Reconstitution of Phaseolus vulgaris alpha-amylase inhibitor from isolated subunits.
The reconstitution of Phaseolus vulgaris alpha-amylase inhibitor from its two kinds of subunits was studied. The inhibitor was reconstituted by quick dilution from its subunits denatured in 6 M guanidine hydrochloride with a concomitant restoration of the inhibitory activity. Various environmental factors had a profound influence on the rate and extent of the reconstitution. Especially low protein concentration and high ionic strength were essential for a high yield of regained activity, and Zn2+ was also effective in promoting reconstitution. The maximum specific activity of reconstituted inhibitor was obtained at a 1:1 molar ratio of alpha-subunit: beta-subunit. Under the optimal conditions obtained, the activity regain was about 60% of the activity of the native inhibitor. Further, this report advances the possibility that the glycan chains of each subunit may be important in proper folding and assembly of the subunit polypeptides.
Higaki
H
H
Department of Agricultural Chemistry, College of Agriculture, University of Osaka Prefecture, Japan.
Yamaguchi
H
H
D001216:Asparagine / Q000737:chemistry; D002240:Carbohydrate Sequence; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D007887:Fabaceae / Q000737:chemistry*; D019791:Guanidine; D006146:Guanidines; D007700:Kinetics; D037102:Lectins / Q000737:chemistry* / Q000302:isolation & purification / Q000494:pharmacology; D008969:Molecular Sequence Data; D037121:Plant Lectins; D010940:Plant Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000494:pharmacology; D010946:Plants, Medicinal; D011134:Polysaccharides / Q000737:chemistry; D011489:Protein Denaturation; D000516:alpha-Amylases / Q000037:antagonists & inhibitors*
D006146:Guanidines; D037102:Lectins; D037121:Plant Lectins; D010940:Plant Proteins; D011134:Polysaccharides; C061510:alpha-amylase inhibitor, Phaseolus vulgaris; D001216:Asparagine; D000516:alpha-Amylases; D019791:Guanidine