LIMITED PROTEOLYSIS OF CHICKEN GIZZARD 5'-NUCLEOTIDASE

被引:4
|
作者
STOCHAJ, U
CRAMER, M
MANNHERZ, HG
机构
[1] UNIV MARBURG,INST ANAT & ZELLBIOL,W-3550 MARBURG,GERMANY
[2] UNIV COLOGNE,INST GENET,W-5000 COLOGNE 41,GERMANY
关键词
5'-NUCLEOTIDASE; LIMITED PROTEOLYSIS; PROTEIN DOMAIN ORGANIZATION; AFFINITY LABELING; N-LINKED CARBOHYDRATES;
D O I
10.1016/0167-4838(92)90413-8
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Chicken gizzard 5'-nucleotidase represents an ectoenzyme which is linked to the plasma membrane via a phosphatidylinositol glycan. We have characterized the possible domain-like organization of 5'-nucleotidase by limited proteolysis. A hydrophobic proteolytic fragment carrying the intact C-terminus, as well as two major hydrophilic products, were identified. We developed procedures for specific radiolabelling of the active center of 5'-nucleotidase. This allowed us to locate the catalytic site within hydrophilic fragments obtained after limited proteolysis. We demonstrate that removal of N-linked carbohydrate chains increases the sensitivity of 5'-nucleotidase to proteolytic attack, indicating that sugar moieties protect against proteolysis. 5'-Nucleotidase represents a binding protein for components of the extracellular matrix. The interaction between 5'-nucleotidase and the laminin/nidogen complex unmasked proteolytic cleavage sites in the C-terminal portion of the enzyme. This resulted in the specific production of a hydrophilic form of 5'-nucleotidase. In summary, we have further charcterized chicken gizzard 5'-nucleotidase: (1) the protein is organized into two domain-like structures, (2) the N-terminal domain harbors the active center; (3) N-linked carbohydrates protect the protein against proteolytic degradation; (4) interaction with components of the extracellular matrix alters the conformation of 5'-nucleotidase.
引用
收藏
页码:327 / 332
页数:6
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