创伤后多脏器功能衰竭患者白细胞流变性 和细胞粘附分子水平的变化

目的：探讨创伤后多器官功能衰竭（MOF）患者白细胞流变性和细胞粘附分子（CAMs）水平的变化。方法：采用DXC-300A型核孔膜红细胞变形能力测定仪、JYJ-Ⅲ型体外血栓血小板粘附两用仪、酶联免疫吸附法（ELISA）,分别检测了36例创伤后MOF患者、31例创伤患者和35例健康人外周血液白细胞变形能力（LD）、白细胞粘附功能（LAF）、白细胞CD18表达、血浆可溶性细胞间粘附分子-1（sICAM-1）和可溶性血管细胞间粘附分子-1（sVCAM—1）的变化。结果：MOF患者白细胞滤过指数（LFI）、白细胞粘附率（LAR）、白细胞CD180表达、sICAM—1,sVCAM—1均明显增高,与对照组和创伤组比较差异有极显著性（F=68．45-116．20,q=12．161—21、374,P〈0．001）,MOF组死亡者各指标变化较存活者更明显（t=6．920—11．665,P〈0,001）。MOF患者LFI和LAR与sICAM—1,sVCAM—1和白细胞CD18表达呈正相关（r=0．691～0．844,P〈0．001）,LFI与LAR呈正相关（r=0．771,P〈0．001）。结论：白细胞流变性和CAMs水平异常参与了MOF的发生,且与病情严重程度有密切关系。     

sICAM-1在多发性硬化症发病机制中的作用探讨

目的：探讨可溶性粘附分子－1（sICAM-1）在多发性硬化症发病机制中的作用。方法：用ELISA（双抗体夹心法）检测15例未经治疗的多发性硬化症患者血清中sICAM-1的水平,以15例正常人作对照。结果：患者血清中sICAM-1水平显著高于正常对照组（P＜0．01）;复发期患者sICAM-1水平显著高于缓解期。结论：sICAM-1可能是中枢神经系统急性炎症的标志之一;血清中sICAM-1水平的测定对临床诊断,预测复发具有一定意义。     

大肠癌患者血清和组织中sIL-2R和sICAM-1水平的变化及意义

目的：探讨可溶性白细胞介素2受体（sIL-2R）和可溶性细胞间粘附分子1（sICAM-1）的水平在大肠癌患者血清和组织中的变化及临床意义。方法：选择本院于2010年1月-2012年12月收治的55例大肠癌患者作为观察组,选取同期收治的41例大肠息肉患者作为对照组,38例健康志愿者作为健康组,应用双抗体夹心ELISA法对上述三组血清和组织中的sIL-2R和sICAM-1水平进行检测,对比分析上述指标的变化情况及其临床意义。结果：观察组血清和组织中sIL-2R水平分别为（210．3±128．5）pmol／L、（235．8±172．4）pmol／L,明显高于对照组与健康组,比较差异均有统计学意义（P〈0．05）,且sICAM-1平均水平为（392．4±133．3）p/μg／L,显著高于对照组和健康组,比较差异亦有统计学意义（P〈0．05）;观察组术后sICAM-2R和sICAM-1水平分别为（85．1±41．7）pmol／L、287.3±106．1（p／ug／L）,均显著低于术前,比较差异有统计学意义（P〈0．05）。结论：大肠癌患者机体中sIL-2R及sICAM-1的水平与疾病的发生和存在有密不可分的关系,故临床中,测定两指标的含量对大肠癌的诊断有一定的临床意义,可将其作为一个较为客观的指标进行检测。     

