Jk. Bielicki et al., GAS-PHASE CIGARETTE-SMOKE INHIBITS PLASMA LECITHIN-CHOLESTEROL ACYLTRANSFERASE ACTIVITY BY MODIFICATION OF THE ENZYMES FREE THIOLS, Biochimica et biophysica acta, L. Lipids and lipid metabolism, 1258(1), 1995, pp. 35-40
Cigarette smoking is associated with an increased risk of premature at
herosclerosis. The underlying mechanisms responsible for this associat
ion are unknown. Recent work from this laboratory has shown that ex vi
vo exposure of plasma to gas-phase cigarette smoke (CS) produces a rap
id inhibition of lecithin-cholesterol acyltransferase (LCAT) activity
and crosslinking of HDL-apolipoproteins. The goal of the present study
was to investigate the mechanism(s) by which CS inhibited LCAT and mo
dified HDL. When dialyzed human plasma (12 ml) was exposed to the gas-
phase of an equivalent of 1/8 of a cigarette (one 'puff) at 15 min int
ervals for 3 h, LCAT activity was reduced by 76 +/- 1% compared to con
trols; supplementation of plasma with glutathione produced a dose-depe
ndent protection of LCAT activity where at the highest concentration (
1 mM) 78% protection was observed. A similar protection was obtained w
ith N-acetyl cysteine (1 mM). In addition to LCAT inhibition, HDL-apol
ipoproteins were crosslinked after 3 h exposure of plasma to CS; cross
linking was reduced by the addition of either glutathione or N-acetyl
cysteine to plasma. The amino compounds N-acetyl lysine, N-acetyl argi
nine, and aminoguanidine failed to protect LCAT and HDL indicating a s
pecificity with regard to the ability of free thiols to buffer the del
eterious components of CS which inhibited LCAT and crosslinked HDL-apo
lipoproteins. Since LCAT contains two free cysteine residues (Cys-31 a
nd -184) near the active site of the enzyme, we tested whether pretrea
tment of plasma with the reversible sulfhydryl modifying compound, 5,5
'-dithiobis-2-nitrobenzoic acid (DTNB), could protect LCAT from CS-ind
uced inhibition. Plasma pretreated with DTNB prior to CS exposure reta
ined an additional 50% of LCAT activity compared to CS treatment in th
e absence of DTNB. These results indicate that covalent modification o
f free cysteine residues of LCAT was, in part, responsible for the inh
ibition of plasma LCAT activity by CS.