Abstract
In investigating the binding of ligands to proteins, many studies have been concerned with answering the fundamental questions proposed by Scatchard (1), that is, “How many” and “How tightly bound?” Although these questions are of utmost importance, the kineticist also wishes to know “How rapidly?” The development of rapid kinetic methodologies has provided many of the tools to answer the latter question (2–4). Suitably designed kinetic experiments may, in many instances, be able to provide answers to the former questions as well. Moreover, kinetic studies may yield mechanistic insight that cannot be elucidated from other approaches.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Scatchard, G. (1949) The attractions of proteins for small molecules and ions. Ann. N. Y. Acad. Sci. 51, 660–672.
Johnson, K. A. (1986) Rapid kinetic analysis of mechanochemical adenosinetriphosphatases. Methods Enzymol. 134, 677–705.
Johnson, K. A. (1992) Transient-state kinetic analysis of enzyme reaction pathways in The Enzymes, 3rd ed, Vol. 20 (Sigman, D. S., ed.), Harcourt Brace Jovanovich, San Diego, pp. 1–60.
Gutfreund, H. (1999) Rapid-flow techniques and their contributions to enzymology. Trends Biochem. Sci. 24, 457–460.
Newton, A. C. and Johnson, J. E. (1998) Protein kinase C: a paradigm for regulation of protein function by two membrane-targeting modules. Biochim. Biophys. Acta 1376, 155–172.
Berridge, M. J. (1990) Calcium oscillations. J. Biol. Chem. 265, 9583–9586.
Werner, M. H., Bielawska, A. E., and Hannun, Y. A. (1992) Multiphasic generation of diacylglycerol in thrombin-activated human platelets. Biochem. J. 282, 815–820.
Johnson, J. E., Giorgione, J., and Newton, A. C. (2000) The C1 and C2 domains of protein kinase C are independent membrane targeting modules, with specificity for phosphatidylserine conferred by the C1 domain. Biochemistry 39, 11,360–11,369.
Orr, J. W. and Newton, A. C. (1992) Interaction of protein kinase C with phosphatidylserine. 1. Cooperativity in lipid binding. Biochemistry 31(19), 4661–4667.
Ananthanarayanan, B., Das, S., Rhee, S. G., Murray, D., and Cho, W. (2002) Membrane targeting of C2 domains of phospholipase C-δ isoforms. J. Biol. Chem. 277, 3568–3575.
Nalefski, E. A. and Falke, J. J. (2002) Use of fluorescence resonance energy transfer to monitor Ca2+-triggered membrane docking of C2 domains. Methods Mol. Biol. 172, 295–303.
Bazzi, M. D. and Nelsestuen, G. L. (1987) Association of protein kinase C with phospholipid vesicles. Biochemistry 26, 115–122.
Beechem, J. M. (1992) Global analysis of biochemical and biophysical data. Methods Enzymol. 210, 37–53.
Nalefski, E. A. and Newton, A. C. (2001) Membrane binding kinetics of protein kinase C βII mediated by the C2 domain. Biochemistry 40, 13,216–13,229.
Gill, S. C. and von Hippel, P. H. (1989) Calculation of protein extinction coefficients from amino acid sequence data. Anal. Biochem. 182(2), 319–326.
Smith, P. K., Krohn, R. I., Hermanson, G. T, and Klenk, D. C. (1985) Measurement of protein using bicinchoninic acid. Anal. Biochem. 150, 76–85.
Bartlett, G. R. (1958) Phosphorus assay in column chromatography. J. Biol. Chem. 234, 466–468.
Arbuzova, A., Wang, J., Murray, D., Jacob, J., Cafiso, D. S., and McLaughlin, S. (1997). Kinetics of interaction of the myristoylated alanine-rich C kinase substrate, membranes, and calmodulin. J. Biol. Chem. 272, 27,167–27,177.
Taylor, J. R. (1997) An Introduction to Error Analysis, 2nd ed, University Science Books, Sausalito, CA.
Mannervik, B. (1982). Regression analysis, experimental error, and statistical criteria in the design and analysis of experiments for discrimination between rival kinetic models. Methods Enzymol. 87, 370–390.
Straume, M. and Johnson, M. L. (1992) Analysis of residuals: criteria for determining goodness-of-fit. Methods Enzymol. 210, 87–105.
Tonomura, B., Nakatani, H., Ohnishi, M., Yamaguchi-Ito, J., and Hiromi, K. (1978) Test reactions for a stopped-flow apparatus. Reduction of 2,6-dichlorophenolindophenol and potassium ferricyanide by L-ascorbic acid. Anal. Biochem. 84, 370–383.
Torok, K. and Trentham, D. R. (1994) Mechanism of 2-chloro-(ε-amino-Lys75)-[6-[4-(N,N-diethylamino)phenyl]-1,3,5-triazin-4-yl]calmodulin interactions with smooth muscle myosin light chain kinase and derived peptides. Biochemistry 33, 12,807–12,820.
Zhao, Z., Rothery, R. A., and Weiner, J. H. (1999) Stopped-flow studies of the binding of 2-n-heptyl-4-hydroxyquinoline-N-oxide to fumarate reductase of Escherichia coli. Eur. J. Biochem. 260, 50–56.
Lacourciere, K. A., Stivers, J. T., and Marino, J. P. (2000) Mechanism of neomycin and Rev peptide binding to the Rev responsive element of HIV-1 as determined by fluorescence and NMR spectroscopy. Biochemistry 39, 5630–5641.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2003 Humana Press Inc., Totowa, NJ
About this protocol
Cite this protocol
Nalefsk, E.A., Newton, A.C. (2003). Use of Stopped-Flow Fluorescence Spectroscopy to Measure Rapid Membrane Binding by Protein Kinase C. In: Newton, A.C. (eds) Protein Kinase C Protocols. Methods in Molecular Biology™, vol 233. Humana Press. https://doi.org/10.1385/1-59259-397-6:115
Download citation
DOI: https://doi.org/10.1385/1-59259-397-6:115
Publisher Name: Humana Press
Print ISBN: 978-1-58829-068-7
Online ISBN: 978-1-59259-397-2
eBook Packages: Springer Protocols