大肠癌患者血清和组织中sIL-2R 和sICAM-1 水平 的变化及意义

目的：探讨可溶性白细胞介素2 受体（sIL-2R）和可溶性细胞间粘附分子1（sICAM-1）的水平在大肠癌患者血清和组织中的 变化及临床意义。方法：选择本院于2010 年1月-2012 年12 月收治的55例大肠癌患者作为观察组,选取同期收治的41 例大肠 息肉患者作为对照组,38例健康志愿者作为健康组,应用双抗体夹心ELISA法对上述三组血清和组织中的sIL-2R 和sICAM-1 水平进行检测,对比分析上述指标的变化情况及其临床意义。结果：观察组血清和组织中sIL-2R 水平分别为（210.3 ± 128.5） pmol/L、（235.8 ± 172.4）pmol/L,明显高于对照组与健康组,比较差异均有统计学意义（P<0.05）,且sICAM-1 平均水平为（392.4± 133.3）p/ug/L,显著高于对照组和健康组,比较差异亦有统计学意义（P<0.05）;观察组术后sIL-2R和sICAM-1 水平分别为（85.1± 41.7）pmol/L、287.3 ± 106.1（p/ug/L）,均显著低于术前,比较差异有统计学意义（P<0.05）。结论：大肠癌患者机体中sIL-2R 及 sICAM-1 的水平与疾病的发生和存在有密不可分的关系,故临床中,测定两指标的含量对大肠癌的诊断有一定的临床意义,可将 其作为一个较为客观的指标进行检测。     

肝硬化患者血小板参数改变的临床意义

目的 观察肝硬化患者血小板参数改变的临床意义.方法 采用美国Coulter-Gen＇s全自动血细胞分析仪,检测90例肝硬化不同Child-pugh分级患者的血小板功能四项指标：血小板计数（PLT）、血小板平均体积（MPV）、血小板压积（PCT）和血小板分布宽度（PDW）;同步检测患者的凝血酶原时间（PT）.结果 肝硬化患者PLT、MPV和PCT均明显低于健康对照组（P＜0.01）;PDW高于对照组（P＜0.05）;PT明显高于对照组（P＜0.01）.结论 上述改变在对评估出血倾向以及肝硬化严重程度均有重要的辅助指导意义.     

血清PCT、sICAM-1联合HMGB1预测急性阑尾炎患儿手术切口感染风险的临床研究

摘要 目的：探讨血清降钙素原（PCT）、可溶性细胞间粘附分子-1（sICAM-1）联合高迁移率族蛋白B1（HMGB1）预测急性阑尾炎（AA）患儿手术切口感染风险的价值。方法：前瞻性选取2020年1月～2022年7月四川大学华西医院小儿外科收治的536例接受腹腔镜手术的AA患儿,根据术后是否发生手术切口感染分为感染组和未感染组。采用酶联免疫吸附法检测血清PCT、sICAM-1、HMGB1水平。采用多因素Logistic回归分析AA患儿手术切口感染的影响因素,采用受试者工作特征（ROC）曲线分析血清PCT、sICAM-1、HMGB1水平对AA患儿手术切口感染的预测价值。结果：536例AA患儿腹腔镜手术后手术切口感染发生率为11.94%（64/536）。与未感染组比较,感染组血清PCT、sICAM-1、HMGB1水平升高（P＜0.05）。多因素Logistic回归分析显示,病程＞24 h、阑尾穿孔、手术操作时间＞60 min、留置腹腔引流和PCT、sICAM-1、HMGB1水平升高为AA患儿手术切口感染的独立危险因素（P＜0.05）。ROC曲线分析显示,血清PCT、sICAM-1联合HMGB1预测AA患儿手术切口感染的曲线下面积（AUC）大于PCT、sICAM-1、HMGB1单独预测。结论：血清PCT、sICAM-1、HMGB1水平升高与AA患儿手术切口感染密切相关,血清PCT、sICAM-1联合HMGB1预测AA患儿手术切口感染的价值较高,可能成为AA患儿手术切口感染的辅助预测指标。     

冠心病病人血清脂联素与可溶性细胞间黏附分子-1的变化及意义

目的：探讨冠状动脉粥样硬化性心脏病（CHD）病人血清中脂联素与可溶性细胞间黏附分子-1（sICAM-1）水平的变化及临床意义。方法：选择冠心病病人59例,其中急性冠脉综合征（ACS）31例,稳定性心绞痛（SAP）28例,采用ELISA法检测病人血清脂联素、sICAM-1的浓度并与20例健康对照比较。结果：ACS组病人血清脂联素的浓度显著低于SAP组和对照组（P〈0．05）．sICAM-1浓度显著高于SAP组和对照组（P〈0．05）,血清脂联素的浓度与sICAM-1水平呈负相关（r=-0．295,P〈0．05）。结论：血清脂联素与sICAM-1水平的变化与冠脉病变的稳定性有关,检测血清中脂联素与slCAM-1对于冠心病的辅助诊断、病情判断、及预防治疗有重要临床意义。     

