Methionine oxidation and inactivation of alpha 1-proteinase inhibitor by Cu2+ and glucose.
The effect of glucose/Cu2+ incubation on (a) pure methionine oxidation, (b) the oxidation of active-site methionine in alpha 1-proteinase inhibitor (alpha 1PI) and (c) the resulting activity and structural changes of this inhibitor was investigated. While no methionine was oxidized during a 24 day, 37 degrees C incubation with 0.01 M EDTA and 100 mM glucose, 64.2% oxidation occurred in 6 days when 0.01 mM Cu2+ was added to the 100 mM glucose. The first-order rate constant for oxidation in 10 mM glucose, 0.01 mM Cu2+ was 0.0218 day-1. Oxidation was inhibited by catalase, but accelerated by ascorbate ion. The active-site methionyl residue of alpha 1PI was oxidized 71.3% after a 4 day incubation in 100 mM glucose, 0.01 mM Cu2+ (pH 7.45), 0.1 M phosphate buffer. The elastase and trypsin inhibiting activities were lowered to 3.1 and 1.5% of control samples during this incubation. The inclusion of 1 mM DETAPAC, a transition metal chelator, resulted in a 98 + % retention of activity. Intrinsic fluorescence (350 nm excitation, 415 nm emission) of alpha 1PI increased 576% over control for the sample incubated in 100 mM glucose, 0.01 mM Cu2+ and SDS-PAGE revealed protein fragment molecular weights of 44.4 and 39.8 kDa. These studies suggest that both methionine oxidation and free radical induced fragmentation contribute to loss of alpha 1PI activity during glucose/Cu2+ incubations.
Hall
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PK
University of Wisconsin Centers, Marshfield.
Roberts
R C
RC
D001665:Binding Sites; D002413:Cations, Divalent; D002614:Chelating Agents; D003300:Copper / Q000494:pharmacology*; D004591:Electrophoresis, Polyacrylamide Gel; D005453:Fluorescence; D005947:Glucose / Q000494:pharmacology*; D006801:Humans; D008715:Methionine / Q000378:metabolism*; D010084:Oxidation-Reduction; D010196:Pancreatic Elastase / Q000037:antagonists & inhibitors; D000515:alpha 1-Antitrypsin / Q000378:metabolism*
D002413:Cations, Divalent; D002614:Chelating Agents; D000515:alpha 1-Antitrypsin; D003300:Copper; D008715:Methionine; D010196:Pancreatic Elastase; D005947:Glucose