Summary
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 °C but only slowly. Donor fractions labeled with acetate from frozen slices exhibited specific transfer (37 °C minus 4 °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 °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.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Balch WE, Dunphy WG, Braell WA, Rothman JE (1984) Reconstitution of the transport of protein between successive compartments of the Golgi measured by the coupled incorporation of N-acetylglucosamine. Cell 39: 405–416
Cross AJ, Crow TJ, Peters TJ (1986) Cortical neurochemistry in Alzheimer-type dementia. Prog Brain Res 70: 153–169
Dunkle S, Reust T, Nowack DD, Waits L, Paulik M, Morré DM, Morré DJ (1992) Temperature and acceptor specificity of cell-free vesicular transfer from transitional endoplasmic reticulum to cis Golgi apparatus. Biochem J 288: 969–976
Farquhar M (1985) Progress in unravelling pathways of Golgi traffic. Annu Rev Cell Biol 1: 447–488
Heape MA, Juguelin H, Boiron F, Cassagne C (1985) An improved one-dimensional thin layer chromatography technique for polar lipids. J Chromatogr 322: 391–395
Mellman I, Simons K (1992) The Golgi complex: in vitro veritas. Cell 68: 829–840
Moreau P, Rodriguez M, Cassagne C, Morré DM, Morré DJ (1991) Transfer and sorting of lipids from endoplasmic reticulum to Golgi apparatus in a cell-free system. J Biol Chem 266: 4322–4328
Morell P, Norton WT (1980) Myelin. Sci Am 242: 88–118
Morré DJ (1982) Intracellular vesicular transport: vehicles, guide elements, mechanisms. In: Weiss D (ed) Axoplasmic transport. Springer, Berlin Heidelberg New York, pp 2–14 (Proceedings in life sciences)
—, Mollenhauer HH, Bracker CE (1971) The origin and continuity of Golgi apparatus. In: Reinert T, Ursprung H (eds) Origin and continuity of cell organelles. Springer, Berlin Heidelberg New York, pp 82–126
—, Kartenbeck J, Franke WW (1979) Membrane flow and interconversions among endomembranes. Biochim Biophys Acta 559: 71–152
—, Penel C, Morré DM, Sandelius AS, Moreau P, Andersson B (1991) Cell-free transfer and sorting of membrane lipids in spinach. Donor and acceptor specificity. Protoplasma 160: 49–64
Nowak DD, Morré DM, Paulik M, Keenan TW, Morré DJ (1987) Intracellular membrane flow: reconstitution of transition vesicle formation and function in a cell-free system. Proc Natl Acad Sci USA 84: 6098–6102
Palade GE (1983) Membrane biogenesis: an overview. Methods Enzymol 96: 29–45
Paulik M, Nowak DD, Morré DJ (1988) Isolation of a vesicular intermediate in the cell-free transfer of membrane from transitional elements of the endoplasmic reticulum to Golgi apparatus cisternae of rat liver. J Biol Chem 263: 17738–17748
Rodriguez M, Moreau P, Paulik M, Lawrence J, Morré DJ, Morré DM (1991) NADH-activated cell-free transfer between Golgi apparatus and plasma membranes of rat liver. Biochim Biophys Acta 1107: 131–138
Rothman JE (1992) Transport between Golgi cisternae. Methods Enzymol 219: 5–12
— (1994) Mechanisms of intracellular protein transport. Nature 372: 55–63
Simons K, Fuller SD (1985) Cell surface polarity in epithelia. Annu Rev Cell Biol 1: 243–288
Van Meer G (1989) Lipid traffic in animal cells. Annu Rev Cell Biol 5: 247–275
Ziegel RF, Dalton AJ (1962) Speculations based on the morphology of the Golgi systems in several types of protein-secreting cells. J Cell Biol 15: 45–54
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Morré, D.M., Ferroli, C. & Hoffman, B. A cell-free approach to the study of membrane trafficking in frozen rat brains potentially applicable to frozen autopsy material. Protoplasma 190, 99–106 (1996). https://doi.org/10.1007/BF01281198
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF01281198