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Mechanism of Membrane Binding by the Bovine Seminal Plasma Protein, PDC-109: A Surface Plasmon Resonance Study

Celestine J. Thomas , V. Anbazhagan ^*, M. Ramakrishnan ^*, Nabil Sultan ^*, Ira Surolia ^* and Musti J. Swamy ^*

^* School of Chemistry, University of Hyderabad, Hyderabad 500 046, India; and Molecular Biophysics Unit, Indian Institute of Science, Bangalore 560 012, India

Correspondence: Address reprint requests to Musti J. Swamy, School of Chemistry, University of Hyderabad, Hyderabad-500 046, India. Tel.: 91-40-301-1071; Fax: 91-40-301-2460/301-0145; E-mail: mjssc{at}uohyd.ernet.in.

Ira Surolia is a summer trainee from Dr. B. R. Ambedkar College, Bangalore 560 045, India.

PDC-109, the major protein of bovine seminal plasma, binds to sperm plasma membranes upon ejaculation and plays a crucial role in the subsequent events leading to fertilization. The binding process is mediated primarily by the specific interaction of PDC-109 with choline-containing phospholipids. In the present study the kinetics and mechanism of the interaction of PDC-109 with phospholipid membranes were investigated by the surface plasmon resonance technique. Binding of PDC-109 to different phospholipid membranes containing 20% cholesterol (wt/wt) indicated that binding occurs by a single-step mechanism. The association rate constant (k₁) for the binding of PDC-109 to dimyristoylphosphatidylcholine (DMPC) membranes containing cholesterol was estimated to be 5.7 x 10⁵ M^-1 s^-1 at 20°C, while the values of k₁ estimated at the same temperature for the binding to membranes of negatively charged phospholipids such as dimyristoylphosphatidylglycerol (DMPG) and dimyristoylphosphatidic acid (DMPA) containing 20% cholesterol (wt/wt) were at least three orders of magnitude lower. The dissociation rate constant (k_-1) for the DMPC/PDC-109 system was found to be 2.7 x 10^-2 s^-1 whereas the k_-1 values obtained with DMPG and DMPA was about three to four times higher. From the kinetic data, the association constant for the binding of PDC-109 to DMPC was estimated as 2.1 x 10⁷ M^-1. The association constants for different phospholipids investigated decrease in the order: DMPC > DMPG > DMPA > DMPE. Thus the higher affinity of PDC-109 for choline phospholipids is reflected in a faster association rate constant and a slower dissociation rate constant for DMPC as compared to the other phospholipids. Binding of PDC-109 to dimyristoylphosphatidylethanolamine and dipalmitoylphosphatidylethanolamine, which are also zwitterionic, was found to be very weak, clearly indicating that the charge on the lipid headgroup is not the determining factor for the binding. Analysis of the activation parameters indicates that the interaction of PDC-109 with DMPC membranes is favored by a strong entropic contribution, whereas negative entropic contribution is primarily responsible for the rather weak interaction of this protein with DMPA and DMPG.