Brain Type II Calcium and Calmodulin-Dependent Protein Kinase: Purification, Characterization and Molecular Cloning

Neurobiology

Bennett, Mark Knowles ; Kennedy, Mary B.

University:California Institute of Technology

Department:Biology

关键词: Neurobiology;   

Others  :  https://thesis.library.caltech.edu/11456/2/Bennett_MK_1986.pdf

美国|英语

来源: Caltech THESIS

【 摘 要 】

A combination of biochemical, immunochemical, and molecular biological techniques have been employed to purify and characterize a rat brain Ca²⁺/calmodulin-dependent protein kinase. The enzyme, named type II Ca²⁺/calmodulin-dependent protein kinase (type II CaM kinase), was identified in rat brain homogenates by its ability to phosphorylate site II on the synaptic vesicle associated protein synapsin I.

Type II CaM kinase has been purified 290 fold over crude homogenates and is found to be composed of multiple copies of two different subunits. Both subunits copurify with kinase activity and are coprecipitated with kinase activity by an anti-kinase monoclonal antibody. The two subunits have molecular weights of 50,000 (α) and 58,000/60,000 (β), and are present in a 3:1 α:β ratio. The type II CaM kinase holoenzyme has a sedimentation coefficient of 16.4 S, a Stokes radius of 95 Å, and a calculated molecular weight of 650,000. A dodecameric holoenzyme consisting of 9 α subunits and 3 β subunits has been proposed. The purified type II CaM kinase phosphorylates several substrates, in addition to synapsin I, at a significant rate, and may therefore be responsible for a number of neuronal responses to Ca²⁺.

The α subunit of type II CaM kinase has a number of biochemical characteristics which are similar to the major protein component of a subcellular fraction which is derived from brain postsynaptic densities (PSDs). A direct comparison between the a subunit of type II CaM kinase and the major PSD protein using immunochemical and biochemical techniques has revealed that they are in fact very similar or identical proteins.

Two approaches have been taken to further characterize the subunits of type II CaM kinase at a molecular level. The first approach has been to isolate cDNA clones which code for the β subunit. A number of clones have been isolated and sequenced. The ammo acid sequence for the β subunit (predicted from the cDNA sequence) is homologous to several other protein kinases. Southern blot analysis with a β subunit cDNA indicates the existence of a type II CaM kinase multigene family. The second approach to the molecular characterization of the type II CaM kinase subunits has been to determine the amino acid sequence of peptides derived from the α subunit. Two regions of α subunit sequence have been determined, and both are found to be homologous to regions of β subunit amino acid sequence deduced from β subunit cDNA clones.

The molecular characterization of neuronal type II CaM kinase in vitro has both provided insight into the possible function of the enzyme in vivo and suggested experimental approaches which may eventually allow its in vivo function to be directly addressed.