Purification and characterization of two cellulases from auxin-treated pea epicotyls.
Two forms of beta-1,4-glucan 4-glucanohydrolase (EC 3.2.1.4) were extracted from growing regions of Pisum sativum epicotyls which had been treated with the auxin, (2,4-dichlorophenoxy)acetic acid. One cellulase is buffer-soluble, the other buffer-insoluble but extractable with high salt concentrations. Both enzymes catalyze endohydrolysis of carboxymethylcellulose with the same pH optimum (5.5 to 6.0). They were purified with the use of DEAE-cellulose chromatography, Sephadex gel filtration, and ultrafiltration. They are distinct proteins as characterized by: electrofocusing and disc gel electrophoresis (pI values = 5.2 and 6.9, respectively); mobility in sodium dodecyl sulfate polyacrylamide gels, fractionation on Sephadex, and sedimentation in the ultracentrifuge (mol wt = approximately 20,000 and 70,000, S values 2.63 and 3.73); and immunological properties. The buffer-soluble enzyme tends to dimerize on purification. Amino acid analyses show that the buffer-soluble enzyme is relatively rich in glycine, alanine, and valine and deficient in cystine, tryosine, and phenylalanine compared to the buffer-insoluble enzyme. The two cellulase activities were generated in approximately equal amounts after auxin treatment. Within 5 days their levels had increased at least 100-fold and they constituted about 0.1% of total cellular protein. Present data indicate that one is not derived from the other.
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002480:Cellulase / Q000302:isolation & purification* / Q000378:metabolism; D002848:Chromatography, DEAE-Cellulose; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004589:Electrophoresis, Disc; D006026:Glycoside Hydrolases / Q000302:isolation & purification*; D006863:Hydrogen-Ion Concentration; D005779:Immunodiffusion; D007210:Indoleacetic Acids / Q000494:pharmacology*; D007525:Isoelectric Focusing; D007700:Kinetics; D008970:Molecular Weight; D010937:Plant Growth Regulators / Q000494:pharmacology*; D010944:Plants / Q000187:drug effects / Q000201:enzymology*; D011487:Protein Conformation; D011817:Rabbits / Q000276:immunology; D013997:Time Factors; D014462:Ultrafiltration
D000596:Amino Acids; D007210:Indoleacetic Acids; D010937:Plant Growth Regulators; D006026:Glycoside Hydrolases; D002480:Cellulase