Calcium-Binding Protein Protocols Calcium-Binding Protein Protocols

Enzymatic Assays 353
dation then selectively reports the activation of NOS’s reductase domain. When
cyt c is included NADPH oxidation should be measured at 337.5 nm to eliminate
interference from cyt c absorption (11).
8. The cyt c reduction assay should be initiated by addition of NADPH. Under these
conditions, the reaction occurs rapidly and is linear for only approx 4 min even
before the addition of CaM. Therefore, CaM must be added quickly to avoid
depletion of NADPH.
9. The cyt c reduction assay requires 5–10 times less NOS than the other assays. If
too much NOS is used, the reaction proceeds too quickly and is linear for too
short a time to follow activation by CaM. Generally, using less NOS allows the
reaction to be linear for several minutes and this allows determination of the effect
of one [CaM] per assay.
10. In the citrulline assay each sample must be corrected for the amount of radioactive
L-Arg that flows through the column and is collected with the L-citrulline
recovery. This is accomplished by subtracting the radioactive counts of a sample
that does not contain NADPH (or subtracting the counts of a reaction which was
stopped at zero time) from all other samples. Citrulline recovery can be determined
with [ 3 H]citrulline (approx 30 µM) under identical conditions. Furthermore,
the citrulline assay can only be used if any contaminating L-Arg in the
NOS sample has been removed by desalting.
4.3. MLCK Assays
11. Alternative substrates to LC20 can be used in this assay: purified myosin II or a
synthetic peptide corresponding, for example, to residues 11–23 of LC20 (with
the sequence KKRPQRATSNVFA), i.e., containing ser19, the site of phosphorylation
by MLCK. If myosin II is used, it is necessary to ensure that the preparation
is not contaminated by CaM, MLCK, or myosin light chain phosphatase.
The CaM activation curve is shifted to the right if the synthetic peptide substrate
is used rather than LC20 or myosin II.
12. Stock MLCK should be diluted just prior to its addition to the assay mixture.
Loss of activity is observed if MLCK is stored in dilute solution.
4.4. CaM Kinase II Assays
13. We copurify CaM kinase II with its substrate caldesmon since the stability of the
enzyme is much greater than it is following their separation.
References
1. Lu, K. P. and Means, A. R. (1993) Regulation of the cell cycle by calcium and
calmodulin. Endocr. Rev. 14, 40–58.
2. Lee, S. H., Kim, J. C., Lee, M. S., Heo, W. D., Seo, H. Y., Yoon, H. W., et al. (1995)
Identification of a novel divergent calmodulin isoform from soybean which has
differential ability to activate calmodulin-dependent enzymes. J. Biol. Chem. 270,
21,806–21,812.