Abstract
Binding of serine protease inhibitors (serpins) to nonprotein ligands such as glycosaminoglycans or phospholipids has been shown to modify their inhibitory activity and—at least in the case of SERPINA5—to mediate serpin internalization into cells. Also phospholipid functions may be altered when bound to serpins or other proteins.
By interacting with phospholipids, serpins might influence a variety of cellular functions. Binding of proteins to phospholipids can be studied by several methods. Here we describe solid-phase assays, in which pure phospholipids are immobilized on nitrocellulose membranes, PVDF membranes, or microtiter plates. Bound proteins are detected with specific antibodies and labeled secondary antibodies. We also describe a method visualizing binding of phospholipids in suspension by non-denaturing polyacrylamide gel electrophoresis (PAGE) followed by Western blotting.
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
Wahlmüller FC (2015) Protein C inhibitor. In: Geiger M, Wahlmüller FC, Furtmüller M (eds) The serpin family. proteins with multiple functions in health and disease. Springer International Publishing, Switzerland, pp 153–177
Ecke S, Geiger M, Binder BR (1997) Heparin binding of protein-C inhibitor - analysis of the effect of heparin on the interaction of protein-C inhibitor with tissue kallikrein. Eur J Biochem 248(2):475–480
Geiger M, Priglinger U, Griffin JH et al (1991) Urinary protein C inhibitor. Glycosaminoglycans synthesized by the epithelial kidney cell line TCL-598 enhance its interaction with urokinase. J Biol Chem 266(18):11851–11857
Pratt CW, Church FC (1992) Heparin binding to protein C inhibitor. J Biol Chem 267(13):8789–8794
Jerabek I, Zechmeister-Machhart M, Binder BR et al (2001) Binding of retinoic acid by the inhibitory serpin protein C inhibitor. Eur J Biochem 268(22):5989–5996
Malleier JM, Oskolkova O, Bochkov V et al (2007) Regulation of protein C inhibitor (PCI) activity by specific oxidized and negatively charged phospholipids. Blood 109(11):4769–4776
Wahlmuller FC, Sokolikova B, Rieger D et al (2014) New lipid interaction partners stimulate the inhibition of activated protein C by cell-penetrating protein C inhibitor. Thromb Haemost 111(1):41–52
Ecke S, Geiger M, Binder BR (1992) Glycosaminoglycans regulate the enzyme specificity of protein-C inhibitor. Ann N Y Acad Sci 667:84–86
Baumgartner P, Geiger M, Zieseniss S et al (2007) Phosphatidylethanolamine critically supports internalization of cell-penetrating protein C inhibitor. J Cell Biol 179(4):793–804
Rehault SM, Zechmeister-Machhart M, Fortenberry YM et al (2005) Characterization of recombinant human protein C inhibitor expressed in Escherichia coli. Biochim Biophys Acta 1748(1):57–65
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2018 Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Wahlmüller, F.C., Mihaly-Bison, J., Geiger, M. (2018). Binding of Serpins to Immobilized Phospholipids and Phospholipids in Suspension. In: Lucas, A. (eds) Serpins. Methods in Molecular Biology, vol 1826. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-8645-3_8
Download citation
DOI: https://doi.org/10.1007/978-1-4939-8645-3_8
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-8644-6
Online ISBN: 978-1-4939-8645-3
eBook Packages: Springer Protocols