Imaoka T, Lynham J A, Haslam R J
J Biol Chem. 1983 Sep 25;258(18):11404-14.
Secretion of platelet granule constituents is closely associated with the phosphorylation of a cytosol polypeptide of Mr = 47,000 that we have called P47 (Haslam, R. J., Lynham, J. A., and Fox, J. E. B. (1979) Biochem. J. 178, 397-406). This polypeptide is a substrate of Ca2+-activated phospholipid-dependent protein kinase (Kawahara, Y., Takai, Y., Minakuchi, R., Sano, K., and Nishizuka, Y. (1980) Biochem. Biophys. Res. Commun. 97, 309-317). Two-dimensional gel electrophoresis of protein from human platelets that had been preincubated with 32Pi demonstrated the presence under control conditions of 2-3 major forms of P47 that contained very little 32P (pI values, 6.6-6.8) and, after induction of secretion with thrombin, their replacement by 7-9 highly labeled phosphorylated forms of P47 (pI values, 6.1-6.5). Native phosphorylated P47 was purified from thrombin-stimulated 32P-labeled platelets by ammonium sulfate fractionation and column chromatography on DEAE-cellulose, phenyl-Sepharose, and hydroxylapatite. The final 32P-labeled product was obtained in a yield of 20-25% and was purified about 400-fold relative to platelet lysate. This material was homogeneous on sodium dodecyl sulfate-polyacrylamide gel electrophoresis but, like the starting material, contained 7-9 separable phosphorylated components with different pI values. Purified phosphorylated P47 had a sedimentation coefficient (s20,w) of 3.57 S and a Stokes radius of 3.33 nm from which an Mr = 49,000 and a frictional ratio (f/f0) of 1.4 were calculated. These findings and failure to detect multimers after treatment of the protein with dimethyl suberimidate indicate that P47 normally exists as a monomer. The 32P-labeled phosphate present in purified P47 had the chemical stability of serine or threonine phosphoesters and analysis indicated the presence of 83% phosphoserine and 17% phosphothreonine. Limited proteolysis of purified 32P-labeled P47 by Staphylococcus aureus V8 protease generated a major unlabeled fragment (Mr = 23,500) and up to six labeled fragments (Mr = 24,700-14,800), the relative amounts of the latter depending on the extent of proteolysis. The same labeled fragments were obtained after proteolysis of each of the major phosphorylated components of P47, suggesting that these represent different phosphorylation states of variants of the same protein and that most or all of the phosphorylation sites are on a single 14,800-Da segment of the protein. The availability of pure native phosphorylated P47 should facilitate investigation of the physiological role of this protein in platelets.
血小板颗粒成分的分泌与一种分子量为47,000的胞质溶胶多肽（我们称之为P47）的磷酸化密切相关（哈斯拉姆，R. J.，林厄姆，J. A.，以及福克斯，J. E. B.（1979年）《生物化学杂志》178卷，397 - 406页）。这种多肽是钙激活的磷脂依赖性蛋白激酶的底物（川原，Y.，高井，Y.，皆口，R.，佐野，K.，以及西冢，Y.（1980年）《生物化学与生物物理学研究通讯》97卷，309 - 317页）。对预先用³²P₁孵育的人血小板蛋白质进行二维凝胶电泳，结果表明在对照条件下存在2 - 3种主要形式的P4₇，其³²P含量极少（等电点值为6.6 - 6.8），在用凝血酶诱导分泌后，它们被7 - 9种高度标记的磷酸化形式的P4₇所取代（等电点值为6.1 - 6.5）。通过硫酸铵分级分离以及在DEAE - 纤维素、苯基 - 琼脂糖和羟基磷灰石上进行柱色谱，从经凝血酶刺激的³²P标记的血小板中纯化出天然磷酸化的P4₇。最终的³²P标记产物的产率为20 - 25%，相对于血小板裂解物纯化了约400倍。该物质在十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳上是均一的，但与起始材料一样，含有7 - 9种可分离的具有不同等电点值的磷酸化成分。纯化的磷酸化P4₇的沉降系数（s₂₀,w）为3.57 S，斯托克斯半径为3.33 nm，据此计算出其分子量为49,000，摩擦比（f/f₀）为1.4。这些发现以及在用亚胺基二甲酯处理该蛋白质后未检测到多聚体，表明P4₇通常以单体形式存在。纯化的P4₇中存在的³²P标记的磷酸盐具有丝氨酸或苏氨酸磷酸酯的化学稳定性，分析表明存在83% 的磷酸丝氨酸和17% 的磷酸苏氨酸。用金黄色葡萄球菌V8蛋白酶对纯化的³²P标记的P4₇进行有限蛋白酶解，产生了一个主要的未标记片段（分子量为23,500）和多达六个标记片段（分子量为24,700 - 14,800），后者的相对量取决于蛋白酶解的程度。在对P4₇的每种主要磷酸化成分进行蛋白酶解后也获得了相同的标记片段，这表明这些片段代表同一蛋白质变体的不同磷酸化状态，并且大多数或所有磷酸化位点都在该蛋白质的一个14,800道尔顿的单一区段上。纯天然磷酸化P4₇的可得性应有助于研究该蛋白质在血小板中的生理作用。
