A quantitative colorimetric method of measuring alkaline phosphatase activity in eukaryotic cell membranes

Alkaline phosphatase (ALP) is glycoprotein structured metalophosphatase with several defined functions. It is present in many tissues of all living beings from bacteria to mammals. The enzyme may catalyse the hydrolysis of various monophosphate esters at alkaline pH. The objective of this study was to quantify ALP functioning particularly in the membranes of eukaryotic cells. The membranes of seven different cells (myeloma cells; hybrid cells; erythroleukaemia cells; lymphocytes and erythrocytes) were tested for ALP activity using a cellular enzyme assay, which is based on the conversion of para-nitrophenylphosphate (p-NPP) to para-nitrophenol and the colorimetric determination of the resulting coloured product. The test system was optimised with respect to substrate concentration, reaction time and the number of cells used as a source of enzyme. The obtained values were converted to quantitative results through a standard curve created using commercial ALP. In order to determine the effect of serum concentration on enzyme activity, 1G2 hybridoma, which is among the cells used in this study and which synthesizes monoclonal antibody against human serum albumin, was produced in different serum concentrations ranging from 0 to 15%.