Characterization of thiol-, aspartyl-, and thiol-metallo-peptidase activities in Madin-Darby canine kidney cells

• Published in: Journal of cellular biochemistry 2000-01, Vol.76 (3), p.478-488
• Main Authors: Oliveira, Vitor, Ferro, Emer S., Gomes, Marcelo D., Oshiro, Maria E.M., Almeida, Paulo C., Juliano, Maria A., Juliano, Luiz
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Animals; Aspartic Acid Endopeptidases - isolation & purification; Aspartic Acid Endopeptidases - metabolism; Catalytic Domain; cathepsin B; cathepsin D; cathepsin L; Cell Line; Chromatography, High Pressure Liquid; Cysteine Endopeptidases - isolation & purification; Cysteine Endopeptidases - metabolism; Dogs; endooligopeptidase; Kidney - cytology; Kidney - enzymology; Male; MDCK cells; Metalloendopeptidases - chemistry; Metalloendopeptidases - isolation & purification; Metalloendopeptidases - metabolism; Oligopeptides - chemistry; Peptide Hydrolases - isolation & purification; Peptide Hydrolases - metabolism; Rats; Substrate Specificity; Testis - enzymology; THIMET
• ISSN: 0730-2312; 1097-4644
• DOI: 10.1002/(SICI)1097-4644(20000301)76:3<478::AID-JCB14>3.0.CO;2-H

We combined fluorogenic substrates or internally quenched fluorescent peptides with specific inhibitors in the pH profile of proteolytic activity experiments in order to detect proteolytic activities in lysates of MDCK cells. Hydrolytic activities related to cathepsin B, L, and D were observed. Serine‐proteinase was not detected; however, we clearly demonstrated the presence of a thiol‐metallo‐endo‐oligopeptidase, also called thimet‐oligopeptidase (TOP). This peptidase from MDCK cells has substrate and inhibitor specificities as well as an activation profile with mercaptoethanol that are indistinguishable from the recombinant rat testis TOP (EC 3.4.24.15). In addition, polyclonal purified antibodies to this enzyme depleted the TOP activity of MDCK cells in whole homogenate. Although we present only preliminary data, TOP is secreted by MDCK cells. The presence of TOP in a phenotype polarized MDCK cells can have special significance in the cytoplasmic selection, transport, or clearance of short peptides due to restriction of the enzyme to sequences from 6 to 17 amino acids. Therefore, the MDCK cell could be a very useful cellular model with which to study some of the suggested TOP biological functions as processing of biological active peptides and antigen presentation. J. Cell. Biochem. 76:478–488, 2000. © 2000 Wiley‐Liss, Inc.