Thiel G, Schmidt W E, Meyer H E, Söling H D
Abteilung Klinische Biochemie, Zentrum Innere Medizin, Universität Göttingen, Federal Republic of Germany.
Eur J Biochem. 1988 Jan 4;170(3):643-51. doi: 10.1111/j.1432-1033.1988.tb13746.x.
Stimulation of secretion in exocrine glands by agonists involving cAMP as second messenger leads to the phosphorylation of the ribosomal protein S6 (protein I) and two other particulate proteins with apparent molecular masses of 24 kDa (protein II) and 22 kDa (protein III) [Jahn, R., Unger, C. & Söling, H. D. (1980) Eur. J. Biochem. 112, 345-352]. This report describes the purification and characterization of protein III. Solubilization studies indicate that protein III is an intrinsic membrane protein. It could be extracted from the endoplasmic reticulum membrane only with Triton X-100, SDS or concentrated formic or acetic acid. The purification of this protein involved extraction of the microsomes with Triton X-100, removal of the detergent by acetone precipitation, extraction of water-soluble proteins, lipids and lipoproteins, and preparative SDS polyacrylamide gel electrophoresis. The protein has a basic pI (greater than 8.7). For determination of the amino acid composition of protein III and for sequencing of its amino-terminal portion, the protein was electroeluted out off the gel, the detergent removed and the protein finally purified by reversed-phase HPLC. Protein III could be phosphorylated in vitro by the catalytic subunit of the cAMP-dependent protein kinase to a degree of approximately 0.14 mol phosphate/mol protein. The only phosphopeptide obtained after in vitro phosphorylation and subsequent tryptic or chymotryptic digestion was identical with the phosphopeptide obtained after stimulation of intact rat parotid gland lobules with isoproterenol. The sequence of this peptide was Lys-Leu-Ser(P)-Glu-Ala-Asp-Asn-Arg. It was confirmed by an analysis of the synthetic peptide following in vitro phosphorylation with cAMP-dependent protein kinase. The first 41 N-terminal residues of protein III were sequenced. So far no sequence homology with other known peptides or proteins could be found.
以环磷酸腺苷（cAMP）作为第二信使的激动剂刺激外分泌腺分泌，会导致核糖体蛋白S6（蛋白I）以及另外两种表观分子量分别为24 kDa（蛋白II）和22 kDa（蛋白III）的颗粒蛋白发生磷酸化[扬恩，R.，翁格尔，C. & 索林，H. D.（1980年）《欧洲生物化学杂志》112卷，345 - 352页]。本报告描述了蛋白III的纯化及特性。增溶研究表明蛋白III是一种内在膜蛋白。它只能用曲拉通X - 100、十二烷基硫酸钠（SDS）或浓甲酸或乙酸从内质网膜中提取出来。该蛋白的纯化过程包括用曲拉通X - 100提取微粒体，通过丙酮沉淀去除去污剂，提取水溶性蛋白、脂质和脂蛋白，以及制备性SDS聚丙烯酰胺凝胶电泳。该蛋白的等电点呈碱性（大于8.7）。为了测定蛋白III的氨基酸组成并对其氨基末端部分进行测序，将蛋白从凝胶中电洗脱出来，去除去污剂，最后通过反相高效液相色谱法对蛋白进行纯化。蛋白III在体外可被cAMP依赖性蛋白激酶的催化亚基磷酸化，磷酸化程度约为0.14摩尔磷酸/摩尔蛋白。体外磷酸化并随后经胰蛋白酶或胰凝乳蛋白酶消化后得到的唯一磷酸肽，与用异丙肾上腺素刺激完整大鼠腮腺小叶后得到的磷酸肽相同。该肽的序列为赖氨酸 - 亮氨酸 - 丝氨酸（磷酸化） - 谷氨酸 - 丙氨酸 - 天冬氨酸 - 天冬酰胺 - 精氨酸。通过用cAMP依赖性蛋白激酶进行体外磷酸化后对合成肽的分析得到了证实。测定了蛋白III的前41个氨基末端残基的序列。到目前为止，尚未发现与其他已知肽或蛋白有序列同源性。
