Casnellie J E, Harrison M L, Pike L J, Hellström K E, Krebs E G
Proc Natl Acad Sci U S A. 1982 Jan;79(2):282-6. doi: 10.1073/pnas.79.2.282.
The particulate fraction from a lymphoma cell line, LSTRA, was found to contain an apparent high level of tyrosine protein kinase activity. When this fraction was incubated with [gamma-32P]ATP in the presence of 10 mM MnCl2, hydrolyzed, and assayed, 70--80% of the radioactivity recovered in phosphoamino acids was in phosphotyrosine. Gel electrophoresis of the proteins showed that a large portion of the 32P was in a single protein with a molecular weight of approximately 58,000. The phosphorylated residue in this protein was identified as phosphotyrosine. Detergent extracts of the particulate fraction from LSTRA cells contained both the Mr 58,000 protein and the enzyme responsible for its phosphorylation. These extracts were found to catalyze the phosphorylation of the tyrosine residue in the synthetic peptide, Ile-Glu-Asp-Asn-Glu-Tyr-Thr-Ala-Arg-Gln-Gly, corresponding to the sequence around the tyrosine that is phosphorylated in pp60src; the Km for the peptide in this reaction was 5 mM. High-performance liquid chromatography was used to assay for this phosphorylation. A second peptide was synthesized that contained two additional arginine residues whose presence permitted the phosphorylation of the peptide to be measured by a simple assay using phosphocellulose paper. The Km for this peptide was 3--4 mM, indicating that the presence of the additional arginine residues did not alter the apparent affinity of the kinase for the peptide.
人们发现，淋巴瘤细胞系LSTRA的微粒部分含有明显高水平的酪氨酸蛋白激酶活性。当该部分在10 mM MnCl₂存在的情况下与[γ-³²P]ATP一起孵育、水解并进行测定时，在磷酸氨基酸中回收的放射性的70%-80%存在于磷酸酪氨酸中。蛋白质的凝胶电泳显示，大部分³²P存在于一种分子量约为58,000的单一蛋白质中。该蛋白质中的磷酸化残基被鉴定为磷酸酪氨酸。LSTRA细胞微粒部分的去污剂提取物既含有分子量为58,000的蛋白质，也含有负责其磷酸化的酶。这些提取物被发现能催化合成肽Ile-Glu-Asp-Asn-Glu-Tyr-Thr-Ala-Arg-Gln-Gly中酪氨酸残基的磷酸化，该肽对应于pp60src中被磷酸化的酪氨酸周围的序列；此反应中该肽的Km为5 mM。使用高效液相色谱法测定这种磷酸化。合成了另一种肽，它含有另外两个精氨酸残基，其存在使得可以通过使用磷酸纤维素纸的简单测定法来测量该肽的磷酸化。该肽的Km为3-4 mM，表明额外精氨酸残基的存在并未改变激酶对该肽的表观亲和力。
