SULFATE AND SIALIC ACID-CONTAINING GLYCOLIPIDS INHIBIT DNA-POLYMERASEALPHA-ACTIVITY

The effects of various glycolipids on the activity of immunoaffinity-p urified calf thymus DNA polymerase alpha were studied in vitro. Preinc ubation with sialic acid-containing glycolipids, such as sialosylparag loboside (SPG), GM3, GM1, and GD1a, and sulfatide (cerebroside sulfate ester, CSE) dose-dependently inhibited the activity of DNA polymerase alpha, while other glycolipids, as well as free sphingosine and ceram ide did not. About 50% inhibition was achieved by preincubating the en zyme with 2.5 muM of CSE, 50 muM of SPG or GM3, and 80 muM of GM1. Inh ibition was noncompetitive with both the DNA template and the substrat e dTTP, as well as with the other dNTPs. Since the inhibition was larg ely reversed by the addition of 0.05% Nonidet P40, these glycolipids m ay interact with the hydrophobic region of the enzyme protein. Apparen tly, the sulfate moiety in CSE and the sialic acid moiety in gangliosi des were essential for the inhibition since neither neutral glycolipid s (i.e., glucosylceramide, galactosylceramide, lactosylceramide) nor a sialo-gangliosides (GA1 and GA2) showed any inhibitory effect. Further more, the ceramide backbone was also found to be necessary for maximal inhibition since the inhibition was largely abolished by substituting the lipid backbone with cholesterol. Increasing the number of sialic acid moieties per molecule further enhanced the inhibition, while elon gating the sugar chain diminished it. It was clearly shown that the N- acetyl residue of the sialic acid moiety is particularly essential for inhibition by both SPG and GM3 because the loss of this residue or su bstitution with a glycolyl residue completely negated their inhibitory effect on DNA polymerase alpha activity.