Purification and characterization of the cytoplasmic domain of human receptorlike protein tyrosine phosphatase RPTP.mu
| Autor: | Zondag Gc, van Etten I, Moolenaar Wh, Martijn F.B.G. Gebbink, Verheijen Mh |
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| Rok vydání: | 1993 |
| Předmět: |
Sf9
Protein tyrosine phosphatase Moths Biology Cytoplasmic part medicine.disease_cause Biochemistry Substrate Specificity Structure-Activity Relationship Cell surface receptor Escherichia coli medicine Animals Humans Point Mutation Phosphotyrosine chemistry.chemical_classification Mutation COS cells Myelin Basic Protein Molecular biology Recombinant Proteins Amino acid Ectodomain chemistry Mutagenesis Site-Directed Tyrosine Muramidase Protein Tyrosine Phosphatases Baculoviridae Gene Deletion |
| Zdroj: | Biochemistry. 32:13516-13522 |
| ISSN: | 1520-4995 0006-2960 |
| DOI: | 10.1021/bi00212a017 |
| Popis: | RPTP mu is a recently described receptor-like protein tyrosine phosphatase (PTP), the ectodomain of which mediates homophilic cell-cell adhesion. The cytoplasmic part contains two homologous PTP-like domains and a juxtamembrane region that is about twice as large as in other receptor-like PTPs. The entire 80-kDa cytoplasmic part of human RPTP mu was expressed in insect Sf9 cells and its enzymatic activity was characterized after purification to electrophoretic homogeneity. In addition, the effects of deletion and point mutations were analyzed following expression in Escherichia coli cells. The purified cytoplasmic part of RPTP mu displays high activity toward tyrosine-phosphorylated, modified lysozyme (Vmax 4500 nmol min-1 mg-1) and myelin basic protein (Vmax 8500 nmol min-1 mg-1) but negligible activity toward tyrosine-phosphorylated angiotensin or the nonapeptide, EDNDpYINASL, that serves as a good substrate for protein tyrosine phosphatase PTP1B. This suggests that RPTP mu and PTP1B have distinct substrate specificities. Catalytic activity is independent of Ca2+ (up to 1 mM) but is strongly inhibited by Zn2+, Mn2+, vanadate, phenylarsenic oxide, and heparin. The first of the two catalytic domains is 5-10 times less active than the expressed catalytic region containing both domains. Mutation of Cys 1095 to Ser in the first catalytic domain abolishes enzymatic activity when analyzed following expression in either E. coli or mammalian COS cells. Deletion of the first 53 amino acids from the juxtamembrane region reduces catalytic activity about 2-fold. |
| Databáze: | OpenAIRE |
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