Substrate specificity, inhibition and enzymological analysis of recombinant human glutamate carboxypeptidase II.
Glutamate carboxypeptidase II (GCPII, EC 3.4.17.21) is a membrane peptidase expressed in a number of tissues such as kidney, prostate and brain. The brain form of GCPII (also known as NAALADase) cleaves N-acetyl-aspartyl glutamate to yield free glutamate. Animal model experiments show that inhibition of GCPII prevents neuronal cell death during experimental ischaemia. GCPII thus represents an important target for the treatment of neuronal damage caused by excess glutamate. In this paper we report expression of an extracellular portion of human glutamate carboxypeptidase II (amino acids 44-750) in Drosophila Schneider's cells and its purification to homogeneity. A novel assay for hydrolytic activity of recombinant human GCPII (rhGCPII), based on fluorimetric detection of released alpha-amino groups was established, and used for its enzymological characterization. rhGCPII does not show dipeptidylpeptidase IV-like activity assigned to the native form of the enzyme previously. Using a complete set of protected dipeptides, substrate specificity of rhGCPII was elucidated. In addition to the previously described substrates, four novel compounds, Ac-Glu-Met, Ac-Asp-Met and, surprisingly, Ac-Ala-Glu and Ac-Ala-Met were identified as substrates for GCPII, and their respective kinetic constants determined. The glycosylation of rhGCPII was found indispensable for the enzymatic activity.
Barinka
Cyril
C
Institute of Organic Chemistry and Biochemistry of the Academy of Science of the Czech Republic, Praha.
Rinnová
Markéta
M
Sácha
Pavel
P
Rojas
Camilo
C
Majer
Pavel
P
Slusher
Barbara S
BS
Konvalinka
Jan
J
D000818:Animals; D000954:Antigens, Surface; D001921:Brain / Q000201:enzymology; D002268:Carboxypeptidases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D018819:Dipeptidyl Peptidase 4 / Q000378:metabolism; D004330:Drosophila; D004789:Enzyme Activation / Q000502:physiology; D015971:Gene Expression Regulation, Enzymologic; D043425:Glutamate Carboxypeptidase II; D006031:Glycosylation; D006801:Humans; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism; D013379:Substrate Specificity; D014407:Tumor Cells, Cultured
D000954:Antigens, Surface; D010446:Peptide Fragments; D011994:Recombinant Proteins; D002268:Carboxypeptidases; D018819:Dipeptidyl Peptidase 4; C473848:FOLH1 protein, human; D043425:Glutamate Carboxypeptidase II