Lipid peroxidation as the mechanism of modification of brain 5'-nucleotidase activity in vitro.
The effect of lipid peroxidation on the Mg2(+)-independent and Mg2(+)-dependent activity of brain cell membrane 5'-nucleotidase was determined and the affinity of the active sites of Mg2(+)-dependent enzyme for 5'-AMP (substrate) and Mg2+ (activator) was examined. Brain cell membranes were peroxidized at 37 degrees C in the presence of 100 microM ascorbate and 25 microM FeCl2 (resultant) for 10 min. The activity of 5'-nucleotidase and lipid peroxidation products (thiobarbituric acid reactive substances) were determined. At 10 min, the level of lipid peroxidation products increased from 0.20 +/- 0.10 to 17.5 +/- 1.5 nmoles malonaldehyde/mg membrane protein. The activity of Mg2(+)-independent 5'-nucleotidase increased from 0.201 +/- 0.020 in controls to 0.305 +/- 0.028 mumol Pi/mg protein/hr in peroxidized membranes. In the presence of 10 mM Mg2+, the activity increased by 5.8-fold in the peroxidized membrane preparation in comparison to 14-fold in control. In peroxidized preparation, the affinity of active site of Mg2(+)-dependent 5'-nucleotidase for 5'-AMP tripled, as indicated by a significant decrease in Km (Km = 95 +/- 2 microM AMP for control; Km = 32 +/- 2 microM AMP for peroxidized). Vmax was significantly reduced from 3.35 +/- 0.16 in control to 1.70 +/- 0.9 mumoles Pi/mg protein in peroxidized membranes. The affinity of the active site for Mg2+ significantly increased (Km = 6.17 +/- 0.37 mM Mg2+ for control; Km = 4.0 +/- 0.31 peroxidized).(ABSTRACT TRUNCATED AT 250 WORDS)
Mishra
O P
OP
Department of Physiology, University of Pennsylvania School of Medicine, Philadelphia 19104.
Delivoria-Papadopoulos
M
M
Cahillane
G
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Wagerle
L C
LC
D015720:5'-Nucleotidase / Q000378:metabolism*; D000249:Adenosine Monophosphate / Q000378:metabolism; D000818:Animals; D001205:Ascorbic Acid / Q000494:pharmacology; D001665:Binding Sites; D001921:Brain / Q000201:enzymology*; D002462:Cell Membrane / Q000201:enzymology; D005296:Ferrous Compounds / Q000494:pharmacology; D015227:Lipid Peroxidation; D008274:Magnesium / Q000494:pharmacology; D008315:Malondialdehyde / Q000378:metabolism; D013552:Swine; D013858:Thiobarbiturates
D005296:Ferrous Compounds; D013858:Thiobarbiturates; D000249:Adenosine Monophosphate; D008315:Malondialdehyde; D015720:5'-Nucleotidase; D008274:Magnesium; C029684:thiobarbituric acid; D001205:Ascorbic Acid; C029451:ferrous chloride