Date of Award
Doctor of Philosophy
Botany and Plant Pathology
In vitro autolytic reactions in Zea mays (hybrid B73 x Mo17) coleoptile cell walls were investigated. Of interest were the polysaccharides which served as substrates, characteristics of the participating enzymes and the mechanism of hydrolysis;During cell wall autolytic reactions, 85 to 90% of the hemicellulosic (beta)-D-glucan, which constituted as much as 117 (mu)g per mg cell wall dry weight, was liberated from cell wall in the form of polymeric and monomeric products. Enzymolysis of the polymeric products with a purified bacterial glucanase specific for mixed-linkage glucans confirmed that (beta)-D-glucan served as the primary substrate for autolytic reactions. Small quantities of galactose were observed; however, other major wall components consisting of xylose and arabinose were unaffected during autolytic reactions;The autolytic enzymes were found to be tenaciously associated with isolated cell wall. Pretreatment of cell wall with detergents and ionic strength failed to significantly affect their autolytic potential. The use of high ionic strength (3 M) facilitated the dissociation of the enzyme-cell wall complex, and reconstitution studies confirmed that the salt-solubilized enzymes included the autolytic enzymes;Purification of the cell wall enzymes disclosed both endo- and exo-glucanases proteins. The former, apparently an endo-(beta)-1,3-glucanase, had a molecular weight of 26,000 daltons and exhibited a pH optimum of 5.5. This enzyme was apparently responsible for initiating the autolytic dissolution of wall glucan. An exo-(beta)-1,3-glucanase with a molecular weight of 60,000 daltons was further purified using a cation-exchange procedure. The enzyme was strongly inhibited by inorganic mercurials and along with an exocellulase appeared to be responsible for the hydrolysis of endo-enzyme-resistant polymeric glucan generated by the endoenzyme. It is believed that the capacity of the endoenzyme to degrade (beta)-D-glucan was dependent on the presence in the glucan of linkage sequences with regions of contiguous (beta),1-3 glucosyl bonds;The results of studies employing mercuric chloride, nojirimycin and glucono-(delta)-lactone suggested that (beta)-glucosidase activity was not required for glucan hydrolysis;Cell wall glucanases have frequently been implicated as having some role in auxin-induced growth. The glucanases participating in autolytic reactions did not appear to be under the direct control of auxin.
Digital Repository @ Iowa State University, http://lib.dr.iastate.edu/
Donald John Huber
Huber, Donald John, "Studies on Zea coleoptile cell wall [beta]-D-glucanases and their native substrates " (1980). Retrospective Theses and Dissertations. 6694.