부산대학교 도서관

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.bbamem.2005.09.014 Byline: Shadi Abu-Baker (a), Xiaoyang Qi (b), Justin Newstadt (a), Gary A. Lorigan (a) Keywords: Saposin C; Dioleoylphosphatidylglycerol; Dioleoylphosphatidylserine; Solid-state NMR spectroscopy; Molecular order parameter; Mixed bilayer Abbreviations: Sap C, Saposin C; S.sub.CD, Molecular Order Parameters; CSA, Chemical Shift Anisotropy; GCase, Glucosylceramidase; CL, Cardiolipin; DOPG, Dioleoylphosphatidylglycerol; DOPS, Dioleoylphosphatidylserine; PC, Phosphoatidyecholines; PG, Phosphatidylglycerol; PS, Phosphatidylserine; DSPG-d70, Distearoyl-d70-phosphatidylglycerol; DMPC, Dimyristylphosphatidylcholine; HCL, Hydrochloric Acid; TFE, 2,2,2 Triflouroethanol; PLL, Poly(l-lysine); NMR, Nuclear Magnetic Resonance; CP-MAS, Cross-Polarization Magic-Angle Spinning; HPLC, High Performance Liquid Chromatography; MALDI-TOF, Matrix Assisted Laser Desorption Ionization Time-of-flight Mass Spectrometry; MLVs, Multilamellar vesicles Abstract: Saposin C (Sap C) is known to stimulate the catalytic activity of the lysosomal enzyme glucosylceramidase (GCase) that facilitates the hydrolysis of glucosylceramide to ceramide and glucose. Both Sap C and acidic phospholipids are required for full activity of GCase. In order to better understand this interaction, mixed bilayer samples prepared from dioleoylphosphatidylglycerol (DOPG) and dioleoylphosphatidylserine (DOPS) (5:3 ratio) and Sap C were investigated using.sup.2H and.sup.31P solid-state NMR spectroscopy at temperatures ranging from 25 to 50 [degrees]C at pH 4.7. The Sap C concentrations used to carry out these experiments were 0 mol%, 1 mol% and 3 mol% with respect to the phospholipids. The molecular order parameters (S.sub.CD) were calculated from the dePaked.sup.2H solid-state NMR spectra of Distearoyl-d70-phosphatidylglycerol (DSPG-d70) incorporated with DOPG and DOPS binary mixed bilayers. The S.sub.CD profiles indicate that the addition of Sap C to the negatively charged phospholipids is concentration dependent. S.sub.CD profiles of 1 mol% of the Sap C protein show only a very slight decrease in the acyl chain order. However, the S.sub.CD profiles of the 3 mol% of Sap C protein indicate that the interaction is predominantly increasing the disorder in the first half of the acyl chain near the head group (C1-C8) indicating that the amino and the carboxyl termini of Sap C are not inserting deep into the DOPG and DOPS mixed bilayers. The.sup.31P solid-state NMR spectra show that the chemical shift anisotropy (CSA) for both phospholipids decrease and the spectral broadening increases upon addition of Sap C to the mixed bilayers. The data indicate that Sap C interacts similarly with the head groups of both acidic phospholipids and that Sap C has no preference to DOPS over DOPG. Moreover, our solid-state NMR spectroscopic data agree with the structural model previously proposed in the literature [X. Qi, G.A. Grabowski, Differential membrane interactions of saposins A and C. Implication for the functional specificity, J. Biol. Chem. 276 (2001) 27010-27017] [1]. Author Affiliation: (a) Department of Chemistry and Biochemistry, Miami University, Oxford, OH 45056, USA (b) Division and Program of Human Genetics, Cincinnati Children's Hospital Medical Center and University of Cincinnati College of Medicine, Cincinnati, OH 45229, USA Article History: Received 15 July 2005; Revised 7 September 2005; Accepted 12 September 2005