Nakazawa M, Hayashi H, Yoshida Y, Manabe K
Department of Biology, Yokohama City University, Japan.
Plant Cell Physiol. 1993 Jan;34(1):83-91.
Peptide fragments were obtained by limited proteolysis with trypsin and Staphylococcus aureus V 8 protease from either the PR or the PFR form of 121-kDa phytochrome purified from etiolated pea (Pisum sativum L.) shoots. Patterns of bands after polyacrylamide gel electrophoresis in the presence of SDS of the digests were different, with some bands appearing preferentially when the digestions were carried out with the PR or the PFR form. Amino-terminal sequences of the fragments were analyzed to determine the exact locations of the amino-termini of the fragments within the amino acid sequence of the apoprotein of pea phytochrome. The amino acid compositions of some of the sequenced fragments were determined in order to confirm the carboxy-terminal amino acids. Three cleavage regions were identified as kinetically favored sites of cleavage of PFR (Arg-746 to Lys-752, around Glu-877 and around Arg-1010), whereas only one was identified for PR (Glu-38 to Arg-62). Regions of Glu-255, Arg-383, Arg-583 to Glu-620 and Lys-1093 to Glu-1115 were also identified as potential sites of proteolytic cleavage in both forms of the phytochrome. Other cleavage sites, the specificities of which have not yet been determined, are Glu-404, Glu-695 and Lys-1045. Surface-exposed parts of phytochrome in the PR and PFR forms are discussed.
通过用胰蛋白酶和金黄色葡萄球菌V 8蛋白酶对从黄化豌豆（Pisum sativum L.）芽中纯化的121-kDa光敏色素的PR或PFR形式进行有限蛋白酶解来获得肽片段。在SDS存在下对消化产物进行聚丙烯酰胺凝胶电泳后的条带模式不同，当用PR或PFR形式进行消化时，一些条带优先出现。分析片段的氨基末端序列以确定片段的氨基末端在豌豆光敏色素脱辅基蛋白氨基酸序列中的精确位置。确定了一些测序片段的氨基酸组成以确认羧基末端氨基酸。确定了三个切割区域为PFR的动力学上有利的切割位点（Arg-746至Lys-752、Glu-877附近和Arg-1010附近），而PR仅确定了一个切割位点（Glu-38至Arg-62）。Glu-255、Arg-383、Arg-583至Glu-620和Lys-1093至Glu-1115区域也被确定为两种形式的光敏色素中潜在的蛋白水解切割位点。其他切割位点（其特异性尚未确定）为Glu-404、Glu-695和Lys-1045。讨论了PR和PFR形式的光敏色素的表面暴露部分。
