PURIFICATION, CHARACTERIZATION, AND BIOSYNTHESIS OF HUMAN ACID CERAMIDASE
PURIFICATION, CHARACTERIZATION, AND BIOSYNTHESIS OF HUMAN ACID CERAMIDASE
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DOI：
10.1074/jbc.270.19.11098
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发表时间：
1995-05-12
影响因子：
4.8
通讯作者：
SANDHOFF, K
中科院分区：
文献类型：
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作者：
BERNARDO, K;HURWITZ, R;SANDHOFF, K
酸性神经酰胺酶（N-acylsphingosine deacylase，EC 3.5.1.23）是催化神经酰胺水解为鞘氨醇和游离脂肪酸的溶酶体酶，其遗传缺陷导致法伯病中神经酰胺积聚。通过在辛基琼脂糖、伴刀豆球蛋白A-Sepharose、蓝色琼脂糖和DEAE-纤维素上的连续层析，从人尿中纯化酶至表观均一性。最终的制备物比起始材料富集了4450倍，产生了类似于50 kDa的多肽，并且可以被还原成类似于13（α）和类似于40（β）kDa的两个亚基。用内切糖苷酶H或肽-N-聚糖酶F处理纯化的酶，将β亚基的分子量分别降低至类似于30-35和类似于27 kDa。相比之下，α亚基的分子量不变，纯化的酶具有149 μ M的表观Km和136 nmol/mg/h的V-max使用N-月桂酰鞘氨醇作为底物。针对纯化的尿酶提出多克隆抗体，并用于研究酸性神经酰胺酶的生物合成。代谢标记的皮肤成纤维细胞的免疫沉淀研究表明，这两个亚基从一个单一的前体类似的55 kDa。一小部分新合成的酸性神经酰胺酶分泌到介质中作为一个单体的47-kDa的蛋白质，表明产生的成熟的异源二聚体酶发生在内体和/或溶酶体室。
Acid ceramidase (N-acylsphingosine deacylase, EC 3.5.1.23) is the lysosomal enzyme catalyzing the hydrolysis of ceramide to sphingosine and free fatty acid, Its inherited deficiency causes ceramide accumulation in Farber's disease. The enzyme was purified to apparent homogeneity from human urine by sequential chromatography on octyl-Sepharose, concanavalin A-Sepharose, blue-Sepharose, and DEAE-cellulose. The final preparation, which was enriched similar to 4450-fold over the starting material, resulted in a polypeptide of similar to 50 kDa and could be reduced into two subunits of similar to 13 (alpha) and similar to 40 (beta) kDa. Treatment of the purified enzyme with endoglycosidase H or peptide-N-glycanase F reduced the molecular mass of the beta subunit to similar to 30-35 and similar to 27 kDa, respectively. In contrast, the molecular mass of the alpha subunit was unchanged, The purified enzyme had an apparent K-m of 149 mu M and a V-max of 136 nmol/mg/h using N-lauroylsphingosine as substrate. Polyclonal antibodies were raised against the purified urinary enzyme and used to investigate the biosynthesis of acid ceramidase. Immunoprecipitation studies on metabolically labeled skin fibroblasts indicated that both subunits arose from a single precursor of similar to 55 kDa. A minor portion of newly synthesized acid ceramidase was secreted into the me dium as a monomeric 47-kDa protein, indicating that generation of the mature heterodimeric enzyme occurred in endosomal and/or lysosomal compartments.