Human erythrocytes glucose-6-phosphate dehydrogenase: Labelling of a reactive lysyl residue by pyridoxal-5′-phosphate

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Abstract

Reaction of glucose-6-phosphate dehydrogenase from human erythrocytes with pyridoxal-5′-phosphate causes 80% loss of activity. The substrate glucose-6-phosphate fully protects the enzyme against this inhibition, which is reversible upon dilution, but becomes irreversible after treatment with NaBH4. We presume that pyridoxal-5′-phosphate forms with the enzyme a Schiff base which is reduced by NaBH4. One mole of N-ϵ-pyridoxyl-lysine is formed per mole of enzyme subunit when the remaining activity reaches its minimal level of 20%.

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    Citation Excerpt :

    The samples were then incubated in the dark at 25 °C with PLP under mild conditions (100-fold molar excess) for 90 min as described elsewhere (18). The Schiff base was reduced by the addition of an equivalent molar amount of NaBH4 with respect to PLP as reported elsewhere (19). The excess of PLP and NaBH4 in the samples was removed by dialysis and equilibrated against 50 mm sodium phosphate buffer, pH 7.0, and 1 mm EDTA.

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