Allen C M, Muth J D, Gildersleeve N
Biochim Biophys Acta. 1982 Jul 20;712(1):33-41. doi: 10.1016/0005-2760(82)90081-9.
The CTP-dependent dolichol kinase from bovine liver microsomes was optimally extracted using either 0.5% sodium deoxycholate or 0.5% Triton X-100 containing 0.5 M NH4Cl. All activity was found in the supernatant fraction following high-speed centrifugation. This fraction was depleted of phospholipid (phospholipid remaining, less than 5% of total) by gel chromatography of the 0.5% deoxycholate extract. This partially purified enzyme was maximally activated 9- or 53-fold over controls in the presence of 0.1% deoxycholate or 0.1% Triton X-100, respectively. Stimulation of the kinase was also observed with mixtures of dimyristoylphosphatidylcholine and deoxycholate. The level of stimulation by these mixtures was up to 20-fold higher than that observed in controls having deoxycholate alone. Dimyristoylphosphatidylcholine alone was not stimulatory. A 1:1 molar ratio of Triton X-100 or deoxycholate to dimyristoylphosphatidylcholine was optimal for enzyme activation. The half-maximum velocity of the dephospholipidated enzyme at 1:1 molar ratio of detergent to dimyristoylphosphatidylcholine was obtained at 150 or 550 microM CTP in the presence of deoxycholate or Triton X-100, respectively. It has been observed, therefore, that dolichol kinase may be extracted from liver microsomes, depleted of endogenous phospholipids and activated by specific molar ratios of detergent to phospholipid.
使用含0.5 M氯化铵的0.5%脱氧胆酸钠或0.5% Triton X - 100可最佳地提取牛肝微粒体中依赖CTP的多萜醇激酶。高速离心后，所有活性均存在于上清液部分。通过对0.5%脱氧胆酸钠提取物进行凝胶色谱，该部分的磷脂被去除（剩余磷脂占总量的比例小于5%）。在分别存在0.1%脱氧胆酸钠或0.1% Triton X - 100的情况下，这种部分纯化的酶相对于对照分别被最大激活9倍或53倍。在二肉豆蔻酰磷脂酰胆碱和脱氧胆酸钠的混合物中也观察到了激酶的刺激作用。这些混合物的刺激水平比仅含脱氧胆酸钠的对照中观察到的刺激水平高20倍。单独的二肉豆蔻酰磷脂酰胆碱没有刺激作用。Triton X - 100或脱氧胆酸钠与二肉豆蔻酰磷脂酰胆碱的1:1摩尔比最有利于酶的激活。在分别存在脱氧胆酸钠或Triton X - 100的情况下，去污剂与二肉豆蔻酰磷脂酰胆碱的摩尔比为1:1时，去磷脂化酶的半最大速度分别在150或550 microM CTP时获得。因此，已经观察到多萜醇激酶可以从肝微粒体中提取，去除内源性磷脂，并通过去污剂与磷脂的特定摩尔比激活。
