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(Na+-K+)-ATPase interaction with L-ascorbic acid. Effect on p-nitrohenylphosphatase partial reaction
G A Miggiano1, G E Martorana, A Mordente
1Istituto di Chimica Biologica, Facoltà di Medicina e Chirurgia, Università Cattolica del S. Cuore, Roma.
The Italian Journal of Biochemistry
|September 1, 1988
Summary
L-ascorbic acid inhibits rabbit kidney (Na+-K+)-ATPase activity by affecting enzyme conformations. Ascorbic acid concentrations influence the kinetic rate, with low levels enhancing and high levels inhibiting enzyme function.
Area of Science:
- Biochemistry
- Enzymology
- Membrane Transport
Background:
- The (Na+-K+)-ATPase is a crucial ion pump in kidney cells, vital for maintaining cellular homeostasis.
- Understanding its regulation by small molecules like L-ascorbic acid is important for cellular function.
- Previous studies have explored various modulators of (Na+-K+)-ATPase activity.
Purpose of the Study:
- To investigate the effect of L-ascorbic acid preincubation on the partial reaction of rabbit kidney (Na+-K+)-ATPase.
- To elucidate the mechanism by which L-ascorbic acid influences enzyme kinetics and conformation.
Main Methods:
- Enzyme kinetics assays measuring p-nitrophenylphosphatase activity.
- Spectroscopic analysis of intrinsic enzyme fluorescence to detect conformational changes.
- Preincubation of purified rabbit kidney (Na+-K+-ATPase) with varying concentrations of L-ascorbic acid.
Main Results:
- L-ascorbic acid preincubation caused a pseudo-first order decay in p-nitrophenylphosphatase activity.
- The pseudosubstrate p-nitrophenylphosphate partially reversed the inhibitory effect of L-ascorbic acid.
- Ascorbic acid concentrations below 0.7 mmol/L enhanced kinetic rates, while higher concentrations inhibited activity.
- L-ascorbic acid altered intrinsic fluorescence, indicating the formation of unique enzyme intermediate forms.
Conclusions:
- L-ascorbic acid induces distinct, destabilized intermediate forms of (Na+-K+)-ATPase.
- These ascorbate-induced forms are different from cation- or phosphate-induced states.
- The enzyme's conformational flexibility is modulated by L-ascorbic acid, impacting its catalytic activity.