GENE 540 Midterm: MPF Kinase Activity
GENETICS 540 – Molecular Structure & Processes
MPF Kinase Activity
Kinase activity assay
• You take whatever sample you want to see if it has kinase
activity and you also add a “model” substrate protein such as
histone H1
• Then you add gamma-32P ATP and then run the reaction
• Then after sometime, if you see 32P-sample, then your sample is now phosphorylated
We are trying to purify MPF from an extract that has a MPF activity
- Run the sample on a polyacrylamide gel and then look at which fractions had which
major proteins, etc…
- They found that when there is an induction of mitosis, you get cyclin B and Cdk1
activity, as well as H1 kinase activity; H1 kinase activity also co-purifies with MPF
- Remember kinases phosphorylates serines, threonines and tyrosines; there are 2 classes
of kinases: one class phosphorylates serines and threonines and the other class
phosphorylates tyrosines; hence its not the amino acid that is the substrate, but rather
the amino acid residue that is sticking out of the protein
- These kinases take the gamma-phosphate from ATP and add it to the appropriate amino
acid residue
- They found that the cdk1 was similar to the cdc2 kinase → they found that MPF
consists of active Cdk1 and cyclin B
All eukaryotes have these 2 proteins that are required to go to M-phase: cdk1 and cyclin B
(regulatory)
o The heterodimer is an S/T protein kinase.
o 4Cyclin is the regulatory subunit.
o 4The catalytic subunit is Cdc2, or
o Cyclin-dependent kinase 1 (Cdk1) 4The Cdk is only active when bound to a cyclin 4Cdk1
does not cycle
o 4Cyclin B accumulates during interphase and
Document Summary
You take whatever sample you want to see if it has kinase activity and you also add a model substrate protein such as histone h1. Then you add gamma-32p atp and then run the reaction: then after sometime, if you see 32p-sample, then your sample is now phosphorylated. We are trying to purify mpf from an extract that has a mpf activity. Run the sample on a polyacrylamide gel and then look at which fractions had which major proteins, etc . They found that when there is an induction of mitosis, you get cyclin b and cdk1 activity, as well as h1 kinase activity; h1 kinase activity also co-purifies with mpf. These kinases take the gamma-phosphate from atp and add it to the appropriate amino acid residue. They found that the cdk1 was similar to the cdc2 kinase they found that mpf consists of active cdk1 and cyclin b.
