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Ca2+/calmodulin-dependent protein kinase II from hen brain. Purification and characterization
R P Gupta1, D M Lapadula, M B Abou-Donia
1Department of Pharmacology, Duke University Medical Center, Durham, NC 27710.
Biochemical Pharmacology
|May 8, 1992
Summary
This study purified hen brain Ca2+/calmodulin-dependent protein kinase II (CaM-kinase II), revealing its subunit composition and broad substrate specificity. The enzyme showed similarities to rat CaM-kinase II, particularly in its alpha subunit.
Area of Science:
- Biochemistry
- Neuroscience
- Enzymology
Background:
- Ca2+/calmodulin-dependent protein kinase II (CaM-kinase II) is a crucial enzyme in neuronal signaling.
- Understanding the properties of CaM-kinase II from different species aids in elucidating its conserved and divergent functions.
- Hen brain CaM-kinase II purification and characterization provide insights into avian brain biochemistry.
Purpose of the Study:
- To purify and characterize Ca2+/calmodulin-dependent protein kinase II (CaM-kinase II) from hen whole brain.
- To determine the subunit composition, substrate specificity, and kinetic properties of hen brain CaM-kinase II.
- To investigate the immunocross-reactivity of hen brain CaM-kinase II with enzymes from other species.
Main Methods:
- Purification of CaM-kinase II using phosphocellulose and calmodulin-Agarose column chromatography.
- Enzyme activity assays using Microtubule associated protein-2 (MAP-2) and other substrates.
- Sodium dodecyl/sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) for subunit analysis.
- Autophosphorylation experiments and kinetic analysis (Km, K0.5).
- Generation of antiserum against the CaM-kinase II alpha subunit for cross-reactivity studies.
Main Results:
- CaM-kinase II was purified 3000-fold with a specific activity of 200 nmol/min/mg.
- The enzyme comprised alpha (46,000 Da) and beta/beta' (55,000/52,000 Da) subunits in a 3:1 ratio.
- Broad substrate specificity was observed, with preferential phosphorylation of myelin basic protein and MAP-2.
- Autophosphorylation reduced enzyme activity and altered alpha-subunit mobility.
- Hen brain CaM-kinase II showed immunocross-reactivity with the alpha subunit of CaM-kinase II from various mammalian species.
Conclusions:
- Purified hen brain CaM-kinase II is a multifunctional enzyme with properties similar to rat brain CaM-kinase II.
- The alpha subunit appears conserved across species, while beta/beta' subunits may exhibit greater divergence.
- These findings contribute to the understanding of CaM-kinase II structure-function relationships and evolutionary conservation.