多囊卵巢综合征肥胖患者血清维生素D、铁蛋白水平、sICAM-1与胰岛素抵抗、糖脂代谢指标的相关性

摘要 目的：探讨多囊卵巢综合征（PCOS）肥胖患者血清维生素D、铁蛋白、可溶性细胞间粘附分子-1（sICAM-1）水平与胰岛素抵抗、糖脂代谢指标的相关性。方法：2018年8月到2021年11月,选择在本院妇科诊治的PCOS患者65例作为研究对象,分为PCOS肥胖组（n=30,体重指数＜28 kg/m²）和PCOS非肥胖组（n=35,体重指数<28 kg/m²）。检测与计算两组清维生素D、铁蛋白、sICAM-1、胰岛素抵抗、糖脂代谢指标并进行相关性分析。结果：两组的血清甲状腺素（T4）、促甲状腺激素（TSH）与泌乳素（PRL）对比差异无统计学意义（P>0.05）,肥胖组的血清促黄体生成素（LH）、促卵泡生成素（FSH）、睾酮（T）水平高于非肥胖组（P<0.05）。肥胖组的血清铁蛋白、sICAM-1水平高于非肥胖组,血清维生素D水平低于非肥胖组（P<0.05）。肥胖组的胰岛素抵抗指数（HOMA-IR）、胰岛素水平（FINS）、甘油三酯（TG）、总胆固醇（TC）、低密度脂蛋白胆固醇（LDL-C）较非肥胖组,高密度脂蛋白胆固醇（HDL-C）低于非肥胖组（P<0.05）。在PCOS肥胖患者中,Pearson分析显示血清维生素D、铁蛋白、sICAM-1与胰岛素抵抗、糖脂代谢指标都存在相关性（P<0.05）。结论：PCOS肥胖患者与非肥胖患者的血清维生素D、铁蛋白、sICAM-1、胰岛素抵抗、糖脂代谢指标水平存在差异,血清维生素D、铁蛋白、sICAM-1水平与胰岛素抵抗、糖脂代谢指标存在相关性。     

血流变学指标及血清学指标在骨折延迟愈合患者中的临床意义

目的:探讨血流变学和血清学指标在骨折延迟愈合患者中的变化及其临床意义。方法:随机选取2010年1月~2016年6月在我院进行手术治疗的骨折延迟愈合及骨折正常愈合患者各90例,分别为观察组与对照组,对比分析两组患者术后第1、8、12周时血清可溶性血管细胞黏附分子1(sVCAM-1)、胰岛素样生长因子1(IGF-1)、血小板衍生生长因子(PDGF)及人可溶性细胞间黏附分子1(sICAM-1)和红细胞刚性指数、红细胞聚集指数、血浆黏度的差异。结果:术后第1、8、12周两组血清学及血流变学各指标整体相比差异均具有统计学意义(均P0.05),且组内两两比较均具有统计学差异(均P0.05)。术后8、12周观察组血清s ICAM-1、sVCAM-1、红细胞刚性指数、红细胞聚集指数、血浆黏度均高于对照组,而血清PDGF、IGF-1均低于对照组,比较差异均具有统计学意义(均P0.05)。结论:骨折患者血清sICAM-1、PDGF、IGF-1、sVCAM-1及红细胞刚性指数、红细胞聚集指数、血浆黏度水平会随着病程进展发生变化,并且血清sICAM-1、sVCAM-1及红细胞刚性指数、红细胞聚集指数、血浆黏度水平的升高,血清PDGF、IGF-1水平的降低可能是引起骨折延迟愈合的重要因素,对于骨折患者的临床治疗具有重要临床意义。     

