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Phosphofructokinase from mollusc muscle is activated by phosphorylation
M Biethinger1, R Hoffmann, H W Hofer
1Faculty of Biology, University of Konstanz, Germany.
Abstract:
Phosphofructokinase was purified from muscle tissue of two different molluscs, edible snails, Helix pomatia (gastropoda), and mussels, Mytilus edulis (bivalvia). Under denaturing conditions, both enzymes had a molecular mass of 82 kDa. In the presence of ATP-Mg2+, the enzymes were rapidly phosphorylated in vitro by the catalytic subunit of cyclic AMP (cAMP)-dependent protein kinase purified from snail muscle and also by the C subunit of protein kinase from bovine heart. The extent of phosphorylation was 0.6 and 0.5 phosphate residues per subunit for the snail and the mussel phosphofructokinase, respectively. Phosphorylation of both phosphofructokinases effected a decrease in ATP inhibition at neutral or slightly acidic pH values and increased the affinity for fructose 6-phosphate. The resulting activation in the presence of suboptimum fructose 6-phosphate concentrations was more distinct for the snail enzyme. In addition, phosphorylated phosphofructokinase from mussels exhibited a marked increase in Vmax when activated by either 5'-AMP or fructose 2,6-bisphosphate.
Insights
This study purified phosphofructokinase from snail and mussel muscles, finding phosphorylation by protein kinase altered enzyme activity and substrate affinity. These findings offer insights into molluscan enzyme regulation.
Area of Science:
- Biochemistry
- Enzymology
- Comparative Physiology
Background:
- Phosphofructokinase (PFK) is a key glycolytic enzyme.
- Molluscan PFK regulation is less understood than in other organisms.
- Enzyme phosphorylation is a common regulatory mechanism.
Purpose of the Study:
- To purify and characterize phosphofructokinase from two distinct mollusc species: Helix pomatia (gastropod) and Mytilus edulis (bivalve).
- To investigate the effects of phosphorylation by cyclic AMP (cAMP)-dependent protein kinase on the kinetic properties of these enzymes.
Main Methods:
- Purification of phosphofructokinase from snail and mussel muscle tissues.
- In vitro phosphorylation assays using purified protein kinase catalytic subunits.
- Analysis of enzyme kinetics, including ATP inhibition, fructose 6-phosphate affinity, and Vmax.
Main Results:
- Both snail and mussel PFKs had a molecular mass of 82 kDa and were phosphorylated by cAMP-dependent protein kinase.
- Phosphorylation decreased ATP inhibition and increased fructose 6-phosphate affinity for both enzymes.
- Mussel PFK showed a marked increase in Vmax upon phosphorylation when activated by 5'-AMP or fructose 2,6-bisphosphate.
Conclusions:
- Phosphorylation significantly modulates the activity and substrate binding of molluscan phosphofructokinases.
- The regulatory mechanisms of PFK in gastropods and bivalves share similarities but also exhibit species-specific differences.
- These findings contribute to understanding metabolic regulation in invertebrates.