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Two-dimensional analysis of proteinase activity.
J Biochem Biophys Methods. 2006 Jun 30; 67(2-3):173-9.JB

Abstract

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.

Authors+Show Affiliations

USDA ARS Grain Marketing and Production Research Center, 1515 College Avenue, Manhattan, KS 66502, USA. bso@ksu.edu

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

16616785

Citation

Oppert, Brenda. "Two-dimensional Analysis of Proteinase Activity." Journal of Biochemical and Biophysical Methods, vol. 67, no. 2-3, 2006, pp. 173-9.
Oppert B. Two-dimensional analysis of proteinase activity. J Biochem Biophys Methods. 2006;67(2-3):173-9.
Oppert, B. (2006). Two-dimensional analysis of proteinase activity. Journal of Biochemical and Biophysical Methods, 67(2-3), 173-9.
Oppert B. Two-dimensional Analysis of Proteinase Activity. J Biochem Biophys Methods. 2006 Jun 30;67(2-3):173-9. PubMed PMID: 16616785.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Two-dimensional analysis of proteinase activity. A1 - Oppert,Brenda, Y1 - 2006/03/29/ PY - 2006/01/09/received PY - 2006/03/01/accepted PY - 2006/4/18/pubmed PY - 2006/7/27/medline PY - 2006/4/18/entrez SP - 173 EP - 9 JF - Journal of biochemical and biophysical methods JO - J Biochem Biophys Methods VL - 67 IS - 2-3 N2 - 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. SN - 0165-022X UR - https://www.unboundmedicine.com/medline/citation/16616785/Two_dimensional_analysis_of_proteinase_activity_ L2 - https://linkinghub.elsevier.com/retrieve/pii/S0165-022X(06)00041-8 DB - PRIME DP - Unbound Medicine ER -