Muschietti J P, Martinetto H E, Coso O A, Farber M D, Torres H N, Flawia M M
Instituto de Investigaciones en Ingeniería Genética y Biología Molecular, Facultad de Ciencias Exactas y Naturales, Buenos Aires, Argentina.
Biochem J. 1993 Apr 15;291 ( Pt 2)(Pt 2):383-8. doi: 10.1042/bj2910383.
G-protein subunits were characterized from Medicago sativa (alfalfa) seedlings. Crude membranes and GTP-Sepharose-purified fractions were electrophoresed on SDS/polyacrylamide gels and analysed by Western blotting with 9193 (anti-alpha common) and AS/7 (anti-alpha t, anti-alpha i1 and anti-alpha i2) polyclonal antibodies. These procedures led to the identification of a specific polypeptide band of about 43 kDa. Another polypeptide reacting with the SW/1 (anti-beta) antibody, of about 37 kDa, was also detected. The 43 kDa polypeptide bound specifically [alpha-32P]GTP by a photoaffinity reaction and was ADP-ribosylated by activated cholera toxin, but not by pertussis toxin. Irradiation of etiolated Medicago sativa protoplast preparations at 660 nm for 1 min produced a maximal increase in the guanosine 5'-[gamma-thio]triphosphate (GTP[35S])-binding rate. After this period of irradiation, the binding rate tended to decrease. The effect of a red-light (660 nm) pulse on the binding rate was reversed when it was immediately followed by a period of far-red (> 730 nm) illumination. These results may suggest that activation of GTP[S]-binding rate was a consequence of conversion of phytochrome Pr into the Ptr form.
从紫花苜蓿（苜蓿）幼苗中对G蛋白亚基进行了表征。将粗膜和经GTP-琼脂糖纯化的组分在SDS/聚丙烯酰胺凝胶上进行电泳，并用9193（抗α通用）和AS/7（抗αt、抗αi1和抗αi2）多克隆抗体通过蛋白质免疫印迹法进行分析。这些步骤鉴定出了一条约43 kDa的特异性多肽条带。还检测到了另一条与SW/1（抗β）抗体反应的约37 kDa的多肽。这条43 kDa的多肽通过光亲和反应特异性结合[α-32P]GTP，并被活化的霍乱毒素进行ADP核糖基化，但不被百日咳毒素作用。对黄化的紫花苜蓿原生质体制备物在660 nm下照射1分钟，可使鸟苷5'-[γ-硫代]三磷酸（GTP[35S]）结合率达到最大增幅。在此照射期后，结合率趋于下降。当红光（660 nm）脉冲后紧接着进行一段远红光（>730 nm）照射时，其对结合率的影响会逆转。这些结果可能表明GTP[S]结合率的激活是光敏色素Pr转化为Ptr形式的结果。
