The interaction of the major acidic bovine seminal plasma protein, PDC-109, with dimyristoylphosphatidylcholine (DMPC) membranes has been investigated by spin-label electron spin resonance spectroscopy. Studies employing phosphatidylcholine spin labels, bearing the spin labels at different positions along the sn-2 acyl chain indicate that the protein penetrates into the hydrophobic interior of the membrane and interacts with the lipid acyl chains up to the 14th C atom. Binding of PDC-109 at high protein/lipid ratios (PDC-109:DMPC = 1:2, w/w) results in a considerable decrease in the chain segmental mobility of the lipid as seen by spin-label electron spin resonance spectroscopy. A further interesting new observation is that, at high concentrations, PDC-109 is capable of (partially) solubilizing DMPC bilayers. The selectivity of PDC-109 in its interaction with membrane lipids was investigated by using different spin-labeled phospholipid and steroid probes in the DMPC host membrane. These studies indicate that the protein exhibits highest selectivity for the choline phospholipids phosphatidylcholine and sphingomyelin under physiological conditions of pH and ionic strength. The selectivity for different lipids is in the following order: phosphatidylcholine approximately sphingomyelin > or = phosphatidic acid (pH 6.0) > phosphatidylglycerol approximately phosphatidylserine approximately and rostanol > phosphatidylethanolamine > or = N-acyl phosphatidylethanolamine >> cholestane. Thus, the lipids bearing the phosphocholine moiety in the headgroup are clearly the lipids most strongly recognized by PDC-109. However, these studies demonstrate that this protein also recognizes other lipids such as phosphatidylglycerol and the sterol androstanol, albeit with somewhat reduced affinity.

No abstract

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 (k(1)) for the binding of PDC-109 to dimyristoylphosphatidylcholine (DMPC) membranes containing cholesterol was estimated to be 5.7 x 10(5) M(-1) s(-1) at 20 degrees C, while the values of k(1) 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 10(7) 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.

The interaction of the major bovine seminal plasma protein, PDC-109 with lipid membranes was investigated by isothermal titration calorimetry. Binding of the protein to model membranes made up of diacyl phospholipids was found to be endothermic, with positive values of binding enthalpy and entropy, and could be analyzed in terms of a single type of binding sites on the protein. Enthalpies and entropies for binding to diacylphosphatidylcholine membranes increased with increase in temperature, although a clear-cut linear dependence was not observed. The entropically driven binding process indicates that hydrophobic interactions play a major role in the overall binding process. Binding of PDC-109 with dimyristoylphosphatidylcholine membranes containing 25 mol% cholesterol showed an initial increase in the association constant as well as enthalpy and entropy of binding with increase in temperature, whereas the values decreased with further increase in temperature. The affinity of PDC-109 for phosphatidylcholine increased at higher pH, which is physiologically relevant in view of the basic nature of the seminal plasma. Binding of PDC-109 to Lyso-PC could be best analysed in terms of two types of binding interactions, a high affinity interaction with Lyso-PC micelles and a low-affinity interaction with the monomeric lipid. Enthalpy-entropy compensation was observed for the interaction of PDC-109 with phospholipid membranes, suggesting that water structure plays an important role in the binding process.

Binding of PDC-109, the major protein of the bovine seminal plasma, to sperm plasma membrane results in an e¥ux of cholesterol and choline phospholipids, a necessary event before capacitation can occur. The selectivity of PDC-109 for di¡erent spin-labelled phospholipids and sterol probes in dimyristoylphosphatidylcholine (DMPC) host matrix has been char-