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We have purified an abundant, 33000-dalton polypeptide (P33) from cultured pith parenchyma tissue of Nicotiana tabacum L. cv. Havana 425. The accumulation of P33 in culture is inhibited by the cytokinin kinetin (N(6)-furfuryl-amino purine). When tissues are subcultured on auxin-containing medium, the P33 content measured by rocket immunoelectrophoresis increases by 10-fold from 9 to 90 μg·mg(-1) soluble protein over a 7-d period. This increase is blocked when kinetin is added to the culture medium. There is strong evidence that P33 is a β-1,3-glucanase (EC 3.2.1.39): i) Purified P33 specificially promotes the endo-type hydrolysis of β-1,3-glucans and has essentially the same moleculear weight, pH optimum, and sensitivitiy to heavy metals as the β-1,3-glucanase isolated by others from tobacco. ii) Glucanase activity is inhibited by specific antibodies against P33. iii) P33 and glucanase activity co-purify and cannot be separated by affinity chromatography using the β-1,3-glucanase substrate pachyman. iv) P33 content and glucanase activity are strongly correlated in tissues grown under inductive and non-inductive conditions. The pattern of glucanase synthesis estimated by [(35)S]methionine incorporation parallels changes in the amount of glucanase. This indicates that cytokinin acts, at least in part, by blocking synthesis of the enzyme. |
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