Increased susceptibility of the sickle cell membrane Ca2+ + Mg(2+)- ATPase
to t-butylhydroperoxide: protective effects of ascorbate and desferal
RB Moore, TM Hulgan, JW Green and LD Jenkins
USA Comprehensive Sickle Cell Center, University of South Alabama, Mobile
36617.
Normal and sickle cell erythrocyte membranes were examined for significant
differences in their ATPase activities, thiobarbituric acid reactive
products formed (measured relative to malondialdehyde), membrane protein
polymerization, and number of protein-free sulfhydryl groups when treated
with 0.5 mmol/L t-butylhydroperoxide (tBHP) for 30 minutes. Isolated sickle
cell membranes treated with tBHP produced significantly greater inhibition
in both their basal and calmodulin- stimulated Ca2+ + Mg(2+)-ATPase
activities (75% inhibition in both cases) compared with that of control
membranes. In addition, there was significantly more malondialdehyde formed
from sickle cell membranes compared with control membranes. Oxidation
caused greater protein polymerization in sickle cell membranes compared
with normal membranes as demonstrated by the formation of high molecular
weight polymers separated on sodium dodecyl sulfate polyacrylamide gels.
The number of free sulfhydryl groups present in spectrin and actin
decreased more in sickle cell membranes as measured by 3H-N-ethyl maleimide
autoradiography and gel scanning. To prevent enzyme inhibition, erythrocyte
membranes were treated with tBHP in the presence of 1 mmol/L ascorbate, a
potential antioxidant, and 1 mmol/L desferal, an iron chelator. Both
ascorbate and desferal added alone with tBHP were effective in preventing
inhibition of the basal and calmodulin- stimulated Ca2+ + Mg(2+)-ATPase
activities in normal membranes, but in sickle cell membranes only the
addition of ascorbate and desferal together offered significant protection.
The enhanced oxidation observed with sickle cell membranes can be mimicked
in normal white membranes by adding hemoglobin, hemin, or ferrous chloride
in the presence of tBHP. In contrast to hemoglobin, ferrous chloride has
the ability to enhance membrane oxidation in the presence of ascorbate with
or without tBHP. Furthermore, the addition of desferal to these membranes
greatly decreased the iron-ascorbate-tBHP oxidation of erythrocyte
membranes as determined by the sustained ATPase activities and the reduced
formation of malondialdehyde. Maximal protection was provided by 1 mmol/L
desferal in the presence of 1 mmol/L ascorbate, although some protection
was observed even at 10 mumol/L, the lowest concentration tested. These
results are discussed in light of the pro- and anti-oxidant effects of
ascorbate in the absence and presence of iron and tBHP.
Volume 79,
Issue 5,
pp. 1334-1341,
03/01/1992
Copyright © 1992 by The American Society of Hematology
