Uriarte M, Stalmans W, Hickman S, Bollen M
Afdeling Biochemie, Fakulteit Geneeskunde, Katholieke Universiteit Leuven, Belgium.
Biochem J. 1993 Jul 1;293 ( Pt 1)(Pt 1):93-100. doi: 10.1042/bj2930093.
A glycoprotein fraction was isolated from rat liver membranes by affinity chromatography on immobilized wheat-germ lectin. Incubation of this fraction with MgATP or MgGTP resulted in a sequential phosphorylation and dephosphorylation of a complex of three polypeptides (118, 128 and 197 kDa on SDS/PAGE) with N-linked sialyloligosaccharides. Each polypeptide was recognized by polyclonal antibodies against recombinant plasma cell differentiation antigen PC-1. The relationship of the 118 kDa and 128 kDa polypeptides with PC-1 was confirmed by observations that they are linked by disulphide bonds into a larger protein, and that they are exclusively phosphorylated on Thr residues. Phosphorylation of p118, p128 and p197 only occurred after a lag period (up to 90 min at 30 degrees C), which lasted until most of the ATP had been converted to adenosine and Pi, with ADP and AMP as intermediate products. The length of the latency period increased with the concentration of initially added ATP (5-1000 microM) and could be prolonged by a second addition of similar concentrations of ATP, ADP, AMP and various nucleotide analogues. Most potent were the alpha beta-methylene derivatives of ADP and ATP. Adenosine was poorly effective. AMP, ADP, and perhaps ATP, emerge as the direct determinants of the latency. After further purification of the lectin-purified membrane fraction on anion-exchange and molecular-sieve columns, the complex of p118, p128 and p197 was still capable of autophosphorylation and dephosphorylation. The dephosphorylation was not affected by classical inhibitors (NaF, okadaic acid, EDTA, EGTA, phenylalanine). It was stimulated about 20-fold by various adenine nucleotides and analogues, with the same order of efficiency as noted for the induction of the latency. A similar stimulation of dephosphorylation was caused by 0.5 mM Na3VO4, which also prevented the phosphorylation of the three polypeptides. The likely explanation for the latency that precedes the phosphorylation of the membrane proteins is that the action of a protein kinase is initially offset by nucleotide-stimulated dephosphorylation.
通过固定化麦胚凝集素亲和层析从大鼠肝细胞膜中分离出一种糖蛋白组分。该组分与MgATP或MgGTP孵育会导致三种多肽（SDS/PAGE上为118、128和197 kDa）与N-连接唾液酸寡糖形成的复合物依次发生磷酸化和去磷酸化。每种多肽都能被抗重组浆细胞分化抗原PC-1的多克隆抗体识别。118 kDa和128 kDa多肽与PC-1的关系通过以下观察得到证实：它们通过二硫键连接成一种更大的蛋白质，并且它们仅在苏氨酸残基上发生磷酸化。p118、p128和p197的磷酸化仅在延迟期（30℃下长达90分钟）后发生，该延迟期一直持续到大部分ATP转化为腺苷和磷酸，ADP和AMP作为中间产物。延迟期的长度随最初添加的ATP浓度（5 - 1000 microM）增加，并且可以通过再次添加相似浓度的ATP、ADP、AMP和各种核苷酸类似物而延长。最有效的是ADP和ATP的αβ-亚甲基衍生物。腺苷效果不佳。AMP、ADP以及可能的ATP是延迟的直接决定因素。在阴离子交换和分子筛柱上对凝集素纯化的膜组分进一步纯化后，p118、p128和p197的复合物仍然能够进行自磷酸化和去磷酸化。去磷酸化不受经典抑制剂（NaF、冈田酸、EDTA、EGTA、苯丙氨酸）影响。它受到各种腺嘌呤核苷酸和类似物的约20倍刺激，刺激效率与延迟诱导的顺序相同。0.5 mM Na3VO4也引起类似的去磷酸化刺激，它还能阻止这三种多肽的磷酸化。膜蛋白磷酸化之前出现延迟的可能解释是，蛋白激酶的作用最初被核苷酸刺激的去磷酸化所抵消。
