Enzymology of Lignification

Spruce ( Picea abies ) seeds contained a soluble β‐glucosidase which was active with 4‐nitrophenyl β‐D‐glucoside but did not catalyse hydrolysis of coniferin (conifery β‐D‐glucoside). In contrast, a cell wall fraction from hypocotyls and roots of spruce seedlings showed good glucosidase activity towards coniferin with a pH optinum Between 4.5 and 5.5. A number of other aryl β‐glucosides were also hydrolysed by these particulate fractions. Michaelis constants and maximal velocities were determined for coniferin (Km =380 and 1300 μ for roots and hypocotyls, respectively) and other aryl glycosides. The cell wall fractions probably contain more than one glycosidase. For 4‐nitrophenyl β‐D‐glucoside and picein (4‐hydroxyacetophenone β‐D‐glucoside) two separate and distinct lines could be drawn for high and low substrate concintrations in Lineweaver‐Burk of Eadie‐Hofstee plots. Attempts to solublize the β‐glucohydrolase activity could be extracted from the hypocotyl particulate fraction by treatment with 0.6 M Nacl. The solublized enzyme was separated on Sepharose 6 B into was fractions with glucosidase activity (glucosidase I and II). Glucosidase I was Purified to apparent homogeneity by repeated gel filtration on Sepharose 6 B. Dodecylsulfate‐gel electrophoris and sedimentation equilibrium measurements showed that glucosidase I is composed of only one polypeptide chain with M r 58 570 SS2400. The properties of glucodidase I with respect to pH and temperature optimum and to substrate specificity towards coniferin and other aryl β‐glucosides are similar to those of the hypocotyol particulate fraction. In contrast, glucosidase II has a 10 times smaller ratio for the relative maximal velocities with coniferin and 4‐nitrophenyl β‐glucosidase than glucosidase I. The presince of cell‐well‐bound β‐glucosidase for coniferin in spruce seedlings is consistent with the view that coniferin participates in lignification.