Van Ness B G, Howard J B, Bodley J W
J Biol Chem. 1978 Dec 25;253(24):8687-90.
We have developed a method for the purification in micromole amounts of the trypsin-derived ADP-ribosyl peptide from diphtheria toxin-modified yeast elongation factor 2 (EF-2). EF-2 was partially purified (15 to 20% purity) by ammonium sulfate precipitation and DEAE-Sephadex chromatography. After [3H]ADP-ribosylation by [3H]nad+ and diphtheria toxin, EF-2 was digested with trypsin and a homogeneous [3H]ADP-ribosyl peptide was isolated by chromatography on DEAE-Sephadex and dihydroxyboryl-substituted cellulose. Based on the amount of ADP-ribose acceptor activity in the crude extract, the overall yield of the peptide was 35%. The yeast peptide contains an unusual amino acid (X) which is the site of ADP ribosylation and is apparently identical to the amino acid reported from rat liver EF-2 by Robinson et al. (Robinson, E. A., Hendriksen, O., and Maxwell, E.S. (1974) J. Biol. Chem. 249, 5088-5093). The sequence of the 15-residue yeast peptide was determined to be: Val-Asn-Ile-Leu-Asp-Val-Thr-Leu-His-Ala-Asp-Ala-Ile-X-Arg. The 11 COOH-terminal residues of this peptide and of the homologous 15-residue peptide reported by Maxwell and co-workers from rat liver EF-2 are identical.
我们开发了一种方法，可从白喉毒素修饰的酵母延伸因子2（EF-2）中纯化出微摩尔量的胰蛋白酶衍生的ADP-核糖基肽。通过硫酸铵沉淀和DEAE-葡聚糖凝胶色谱法对EF-2进行了部分纯化（纯度为15%至20%）。在用[³H]NAD⁺和白喉毒素进行[³H]ADP-核糖基化后，用胰蛋白酶消化EF-2，并通过DEAE-葡聚糖凝胶色谱和二羟基硼基取代纤维素色谱分离出一种均一的[³H]ADP-核糖基肽。根据粗提物中ADP-核糖受体活性的量，该肽的总产率为35%。酵母肽含有一种不寻常的氨基酸（X），它是ADP核糖基化的位点，显然与罗宾逊等人报道的大鼠肝脏EF-2中的氨基酸相同（罗宾逊，E.A.，亨德里克森，O.，和麦克斯韦，E.S.（1974年）《生物化学杂志》249，5088 - 5093）。确定了这种15个残基的酵母肽的序列为：Val-Asn-Ile-Leu-Asp-Val-Thr-Leu-His-Ala-Asp-Ala-Ile-X-Arg。该肽的11个羧基末端残基与麦克斯韦及其同事报道的大鼠肝脏EF-2的同源15个残基肽的相应残基相同。
