Determination of solute accumulation in chloroplasts by rapid centrifugal transfer through silicone fluid layers
✍ Scribed by R.E. Gaensslen; Richard E. McCarty
- Publisher
- Elsevier Science
- Year
- 1972
- Tongue
- English
- Weight
- 648 KB
- Volume
- 48
- Category
- Article
- ISSN
- 0003-2697
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✦ Synopsis
Winkler et al. (
1
) have discussed some methods used in the determination of the transport of small molecules and ions in cells and subcellular organelles. These methods include (A) filtration of the suspension and determination of the radioactivity in the filtrate or on the filter; (B) centrifugation and determination of the radioactivity in t'he pellet or supernatant fluid; and (C) centrifugation of the biological material through an inert layer whose density is greater than that of the medium.
Filtration methods are not readily applicable to the study of lightdependent solute accumulation in chloroplasts because the chloroplasts clog the filter and because the accumulated solutes are rapidly lost from the chloroplasts in the dark. Cent,rifugation techniques have been applied to the study of ion upt)ake in illurllinated chloroplwts. Deamer and Packer (2) layered the chloroplast suspension on a 4% dextran subphase which also contained 5 mM AgN(& and 0.1 M KaNo,. After illumination the chloroplasts were pelleted within 10 see by ccntrifugation. This method, however, gives only a senliq"antitativc estimate of uptake. Further, the method was not applicable to the study of amine uptake and no measurements of the amount of suspending medium which may contaminate the chloroplast pellets were made.
Centrifugation of mitochondria through silicone fluid layers whose density is greater tha,n that of the medium has been used by several investigators to study solute transport 13-6). Heldt and Rapley used a similar technique to measure the permeability of spinach chloroplasts to water and a number of metaholites (7,8), In this paper, we describe 'Taken, in part, from a thesis submitted by R.E.G. to Cornell University in partial fulfillment of the requirements for the degree of Doctor of Philosophy.