Chloroplast Suspension Protocols

Price, Carl A price at OCELOT.RUTGERS.EDU
Thu Jul 20 10:31:30 EST 1995


Ben Levin asks about a "protocol for separating chloroplasts (centrifugation,
yes?) and keeping them for biochemical analysis, i.e. observing various
environmental (chemical, photo-illumination) effects on fine structure."
     
     I can provide some history: 
     Prior to 1974, one had the choice of chloroplasts that were physiologically
active or pure, but not both. Differential centrifugation would yield
preparations active in photosynthesis or protein synthesis, but they were
contaminated with broken chloroplasts, naked thylakoids, and other cell
fragments. (Researchers interested only in the light reactions of photosynthesis
preferred to work with preparations of thylakoids, and, since they rarely
bothered to examine their ultrastructure, routinely referred to them as
"chloroplasts.") Intact chloroplasts could be isolated by isopycnic
sedimentation in sucrose gradients, but the combination of high osmotic
potential contributed by the sucrose and high hydrostatic pressure created by
the centrifugal field generally rendered them photosynthetically inactive. 
     Following a search for suitable gradient materials that would not
contribute to the osmotic potential, we were able to isolate pure,
photosynthetically active chloroplasts from spinach by sedimentation in
silica-sol gradients (Morgenthaler et al., 1974, 1975). We later adapted the
procedure to prepare active chloroplasts from Euglena gracilis (Ortiz et al.,
1980).  
     Our initial choice of silica sol was Ludox AM (DuPont), an industrial
silica stabilized with alumina. Subsequently, Hakan Pertoft and Torvard Laurent
(1977) hit upon the idea of stabilizing the silica with an electrostatic shield
of polyvinylpyrrolidone. Pharmacia marketed "Pertoft's colloid" as "Percoll."
While much more expensive than Ludox AM, Percoll is more stable and does not
require prior purification.
     We have published a number of protocols for the isolation of chloroplasts
and other kinds of plastids in silica-sol gradients (e.g., Price & Reardon,
1982; Price et al., 1994; Price et al., 1995).
         
     Morgenthaler, J.-J., C.A. Price, J.M. Robinson & M. Gibbs. 1974.
Photosynthetic activity of spinach chloroplasts after isopycnic centrifugation
in gradients of silica. Plant Physiol. 54:532-534.
     Morgenthaler, J.-J., M.P.F. Marsden & C.A. Price. 1975. Factors affecting
the separation of photosynthetically competent chloroplasts in gradients of
silica sols. Arch. Biochem. Biophys. 168:289-301.
     Ortiz, W, EM Reardon & CA Price, 1980. Preparation of chloroplasts from
Euglena highly active in protein synthesis. Plant Physiol. 66: 291-294. 
     Pertoft, H., T.C. Laurent. 1977. Isopycnic separation of cells and cell
organelles by centrifugation in modified colloidal silica gradients. pp. 25-65
in: Methods of Cell Separation, vol. 1, N. Catsimpoolas, ed. Plenum. 
     Price, C. A. & E. M. Reardon. 1982. Isolation of chloroplasts for protein
synthesis from spinach and Euglena gracilis by centrifugation in silica sols.
pp. 189-209, in: Methods in Chloroplast Molecular Biology, M. Edelman, R. B.
Hallick & N.-H. Chua, eds. Elsevier Biomedical 1141 pp.
     Price, C.A., N. Hadjeb, L. Newman, E.M. Reardon. 1994. Isolation of
chloroplasts and chloroplast DNA,  D4:1Ð15 in: Plant Molecular Biology Manual,
S. Gelvin & R. Schilperoort, eds., Kluwer, Dordrecht.
     Price, C.A., N. Hadjeb, L.A. Newman, E.M. Reardon. 1995. Chromoplasts, in:
Plant Cell Biology, D.W. Galbraith, D.P. Bourque, H.J. Bohnert, eds., Academic
Press. in press.
     
     
     




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