Identification of ras and ras-related low-molecular-mass GTP-binding proteins associated with rat lung lamellar bodies.
Identification of ras and ras-related low-molecular-mass GTP-binding proteins associated with rat lung lamellar bodies.
复制标题
鉴定与大鼠肺板层体相关的 ras 和 ras 相关低分子量 GTP 结合蛋白。
DOI：
10.1165/ajrcmb/6.3.253
复制
发表时间：
1992
影响因子：
6.4
通讯作者：
Dickey,BF
中科院分区：
文献类型：
--
作者：
Rubins,JB;Panchenko,M;Shannon,TM;Dickey,BF
材料和方法材料雄性无病原体的Sprague-Dawley大鼠（约200 g）获自Charles River Laboratories（威尔明顿，MA）。[a-32 P] GTP（3，000 Ci/mmol）购自阿默舍姆（阿灵顿高地，IL）。单克隆抗体表面活性剂脱辅基蛋白A（SP-A）是大卫Phelps博士（哈佛大学，波士顿，MA）赠送的。抗G1和G2 α亚基和抗β 336亚基的抗体由Janet Robishaw博士（Geisinger Clinic，丹维尔，PA）提供.抗ras抗体是市售的单克隆抗体（RAS 10; NEN，Boston，~ A），其识别哺乳动物H-、N-和K-ras p21蛋白。抗res单克隆抗体Y13-259来自Jennifer Coburn（Tufts University，Boston，MA）。抗rab 3A单克隆抗体由Ian Macara博士（罗切斯特大学，罗切斯特，纽约州）提供。抗酵母Sec 4蛋白的兔抗血清由Peter Novick博士（耶鲁大学，纽黑文，康涅狄格州）提供。ADP-核糖基化因子（ARF/CF，一种支持霍乱毒素催化的Gs的ADP核糖基化的胞质19-kD LMM-GBP）和肉毒杆菌外切酶C3的抗体由Michael Gill博士（Tufts University，Boston，MA）慷慨提供，并且先前已经描述（16）。蛋白质定量和分离通过Peterson（17）描述的Lowry方法的改进来测定蛋白质，使用牛血清白蛋白作为标准。根据Laemmli（18）的方法，通过不连续十二烷基硫酸钠聚丙烯酰胺凝胶电泳（S~ S-PAGE）分离蛋白质，使用分子量标准品磷酸化酶B，95.5 kD;谷氨酸脱氢酶，55 kD;乳酸脱氢酶，36 kD;碳酸酐酶，29 kD;和乳球蛋白，18.4 kD（Diversified Biotech，Newton，MA）。
Materials and MethodsMaterials Male pathogen-free Sprague-Dawley rats (approximately 200 g) were obtained from Charles River Laboratories (Wilmington, MA).[a-32P] GTP (3,000 Ci/mmol) was purchased from Amersham (Arlington Heights, IL). Monoclonal antibody~ o surfactant apoprotein A (SP-A) was a gift from Dr. DaVId Phelps (Harvard University, Boston, MA). Antibodies to G, and G, a subunits and to the {336 subunit were provided by Dr. Janet Robishaw (Geisinger Clinic, Danville, PA). The anti-ras antibody was a commercially available monoclonal antibody (RAS 10; NEN, Boston,~ A) that reco~ nizes mammalian H-, N-, and K-ras p21 proterns. The anti-res monoclonal antibody Y13-259 was from Jennifer Coburn (Tufts University, Boston, MA). The anti-rab 3Amonoclonal antibody was provided by Dr. Ian Macara (University of Rochester, Rochester, NY). Rabbit antiserum against the yeast Sec4 protein was provided by Dr. Peter Novick (Yale University, New Haven, CT). Antibody to ADP-ribosylation factor (ARF/CF, a cytosolic 19-kD LMM-GBP that supports cholera toxin-catalyzed ADP ribosylation of Gs) and botulinum exoenzyme C3 were graciously provided by Dr. Michael Gill (Tufts University, Boston, MA) and have been previously described (16). All other reagents were the highest grade available, obtained from commercial sources.Protein Quantitation and Separation Protein was assayed by the modification of the Lowry method described by Peterson (17), using bovine serum albumin as a standard. Proteins were separated by discontinuous sodium dodecyl sulfate polyacrylamide gel electrophoresis (S~ S-PAGE) according to the method of Laemmli (18), using the molecular weight standards phosphorylase B, 95.5 kD; glutamate dehydrogenase, 55 kD; lactate dehydrogenase, 36 kD; carbonic anhydrase, 29 kD; and lactoglobulin, 18.4 kD (Diversified Biotech, Newton, MA).