胰岛素受体数增加3.5倍。应用抗酪氨酸磷酸酯抗体,对胰岛素作用前后的转化与未转化淋巴细胞内,酪氨酸残基磷酸化的蛋白进行了鉴定,结果表明:除了95kD受体β亚基自身磷酸化外,45kD蛋白质也明显磷酸化,我们命名它为PP45。我们认为PP45可能是淋巴细胞中胰岛素受体酪氨酸蛋白激酶的主要内源性废物,它的磷酸化是胰岛素信息传递过程级联反应的初始步骤。     

重组白细胞介素-2纯化过程中影响活性的因素探讨

<正> 白细胞介素-2(Interleukin-2,IL-2)是一种重要的免疫调节剂,具有促进T细胞增殖和分化,调节NK细胞活性,诱导LAK等功能,在肿瘤治疗上有广泛的应用前景。我们的前文就重组菌中IL-2包含体的制备、性质及重组rIL-2的纯化进行了研究,本文就重组rIL-2纯化过程中影响其活性的有关因素进行了探讨。     

肌钙蛋白C(Tropnin C)的提纯与鉴定

<正> 肌钙蛋白C(TnC)是肌钙蛋白复合体的钙结合亚基,系骨骼肌和心肌收缩系统的触发因子。研究表明TnC与钙调素(CaM)类似,具有EF手结构和四个特殊的钙结合区。TnC与Ca~(2+)、Mg~(2+)等金属离子的结合可发生构型变化及由此导致的体内一系列酶活性等生理功能的变化。提纯TnC是进行其诸方面研究的基础。     

恒河猴心房利钠多肽的分离、纯化及活性研究

<正> 1983年以来,人们相继从人和大鼠等动物的心房中分离到心房利钠多肽(ANP)。但对非人灵长类动物——恒河猴的ANP了解甚少。由于ANP对机体循环系统的调节起着重要作用,其强大的降压、利钠、利尿的生理功能,在心血管疾病的防治方面具有重要意义。我们曾报导过恒河猴心房肌细胞中存在着大量的ANP样物质,随后对此物质进行了分离、纯化及活性检测,现简报如下。     

6_m2细胞转化与肌醇磷脂代谢相关性研究

肌醇磷脂代谢与V-mos癌基因转化细胞的相关性,迄今为止未见报导。本文用6m2细胞(Moloney鼠类肉瘤病毒(含V-mos)温度敏感突变株(MoMuSVts110)转化的NRK细胞)为模型,探讨了肌醇磷脂代谢与细胞转化的相关性。在33℃ (转化型温度)时,细胞内PIP(磷脂酰肌醇-4-磷酸)含量明显高于39℃(正常型温度),显示出转化型6m2细胞中存在一个提高的PI激酶活性。同时可见DG(二酰甘油)和IP_3(肌醇三磷酸)含量和蛋白激酶C(PKC)活性均明显高于正常型细胞。当细胞由39℃转至33℃10min,PIP、DG、IP_3含量和PKC活性均明显增加,并伴随有PKC活性由胞质向质膜上的转移。实验结果表明肌醇磷脂代谢参与了6m2细胞转化过程。文中对其作用机理进行了讨论。     

用固定化方法研究超氧化物歧化酶的活性与结构的关系

 采用吸附、包埋、共价交联等方法固定化超氧化物歧化酶(SOD)所得固定化酶活力回收都不高,表明酶的催化反应速率受超氧阴离子自由基(O_2~-)扩散速率的控制。用海藻酸钠包埋SOD,固定化酶不表现活力,破固定化后所得的糊状物却有很高的活力。用戊二醛交联所得的固定化酶活力回收率也很低,表明ε-NH_3~+的正电荷是酶活力所必需。     

The MAS-encoded processing protease of yeast mitochondria. Interaction of the purified enzyme with signal peptides and a purified precursor protein

The matrix of yeast mitochondria contains a chelator-sensitive protease that removes matrix-targeting signals from most precursor proteins transported into this compartment. The enzyme consists of two nonidentical subunits that are encoded by the nuclear genes MAS1 and MAS2. With the aid of these cloned genes, we have now overexpressed the active holoenzyme in yeast, purified it in milligram amounts, and studied its biochemical and physical properties. Atomic absorption analysis shows that the purified enzyme lacks significant amounts of zinc, manganese, or cobalt; if none of these metal ions is added during the assay, the enzyme is catalytically inactive but can still cleave substoichiometric amounts of substrate. The amino-terminal sequences of the two mature subunits were determined; comparison with the deduced amino acid sequences of the corresponding precursors revealed that the MAS1 and MAS2 subunits are synthesized with prepeptides composed of 19 and 13 residues, respectively, which have similar sequences. The enzyme is inhibited competitively by chemically synthesized matrix-targeting peptides; the degree of inhibition correlates with the peptides' targeting efficacy. Matrix-targeting peptides containing the cleavage site of the corresponding authentic precursor protein are cleaved correctly by the purified enzyme. A purified artificial precursor protein bound to the holoenzyme can be photocross-linked to the MAS2 subunit.     

The cDNA for rat selenoprotein P contains 10 TGA codons in the open reading frame

Selenoprotein P is a plasma protein recently purified and characterized as containing 7.5 +/- 1.0 selenium atoms/molecule as selenocysteine. In rats maintained on a defined diet containing nutritionally adequate amounts of selenate as the sole selenium source, over half the selenium in plasma is accounted for by selenoprotein P. Its cDNA has been cloned from a rat liver library and sequenced. The sequence is highly unusual, containing 10 TGA codons in its open reading frame prior to the TAA termination codon. TGA designates selenocysteine in other selenoproteins, and limited peptide sequencing that included the amino acids encoded by two of the TGA codons verified that they correspond to selenocysteine. The deduced 366-amino acid sequence is histidine- and cysteine-rich and contains 9 of its selenocysteines in the terminal 122 amino acids. Comparison of the deduced amino acid sequence of selenoprotein P with those of other selenoprotein reveals no significant similarities. Selenoprotein P represents a new class of selenoproteins and is the first protein described with more than 1 selenocysteine in a single polypeptide chain. The primary structure of selenoprotein P suggests that it might be responsible for some of the antioxidant properties of selenium.     

Freezing of phosphocholine headgroup in fully hydrated sphingomyelin bilayers and its effect on the dynamics of nonfreezable water at subzero temperatures.

Differential scanning calorimetry (DSC) and nuclear magnetic resonance (NMR) spectroscopy are applied to characterize the nonfreezable water molecules in fully hydrated D2O/sphingomyelin at temperatures below 0 degrees C. Upon cooling, DSC thermogram displays two thermal transitions peaked at -11 and -34 degrees C. The high-temperature exothermic transition corresponds to the freezing of the bulk D2O, and the low-temperature transition, which has not previously been reported, can be ascribed to the freezing of the phosphocholine headgroup in the lipid bilayer. The dynamics of nonfreezable water are also studied by 2H NMR T1 (spin-lattice relaxation time) and T2e (spin-spin relaxation time obtained by two pulse echo) measurements at 30.7 MHz and at temperatures down to -110 degrees C. The temperature dependence of the T1 relaxation time is characterized by a distinct minimum value of 2.1 +/- 0.1 ms at -30 degrees C. T2e is discontinuous at temperature around -70 degrees C, indicating another freezing-like event for the bound water at this temperature. Analysis of the relaxation data suggest that nonfreezable water undergoes both fast and slow motions at characteristic NMR time scales. The slow motions are affected when the lipid headgroup freezes.