可溶性细胞间粘附分子在慢性肾脏病中的变化

目的:研究慢性肾病(CKD)患者血清可溶性细胞粘附分子-1(soluble intercellular adhesionmolecule-1,sICAM)的变化与临床意义。方法:用双抗体夹心ELISA方法,对52例CKD患者及20例健康对照人群的sICAM-1水平进行检测分析。52例CKD患者中,其中27例为CRF血液透析患者;25例肾功能正常CKD患者。结果:CKD组患者sICAM-1水平明显高于对照组(105.42±61.95)(P<0.01);肾功能正常CKD组和CKD-CRF组sICAM-1水平均显著高于对照组(P<0.01);CRF组sICAM-1水平明显低于肾功能正常CKD组(P<0.01);但高于对照组(84.80±19.61/164.08±70.66/54.61±5.48)(P<0.01)。结论:sICAM-1水平在慢性肾脏病中明显升高,CRF组病人sICAM-1水平低于CKD肾功能正常患者,提示透析过程中可能有sICAM溢出,吸附并丢失入透析液中(1),或可能是肾纤维化为主的病变使sICAM-1表达下降。     

几类异质小麦雄性不育系育性恢复性的细胞遗传学研究

系统调查了4类异质(粘果、易变、偏凸、二角山羊草细胞质)1BL/lRS、非1BL/1RS小麦雄性不育系与其恢复系杂种F减数分裂中期Ⅰ出现单价体细胞频率,以及后期Ⅰ出现落后染色体和染色体桥细胞频率,并对中、后期染色体变异率与杂种F自交结实率进行了相关分析.结果表明(1)1BL/1RS型杂种在中期Ⅰ、后期Ⅰ染色体变异率要明显高于非1BL/1RS杂种;(2)4类异源细胞质在非1BL/1RS杂种中有着明显提高单价体细胞频率的作用;(3)在1BL/1RS杂种中,1B@1BL/1RS杂合核型染色体联会松弛,对单价体频率的影响远大于异源细胞质的影响;(4)1BL/1RS型杂种自交结实率与中期出现单价体细胞频率不直接相关,而与后期出现落后染色体和染色体桥细胞的频率呈高度负相关;(5)非1BL/1RS型杂种在减数分裂中、后期染色体行为相对稳定,易恢复且恢复度高,很有实际利用价值.     

Stability of structures of the epsilon subunit and terminator of thermophilic ATPase

F1-type ATPase is the central enzyme for ATP synthesis in most organisms. Because of the extreme reconstitutability of thermophilic ATPase (TF1) and diversity of the minor subunits of F1 type ATPase, an operon coding for TF1 was isolated from DNA of thermophilic bacterium PS3, and its terminal region containing the epsilon subunit (TF1 epsilon) and terminator was sequenced. The primary structure of the epsilon subunit (Mr = 14 333) was deduced from the nucleotide sequence (396 base-pairs) and amino-acid sequence of its amino terminus. The conclusions drawn from the results are as follows. Homologies: TF1 epsilon shows only 6% homology with the epsilon subunits of eight species reported, but 50% homology with Escherichia coli epsilon and 41% with chloroplast. The residues having a tendency to form reverse turns (Gly, Pro and Tyr) and His are relatively well conserved. Unlike some F1 epsilon types TF1 epsilon has no ATPase inhibitor activity and is not homologous with ATPase inhibitor. TF1 epsilon is essential to connect F1 to F0, like the b subunit, and is weakly homologous with the b subunit of F0F1. The cause of 3 beta: 1 epsilon subunit stoichiometry: The ribosome binding sequence of TF1 epsilon is TAGGN7, which is incomplete compared with that of TF1 beta. The codon usage for TF1 epsilon is similar to that for TF1 epsilon. The cause of stability of TF1 epsilon and its gene: There are 18 ionic groups at the putative reverse turns and the N- and C-termini of TF1 epsilon, but only 10 ionic groups in the corresponding sites of E. coli epsilon subunit. These ionic groups enhance the external polarity of TF1 epsilon and may intensify subunit-subunit interaction. There is a terminator at the 3' end of the TF1 epsilon gene, which is stabilized by a long (13 base-pairs) stem.     

