Calcium-Binding Protein Protocols Calcium-Binding Protein Protocols

340 Walsh et al.
verts CaM from an activator to a competitive antagonist of NOS (4). A great
deal can be learned, therefore, from studying the effect of CaM mutants and
isoforms on the activation of various CaM target enzymes.
In this chapter, we describe assay methods for such a comparison using five
CaM target enzymes: PDE, CaN, NOS, myosin light chain kinase (MLCK),
and Ca 2+ /CaM-dependent protein kinase II (CaM kinase II). CaM-dependent
PDE catalyzes the hydrolysis of cAMP and cGMP to the corresponding 5'-nucleoside
monophosphates, thereby terminating cyclic nucleotide signaling (5). This
enzyme, therefore, represents an important point of cross-talk between Ca 2+
and cyclic nucleotide signaling pathways. CaN is a Ca 2+ /CaM-dependent protein
serine/threonine phosphatase with a relatively narrow substrate specificity
(6). It has diverse regulatory roles, e.g., T-lymphocyte activation, regulation of
neurotransmitter release, and modulation of long-term changes in synaptic plasticity.
It is the target of the immunosuppressive drugs, FK506 and cyclosporin
A. NOS catalyzes the formation of the intercellular messenger nitric oxide (NO)
from L-arginine. NO is a major regulator in the nervous, immune, and cardiovascular
systems (7). There are two classes of NOS: constitutive and inducible.
Constitutive NOS is regulated by Ca 2+ -dependent interaction with CaM,
whereas inducible NOS contains tightly bound CaM that is not dissociated by
chelation of Ca 2+ ions. MLCK plays a key role in the regulation of smooth
muscle contraction and nonmuscle motility via the specific phosphorylation of
myosin II (8). Finally, CaM kinase II, unlike MLCK, has a large number of
substrates and is, therefore, involved in the regulation of diverse physiological
processes including synaptic transmission, secretion, and gene expression (9).
For activation of PDE, CaN, and NOS by CaM, we describe continuous
assays that allow enzymatic activity to be monitored by changes in fluorescence
or absorption upon enzyme activation. For activation of MLCK, CaM
kinase II, and NOS by CaM, we describe radioisotope-based assays that follow
incorporation of 32 P from [γ- 32 P]ATP into the 20-kDa light chain of myosin II
(LC 20), incorporation of 32 P from [γ- 32 P]ATP into caldesmon and conversion
of L-[ 14 C] arginine to L-citrulline and NO, respectively.
For most of these enzymes, CaM binds and removes a pseudosubstrate
(autoinhibitory) domain from the enzyme’s active site resulting in enzyme
activation. CaM activation of NOS is more complicated (10,11). CaM binds
NOS between its N-terminal oxygenase domain and its C-terminal reductase
domain and it stimulates NADPH oxidation and reduction of bound flavin in
NOS’s reductase domain (11). CaM also facilitates electron transfer from the
reductase domain to the heme-containing oxygenase domain, resulting in
the conversion of L-Arg to NO and L-citrulline. Finally, CaM can stimulate the
intermolecular transfer of electrons from NOS’s reductase domain to exogenous
electron acceptors like cytochrome c (cyt c). Thus, for activation of NOS