Nuclear magnetic resonance study of the impact of ribose 2′-O-methylation on the aqueous solution conformation of cytidylyl-(3′ → 5′)-cytidine

In order to obtain information about the conformational characteristics at the nearestneighbor level in the 2′-O-methylated region of t-RNA, as well as in the bizarre 5′-terminus of eucaryotic mRNA, a detailed nuclear magnetic resonance study of 2′-O-methyl-cytidylyl-(3′ → 5′)-cytidine (CmpC) was conducted. Proton spectra were recorded at 270 MHz in the Fourier mode in D₂O solutions, 0.01M, pD 7.3 in the temperature range 5–80°C. Complete accurate sets of nmr parameters were derived for each of the nucleotidyl units by a combination of homo-nuclear decouplings and simulation iteration methods. The data were translated into conformational parameters using procedures developed in earlier studies from these laboratories. It is shown that the ribofuranose ring exists at a ²E ? ³E equilibrium with clear preference [(75–80)%] for the ³E mode. The C(4′)-C(5′) and C(5′)-O(5′) bonds form a stable conformational network with outspoken preference for conformers in which Ψ₁, Ψ₂ ? 60° and ?₂ ? 180°. The orientation of the 3′-phosphate and 2′-O-methyl groups is such that ?₁′ ? 210° and ?″ ? 60°. The phosphodiester bonds are flexible and shift trends for base, H(1′), and H(5″) suggest the existence of a conformational blend of right-handed stack (g^?g^?), left-handed stack (g⁺g⁺), and unstacked arrays (tg^? and tg⁺). Elevation of temperature perturbs the ²E ? ³E equilibrium accompanied with modest depopulation of ψ₁, ψ₂ ? 60° and ?₂ ? 180° conformers. The major effect of elevation of temperature is in the increase of unstacked arrays at the expense of g^?g^? and g⁺g⁺ conformers. The shift trend of Cmp-H(3′) with temperature shows that torsional variation about O(3′)-P is facilitated by increase in temperature and the preferred rotamer about O(3′)-P in the unstacked form is t (ω₁′ = 180°). A detailed comparison of the aqueous solution conformations of CpC and CmpC reveals that 2′-O-methylation causes: (i) a reduction in the magnitude of _χ1; (ii) an increase in the population of ³E pucker at the 3′-nucleotidyl unit; and (iii) modest perturbations in the O(3′)-P and P-O(5′) bond conformations. Comparison of the aqueous solution conformations of AmpA and CmpC makes clear that the conformational properties of pyrimidine-pyrimidine and purine-purine dimers which carry a 2′-O-methylated 3′-nucleotidyl unit are significantly different.     

Cis-Trans equilibrium and kinetic studies of acetyl-L-proline and glycyl-L-proline

By means of carbon-13 nmr (at 25 MHz) the trans/cis conformer ratio in glycyl-L -proline has been measured in aqueous (D₂O) solution over the temperature range 33–96°C. It is found that ΔH⁰ = ?4.2 kJ/mole and ΔS⁰ = ?9.7 J/mole/K. Measurements of the T₁ values for the proline ring carbons yielded values consistent with a fast puckering process involving both the β- and γ-carbons. Measurements of the rate of cis-trans conformational interconversion in glycyl-L -proline, using complete line-shape analysis for the glycyl α-carbon resonance, gave values for the trans → cis isomerization as follows: ΔH^≠ = 83.5 ± 0.2 kJ/mole; ΔS^≠ = 0.0 ± 10 J/mole/K. A more approximate determination from coalescence temperature observations gave a value of ΔG^≠ of 82.0 ± 0.4 kJ/mole for this process in acetyl-L -proline in aqueous solution. The presence of 12M NaSCN lowered this barrier by ca. 2.6 kJ/mole. Such measurements are relevant to present theoretical models of the denaturation-renaturation processes in proteins, in which proline residues may play a key role.     

