TY - JOUR
T1 - Sulfate- and sialic acid-containing glycolipids inhibit DNA polymerase α activity
AU - Simbulan, Cynthia Marie G.
AU - Taki, Takao
AU - Tamiya-koizumi, Keiko
AU - Suzuki, Motoshi
AU - Savoysky, Ericka
AU - Shoji, Mami
AU - Yoshida, Shonen
N1 - Funding Information:
for helpful discussions and also to Ms. T. Tomita and S. Furumoto for their excellent technical assistance. This work was supported in part by the Grant-in-Aid for Cancer Research and for Scientific Research from the Ministry of Education, Science and Culture of Japan.
PY - 1994/3/16
Y1 - 1994/3/16
N2 - The effects of various glycolipids on the activity of immunoaffinity-purified calf thymus DNA polymerase α were studied in vitro. Preincubation with sialic acid-containing glycolipids, such as sialosylparagloboside (SPG), GM3, GM1, and GD1a, and sulfatide (cerebroside sulfate ester, CSE) dose-dependently inhibited the activity of DNA polymerase α, while other glycolipids, as well as free sphingosine and ceramide did not. About 50% inhibition was achieved by preincubating the enzyme with 2.5 μM of CSE, 50 μM of SPG or GM3, and 80 μM of GM1. Inhibition was noncompetitive with both the DNA template and the substrate dTTP, as well as with the other dNTPs. Since the inhibition was largely reversed by the addition of 0.05% Nonidet P40, these glycolipids may interact with the hydrophobic region of the enzyme protein. Apparently, the sulfate moiety in CSE and the sialic acid moiety in gangliosides were essential for the inhibition since neither neutral glycolipids (i.e., glucosylceramide, galactosylceramide, lactosylceramide) nor asialo-gangliosides (GA1 and GA2) showed any inhibitory effect. Furthermore, 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 elongating 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 substitution with a glycolyl residue completely negated their inhibitory effect on DNA polymerase α activity.
AB - The effects of various glycolipids on the activity of immunoaffinity-purified calf thymus DNA polymerase α were studied in vitro. Preincubation with sialic acid-containing glycolipids, such as sialosylparagloboside (SPG), GM3, GM1, and GD1a, and sulfatide (cerebroside sulfate ester, CSE) dose-dependently inhibited the activity of DNA polymerase α, while other glycolipids, as well as free sphingosine and ceramide did not. About 50% inhibition was achieved by preincubating the enzyme with 2.5 μM of CSE, 50 μM of SPG or GM3, and 80 μM of GM1. Inhibition was noncompetitive with both the DNA template and the substrate dTTP, as well as with the other dNTPs. Since the inhibition was largely reversed by the addition of 0.05% Nonidet P40, these glycolipids may interact with the hydrophobic region of the enzyme protein. Apparently, the sulfate moiety in CSE and the sialic acid moiety in gangliosides were essential for the inhibition since neither neutral glycolipids (i.e., glucosylceramide, galactosylceramide, lactosylceramide) nor asialo-gangliosides (GA1 and GA2) showed any inhibitory effect. Furthermore, 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 elongating 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 substitution with a glycolyl residue completely negated their inhibitory effect on DNA polymerase α activity.
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U2 - 10.1016/0167-4838(94)90093-0
DO - 10.1016/0167-4838(94)90093-0
M3 - Article
C2 - 8142486
AN - SCOPUS:0028214445
SN - 0167-4838
VL - 1205
SP - 68
EP - 74
JO - Biochimica et Biophysica Acta (BBA)/Protein Structure and Molecular
JF - Biochimica et Biophysica Acta (BBA)/Protein Structure and Molecular
IS - 1
ER -