King S M, Marchese-Ragona S P, Parker S K, Detrich H W
Department of Biology, Northeastern University, Boston, Massachusetts 02115, USA.
Biochemistry. 1997 Feb 11;36(6):1306-14. doi: 10.1021/bi962229q.
Adaptive compensation of enzymatic activities is common among cold-living poikilotherms. Their enzymes often demonstrate higher activities at low temperatures than do homologs from temperate or thermophilic species. To understand the molecular features necessary for cold adaptation of microtubule motor proteins, we have initiated studies of the flagellar dynein ATPases of Antarctic fishes (body temperature range = -1.8 to +2 degrees C). Dyneins were isolated by high-salt extraction of demembranated sperm axonemes from the Antarctic yellowbelly rockcod, Notothenia coriiceps. Although solubilization of inner arms was incomplete, an inner arm dynein was recognized as a discrete complex containing one major dynein heavy chain (DHC) and sedimenting through sucrose gradients at approximately 12 S. Like inner arm dyneins from Chlamydomonas, the fish complex contained an actin-immunoreactive protein of 43 kDa and a 30-kDa protein. One isoform of the inner arm DHC gene family of N. coriiceps was detected by the polymerase chain reaction, and Southern analysis established that this DHC gene is present at one copy per haploid genome. Outer arm dynein was extracted quantitatively by high-salt treatment, contained two DHCs (one major, one minor), and sedimented through sucrose gradients as a polydisperse, aggregating system. Associated with the outer arm DHCs were five presumptive intermediate chains (ICs) of 66-91 kDa, immunologically defined by their cross-reactivity to four monoclonal antibodies specific for ICs from other organisms. The basal (non-microtubule-stimulated) specific ATPase activities of the N. coriiceps inner and outer arm dyneins were approximately 0.07 and approximately 0.04 micromol of P(i) min(-1) mg(-1), respectively, at 0 degrees C, attained their maxima (approximately 0.1 micromol of P(i) min(-1) mg(-1)) at 9 and 19 degrees C, respectively, and at higher temperatures declined substantially. Furthermore, the activities of the fish dyneins at temperatures < or = 15 degrees C were significantly larger than that of outer arm dynein from the mesophile Tetrahymena. These results suggest that the greater catalytic efficiencies of N. coriiceps inner and outer arm dyneins at low temperatures are due to enhanced polypeptide flexibility in the active sites of their protein subunits. We conclude that temperature adaptation of flagellar dyneins from Antarctic fishes is compatible with substantial conservation of primary and quaternary structure.
酶活性的适应性补偿在低温生活的变温动物中很常见。它们的酶在低温下通常比来自温带或嗜热物种的同源酶表现出更高的活性。为了了解微管运动蛋白冷适应所需的分子特征，我们开始了对南极鱼类（体温范围=-1.8至+2摄氏度）鞭毛动力蛋白ATP酶的研究。通过高盐提取南极黄腹岩鳕（Notothenia coriiceps）去膜精子轴丝来分离动力蛋白。尽管内臂的溶解不完全，但一种内臂动力蛋白被识别为一个离散的复合物，包含一条主要的动力蛋白重链（DHC），并在蔗糖梯度中以约12 S的沉降系数沉降。与衣藻的内臂动力蛋白一样，鱼类复合物包含一种43 kDa的肌动蛋白免疫反应性蛋白和一种30 kDa的蛋白。通过聚合酶链反应检测到了南极黄腹岩鳕内臂DHC基因家族的一种同工型，Southern分析表明该DHC基因在每个单倍体基因组中以一个拷贝存在。外臂动力蛋白通过高盐处理被定量提取，包含两条DHC（一条主要的，一条次要的），并作为多分散的聚集体系在蔗糖梯度中沉降。与外臂DHC相关的是五条推定的66-91 kDa中间链（IC），通过它们与针对其他生物IC的四种单克隆抗体的交叉反应在免疫学上得以定义。南极黄腹岩鳕内臂和外臂动力蛋白在0摄氏度时的基础（非微管刺激）特异性ATP酶活性分别约为0.07和约0.04微摩尔Pi·分钟-1·毫克-1，分别在9摄氏度和19摄氏度时达到最大值（约0.1微摩尔Pi·分钟-1·毫克-1），在更高温度下大幅下降。此外，在温度≤15摄氏度时，鱼类动力蛋白的活性显著高于嗜温生物嗜热四膜虫的外臂动力蛋白。这些结果表明，南极黄腹岩鳕内臂和外臂动力蛋白在低温下更高的催化效率是由于其蛋白质亚基活性位点多肽灵活性的增强。我们得出结论，南极鱼类鞭毛动力蛋白的温度适应性与一级和四级结构的大量保守是相容的。