X-ray structure of the Ca2+-binding interaction domain of C1s. Insights into the assembly of the C1 complex of complement

C1, the complex that triggers the classical pathway of complement, is assembled from two modular proteases C1r and C1s and a recognition protein C1q. The N-terminal CUB1-EGF segments of C1r and C1s are key elements of the C1 architecture, because they mediate both Ca2+-dependent C1r-C1s association and interaction with C1q. The crystal structure of the interaction domain of C1s has been solved and refined to 1.5 A resolution. The structure reveals a head-to-tail homodimer involving interactions between the CUB1 module of one monomer and the epidermal growth factor (EGF) module of its counterpart. A Ca2+ ion is bound to each EGF module and stabilizes both the intra- and inter-monomer interfaces. Unexpectedly, a second Ca2+ ion is bound to the distal end of each CUB1 module, through six ligands contributed by Glu45, Asp53, Asp98, and two water molecules. These acidic residues and Tyr17 are conserved in approximately two-thirds of the CUB repertoire and define a novel, Ca2+-binding CUB module subset. The C1s structure was used to build a model of the C1r-C1s CUB1-EGF heterodimer, which in C1 connects C1r to C1s and mediates interaction with C1q. A structural model of the C1q/C1r/C1s interface is proposed, where the rod-like collagen triple helix of C1q is accommodated into a groove along the transversal axis of the C1r-C1s heterodimer.     

Structure of the carbohydrate moieties of bovine rhodopsin.

The sugar chains of bovine rhodopsin were released from the polypeptide moiety by hydrazinolysis and reduced with NaB[3H]4 after N-acetylation. The radioactive oligosaccharides thus obtained were fractionated into three components by paper chromatography. The structures of these components were elucidated as GlcNAc beta 1 leads to 2Man alpha 1 leads to 3 (Man alpha 1 leads to 6)Man beta 1 leads to 4GlcNAc beta 1 leads to 4GlcNAc, GlcNAc beta 1 leads to 2Man alpha 1 leads to 3(Man alpha 1 leads to 3 and 6 Man alpha 1 leads to 6)Man beta leads to 4GlcNAc beta 1 leads to 4GlcNAc, and GlcNAc beta 1 leads to 2Man alpha 1 leads to 3(Man alpha 1 leads to 3 (Man alpha 1 leads to 6)Man alpha 1 leads to 6)Man beta 1 leads to 4GlcNAc beta 1 leads to 4GlcNAc, by sequential exoglycosidase digestion, methylation analysis, and endo-beta-N-acetylglucosaminidase D digestion. The unusual features of the sugar chains of rhodopsin molecule seem to support the proposed processing pathway for the biosynthesis of asparagine-linked sugar chains of glycoproteins.     

Kinetic properties of binding of myosin subfragment-1 with F-actin in the absence of nucleotide

The rate constant for the binding of myosin subfragment-1 (S-1) with F-actin in the absence of nucleotide, k1, and that for dissociation of the F-actin-myosin subfragment-1 complex (acto-S-1), k-1, were measured independently. The rate of S-1 binding with F-actin was measured from the time course of the change in the light scattering intensity after mixing S-1 with various concentrations of F-actin and k1 was found to be 2.55 X 10(6) M-1 X S-1 at 20 degrees C. The dissociation rate of acto-S-1 was determined using F-actin labeled with pyrenyl iodoacetamide (Pyr-FA). Pyr-FA, with its fluorescence decreased by binding with S-1, was mixed with acto-S-1 complex and the rate of displacement of F-actin by Pyr-FA was measured from the decrease in the Pyr-FA fluorescence intensity. The k-1 value was calculated to be 8.5 X 10(-3) S-1 (or 0.51 min-1). The value of the dissociation constant of S-1 from acto-S-1 complex, Kd, was calculated from Kd = k-1/k1 to be 3.3 X 10(-9) M at 20 degrees C. Kd was also measured at various temperatures (0-30 degrees C), and the thermodynamic parameters, delta G degree, delta H degree, and delta S degree, were estimated from the temperature dependence of Kd to be -11.3 kcal/mol, +2.5 kcal/mol, and +47 cal/deg . mol, respectively. Thus, the binding of the myosin head with F-actin was shown to be endothermic and entropy-driven.     

