Ethanolamine base exchange in astrocyte primary cultures: localization and developmental studies

Abstract

The enzymatic activities of ethanolamine base exchange (EBEE) and CDP-ethanolamine: 1,2-diacylglycerol ethanolamine phosphotransferase (EPT) were investigated during the growth of rat astrocyte primary cultures. From the 16th day, cells ceased to divide (2.0 X 10(6) cells per culture dish); the total phospholipid (PL) content increased 1.5 fold between the 16th and 24th day (0.20 to 0.30 mumol per mg protein) but the amount of ethanolamine phospholipid (28% of PL content) remained constant. Whereas the specific activity (pmol/min X mg protein) of EPT reached a plateau at 16 days in culture and remained constant (400) thereafter, that of EBEE increased up to the 19th day (190) and decreased gradually to a basal level (75) at the 24th day. EBEE activity was not detected in plasma membranes isolated from 16, 19 and 24 days astrocyte cultures. Sub-cellular fractionation and determination of EBEE specific activities showed that the 104 X 10(3) g fraction (P4) was 4.8 and 8.8 fold enriched at the 16th day and 24th day respectively as compared to the whole cell homogenate (50 and 75). The 7 X 10(3) g (P2) and 17 X 10(3) g (P3) fractions were 8.4 and 7.0 fold enriched respectively at the 19 day in culture. The percentages of the enzymatic activity in the different subcellular fractions were 30, 57.2 and 25.7 for P2 and 39.2, 2.6 and 39.8 for P4 at 16, 19 and 24 days in culture respectively. The activity remained constant in P3 (23%) and was negligible in P1 (6%). Ultrastructural studies revealed that P2 and P3 were enriched in mitochondria while P4 contained essentially microsomes.(ABSTRACT TRUNCATED AT 250 WORDS)