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 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 NMT was genuinely associated with the cell membranes. The membrane-bound enzyme behaved like a peripheral membrane protein. Permeabilization HeLa cells with 50 microM digitonin resulted in the release of 90-93% of lactate dehydrogenase compared to 73-85% of NMT, again suggesting that the majority of the enzyme is cytosolic, but that some may be associated with cell membranes or organelles.
McIlhinney
R A
RA
Anatomical Neuropharmacology Unit, Medical Research Council, Oxford, England.
McGlone
K
K
D000217:Acyltransferases / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D000595:Amino Acid Sequence; D000918:Antibody Specificity; D002458:Cell Fractionation; D002462:Cell Membrane / Q000201:enzymology; D002463:Cell Membrane Permeability; D003600:Cytosol / Q000201:enzymology*; D004072:Digitonin; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D008970:Molecular Weight; D015388:Organelles / Q000201:enzymology; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis
D011993:Recombinant Fusion Proteins; D000217:Acyltransferases; C050480:glycylpeptide N-tetradecanoyltransferase; D004072:Digitonin