Two-dimensional analysis of proteinase activity.
A method was developed to separate proteinases in a complex mixture in two dimensions followed by activity detection using class specific substrates. Using this method, serine proteinase activity was evaluated in gut extracts from a stored-product pest, Plodia interpunctella. With the substrate n-alpha-benzoyl-l-arginine rho-nitroanilide, three major groups of at least six trypsin-like activities were identified, consisting of proteinases with estimated molecular masses of 25-27, 40-41, and 289 kDa, and all with an acidic pI of 4.7-5.5. With the substrate, n-succinyl-ala-ala-pro-phenylalanine rho-nitroanilide, two groups of at least five chymotrypsin-like activities were detected, with estimated molecular masses of 28 and 192 kDa and pI values ranging from 6.1 to 7.3. Using the 2-DE activity blot method, information was obtained on the relative number and physical properties of serine proteinases in a mixture of insect gut proteinases without prior fractionation.
Oppert
Brenda
B
USDA ARS Grain Marketing and Production Research Center, 1515 College Avenue, Manhattan, KS 66502, USA. bso@ksu.edu
D000818:Animals; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel; D007525:Isoelectric Focusing / Q000379:methods; D009036:Moths / Q000201:enzymology*; D012697:Serine Endopeptidases / Q000032:analysis*
D012697:Serine Endopeptidases