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ARS Home » Plains Area » Manhattan, Kansas » Center for Grain and Animal Health Research » Stored Product Insect and Engineering Research » Research » Publications at this Location » Publication #373583

Research Project: Impacting Quality through Preservation, Enhancement, and Measurement of Grain and Plant Traits

Location: Stored Product Insect and Engineering Research

Title: New glutamine-containing substrates for the assay of cysteine peptidases from the C1 papain family

item FILIPPOVA, IRINA - Moscow State University
item DVORYAKOVA, ELENA - Moscow State University
item SOKOLENKO, NIKOLAY - Moscow State University
item SIMONYAN, TATIANA - Moscow State University
item TERESHCHENKOVA, VALERIIA - Moscow State University
item ZHIGANOV, NIKITA - Moscow State University
item DUNAEVSKY, YAKOV - Moscow State University
item BELOZERSKY, MIKHAIL - Moscow State University
item Oppert, Brenda
item ELPIDINA, ELENA - Moscow State University

Submitted to: Frontiers in Molecular Biosciences
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 7/7/2020
Publication Date: 10/22/2020
Citation: Filippova, I.Y., Dvoryakova, E.A., Sokolenko, N.I., Simonyan, T.R., Tereshchenkova, V.F., Zhiganov, N.I., Dunaevsky, Y.E., Belozersky, M.A., Oppert, B.S., Elpidina, E.N. 2020. New glutamine-containing substrates for the assay of cysteine peptidases from the C1 papain family. Frontiers in Molecular Biosciences. 7:578758.

Interpretive Summary: Cysteine peptidases are important enzymes in biological systems, involved in regulating biological processes, but also implicated in disease. The ability to study these enzymes is dependent on sensitive and selective substrates. New enzyme substrates were designed with the amino acid glutamine substituted for the traditional alanine in the critical site of enzyme recognition are more efficient and selective. When we combined these new substrates with commercially available substrates, we were able to easily and quickly differentiate cysteine peptidases involved in many biological reactions. These new substrates were demonstrated in the identification of cysteine peptidases in an analysis of a multi-enzyme digestive complex of beetle stored product pests.

Technical Abstract: New substrates with glutamine in the P1-position are introduced for the assay of peptidases from the C1 papain family, with a general formula of Glp-Phe-Gln-X, where Glp is pyroglutamyl and X is pNA (p-nitroanilide) or AMC (4-amino-7-methylcoumaride). The substrates have a simple structure, and C1 cysteine peptidases of various origins cleave them with high efficiency. The main advantage of the substrates is their selectivity for cysteine peptidases of the C1 family. Peptidases of other clans, including serine trypsin-like peptidases, do not cleave glutamine-containing substrates. We demonstrate that using Glp-Phe-Gln-pNA in combination with a commercially available substrate, Z-Arg-Arg-pNA, provided differential determination of cathepsins L and B. In terms of specific activity and kinetic parameters, the proposed substrates offer improvement over the previously described alanine-containing prototypes. The efficiency and selectivity of the substrates was demonstrated by the example of chromatographic and electrophoretic analysis of a multi-enzyme digestive complex of stored product pests from the Tenebrionidae family.