Wu J K, Sheffield W P, Blajchman M A
Canadian Red Cross Society Blood Transfusion Service, McMaster University, Hamilton, Ontario.
Thromb Haemost. 1992 Sep 7;68(3):291-6.
The homology between antithrombin III (AT-III) of mouse, of man, and that of other species was investigated. Preliminary experiments showed that mouse AT-III inhibited human alpha-thrombin efficiently (second order rate constant [K2nd] 5.8 x 10(3) M-1 s-1) as compared to human AT-III (K2nd 6.7 x 10(3) M-1), but was not recognized on immunoblots by antibodies that recognized both human and rabbit AT-III. In order to compare AT-III from different species at the molecular level, a cDNA clone for murine AT-III was isolated from a lambda ZAP mouse liver cDNA library on the basis of hybridization to a rabbit AT-III cDNA probe. The 1509 bp murine AT-III cDNA consists of a 1398 bp open reading frame, preceded by a 15 bp 5' untranslated region, followed by a 75 bp 3' untranslated region. The deduced primary protein structure consists of a 32 amino acid signal sequence, with a mature portion of 433 residues. Mature murine AT-III is 89% identical to its human counterpart, 86% identical to bovine AT-III, and 82% identical to that of the rabbit. Constructs lacking the nucleotides encoding the signal sequence were engineered and expressed in a cell-free system. The resulting 47 kDa non-glycosylated translation product was capable of being cleaved by human alpha-thrombin, of forming SDS-stable complexes with the protease, and of binding to immobilized heparin. Isolation of the murine AT-III cDNA will make feasible molecularly defined experiments with murine AT-III in the mouse system.
对小鼠、人类及其他物种的抗凝血酶III（AT-III）之间的同源性进行了研究。初步实验表明，与人类AT-III（二级速率常数[K2nd] 6.7×10³ M⁻¹）相比，小鼠AT-III能有效抑制人类α-凝血酶（二级速率常数[K2nd] 5.8×10³ M⁻¹ s⁻¹），但在免疫印迹上不能被识别人类和兔AT-III的抗体所识别。为了在分子水平上比较不同物种的AT-III，基于与兔AT-III cDNA探针的杂交，从λZAP小鼠肝脏cDNA文库中分离出小鼠AT-III的cDNA克隆。1509 bp的小鼠AT-III cDNA由一个1398 bp的开放阅读框组成，前面是一个15 bp的5'非翻译区，后面是一个75 bp的3'非翻译区。推导的一级蛋白质结构由一个32个氨基酸的信号序列组成，成熟部分有433个残基。成熟的小鼠AT-III与其人类对应物的同源性为89%，与牛AT-III的同源性为86%，与兔AT-III的同源性为82%。构建了缺失编码信号序列核苷酸的结构，并在无细胞系统中进行表达。产生的47 kDa非糖基化翻译产物能够被人类α-凝血酶切割，能与蛋白酶形成SDS稳定复合物，并能与固定化肝素结合。小鼠AT-III cDNA的分离将使在小鼠系统中对小鼠AT-III进行分子定义的实验成为可能。
