Ghosal D, Ankrapp D, Keenan T W
Department of Biochemistry and Nutrition, Virginia Polytechnic Institute and State University, Blacksburg 24061.
Biochim Biophys Acta. 1993 Jul 1;1168(3):299-306. doi: 10.1016/0005-2760(93)90186-d.
Secretion of milk lipid globules is achieved through encapsulation of triacylglycerol-rich lipid droplets in a specialized region of apical plasma membrane of mammary epithelial cells. A class of low molecular mass GTP-binding proteins were associated tightly with the lipid globule membrane, and these proteins appeared to change from peripheral to integral membrane proteins during intracellular growth and transit of lipid globule precursors. Inclusion of GTP or GTP gamma S in incubation medium stimulated secretion of lipids from primary cultures of permeabilized rat mammary epithelial cells. Six polypeptides with molecular masses between 28 and 21 kDa were detected by ability to bind GTP gamma S following separation of lipid-globule-associated proteins by SDS-PAGE and transblotting onto nitrocellulose. That all of these polypeptides were distinct immunologically from the archetype ras was evident from lack of immunoreactivity with p21 ras G-protein monoclonal antibody in Western blots. This monoclonal antibody bound to a 23 kDa polypeptide of lipid droplets that was not detected with the GTP gamma S binding assay. A 25 kDa component of milk lipid globules was a potent substrate for ADP-ribosylation by botulinum toxin C3, but cholera toxin was much less effective, suggesting that this component may belong to the rac class of G-proteins. The 21 kDa component was related immunologically to ADP ribosylation factor.
乳脂球的分泌是通过富含三酰甘油的脂滴在乳腺上皮细胞顶端质膜的特定区域被包裹来实现的。一类低分子量的GTP结合蛋白与脂球膜紧密相关，并且在脂球前体的细胞内生长和转运过程中，这些蛋白似乎从外周膜蛋白转变为整合膜蛋白。在孵育培养基中加入GTP或GTPγS可刺激通透化大鼠乳腺上皮细胞原代培养物中的脂质分泌。通过SDS-PAGE分离脂球相关蛋白并转印到硝酸纤维素膜上后，根据结合GTPγS的能力检测到六种分子量在28至21 kDa之间的多肽。在蛋白质印迹中，这些多肽与原型ras在免疫学上均不同，这从它们与p21 ras G蛋白单克隆抗体缺乏免疫反应性可以明显看出。这种单克隆抗体与脂滴的一种23 kDa多肽结合，而该多肽在用GTPγS结合试验中未被检测到。乳脂球的一种25 kDa成分是肉毒杆菌毒素C3进行ADP核糖基化的有效底物，但霍乱毒素的作用则小得多，这表明该成分可能属于rac类G蛋白。21 kDa成分在免疫学上与ADP核糖基化因子相关。