Structural characterization of glycolipids of rat small intestine having one to eight hexoses in a linear sequence

Eight different fractions containing glycolipids with 1 to 8 hexoses in a linear sequence were isolated from rat small intestine. The structure of the major components was established by mass spectrometry and proton nuclear magnetic resonance spectroscopy of the permethylated and permethylated-reduced (LiAlH₄) derivatives and by gas-liquid chromatography of degradation products of the native and permethylated or permethylated-reduced glycolipids. The major compounds were glucosylceramide, lactosylceramide, globotriaosylceramide, and a novel tetrahexosylceramide with the structure Gal α 1 → 3Galα1 → 4Galβ1 → 4Glcβ1 → 1Cer. In addition four minor compounds having five to eight hexoses were identified with the probable structures Galα1 → 3Galα1 → 3Galα1 → 4Galβ1 → 4Glcβ1 → 1Cer, Galα1 → 3Galα1 → 3Galα1 → 3Galα1 → 4Galβ1 → 4Glcβ1 → 1Cer, Gal1 → 3Gal1 → 3Gal1 → 3Gal1 → 3Gal1 → 4Gal1 → 4Glc1 → 1Cer, and Gal1 → 3Gal1 → 3Gal1 → 3Gal1 → 3Gal1 → 3Gal1 → 4Gal1 → 4Glc1 → 1Cer. In the pentahexosylceramide fraction a novel fucolipid was also present having the probable structure Fucα1 → 2Galα1 → 3Galα1 → 4Galβ1 → 4Galβ1 → 1Cer. The lipophilic part of the glycolipids was composed of trihydroxy 18:0 and dihydroxy 18:1 long-chain bases in combination with nonhydroxy and hydroxy 16:0–24:0 fatty acids. Glycolipid studies of isolated mucosal epithelial cells and the nonepithelial intestinal residue revealed a specific cell distribution of these hexosyl compounds. The two major components, glucosylceramide and globotriaosylceramide, were mainly located in the epithelial cells together with small amounts of lactosylceramide and tetrahexosylceramide. The epithelial cells practically lacked however the penta- to octahexosylceramides. The nonepithelial residue contained all hexosyl compounds. The fucolipid was exclusively present in the epithelial cells.     

TPP1基因慢病毒干扰载体的构建及鉴定

目的:构建抑制TPP1基因的短发夹RNA(sh RNA)干扰载体。方法:以人的TPP1基因为靶序列,设计并合成sh RNA序列TPP1-si1和TPP1-si2,分别与RNA干扰慢病毒载体pll3.7连接,双酶切鉴定质粒得到阳性克隆pll-TPP1-si1和pll-TPP1-si2,经测序正确后将其与慢病毒包装载体(RRE、REV、VSVG)共转染293T细胞,进行病毒的包装,将得到的病毒感染稳定高表达外源TPP1蛋白的HT1080细胞,通过Western印迹检测其对TPP1蛋白表达的抑制效果,并进行比较;将抑制效果好的病毒感染高表达外源Pot1蛋白的Hep G2细胞,检测Pot1蛋白在内源TPP1被敲低的情况下能否在端粒定位。结果:经双酶切验证,外源片段成功插入载体pll-TPP1-si1和pll-TPP1-si2中;pll-TPP1-si1和pll-TPP1-si2均能明显抑制TPP1的表达,其中pll-TPP1-si1的抑制效果最好;pll-TPP1-si1病毒感染高表达外源Pot1蛋白的Hep G2细胞,经免疫荧光鉴定能够有效抑制内源TPP1的表达,使Pot1不再端粒定位。结论:构建的抑制TPP1基因的sh RNA干扰载体能有效抑制TPP1的表达,为端粒蛋白TPP1功能的研究奠定了实验基础。     

Synthesis and characterization of deoxy analogues of diphytanylglycerol phospholipids

Novel analogues of diphytanyl phospholipids, 2,3-diphytanyl sn-1-glycerol-1-phosphoryl-1'-(1',3'-propanediol) (dPG), 2,3-diphytanyl-sn-glycerol-1-phosphoryl-1'-propanol (ddPG) and 2,3-diphytanyl-sn-glycerol-1-phosphoryl-1'-(1',3'-propanediol-3'-p hosphate) (dPGP), were synthesized according to modifications of previously published procedures. The samples were TLC and analytically pure and were characterized by 13C- and 1H-NMR and negative FAB/MS. The pK values of dPGP in aqueous dispersions or in methanol/water (1:1, v/v) were determined by potentiometric titration and compared with those of 2,3-diphytanyl-sn-glycerol-1-phosphoryl-3'-sn-glycerol-1'-phosphat e (PGP). The dissociation constant of the third ionizable POH group of dPGP was more than 2 pK units higher than that of PGP, indicating that the free glycerol hydroxyl group plays an important role in headgroup conformation and stabilization, perhaps through hydrogen bonding with the phosphate group(s).     

