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.