IMMUNOCYTOCHEMICAL CHARACTERIZATION AND SUBCELLULAR-LOCALIZATION OF HUMAN MYRISTOYL-COA - PROTEIN N-MYRISTOYLTRANSFERASE IN HELA-CELLS

Antisera have been raised to three synthetic peptides based on the seq uence of human myristoyl-CoA:protein N-myristoyl transferase (NMT) and to the purified enzyme following its expression in Escherichia coli. These antisera have been affinity purified and shown to react both wit h the E. coli expressed human NMT, and specifically with a protein of molecular weight of 63 kDa in immunoblots of the human cell line HeLa. The affinity purified antibodies have also been used to localize NMT in methanol/acetone permeabilized HeLa cells by immunofluorescent stai ning. The immunofluorescence showed a diffuse staining pattern through out the cell, suggesting that the enzyme is predominantly cytosolic. T his was confirmed by determining the distribution of NMT activity in d ifferent subcellular fractions of HeLa cells. Over 90% of NMT enzymati c activity was released from cell lysates during either hypotonic or i sotonic homogenization. However, a small amount of enzymatic activity remained associated with cell membranes, despite extensive washing; an d this was confirmed by immunoblot analysis of these membranes for NMT . In comparison, over 99.5% of lactate dehydrogenase activity was rele ased under the same conditions, which suggests that the NMT was genuin ely associated with the cell membranes. The membrane-bound enzyme beha ved like a peripheral membrane protein. Permeabilization of HeLa cells with 50 mu M digitonin resulted in the release of 90-93% of lactate d ehydrogenase compared to 73-85% of NMT, again suggesting that the majo rity of the enzyme is cytosolic, but that some may be associated with cell membranes or organelles. (C) 1996 Academic Press, Inc.