Protein acylation in the cardiac muscle like cell line, H9c2.
Besides serving as oxidisable substrates, fatty acids (FA) are involved in co- and post-translational modification of proteins (protein acylation). Despite the high rate of fatty acid utilisation in the heart, information on protein acylation in cardiac muscle is scarce. To explore this subject in more detail, we used the H9c2 cell line as an experimental model. After incubation with 3H-palmitate or 3H-myristate, cells were lysed and proteins precipitated, followed by extensive delipidation. The delipidated proteins were subjected to SDS-PAGE and transferred to nitro-cellulose prior to autoradiography. In addition, TLC was used to separate the various lipid classes. The first aspect we addressed was the extent of protein acylation as a function of time, relative to fatty acid incorporation into various lipid classes. Cells were incubated for 30 min, 1 h and 2 h with 100 microCi palmitate (PA, 2.3 nmol) or 125 microCi myristate (MA, 2.5 nmol). Palmitoylation increased from 0.48 +/- 0.25 to 1.25 +/- 0.56 microCi/mg protein between 30 min to 2 h, while myristoylation increased from 0.25 +/- 0.12 to 0.77 +/- 0.36 microCi/mg protein. Furthermore, delipidated proteins subjected to autoradiography showed that a set of distinct proteins was labelled with 3H-palmitate. Incorporation into phospholipids (PL) increased from 40-60% of the total amount of radio-labelled PA or MA supplied between 30 min and 2 h. Only the FA pool differed between MA and PA, with a higher FA content present after incubations with MA. Second, we investigated palmitoylation and incorporation into cellular lipids as a function of the amount of PA applied. Palmitoylation showed saturation at high PA concentrations. The percentage incorporation of 3H-PA in the various lipids depended on the amount of PA added: a decline in the PL pool with a concomitant increase in the size of the diacylglycerol pool at high PA concentrations. Third, inhibition of palmitoylation by cerulenin and tunicamycin was investigated. While both were able to inhibit palmitoylation, cerulenin also inhibited the incorporation of PA into various lipid classes, indicating differences in inhibitory action.
Hasselbaink
Danny M
DM
Department of Physiology, Cardiovascular Research Institute Maastricht, Maastricht University, Maastricht, The Netherlands.
Roemen
Theo H M
TH
van der Vusse
Ger J
GJ
D000215:Acylation; D000818:Animals; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D002460:Cell Line; D002569:Cerulenin / Q000494:pharmacology; D005227:Fatty Acids / Q000737:chemistry / Q000378:metabolism*; D050356:Lipid Metabolism; D032383:Myocytes, Cardiac / Q000166:cytology / Q000378:metabolism*; D011506:Proteins / Q000378:metabolism*; D051381:Rats; D014316:Tritium / Q000378:metabolism; D014415:Tunicamycin / Q000494:pharmacology
D000900:Anti-Bacterial Agents; D005227:Fatty Acids; D011506:Proteins; D014316:Tritium; D014415:Tunicamycin; D002569:Cerulenin