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 least seven individual enzymically active proteins (isomers, numbered 1-7) on isoelectric focusing or column chromatofocusing. At their isoelectric 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 conditions resulted in one protein band. When substrate (amylopectin or pullulan) was included in the gel, the native enzyme as well as any of the individual isomers separated into two (sometimes three) bands ('substrate-induced forms', numbered I-III) with different specific activities, dissociation constants of the enzyme-substrate complexes and activation energies. Each substrate-induced form produced the whole set of seven isomers on isoelectric focusing. The specific activity of the total enzyme reflected the relative proportions of the substrate-induced forms. To some extent the relative proportions, as determined by crossed immunoelectrophoresis, could be shifted in favour of the more or the less active forms by reduction with dithiothreitol, and gentle oxidation respectively. Activation by dithiothreitol did not alter the mode of action of the enzyme but only increased the velocity of substrate degradation and extended its activity into the pH range of the chloroplast. As a consequence of isomer interconversion, microheterogeneity could serve to regulate pullulanase activity in a biochemical manner that shares some features with allosteric regulation.
Henker
A
A
Lehrstuhl für Pflanzenphysiologie, Universität Bayreuth, Universitätsstrasse 30, D 95440 Bayreuth, Germany.
Schindler
I
I
Renz
A
A
Beck
E
E
D000494:Allosteric Regulation / Q000502:physiology; D000687:Amylopectin / Q000494:pharmacology; D002736:Chloroplasts / Q000201:enzymology; D004229:Dithiothreitol / Q000494:pharmacology; D006026:Glycoside Hydrolases / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D007122:Immunoelectrophoresis; D007525:Isoelectric Focusing; D007526:Isoelectric Point; D007527:Isoenzymes / Q000737:chemistry*; D007536:Isomerism; D007700:Kinetics; D010084:Oxidation-Reduction; D010940:Plant Proteins / Q000737:chemistry; D018724:Spinacia oleracea / Q000201:enzymology*
D007527:Isoenzymes; D010940:Plant Proteins; D000687:Amylopectin; D006026:Glycoside Hydrolases; C100181:pullulanase; D004229:Dithiothreitol