Surface density as a significant parameter for the enzymatic activity of two forms of alkaline phosphatase immobilized on phospholipid Langmuir-Blodgett films.
Rat osseous plate alkaline phosphatase, a glycosylphosphatidylinositol (GPI)-anchored phosphomonohydrolase, was immobilized on Langmuir-Blodgett (LB) films. Enzyme solubilization either with polyoxyethylene-9-lauryl ether or with a glycosylphosphatidylinositol-specific phospholipase C resulted in a GPI-anchor-containing and a GPI-anchor-depleted form, respectively. Both forms were adsorbed on dimyristoylphosphatidic acid LB films and restricted to the outermost layer. The surface density and enzyme activity were determined using a quartz crystal microbalance and p-nitrophenylphosphatase activity, respectively. The detergent-solubilized form was co-spread with dimyristoylphosphatidic acid on the air/water interface and transferred to solid supports, providing an enzyme maximum surface density of 530 ng/cm2. Maximal phosphohydrolytic activity, corresponding to 43% of that observed in homogeneous medium, was obtained at a surface density of 179 ng/cm2. The phospholipase C-solubilized form was adsorbed directly from solution, reaching a maximum surface density of 1541 ng/cm2, although the phosphomonohydrolase activity was 10 times lower than that obtained for the anchor-containing form. The combined analysis of surface density and enzymatic activity suggests that the alignment of the protein molecules on the LB lipid films induced by the glycosylphosphatidylinositol anchor facilitates the access of the substrate to the active site. This access is hampered by increasing enzyme surface densities and depends on a specific orientation of the adsorbed enzyme.
Caseli
Luciano
L
Departamento de Química, Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto, Universidade de São Paulo, 14040-901 Ribeirão Preto, São Paulo, Brazil.
Furriel
Rosa Prazeres Melo
RP
de Andrade
José Fernando
JF
Leone
Francisco Assis
FA
Zaniquelli
Maria Elisabete Darbello
ME
D000469:Alkaline Phosphatase / Q000737:chemistry*; D000818:Animals; D004800:Enzymes, Immobilized / Q000737:chemistry*; D008051:Lipid Bilayers / Q000737:chemistry*; D010743:Phospholipids / Q000737:chemistry*; D051381:Rats; D010738:Type C Phospholipases / Q000737:chemistry*
D004800:Enzymes, Immobilized; D008051:Lipid Bilayers; D010743:Phospholipids; D000469:Alkaline Phosphatase; D010738:Type C Phospholipases