DITYROSINE AND TYROSINE OXIDATION-PRODUCTS ARE ENDOGENOUS MARKERS FORTHE SELECTIVE PROTEOLYSIS OF OXIDATIVELY MODIFIED RED-BLOOD-CELL HEMOGLOBIN BY (THE 19-S) PROTEASOME
C. Giulivi et Kja. Davies, DITYROSINE AND TYROSINE OXIDATION-PRODUCTS ARE ENDOGENOUS MARKERS FORTHE SELECTIVE PROTEOLYSIS OF OXIDATIVELY MODIFIED RED-BLOOD-CELL HEMOGLOBIN BY (THE 19-S) PROTEASOME, The Journal of biological chemistry, 268(12), 1993, pp. 8752-8759
Cells exposed to oxidative stress have been shown previously to exhibi
t both protein oxidation and increased proteolysis. Experiments conduc
ted with purified proteins in vitro have indicated that oxidatively mo
dified proteins may be selectively degraded by intracellular proteases
, but a definitive cause-and-effect relationship has not been demonstr
ated previously in intact cells. Several investigators have proposed t
hat oxidatively modified proteins are selectively degraded within cell
s, but the possibility that oxidants may activate intracellular protea
ses (directly or indirectly) to catalyze the indiscriminate degradatio
n of undamaged proteins has not been discounted. Armed with the knowle
dge that dityrosine is a specific product of protein oxidation, we und
ertook a series of experiments to test the hypothesis that oxidized pr
oteins undergo selective intracellular degradation. Our results demons
trate that dityrosine is produced in the hemoglobin molecule when red
blood cells are exposed to a continuous flux of hydrogen peroxide (H2O
2). The dityrosine so produced is only released from the hemoglobin by
proteolysis and is stable to prolonged incubation with cell extracts.
Inhibitors of proteolysis have no effect on dityrosine production but
do effectively prevent dityrosine release. Proteasome (the 670-kDa mu
lticatalytic proteinase complex, that we have previously called macrox
yproteinase or MOP (Pacifici, R. E., Salo, D. C., and Davies, K. J. A.
(1989) Free Radical Biol. & Med. 7, 521-526; Salo, D. C., Pacifici, R
. E., Lin, S. W., Giulivi, C., and Davies, K. J. A. (1990) J. Biol. Ch
em. 265, 11919-11927; Pacifici, R. E., and Davies, K. J. A. (1991) Ger
ontology 37, 166-180) appears responsible for dityrosine release durin
g the selective degradation of oxidatively modified proteins in red bl
ood cells and red cell extracts. We conclude that the elevated rates o
f proteolysis observed in response to oxidative stress do, indeed, ref
lect selective degradation of oxidatively modified (damaged) proteins.
Despite a relatively low production rate, dityrosine has a high fluor
ometric quantum yield and is, of course, a specific product of protein
oxidation. As an apparently stable metabolic end product, dityrosine
may prove to be an extremely valuable (cellular or urinary) marker or
index of organismal oxidative stress.