壳聚糖固定化木瓜蛋白酶的研究

以壳聚糖为载体,成二醛为交联剂将木瓜蛋白酶固定化。5％戊二醛在4-6℃下处理载体5h,加酶液(3.5mg/mL蛋白,pH7.2)固定12h,活力回收达32％,作用于酪蛋白的半衰期为36天,其表观K_m(酪蛋白)值为0.075％(W/V),溶液酶的K_m值为0.086％;最适pH7.0～7.5,溶液酶为7.0～8.5。固定化酶在pH8.5以下,溶液酶在9.0以下活力稳定。固定化酶在45℃以下,溶液酶在75℃以下稳定。用6mol/L脲洗脱固定化酶4次(5.5h)活力仍有54.5％。用固定化酶处理啤酒浊度比对照下降了1.5-3.7倍,蛋白质含量下降了44％,冷藏(4℃)120天无冷混浊现象发生并保持了啤酒原有风味和理化性状。     

减压病山羊纤维蛋白原肽链结构与空间构象的研究

本文报道了减压病山羊纤维蛋白原(FG)结构变化及减压病(DCS)气-血界面活性引起凝血反应的作用。雄性山羊15只,加压-减压发生Ⅰ或Ⅱ型DCS,采静脉血用冷乙醇提取血浆FG。经SDS-PG电泳和CM_(22)-色谱分离S-磺酸化FG,发现FG裂解肽段——带4和X、Y峰,(正常对照组无);经HPLC和组分分析发现FG含量和FG氨基酸残基数明显减少(P<0.05),表明FG参入凝血反应其肽链发生裂解。又经圆二色谱分析发现FG α-hilex％明显下降(P<0.01)。DCS山羊FG结构的改变,证实了气-血界面活性作用引起凝血反应。     

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。     

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。     

心磷脂-细胞色素C体系CD谱的研究

 线粒体内膜中含有特异的心磷脂是细胞色素C氧化酶活性的必需脂。本工作测定了心磷脂脂质体对细胞色素C溶液园二色(CD)谱的影响,发现心磷脂可引起血色素铁的氧化,并使其轴向配位场强的对称性下降。提示心磷脂可能参与酶和底物之间的电子转移过程。     

用亲和层析技术分离纯化天冬氨酸酶

<正> 亲和层析技术做为现代生化实验室常用的提纯方法,具有专一性强,活力损失小,分离步骤少等优点。天冬氨酸酶是重要的酶制剂,以往见报的分离纯化工艺步骤繁琐,活力损失大。本文报道的新工艺是用聚乙二醇(PEG)/磷酸钾双水相抽提;DEAE-sophadex A-50柱层析除PEG和部分杂蛋白;环氧氯丙烷活化载体,底物作配基的亲和层析技术获得了电泳纯的天冬氨酸酶。     

Purification and characterization of a pp60c-src-related tyrosine kinase that effectively phosphorylates a synthetic peptide derived from p34cdc2.

A protein tyrosine kinase has been purified from the particulate fraction of bovine spleen to a specific activity of 0.217 mumol/min/mg at 100 microM ATP and 3 mM [Val5] angiotensin II. Both the angiotensin phosphorylation activity and immunoreactivity towards an antibody preparation raised against a synthetic peptide containing the autophosphorylation site of pp60c-src, Cys-src(403-421), were monitored during the purification. The purified sample displayed three closely spaced protein bands with molecular weights of 50-55 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. All bands could be phosphorylated exclusively on tyrosine residues under autophosphorylation conditions. All reacted on immunoblots with an antibody raised against a synthetic peptide corresponding to the consensus autophosphorylation site of members of the pp60c-src family of tyrosine kinases. Tryptic phosphopeptide maps of the three proteins were essentially indistinguishable. The results suggest that the purified enzyme preparation contained mainly three closely related pp60c-src-family protein tyrosine kinases or a pp60src-family protein tyrosine kinase modified posttranslationally to give three closely spaced protein bands on sodium dodecyl sulfate gel. Neither of these proteins appears to be pp60c-src or p56lck. The spleen protein tyrosine kinase was found to phosphorylate a p34cdc2 kinase peptide, Cys-cdc2(8-20), which contained the regulatory tyrosine residue Tyr-15 about 20 times better than [Val5]angiotensin II or Cys-src(403-421) peptide at a peptide substrate concentration of 1 mM. In contrast, epidermal growth factor receptor kinase partially purified from A431 cells did not show preference for Cys-cdc2(8-20) as its substrate. Although Cys-cdc2(8-20) contained two tyrosine residues, only the tyrosine corresponding to Tyr-15 in p34cdc2 was phosphorylated by the spleen tyrosine kinase. The observation suggests that the primary structure surrounding Tyr-15 of p34cdc2 contains substrate structural determinants specific for the spleen tyrosine kinase.