Immunocytochemical characterization and subcellular localization of human myristoyl-CoA: protein N-myristoyltransferase in HeLa cells

Experimental Cell Research
R A McIlhinney, K McGlone

Abstract

Antisera have been raised to three synthetic peptides based on the sequence 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 with 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 staining. The immunofluorescence showed a diffuse staining pattern throughout the cell, suggesting that the enzyme is predominantly cytosolic. This was confirmed by determining the distribution of NMT activity in different subcellular fractions of HeLa cells. Over 90% of NMT enzymatic activity was released from cell lysates during either hypotonic or isotonic homogenization. However, a small amount of enzymatic activity remained associated with cell membranes, despite extensive washing, and this was confirmed by immunoblot analysis of these membranes for NMT. In comparison, over 99.5% of lactate dehydrogenase activity was released under the same conditions, which suggests that the N...Continue Reading

Citations

Nov 26, 2015·Biochemical and Biophysical Research Communications·Elzbieta DudekMarek Michalak
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