Protein synthesis in yeast. Isolation of variant forms of elongation factor 1 from the yeast Saccharomyces cerevisiae

Two species of the elongation factor 1 (EF-1) differing in molecular weight, subunit composition, and isoelectric point have been isolated from cell-free extracts of the yeast Saccharomyces cerevisiae. The ratio of these two forms of EF-1 activity (EF-1 alpha and EF-1H) seem to vary in different strains and upon the growth phase from which the cells have been isolated. The log phase cells of a protease negative yeast strain EJ101 show a distribution of EF-1 alpha and EF-1H in the ratio of 3:1. Another laboratory yeast strain, D-587-4B, shows a distribution pattern of 4:1. The two forms of EF-1 are completely separable by ion exchange, gel permeation, and hydrophobic and affinity chromatography. Yeast EF-1 alpha is a single polypeptide of molecular weight 50,000 and has an isoelectric point of 8.9. The newly identified form of the yeast EF-1 (EF-1H) has a molecular weight of 200,000. The isoelectric point of this protein is around 5.5. Electrophoresis of the partially purified EF-1H in polyacrylamide gel containing sodium dodecyl sulfate indicates the presence of three nonidentical polypeptides having molecular weights of 50,000, 47,000, and 33,000. The three polypeptides are present in the ratio of 2:1:1. EF-1H is readily converted to EF-1 alpha and EF-1 beta gamma on anion exchange columns. The 50,000 dalton component of EF-1H immunologically cross-reacts with the antibody to EF-1 alpha. The other two polypeptides do not. On the basis of molecular weight, EF-1H is 2-3-fold more active than EF-1 alpha in poly(U)-dependent polyphenylalanine synthesis. EF-1H exchanges nucleotide (GDP----GTP) at a faster rate than EF-1 alpha. Both EF-1 alpha and EF-1H exhibit similar binding constants for GDP and GTP although the affinity of EF-1 alpha for guanine nucleotides is several-fold higher than that of EF-1H. The 33,000-dalton component of EF-1H appears to be functionally analogous to EF-1 beta (Ts) isolated from other eukaryotic sources. The function of EF-1 gamma is unknown.     

Cryoenzymic studies on actomyosin ATPase. Evidence that the degree of saturation of actin with myosin subfragment 1 affects the kinetics of the binding of ATP

The initial steps of actomyosin subfragment 1 (acto-S1) ATPase (dissociation and binding of ATP) were studied at -15 degrees C with 40% ethylene glycol as antifreeze. The dissociation kinetics were followed by light scattering in a stopped-flow apparatus, and the binding of ATP was followed by the ATP chase method in a rapid-flow quench apparatus. The data from the chase experiments were fitted to E + ATP in equilibrium (K1) E.ATP----(k2) E*ATP, where E is acto-S1 or S1. The kinetics of the binding of ATP to acto-S1 were sensitive to the degree of saturation of the actin with S1. There was a sharp transition with actin nearly saturated with S1: when the S1 to actin ratio was low, the kinetics were fast (K1 greater than 300 microM, k2 greater than 40 s-1); when it was high, they were slow (K1 = 14 microM, k2 = 2 s-1). With S1 alone K1 = 12 microM and k2 = 0.07 S-1. With acto heavy meromyosin (acto-HMM) the binding kinetics were the same as with saturated acto-S1, regardless of the HMM to actin ratio. The dissociation kinetics were independent of the S1 to actin ratio. Saturation kinetics were obtained with Kd = 460 microM and kd = 75 S-1. The data for the saturated acto-S1 could be fitted to a reaction scheme, but for lack of structural information the abrupt dependence of the ATP binding kinetics upon the S1 to actin ratio is difficult to explain.(ABSTRACT TRUNCATED AT 250 WORDS)