title: Monomeric 55-kDa guanidinobenzoatase switches to a serine proteinase activity upon tetramerization. Tetrameric proteinase SP 220 K appears as the native form.
abstract: Guanidinobenzoatases are cell surface enzymes present in cells capable of migration or remodeling. The guanidinobenzoatase purified to homogeneity from human renal carcinoma did not display gelatinase activity under the 55-kDa form (Poustis-Delpont, C., Descomps, R., Auberger, P., Delque-Bayer, P., Sudaka, P., and Rossi, B. (1992) Cancer Res. 52, 3622-3628). We bring new insights into the structure-activity relationships of this enzyme using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, [3H]diisopropyl fluorophosphate labeling, gelatin zymography, and immunodetection using a polyclonal antibody raised against the 55-kDa entity. Upon aggregation into a 220-kDa form, the enzyme exhibited [3H]diisopropyl fluorophosphate labeling and diisopropyl fluorophosphate-inhibitable gelatinase activity whereas its capability to cleave p-nitrophenyl p'-guanidinobenzoate as a substrate was abolished. Thus, the guanidinobenzoatase property appears as a feature of a 55-kDa inactive form of a serine proteinase subunit. After boiling in the presence of sodium dodecyl sulfate (3% w/v), the 220-kDa entity subjected to SDS-polyacrylamide gel electrophoresis could be dissociated into a 55-kDa protein as shown by silver staining. The resulting 55-kDa band remained [3H]diisopropyl fluorophosphate-labeled and reacted with anti-55-kDa guanidinobenzoatase antibodies, strongly suggesting that the 220-kDa proteinase was a noncovalently associated tetramer. Interestingly, Triton X-100 extracts of renal carcinoma plasma membranes exhibited a 220-kDa serine proteinase activity, as expressed in gelatin zymography, which was barely detectable in the non-tumoral counterpart. It is noteworthy that an anti-55-kDa guanidinobenzoatase reactive 220-kDa species was also observed in renal carcinoma plasma membranes extracts as assessed by Western blot, whereas it was hardly visible in the non-tumoral counterpart. No signal was immunodetected at M(r) 55,000 in renal carcinoma and kidney cortex membranes in Western blot experiments. Taken together, our data support the idea that the enzyme is expressed under its tetrameric form in the membrane. The purified enzyme is able to exhibit a serine proteinase activity when it recovers its native tetrameric form. This high molecular weight tetrameric proteinase SP 220 K appears as a new member of the cell surface serine protease family.
authors.affiliation: Laboratoire de Biochimie, Faculté de Médecine, Nice, France.
authors.forename: C
authors.initials: C
authors.lastname: Poustis-Delpont
authors.forename: S
authors.initials: S
authors.lastname: Thaon
authors.forename: P
authors.initials: P
authors.lastname: Auberger
authors.forename: C
authors.initials: C
authors.lastname: Gerardi-Laffin
authors.forename: P
authors.initials: P
authors.lastname: Sudaka
authors.forename: B
authors.initials: B
authors.lastname: Rossi
mesh_terms: D000818:Animals; D000906:Antibodies / Q000302:isolation & purification; D015153:Blotting, Western; D002265:Carboxylic Ester Hydrolases / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D002292:Carcinoma, Renal Cell / Q000201:enzymology*; D002462:Cell Membrane / Q000201:enzymology; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D005260:Female; D006801:Humans; D007672:Kidney Cortex / Q000201:enzymology*; D007680:Kidney Neoplasms / Q000201:enzymology*; D046911:Macromolecular Substances; D008565:Membrane Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D008970:Molecular Weight; D011817:Rabbits / Q000276:immunology; D012697:Serine Endopeptidases / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*
chemical_list: D000906:Antibodies; D046911:Macromolecular Substances; D008565:Membrane Proteins; D002265:Carboxylic Ester Hydrolases; C037288:guanidinobenzoate esterase; D010450:Endopeptidases; C108563:SP220K cell surface matrix proteinase; D012697:Serine Endopeptidases