Some structural characteristics of linamarase from cassava (Manihot esculenta Crantz.)

Abstract

Three isozyme of cassava petiole linamarase were used in two types of studies : structural study and subunit interaction study. Native isozymes were insensitive to the following protease: trypsin pronase, thermolysin, papain, subtilisin, collagenase and bromelain. After denaturation, they became sensitive to trypsin, pronase and thermolysin and yielded similar proteolytic fragments suggesting a simlar protein chain among the isozymes. using TNBS to modify lysine residues, the native and denatured forms of the isozyme pI 4.3 were found to have 179 and 199 mole lysines/mole isozyme respectively. Similarly, the native and denatured forms the isozyme pI 2.9 contained 88 and 100 moles/mole respectively. In the presence of a competitive inhibitor, the number of modified lysines of the native isozymes were not changed. The data indicated that most lytic site. The isozymes were probably glycoproteins since they can be retained by a Con A-Sepharose column. The anti-linamarase serum was able to recognize the modified isozymes except isozyme pI 2.9 but could not recognize the heat-denatured isozymes. Guanidine hydrochloride dissociated the enzyme into smaller forms and caused inactivation also. Complete inactivation was observed by 3M GuHC1. However, the isozymes were stable in SDS up to 20% at room temperature for 30 min. In SDS-PAGE containing 1% SDS, active forms of different sizes were found. Treatment with β-mercaptosthanol did not dissociate the enzyme into subunits but heat treatment caused the dissociation and the complete loss of the activity.