Related Experiment Videos
Affinity elution of pyruvate kinase from phosphocellulose
Acta Biologica Et Medica Germanica
|January 1, 1975
Summary
Researchers purified pyruvate kinase from ascites tumor cells using affinity elution chromatography. This method selectively desorbs the enzyme using specific ligands like phosphoenolpyruvate, yielding highly active M-type pyruvate kinase.
Area of Science:
- Biochemistry
- Enzymology
- Protein Purification
Background:
- Pyruvate kinase is a crucial enzyme in glycolysis.
- Purification of pyruvate kinase from ascites tumor cells presents challenges.
- Specific elution techniques are needed for efficient enzyme isolation.
Purpose of the Study:
- To develop an efficient purification method for pyruvate kinase from ascites tumor cells.
- To investigate the efficacy of affinity elution chromatography for enzyme purification.
- To optimize conditions for selective enzyme desorption.
Main Methods:
- Affinity elution chromatography using phosphocellulose as the ion-exchanger.
- Elution of pyruvate kinase with low concentrations of specific ligands: phosphoenolpyruvate, fructose 1,6-bisphosphate, adenosine 5'-diphosphate, and pyrophosphate.
- Determination of optimal conditions for facilitated desorption, selectivity, and recovery.
Main Results:
- Pyruvate kinase was effectively eluted from phosphocellulose by low ligand concentrations.
- Affinity elution chromatography demonstrated high selectivity and recovery.
- Affinity elution with phosphoenolpyruvate proved highly effective for M-type pyruvate kinase preparation.
- An almost homogeneous preparation of pyruvate kinase with a specific activity of 420 was obtained.
Conclusions:
- Affinity elution chromatography is a highly effective method for purifying pyruvate kinase from ascites tumor cells.
- The method allows for selective desorption of the enzyme based on specific ligand interactions.
- This technique enables the preparation of highly active M-type pyruvate kinase.