Spilburg C A, Cox D G, Wang X, Bernat B A, Bosner M S, Lange L G
CV Therapeutics, Palo Alto, California 94304, USA.
Biochemistry. 1995 Nov 28;34(47):15532-8. doi: 10.1021/bi00047a019.
All mammalian pancreatic cholesterol esterases (CEase) bind to membrane-associated heparin at a single site on the intestinal brush border membrane with a dissociation constant of 100 nM. While the enzyme is bound to the membrane, the activity of the human and bovine enzymes is enhanced 2-fold when compared to the activity of the enzyme in solution. On the other hand, soluble heparin potently inhibits the human CEase-catalyzed hydrolysis of cholesterol oleate with an IC50 of 2 x 10(-4) mg/mL, a value that is about 10(4) times more potent than that found with the bovine enzyme. The C-terminal portion of the human enzyme contains 16 proline-rich repeats of 11 amino acids each, while that from other species contains only a few of these repeat units. To determine if the unique human C-terminus is responsible for this inhibition, two chimeras containing either the human N-terminus (residues 1-445) and the bovine C-terminus (residues 446-557), HB, or the bovine N-terminus (residues 1-445) and the human C-terminus (residues 446-722), BH, were prepared. The cholesterol oleate hydrolytic activity of these chimeras was similar to that for the recombinant human and bovine enzymes. Importantly, each chimera was inhibited by heparin with IC50 values of 0.03 and 0.1 mg/mL for HB and BH, respectively. These intermediate IC50 values indicate that human CEase has two structural regions that contribute to is unique inhibition by this sulfated glycosaminoglycan, and these could regulate cholesterol uptake in humans.
所有哺乳动物的胰腺胆固醇酯酶（CEase）在肠道刷状缘膜上的单个位点与膜相关肝素结合，解离常数为100 nM。当该酶与膜结合时，与人及牛酶在溶液中的活性相比，其活性提高了2倍。另一方面，可溶性肝素能有效抑制人CEase催化的胆固醇油酸酯水解，IC50为2×10⁻⁴mg/mL，该值比牛酶的抑制活性强约10⁴倍。人酶的C末端部分包含16个富含脯氨酸的重复序列，每个重复序列由11个氨基酸组成，而其他物种的该部分仅含有少数几个这样的重复单元。为了确定人酶独特的C末端是否导致这种抑制作用，制备了两种嵌合体，一种包含人N末端（第1 - 445位氨基酸）和牛C末端（第446 - 557位氨基酸），即HB，另一种包含牛N末端（第1 - 445位氨基酸）和人C末端（第446 - 722位氨基酸），即BH。这些嵌合体的胆固醇油酸酯水解活性与重组人酶和牛酶的活性相似。重要的是，肝素对每种嵌合体均有抑制作用，HB和BH的IC50值分别为0.03和0.1 mg/mL。这些中间IC50值表明，人CEase有两个结构区域导致其被这种硫酸化糖胺聚糖独特抑制，这两个区域可能调节人体的胆固醇摄取。
