A CELL-FREE APPROACH TO THE STUDY OF MEMBRANE TRAFFICKING IN FROZEN RAT BRAINS POTENTIALLY APPLICABLE TO FROZEN AUTOPSY MATERIAL

摘要

A cell-free transfer system was used to measure capacity of brain membranes to support membrane renewal. To study transfer in brain, radiolabeled donor microsome fractions were prepared using brain slices from rats or frozen human brain autopsy specimens. Acceptor fractions, prepared from fresh or frozen rat brain or frozen human brain autopsy specimens, were immobilized on nitrocellulose. The complete reconstituted transfer system contained ATP plus ATP-regenerating system (or NADH) as a source of energy and brain cytosol. Slices of frozen brain incorporated acetate into membrane lipids with approximately the same efficiency as fresh brains. This efficiency declined with storage at 4 degrees C but only slowly. Donor fractions labeled with acetate from frozen slices exhibited specific transfer (37 degrees C minus 4 degrees C) of labeled membrane lipids with efficiencies comparable to fresh. The acceptor fraction could be prepared either from fresh or frozen material. Transfer was on the average two-fold stimulated by ATP at 37 degrees C compared to no ATP. Transfer also was stimulated by NADH. Analysis of linear transfer rates between 0 and 30 min revealed no significant effect of delay time or of time of prolonged storage on transfer efficiency beyond an initial decline of ca. 25% observed within the first two weeks after freezing. A decline of transfer was obtained with brains as the animals aged. [References: 21]