HELA CELL P L A S M A M E M B R A N E S I. 5'-Nucleotidase and Ouabain-Sensitive ATPase as Markers for Plasma Membranes

A method for the p repa ra t ion of H e L a cell p l a sma m e m b r a n e ghosts is descr ibed. The pur i ty of the p l a sma m e m b r a n e f ract ion was examined by phase con t ras t and e lect ron microscopy , by chemica l analysis , and by assay of m a r k e r enzymes. Da ta on the compos i t ion of the p l a sma m e m b r a n e f ract ion are given. It was observed tha t the d is t r ibut ion pa t te rn of 5 ' -nuc leot idase act ivi ty among the subcel lu lar f ract ions differed from tha t of ouabain-sens i t ive A T Pa se . In addi t ion, the specific act ivi ty o f 5 ' -nuc leot idase did not follow the d is t r ibut ion of the m e m b r a n e ghosts. Thus, this enzyme would seem unsui table as a p l a sma m e m b r a n e marke r . A comple te ba lance sheet for m a r k e r enzyme act ivi t ies dur ing the f rac t iona t ion is necessary for the ca lcula t ion of increase in specific act ivi ty because the act ivi t ies o f both 5 ' -nuc leot idase and ouabain-sens i t ive A T P a s e might change dur ing the f rac t iona t ion procedures . The purification of plasma membranes is usually evaluated by the activity of marker enzymes, especially 5'-nucleotidase and ouabain-sensitive ATPase (1). 5'-Nucleotidase was shown by Essner et al. (2) to be related to the plasma membrane in rat hepatocytes and has subsequently been used in the estimation of the purity of plasma membranes isolated from several other cell types including cells in tissue culture (3-5). We report a novel method for the purification of HeLa cell plasma membranes and demonstrate that the distribution of the activity of 5'-nucleotidase over the fractions differs from that of ouabain-sensitive ATPase. It seems that 5'-nucleotidase activity is not limited to the plasma membrane in HeLa cells. We also show that the presence of EDTA during the isolation of plasma membranes constitutes a source of error in the evaluation of the purification obtained. MATERIALS AND METHODS