HUMAN LYSOSOMAL SPHINGOMYELINASE - SUBSTRATE EFFICACY OF APOLIPOPROTEIN SPHINGOMYELIN COMPLEXES
HUMAN LYSOSOMAL SPHINGOMYELINASE - SUBSTRATE EFFICACY OF APOLIPOPROTEIN SPHINGOMYELIN COMPLEXES
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DOI：
10.1021/bi00363a036
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发表时间：
1986-07-29
期刊：
影响因子：
2.9
通讯作者：
MORRISETT, JD
中科院分区：
文献类型：
--
作者：
AHMAD, TY;BEAUDET, AL;MORRISETT, JD
已经研究了人载脂蛋白刺激人皮肤成纤维细胞的鞘磷脂酶水解鞘磷脂（SM）。载脂蛋白 A-I、A-II、B、C-I 和 E 不会将鞘磷脂水解增强至高于对照水平。相反，apoC-II 通过 .apprx 刺激鞘磷脂水解。 2.5倍。 ApoC-III 是最有效的脱辅基蛋白激活剂，可刺激水解 3-4 倍。对于携带 0、1 或 2 个唾液酸残基的三种不同亚型，ApoC-III 刺激没有显着差异。该脱蛋白肽的氨基末端一半 C-III(I-40) 不与磷脂表面结合，因此不会激活鞘磷脂酶。相比之下，羧基末端的一半，C-III(41-79)，与磷脂表面强烈结合，刺激鞘磷脂水解至与完整、全长脱辅基蛋白产生的水平相同的水平。鞘磷脂囊泡与apoC-III比例增加的孵育导致形成apoC-III:SM比率增加和半径减小的复合物。 1:50 (mol/mol) 复合物中鞘磷脂的水解比 1:200 (mol/mol) 复合物中的鞘磷脂水解高出 2 倍以上。 1:50复合物中的蛋黄鞘磷脂的水解速率在凝胶.fwdarw处是最大的[0.9μmol h-1(毫克蛋白质)-1]。配合物的液晶相转变温度(Tm)(40°C)。在较高或较低温度下水解速率显着下降。对低于、等于和高于 Tm 的表观 Km 和 Vmax 值的测定表明，鞘磷脂水解的温度依赖性主要归因于 Vmax 的变化。这些数据表明，apoC-III 通过将有组织的脂质基质分解成更小的单元来增强鞘磷脂的水解，这些单元可以更容易地容纳在酶的活性位点。
Human apolipoprotein stimulation of sphingomyelin (SM) hydrolysis by sphingomyelinase from human skin fibroblasts has been studied. Apolipoproteins A-I, A-II, B, C-I, and E do not enhance sphingomyelin hydrolysis above control levels. In contrast, apoC-II stimulates sphingomyelin hydrolysis by .apprx. 2.5-fold. ApoC-III, the most potent apoprotein activator, stimulates hydrolysis by 3-4-fold. ApoC-III stimulation is not significantly different for the three different isoforms which carry 0, 1, or 2 sialic acid residues. The amino-terminal half of this apoprotien, C-III(I-40), which does not bind to phospholipid surfaces, does not activate sphingomyelinase. In contrast, the carboxyl-terminal half, C-III(41-79), which strongly binds to phospholipid surfaces, stimulates sphingomyelin hydrolysis to the same level as that produced by the intact, full-length apoprotein. Incubation of sphingomyelin vesicles with increasing proportions of apoC-III results in the formation of complexes of increasing apoC-III:SM ratio and decreasing radius. The hydrolysis of sphingomyelin in the 1:50 (mol/mol) complex was more than 2-fold greater than that of the 1:200 (mol/mol) complex. The rate of hydrolysis of egg yolk sphingomyelin in the 1:50 complex was maximal [0.9 .mu.mol h-1 (mg of protein)-1] at the gel .fwdarw. liquid-crystalline phase transition temperature (Tm) of the complex (40.degree. C). The rate of hydrolysis fell markedly at either higher or lower temperature. Determination of the apparent Km and Vmax values below, at, and above Tm indicated that the temperature dependence of sphingomyelin hydrolysis was attributable primarily to changes in Vmax. These data suggest that apoC-III enhances the hydrolysis of sphingomyelin by breaking down the organized lipid matrix into much smaller units which can be more easily accommodated at the active site of the enzyme.