PURIFICATION AND POLYPEPTIDE COMPOSITION OF DYNEIN ATPASES FROM CHLAMYDOMONAS FLAGELLA
PURIFICATION AND POLYPEPTIDE COMPOSITION OF DYNEIN ATPASES FROM CHLAMYDOMONAS FLAGELLA
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DOI：
10.1002/cm.970020604
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发表时间：
1982-01-01
影响因子：
--
通讯作者：
WITMAN, GB
中科院分区：
文献类型：
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作者：
PFISTER, KK;FAY, RB;WITMAN, GB
用 0.6 M KCl 提取莱茵衣藻分离的脱膜鞭毛轴丝，溶解了总轴丝 Mg2+ 或 Ca2+-ATP 酶活性的 77-92%，在蔗糖密度梯度中沉淀为 18S 和 12S 峰。这 2 个峰的 ATPase 通过羟基磷灰石 (HAP) 柱色谱进一步纯化。从 HAP 柱洗脱的 18S 峰的 ATP 酶活性为与蛋白质峰一致的单峰。 HAP纯化的18S ATP酶具有.apprx的比活性。 2.0.+-. 0.5.mu.mol Pi 水解分钟/毫克并且与.apprx的4个高MW (HMW)多肽相关。 310,000-340,000道尔顿，2个78,000和69,000道尔顿的中间MW(IMW)多肽和8个7800-19,600道尔顿的低MW(LMW)多肽。当 12S 蔗糖梯度峰与拖尾肩一起在 HAP 上进行色谱分析时，根据相关多肽的沉降特性，ATP 酶活性在指定为 12S 和 10.5S 的 2 个峰中洗脱。 12S 峰含有单个动力蛋白 ATP 酶，其比活性为 .apprx。 0.6.+-. 0.3 .mu.mol Pi 水解 min/mg 并与 .apprx 相关。 330,000、21,700 和 18,100 道尔顿多肽。 10.5S峰含有多种HMW、IMW和LMW多肽；其中，1 个 HMW 多肽和 1 个 28,700 道尔顿多肽与 ATP 酶活性密切相关。纯化的 ATP 酶没有共同的多肽；因此，每个都代表一个离散的动力蛋白。基于纯化级分中回收的蛋白质，18S动力蛋白代表.apprx。总轴丝蛋白的9.2%； 12S动力蛋白代表.apprx。轴丝蛋白的4.7%。
Extraction of isolated, demembranated flagellar axonemes of C. reinhardii with 0.6 M KCl solubilized 77-92% of the total axonemal Mg2+ or Ca2+-ATPase activity, which sedimented as 18S and 12S peaks in sucrose density gradients. The ATPase of these 2 peaks were further purified by hydroxylapatite (HAP) column chromatography. The ATPase activity of the 18S peak eluted from the HAP column as a single peak coinciding with the protein peak. The HAP purified 18S ATPase had a specific activity of .apprx. 2.0 .+-. 0.5 .mu.mol Pi hydrolyzed min/mg and was associated with 4 high MW (HMW) polypeptides of .apprx. 310,000-340,000 daltons, 2 intermediate MW (IMW) polypeptides of 78,000 and 69,000 daltons and 8 low-MW (LMW) polypeptides of 7800-19,600 daltons. When the 12S sucrose gradient peak together with a trailing shoulder were chromatographed on HAP, the ATPase activity was eluted in 2 peaks designated 12S and 10.5S on the basis of the sedimentation properties of their associated polypeptides. The 12S peak contained a single dynein ATPase having a specific activity of .apprx. 0.6 .+-. 0.3 .mu.mol Pi hydrolyzed min/mg and associated with .apprx. 330,000-, 21,700- and 18,100-dalton polypeptides. The 10.5S peak contained several HMW, IMW and LMW polypeptides; of these, 1 HMW polypeptide and one 28,700-dalton polypeptide correlated well with the ATPase activity. The purified ATPases had no polypeptides in common; each therefore represents a discrete dynein. Based on protein recovered in the purified fractions, 18S dynein represents .apprx. 9.2% of the total axonemal protein; 12S dynein represents .apprx. 4.7% of the axonemal protein.