Alkaline Phosphatase in Red Cell Membrane: Interconnection of Activities and Membrane Lipid Fluidity

Abstract

A p-nitrophenylphosphate hydrolyzing enzyme bound to the human red cell membrane was studied over a temperature range of 12–30°C under conditions of high pH (9.8) and high ionic strength (1 M diethanolamine/HC1 buffer) which were recommended for determination of alkaline phosphatase activity in human serum. This enzymatic activity revealed some remarkable characteristics, in particular concerning its behaviour towards thiol reagents. Thus it is completely inactivated by dithiothreitol, L(+) cysteine and 2-mercaptoethanol, while it is only slightly inhibited by mersalyl, and not affected by the SH-oxidizing agent diamide. Furthermore, the enzyme appearas not to be identical with the p-nitrophenylphosphatase activity, which was studied previously at lower pH and lower ionic strength by other authors.

For each temperature, K_m and v_max were determined from Lineweaver-Burk plots. Discontinuities at about 17–18° C were found for the temperature dependencies of the enzyme’s activities as well as of the enzyme’s Michaelis constants.

These discontinuities thus occurred at the same temperature at which the phase transition of the red cell membrane lipids was reported.

Moreover, electron spin resonance investigations were carried out, using a stearic acid spin label for polar membrane regions. In accordance with previous findings, application of an order parameter disclosed discontinuities at a similar temperature range.