A. Hernandez-hernandez et al., Oxidative inactivation of human and sheep platelet membrane-associated phosphotyrosine phosphatase activity, FREE RAD B, 26(9-10), 1999, pp. 1218-1230
Incubation of human or sheep platelet crude membranes with xanthine oxidase
/hypoxanthine in the presence of Fe2+/ADP inactivated phosphotyrosine phosp
hatase (PTPase, protein-tyrosine-phosphate-phosphohydrolase, EC 3.1.3.48) a
ctivity in a time-dependent manner, this inhibition being significant withi
n 5 min of treatment. The dynamics of protein thiols differed depending on
the platelet species, but in any case decreases in protein thiols were only
visible 20-45 min after the start of the treatment. The inhibition of PTPa
se activity in general showed good a correlation with the production of thi
obarbituric acid-reactive substances (TBARS). The results with several anti
oxidants suggest that the inhibition of PTPase activity is related to the g
eneration of alkoxyl and/or peroxyl radicals, Furthermore, the formation of
fluorescent products and changes in amino groups were observed only after
long incubation times with the oxidizing agents, these fluorescent products
and the residual enzyme activity remaining in the membrane fraction. Treat
ment of platelet membranes with trans-2-nonenal and n-heptaldehyde, but not
with acetaldehyde, also inhibited membrane-associated PTPase activity. How
ever, the amount of protein thiols was reduced only by treatment with trans
-2-nonenal. Fluorescence product formation was always higher with trans-2-n
onenal, these products being mainly located in the protein fraction. The re
sults with aldehydes suggest that secondary degraded products of lipid hydr
operoxides affect PTPase activity. Kinetic studies of PTPase activity indic
ated that with all treatments enzyme inhibition is mainly due to a decrease
in the V-max value. The results of fluorescence anisotropy measurements of
labeled platelet membranes did not support the notion of a contribution of
the lipid organization to peroxidation-mediated PTPase inhibition. All the
above results indicate that platelet membrane-associated PTPase inhibition
due to treatment with xanthine oxidase/hypoxanthine in the presence of Fe2
+/ADP is a very complex, time-dependent process, and that it is probably re
lated, at least after long periods of peroxidation, to changes in protein t
hiols and amino groups, We predict that the sensitivity of PTPase to lipid
peroxidation must be physiologically relevant because of the increasing imp
ortance of tyrosine phosphorylation in signal transduction, in general, and
in platelet activation and aggregation in particular. (C) 1999 Elsevier Sc
ience Inc.