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Published on: August 27, 2016
Purification and characterization of a possible protooncogene fyn product, p59fyn, from a rat brain particulate
1Department of Biochemistry, Hiroshima University School of Medicine.
Abstract:
Four tyrosine-protein kinases that reacted with antibodies specific to p62c-yes, p60c-src, p60c-src+, and p59fyn, respectively, were solubilized from a rat brain particulate fraction and separated by casein-Toyopearl column chromatography. Possible p59fyn, with a pI of 6.5, was purified 490-fold as a single 59-kDa protein band on SDS-PAGE. The purified enzyme contained almost no phosphotyrosine residues but was autophosphorylated with Mg2+. ATP exclusively at tyrosine residues, with a concomitant increase in the kinase activity toward tyrosine-glutamate (1:4) copolymers. The rate of the copolymer phosphorylation was proportional to the square of the enzyme concentration, suggesting activation through intermolecular catalysis. In the presence of Mn2+, however, the reaction showed a first-order dependence on the enzyme concentration.
Insights
Researchers purified a rat brain tyrosine-protein kinase, p59fyn, and found its activity increases with enzyme concentration, suggesting intermolecular catalysis. This discovery offers insights into kinase regulation mechanisms.
Area of Science:
- Biochemistry
- Molecular Biology
- Neuroscience
Background:
- Tyrosine-protein kinases play crucial roles in cellular signaling pathways.
- Identifying and characterizing specific kinases, such as those in the src-family, is essential for understanding their functions.
- Rat brain is a rich source of diverse kinase enzymes.
Purpose of the Study:
- To solubilize and separate tyrosine-protein kinases from rat brain particulate fractions.
- To purify and characterize a specific kinase, p59fyn.
- To investigate the catalytic properties and activation mechanisms of purified p59fyn.
Main Methods:
- Solubilization of kinases from rat brain particulate fraction.
- Separation using casein-Toyopearl column chromatography.
- Purification and characterization by SDS-PAGE and isoelectric focusing.
- Enzyme kinetics studies using Mg2+, Mn2+, ATP, and tyrosine-glutamate copolymers.
Main Results:
- Four tyrosine-protein kinases, including p59fyn, were identified.
- p59fyn was purified 490-fold as a 59-kDa protein.
- Purified p59fyn autophosphorylated on tyrosine residues and showed increased kinase activity.
- Enzyme concentration dependence suggested intermolecular catalysis with Mg2+ and first-order kinetics with Mn2+.
Conclusions:
- p59fyn is a tyrosine-protein kinase present in rat brain.
- The kinase exhibits distinct activation mechanisms dependent on divalent cations (Mg2+ vs. Mn2+).
- Intermolecular catalysis may be involved in the activation of p59fyn under specific conditions.
