Nabil Ali Mohammed Sultan

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

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.

Purification and physicochemical characterization of two galactose-specific isolectins from the seeds of Trichosanthes cordata

A galactose‐specific lectin has been purified from the seeds of Trichosanthes cordata by affinity chromatography on crosslinked guar gum. The affinity‐eluted lectin could be resolved into two isolectins, TCA‐I and TCA‐II by ion‐exchange chromatography on DEAE cellulose. The molecular weights of the isolectins were determined as 59 and 52 kDa by SDS‐PAGE. TCA‐I is a heterodimer in which the two subunits with masses of 32 and 27 kDa, are covalently connected by disulfide bonds. TCA‐I and TCA‐II are glycoproteins with 6.2% and 6.8% covalently bound neutral sugar, respectively. CD spectroscopic studies indicate that the two isolectins are very similar in secondary structure and contain about 8 to 10% α‐helix, 37–38% β‐sheet, 20% β‐turns, and 32–33% unordered structures. These isolectins have similar carbohydrate specificities as revealed by hemagglutination‐inhibition assays. Carbohydrate specificity, subunit size and composition, and secondary structure of TCA isolectins suggest close similarity to type‐II ribosome inactivating proteins. The agglutination activity of TCA‐I was found to be highest in the pH range 7.0–8.0. The lectin activity was unaffected between 0 and 40°C, but decreased dramatically above 40°C. Association constant for the interaction of TCA‐I with lactose was determined by monitoring ligand‐induced changes in the protein intrinsic fluorescence characteristics as 7.42 × 103 M−1 at 25°C. The exposure and accessibility of the tryptophan residues of TCA‐I and the effect of ligand binding on them have been probed by quenching studies employing neutral and ionic quenchers.

Galactose-Specific Seed Lectins from Cucurbitaceae

Lectins, the carbohydrate binding proteins have been studied extensively in view of their ubiquitous nature and wide-ranging applications. As they were originally found in plant seed extracts, much of the work on them was focused on plant seed lectins, especially those from legume seeds whereas much less attention was paid to the lectins from other plant families. During the last two decades many studies have been reported on lectins from the seeds of Cucurbitaceae species. The main focus of the present review is to provide an overview of the current knowledge on these proteins, especially with regard to their physico-chemical characterization, interaction with carbohydrates and hydrophobic ligands, 3-dimensional structure and similarity to type-II ribosome inactivating proteins. The future outlook of research on these galactose-specific proteins is also briefly considered.

Fluorescence studies on the interaction of hydrophobic ligands with Momordica charantia (bitter gourd) seed lectin

The interaction of Momordica charantia (bitter gourd) seed lectin (MCL) with several nucleic acid bases has been investigated by monitoring changes induced in the protein fluorescence by ligand binding. Values of the binding constant, K(a) were obtained as 1.1 x 10(4), 1.56 x 10(4) and 2.2 x 10(3) M(-1) for adenine, cytosine and uracil, respectively. In addition, binding of 8-anilinonaphthalene 1-sulfonate (ANS) with MCL was investigated by fluorescence spectroscopy. Interaction with MCL at low pH results in a large enhancement of the fluorescence intensity of ANS with a concomitant blue shift in the emission lambda(max), whereas at neutral and basic pH changes in both fluorescence intensity and emission maximum were very small, clearly suggesting that the MCL-ANS interaction is stronger at lower pH values. When excited at 295 nm in the presence of ANS, the protein fluorescence decreased with a concomitant increase in the emission intensity of ANS, suggesting resonance energy transfer from the tryptophan residues of MCL to ANS. Gel filtration profiles of MCL at pH values 2.0 and 7.4 are similar indicating that the tetrameric nature of MCL is retained even at low pH. Addition of lactose or adenine to MCL-ANS mixture did not alter the change in ANS fluorescence suggesting that lactose, adenine and ANS bind to MCL at independent and non-interacting sites. These results are relevant to understanding the functional role of MCL in the parent tissue.