PROTEIN HETEROGENEITY OF SPINACH PULLULANASE RESULTS FROM THE COEXISTENCE OF INTERCONVERTIBLE ISOMERIC FORMS OF THE MONOMERIC ENZYME

Purified pullulanase (starch-debranching enzyme, R-enzyme, EC 3.2.1.41 ) from spinach (Spinacia oleracea L.) chloroplasts separated into at l east seven individual enzymically active proteins (isomers, numbered 1 -7) on isoelectric focusing or column chromatofocusing. At their isoel ectric points (between pH 4.7 and 5.2) these forms were rather stable. At slightly alkaline pH, each converted into the whole set of isomers . PAGE of the purified enzyme under denaturing or non-denaturing condi tions resulted in one protein band. When substrate (amylopectin or pul lulan) was included in the gel, the native enzyme as well as any of th e individual isomers separated into two (sometimes three) bands ('subs trate-induced forms', numbered I-III) with different specific activiti es, dissociation constants of the enzyme-substrate complexes and activ ation energies. Each substrate-induced form produced the whole set of seven isomers on isoelectric focusing. The specific activity of the to tal enzyme reflected the relative proportions of the substrate-induced forms. To some extent the relative proportions, as determined by cros sed immunoelectrophoresis, could be shifted in favour of the more or t he less active forms by reduction with dithiothreitol, and gentle oxid ation respectively. Activation by dithiothreitol did not alter the mod e of action of the enzyme but only increased the velocity of substrate degradation and extended its activity into the pH range of the chloro plast. As a consequence of isomer interconversion, microheterogeneity could serve to regulate pullulanase activity in a biochemical manner t hat shares some features with allosteric regulation.