Structure/activity relationship of placental alkaline phosphatase (PLAP) submitted to denaturing conditions like high temperature and high hydrostatic pressures.

CLOP, E. M.; BONAFE, C.F.S; PERILLO, M.A.

villa Carlos Paz

Congreso; XLII Reunión Anual de la Sociedad Argentina de Biofísica.; 2013

Sociedad Argentina de Biofísica.

In a previous work we investigated the correlation between the catalytic activity of PLAP (GPI-anchored protein) and the organization of the molecular environment in model membranes at different lateral packings and in presence of interfacial molecular crowded milieu. We showed that PLAP activity was capable to probe all these different media. To gain further insights into the mechanism of this environmental tuning we explore the kinetics of PLAP submitted to different denaturing conditions, such as the effect of high hydrostatic pressure (in presence of urea) and high temperatures. The state of the protein was followed by tryptophan intrinsic fluorescence. A multiple unfolded intermediates were found while Vmax showed a maximum at low denaturing pressure conditions and moderate temperatures. Also a cooperative behavior was found in the catalytic activity at high urea concentrations. These results showed that partially unfolded PLAP is capable to exhibit catalytic activity, not necessarily diminishing its efficiency and plausible by changing its kinetic mechanism. The tune of catalysis in heterogeneous media may reflect not only diffusional/motional restrictions but also PLAP structure stability in these environments.