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Plant Physiology 64:1005-1011 (1979)
© 1979 American Society of Plant Biologists

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Articles

Isolation and Characterization of Concanavalin A-labeled Plasma Membranes of Carrot Protoplasts 1

Wendy F. Boss2 and Albert W. Ruesink3

a Department of Biology, Indiana University, Bloomington, Indiana 47401

The plasma membranes of protoplasts released from carrot suspension culture cells were labeled with [14C]acetyl-concanavalin A. After homogenization a single labeled membrane fraction was isolated in a continuous isopycnic Renografin gradient. The labeled membranes peaked at an apparent density of 1.14 grams per cubic centimeter between the Golgi fraction at a density of 1.11 grams per cubic centimeter as determined by latent IDPase activity and the mitochondria at a density of 1.16 grams per cubic centimeter as determined by the cytochrome c oxidase activity. This method provided a very discrete peak of putative plasma membrane. On discontinuous Renografin gradients a relatively pure fraction of labeled plasma membranes could be readily isolated at the 1.122 to 1.146 grams per cubic centimeter interface. The labeled fraction was enriched in both an ATPase (pH 6.5) and a glucan synthetase with a pH optimum of 6.5 whose activity was promoted by magnesium and cellobiose. Enzyme activities were not altered by the membrane label.

2 Present address: Department of Botany, North Carolina State University, Raleigh, North Carolina 27607.

3 To whom all correspondence should be addressed.

1 Supported by National Science Foundation Grants GB 39950 and PCM 76-15222. Represents part of a thesis presented by W. J. Boss to the Graduate School of Indiana University in partial fulfillment of the requirements for the Ph.D. degree.






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