Starace D M, Knox B E
Department of Biochemistry and Molecular Biology, SUNY Health Science Center, 750 East Adams Street, Syracuse, NY 13210, USA.
Exp Eye Res. 1998 Aug;67(2):209-20. doi: 10.1006/exer.1998.0507.
The short wavelength visual pigment from Xenopus responsible for vision in the blue/violet portion of the spectrum was characterized by sequence spectroscopic analysis. The amino acid sequence was deduced by sequencing clones isolated by reverse transcription PCR, from retinal cDNA and genomic libraries. The gene contains 5 exons spanning 8.4 kb of genomic DNA and produces an mRNA of 2.4 kb in length. The deduced amino acid sequence predicts a protein of 347 amino acids with 76-78% identity to other short wavelength opsins. The mRNA encoding the Xenopus violet pigment was detected using in situ hybridization in cones, comprising a few percent of the total photoreceptors in the adult retina. The Xenopus violet opsin cDNA, modified to contain an epitope from the carboxyl terminus of bovine rhodopsin, was expressed in COS1 cells by transient transfection and analysed by UV-visible absorption spectroscopy. The protein expressed in COS1 cells migrated at 34 kD and was glycosylated at a single site in the amino terminus, exhibiting a diffuse pattern on SDS PAGE similar to bovine rhodopsin expressed in COS1 cells. Following incubation with 11-cis retinal, a light-sensitive pigment was formed with the lambdamax=425+/-2 nm. A Schiff base linkage between retinal and the violet opsin was demonstrated by acid denaturation. Xenopus violet opsin was sensitive to hydroxylamine in the dark, reacting with a half-time of 5 min at room temperature. This is the first group S pigment for amphibians. The pigment was expressed and purified from COS1 cells in a form that has permitted for the first time determination of the extinction coefficient, reactivity to hydroxylamine and presence of a Schiff base.
通过序列光谱分析对非洲爪蟾负责光谱蓝/紫部分视觉的短波视觉色素进行了表征。通过对逆转录PCR从视网膜cDNA和基因组文库中分离的克隆进行测序，推导出了氨基酸序列。该基因包含5个外显子，跨越8.4 kb的基因组DNA，产生长度为2.4 kb的mRNA。推导的氨基酸序列预测该蛋白有347个氨基酸，与其他短波视蛋白的同源性为76 - 78%。使用原位杂交在视锥细胞中检测到编码非洲爪蟾紫色色素的mRNA，视锥细胞占成年视网膜中总光感受器的百分之几。将非洲爪蟾紫色视蛋白cDNA进行修饰，使其包含来自牛视紫红质羧基末端的一个表位，通过瞬时转染在COS1细胞中表达，并通过紫外-可见吸收光谱进行分析。在COS1细胞中表达的蛋白迁移率为34 kD，在氨基末端的单个位点进行糖基化，在SDS - PAGE上呈现出与在COS1细胞中表达的牛视紫红质相似的弥散模式。与11 -顺式视黄醛孵育后，形成了一种λmax = 425±2 nm的光敏色素。通过酸变性证明了视黄醛与紫色视蛋白之间的席夫碱连接。非洲爪蟾紫色视蛋白在黑暗中对羟胺敏感，在室温下反应的半衰期为5分钟。这是两栖动物的首个S组色素。该色素以一种首次允许测定消光系数、对羟胺的反应性和席夫碱存在形式从COS1细胞中表达并纯化出来。
