The lectin from the mushroom Pleurotus ostreatus: a phosphatase-activating protein that is closely associated with an alpha-galactosidase activity. A part of this paper has been presented as a preliminary report at the 17th Interlec. Meeting 1997 in Würzburg, Germany.
The lectin from the mushroom Pleurotus ostreatus described earlier [F. Conrad, H. Rüdiger, The lectin from Pleurotus ostreatus: purification, characterization and interaction with a phosphatase, Phytochemistry 36 (1994) 277-283] was further characterized. Determination of the isoelectric point by capillary electrophoresis gave a value of 7.6. The dissociation constant of the lectin-alpha-lactose-1-phosphate complex determined by capillary electrophoresis is 3 mM. The activation of an endogenous phosphatase by the lectin as found earlier for the pseudosubstrate p-nitrophenylphosphate was confirmed also for naturally occurring substrates as ADP and ATP. We observed that at all purification steps the lectin is accompanied by an alpha-galactosidase activity. Both activities could neither be resolved by electrophoresis under non-denaturing conditions nor by affinity chromatography. However, carbohydrate binding by the lectin and carbohydrate processing by the enzyme are not due to the same site since: (i) the lectin accepts both alpha- and beta-glycosides whereas the enzyme activity is restricted to the alpha-anomer; (ii) the interaction with erythrocytes leads to a stable agglutinate, i.e. no 'clot-dissolving activity' [C.N. Hankins, J.I. Kindinger, L.M. Shannon, Legume alpha-galactosidases which have hemagglutinin properties, Plant Physiol. 65 (1980) 618-622] is observed; (iii) the alpha-galactosidase activity is inhibited by galactose but not by a beta-galactoside. Therefore, lectin and enzymatic activities are either properties of two tightly associated proteins, or of just one molecule. The kinetic parameters of the lectin-associated alpha-galactosidase activity for p-nitrophenyl-alpha-galactopyranoside are: K(M)=2.5 mM, k(cat)=66 s(-1), and K(I)=20mM for the inhibitor D-galactose.
Brechtel
R
R
Institut für Pharmazie und Lebensmittelchemie der Universität Würzburg, Am Hubland, D-97074, Würzburg, Germany
Wätzig
H
H
Rüdiger
H
H