Neumann E M
Department of Physiology, Carlsberg Laboratory, Copenhagen Valby.
Carlsberg Res Commun. 1988;53(7):395-409. doi: 10.1007/BF02904467.
The plastoquinone-binding polypeptide D-2 of the reaction center complex of photosystem II in barley has been expressed in vitro using the expression vector pGem3 containing the psbD gene of chloroplast DNA as template. The full length D-2 polypeptide with an apparent mol. weight of 32 kD is a major product when mRNA is transcribed with SP6 RNA polymerase from an insert containing the psbD gene with a 36 bp 5' leader sequence and a 3' tail of 99 bp downstream of the stop codon. Translation of the mRNA had to be done in a rabbit reticulocyte lysate. Translation also occurred from the internal AUG codons giving rise to truncated D-2 polypeptides with sizes of 30, 25 and 17 kD. They are immunoprecipitable with antisera either raised against amino acid residues 235 to 241 or against 345 residues of the D-2 polypeptide. A truncated translation product of 12 kD probably initiated at codon 247 is not recognized by either antiserum. An additional immunoprecipitable protein with an apparent molecular weight of 46 kD is observed by SDS-PAGE and is interpreted as a homomeric aggregate of D-2 polypeptides. Upon addition of methanol solubilized 2,6 dichloro-p-benzoquinone or other quinones a preferential translation of the full-length and truncated D-2 polypeptides is observed. The use of in vitro synthesized D-2 polypeptides for studies of binding quinones, other electron carriers and chlorophyll chromophores is discussed.
利用以叶绿体DNA的psbD基因为模板的表达载体pGem3，在体外表达了大麦光系统II反应中心复合物的质体醌结合多肽D-2。当用SP6 RNA聚合酶从一个含有psbD基因的插入片段转录mRNA时，该插入片段带有36 bp的5'前导序列和终止密码子下游99 bp的3'尾，表观分子量为32 kD的全长D-2多肽是主要产物。mRNA的翻译必须在兔网织红细胞裂解物中进行。从内部AUG密码子也会发生翻译，产生大小为30、25和17 kD的截短D-2多肽。它们可以被针对D-2多肽的235至241位氨基酸残基或345位氨基酸残基产生的抗血清免疫沉淀。一个可能起始于密码子247的12 kD截短翻译产物不能被任何一种抗血清识别。通过SDS-PAGE观察到一种表观分子量为46 kD的额外免疫沉淀蛋白，被解释为D-2多肽的同聚体聚集体。加入甲醇溶解的2,6-二氯对苯醌或其他醌后，观察到全长和截短D-2多肽的优先翻译。讨论了使用体外合成的D-2多肽研究醌、其他电子载体和叶绿素发色团的结合。
