Abstract
Protoplasts from infected and uninfected cells were isolated from the central nitrogen fixing tissue of French bean (Phaseolus vulgaris L. cv Contender) root nodules. Successive filtrations allowed the separation of the infected cells, whereas the small uninfected cells were isolated on a discontinuous Percoll gradient. Higher yields of intact protoplasts were obtained from young (4-week-old) nodules whereas no protoplasts could be isolated from the oldest nodules. When proteolysis was determined in the cytosolic fraction of both infected and uninfected cells, at pH 5.0 and 8.0, with leghemoglobin or azocasein as substrate, activity was present only in infected cell protoplasts and increased with nodule age. A protease with an acidic pH optimum, mainly responsible for this increasing activity, was highly purified from senescing nodules by electro-elution after nondenaturing polyacrylamide gel electrophoresis and used to produce polyclonal antibodies. Western blots of nodule protein separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and probed with purified anti-protease immunoglobulin G showed the molecular mass of the protease to be 58 kilodaltons. Blots also confirmed that protease protein was located in infected cell protoplasts only, regardless of nodule age.
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