Erusalimsky J D, Brooks S F, Herget T, Morris C, Rozengurt E
Imperial Cancer Research Fund, Lincoln's Inn Fields, London, United Kingdom.
J Biol Chem. 1991 Apr 15;266(11):7073-80.
The complete amino acid sequence of 80 K, the major acidic protein kinase C (PKC) substrate of rat brain, was deduced from a cDNA nucleotide sequence. An open reading frame of 927 bases predicted a protein of 309 amino acid residues (Mr = 29,796, pI = 4.06). 58% of the deduced protein sequence was confirmed by Edman degradation of peptides generated by proteolysis of purified 80 K. The absence of internal methionine residues in the deduced amino acid sequence was confirmed by the inability to cleave 80 K with CNBr. Antiserum raised against a synthetic peptide corresponding to residues 298-309 of the predicted amino acid sequence recognizes the 80-kDa polypeptide in Western blots. The protein shows 65% sequence identity with a closely related PKC substrate from bovine brain. Genomic Southern blot analysis using a probe corresponding to a segment of the 80 K gene devoid of introns showed one major band. Northern blot analysis of rat brain RNA reveals a prominent transcript of 2.2 kilobases which hybridizes to 80 K cDNA. The amino acid composition and hydropathicity plot suggest an extended structure with no hydrophobic domains. The amino acid sequence showed many short repeats as well as several potential phosphorylation sites, five of which were for PKC, one was for both PKC and cyclic AMP-dependent protein kinase, and one for casein kinase II, and potential glycosylation sites. Indeed, carbohydrate moieties were detected on electroblots of purified 80 K using both a specific glycan stain and Galanthus nivalis plant lectin which binds to terminal D-mannose in the glycan moiety. This is the first time that this major PKC substrate has been identified as a glycoprotein.
大鼠脑主要酸性蛋白激酶C（PKC）底物80K的完整氨基酸序列由cDNA核苷酸序列推导得出。一个927个碱基的开放阅读框预测出一个由309个氨基酸残基组成的蛋白质（Mr = 29,796，pI = 4.06）。通过对纯化的80K进行蛋白水解产生的肽段进行埃德曼降解，证实了推导的蛋白质序列的58%。推导的氨基酸序列中不存在内部甲硫氨酸残基，这一点通过用溴化氰不能切割80K得到证实。针对与预测氨基酸序列的298 - 309位残基相对应的合成肽产生的抗血清在蛋白质印迹中识别80 kDa多肽。该蛋白质与来自牛脑的密切相关的PKC底物显示出65%的序列同一性。使用对应于80K基因无内含子片段的探针进行基因组Southern印迹分析显示一条主要条带。对大鼠脑RNA进行Northern印迹分析揭示了一个2.2千碱基的突出转录本，它与80K cDNA杂交。氨基酸组成和亲水性图谱表明该结构呈伸展状，没有疏水结构域。氨基酸序列显示出许多短重复序列以及几个潜在的磷酸化位点，其中五个是PKC的位点，一个是PKC和环磷酸腺苷依赖性蛋白激酶共有的位点，一个是酪蛋白激酶II的位点，还有潜在的糖基化位点。实际上，使用特异性聚糖染色剂和与聚糖部分末端D - 甘露糖结合的雪花莲植物凝集素在纯化的80K的电印迹上检测到了碳水化合物部分。这是首次将这种主要的PKC底物鉴定为糖蛋白。
