31P-NMR investigation of magnetically oriented rod outer segments. Spectral analysis and identification of individual phospholipids.
A 31P-NMR study of magnetically oriented bovine rod outer segments is presented. We demonstrate that carefully isolated bovine rod outer segments retain the capacity to orient in a magnetic field. Maximal orientation (85-90%) is achieved at field strengths over 4.7 T in the NMR spectrometer. The lineshape of the 'oriented spectra' is totally different from the 'bilayer lineshape' of randomly oriented photoreceptor membranes. The oriented spectra consist of two phospholipid peaks, a major low-field peak (75-80% of the total intensity) near 30 ppm, and a minor high-field peak near - 14 ppm as well as two sharp metabolite peaks around 0 ppm. The phospholipid peaks are a composite of three narrower partially resolved resonances assigned to the individual phospholipid classes phosphatidylserine, phosphatidylcholine and phosphatidylethanolamine. Based on the morphology and magnetic anisotropy of the rod outer segment, the major phospholipid peak is attributed to the flat part of the disk membranes while the phospholipids of the plasma membrane are thought to contribute only to the minor peak. Disk rim phospholipids and non-oriented material contribute to the minor peak and, in addition, contribute some intensity to the middle part of the spectrum. The phospholipid class composition of the major peak is estimated by spectral simulation and is consistent with the phospholipid class composition of rod outer segment membranes. Hence, 31P analysis of oriented rod outer segments resolves the main phospholipids in at least two different membrane pools in the rod outer segment and allows the differential investigation of these pools. Most of the mobile phosphate metabolite intensity resides in the Pi peak at 3.5 ppm. A slight shift in the Pi resonance position indicates a 0.2 pH unit acidification upon illumination of rhodopsin. The absence of detectable nucleotide resonances, when compared with chemical analysis, indicates that the majority of the nucleotide population present is rather immobile and probably bound to the membranes.
L C
LC
Mollevanger
E A
EA
Dratz
B
B
De Kruijff
C W
CW
Hilbers
W J
WJ
De Grip
D000818:Animals; D002417:Cattle; D009682:Magnetic Resonance Spectroscopy; D008563:Membrane Lipids / Q000032:analysis; D010743:Phospholipids / Q000032:analysis*; D010758:Phosphorus; D010786:Photoreceptor Cells / Q000032:analysis*; D012374:Rod Cell Outer Segment / Q000032:analysis* / Q000648:ultrastructure
D008563:Membrane Lipids; D010743:Phospholipids; D010758:Phosphorus