Speziale M V, Detwiler T C
Department of Biochemistry, State University of New York Health Science Center, Brooklyn 11203.
J Biol Chem. 1990 Oct 15;265(29):17859-67.
The free thiols of platelet thrombospondin (TSP) were derivatized with labeled N-ethylmaleimide (NEM) or iodoacetamide (IAM). When Ca2+ was chelated with EDTA, 2.9 mol of NEM or 2.6 mol of IAM reacted/mol of native TSP. No additional thiols were found after denaturation with urea. Since TSP has three apparently identical polypeptide chains, this suggests one free thiol/polypeptide chain. Ca2+ protected all of the thiols from reaction with IAM. In Ca2+ about half the thiols reacted normally with NEM and the others were unreactive, indicating that the thiols of TSP are not identical. The number of reactive thiols as a function of [Ca2+] revealed a sigmoidal curve with a transition midpoint of 207 microM. The ability of analogs of NEM to compete for derivatization of the thiols with labeled NEM was greater with larger, more hydrophobic agents. Gel electrophoretic separation of labeled TSP that had been partially digested with thrombin and trypsin indicated that some of the label was in the C-terminal tryptic fragment but that most was in the adjacent trypsin-sensitive region. After cyanogen bromide cleavage of the labeled and reduced protein, four labeled fractions were obtained from a gel filtration column. With subsequent combinations of tryptic digestion and reversed-phase high performance liquid chromatography, labeled peptides were purified from these four fractions, and the amino acid sequences were determined. Twelve labeled cysteines were identified, each with a specific radioactivity less than that of the thiol labeling reagent, indicating that only a fraction of that cysteine in a population of TSP molecules was a free thiol at the time of derivatization. While 2 labeled cysteines are in the non-repeating C-terminal portion of the molecule, the other 10 labeled cysteines are in the adjacent trypsin-sensitive type 3 repeats proposed (Lawler, J., and Hynes, R. O. (1986) J. Cell. Biol. 103, 1635-1648) as the calcium-binding region of the molecule. The disulfide bonds most sensitive to reduction by dithioerythritol were also stabilized by Ca2+, implying location in the Ca2(+)-sensitive part of the molecule. It is proposed that one equivalent of free thiol/polypeptide chain is distributed among 12 different cysteine residues through an intramolecular thioldisulfide isomerization.
血小板凝血酶敏感蛋白（TSP）的游离巯基用标记的N - 乙基马来酰亚胺（NEM）或碘乙酰胺（IAM）进行衍生化。当Ca²⁺与EDTA螯合时，每摩尔天然TSP有2.9摩尔NEM或2.6摩尔IAM发生反应。用尿素变性后未发现额外的巯基。由于TSP有三条明显相同的多肽链，这表明每条多肽链有一个游离巯基。Ca²⁺保护所有巯基不与IAM反应。在Ca²⁺存在下，约一半的巯基能正常与NEM反应，其余的则无反应，这表明TSP的巯基并不相同。反应性巯基的数量作为[Ca²⁺]的函数呈现出一条S形曲线，转变中点为207微摩尔。NEM类似物与标记的NEM竞争巯基衍生化的能力，对于更大、更疏水的试剂更强。对经凝血酶和胰蛋白酶部分消化的标记TSP进行凝胶电泳分离表明，一些标记位于C末端胰蛋白酶片段中，但大多数位于相邻的对胰蛋白酶敏感的区域。对标记并还原的蛋白质进行溴化氰裂解后，从凝胶过滤柱上获得了四个标记组分。随后通过胰蛋白酶消化和反相高效液相色谱的组合，从这四个组分中纯化出标记肽段，并测定了氨基酸序列。鉴定出了12个标记的半胱氨酸，每个的比放射性都低于巯基标记试剂，这表明在衍生化时，TSP分子群体中的半胱氨酸只有一部分是游离巯基。虽然有2个标记的半胱氨酸位于分子的非重复C末端部分，但另外10个标记的半胱氨酸位于相邻提议的对胰蛋白酶敏感的3型重复序列中（Lawler，J.，和Hynes，R. O.（1986）J. Cell. Biol. 103，1635 - 1648），该区域被认为是分子的钙结合区域。对二硫苏糖醇还原最敏感的二硫键也被Ca²⁺稳定，这意味着它们位于分子对Ca²⁺敏感的部分。有人提出，每条多肽链中的一当量游离巯基通过分子内硫醇 - 二硫键异构化分布在12个不同的半胱氨酸残基之间。
