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Electrophoretic analysis of proteinases in sodium dodecyl sulfate-polyacrylamide gels containing copolymerized radiolabeled protein substrates: application to proenkephalin processing enzymes.
Anal Biochem. 1990 Oct; 190(1):141-6.AB

Abstract

A novel method is described for the zymographic analysis of proteinases in sodium dodecyl sulfate-polyacrylamide gels containing copolymerized radiolabeled protein substrates such as [35S]methionine-labeled proenkephalin or 125I-labeled proinsulin. After electrophoresis the enzyme is reactivated and cleaves the radiolabeled in situ substrate into smaller peptides. These small peptides are able to diffuse out of the gel, leaving clear areas against a dark background when visualized by autoradiography. The technique can be used to detect as little as 200 fg of trypsin using only 50 ng (1.25 microCi) of [35S]proenkephalin. Soluble- and membrane-bound adrenal trypsin-like enzyme were isolated from bovine adrenal chromaffin granules. Both proteinases cleaved [35S]methionine-labeled proenkephalin but not 125I-labeled proinsulin. Moreover, both had a Mr of approximately 30,000. The potential of this technique for general use is discussed. An additional method using the synthetic fluorogenic substrate t-butoxycarbonyl Glu-Lys-Lys aminomethylcoumarin is also described.

Authors+Show Affiliations

Irvine JW
Department of Biochemistry and Molecular Biology, Louisiana State University Medical Center, New Orleans 70112.
Roberts SF
No affiliation info available
Lindberg I
No affiliation info available

MeSH

Adrenal MedullaAnimalsAutoradiographyCattleChromaffin GranulesChymotrypsinElectrophoresis, Polyacrylamide GelEndopeptidasesEnkephalinsKallikreinsMethionineMicrochemistryProtein PrecursorsProteinsSerine EndopeptidasesSilverSodium Dodecyl SulfateSubstrate SpecificitySulfur RadioisotopesTrypsin

Pub Type(s)

Comparative Study
Journal Article
Research Support, U.S. Gov't, P.H.S.

Language

eng

PubMed ID

2285141

Citation

Irvine, J W., et al. "Electrophoretic Analysis of Proteinases in Sodium Dodecyl Sulfate-polyacrylamide Gels Containing Copolymerized Radiolabeled Protein Substrates: Application to Proenkephalin Processing Enzymes." Analytical Biochemistry, vol. 190, no. 1, 1990, pp. 141-6.
Irvine JW, Roberts SF, Lindberg I. Electrophoretic analysis of proteinases in sodium dodecyl sulfate-polyacrylamide gels containing copolymerized radiolabeled protein substrates: application to proenkephalin processing enzymes. Anal Biochem. 1990;190(1):141-6.
Irvine, J. W., Roberts, S. F., & Lindberg, I. (1990). Electrophoretic analysis of proteinases in sodium dodecyl sulfate-polyacrylamide gels containing copolymerized radiolabeled protein substrates: application to proenkephalin processing enzymes. Analytical Biochemistry, 190(1), 141-6.
Irvine JW, Roberts SF, Lindberg I. Electrophoretic Analysis of Proteinases in Sodium Dodecyl Sulfate-polyacrylamide Gels Containing Copolymerized Radiolabeled Protein Substrates: Application to Proenkephalin Processing Enzymes. Anal Biochem. 1990;190(1):141-6. PubMed PMID: 2285141.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Electrophoretic analysis of proteinases in sodium dodecyl sulfate-polyacrylamide gels containing copolymerized radiolabeled protein substrates: application to proenkephalin processing enzymes. AU - Irvine,J W, AU - Roberts,S F, AU - Lindberg,I, PY - 1990/10/1/pubmed PY - 1990/10/1/medline PY - 1990/10/1/entrez SP - 141 EP - 6 JF - Analytical biochemistry JO - Anal Biochem VL - 190 IS - 1 N2 - A novel method is described for the zymographic analysis of proteinases in sodium dodecyl sulfate-polyacrylamide gels containing copolymerized radiolabeled protein substrates such as [35S]methionine-labeled proenkephalin or 125I-labeled proinsulin. After electrophoresis the enzyme is reactivated and cleaves the radiolabeled in situ substrate into smaller peptides. These small peptides are able to diffuse out of the gel, leaving clear areas against a dark background when visualized by autoradiography. The technique can be used to detect as little as 200 fg of trypsin using only 50 ng (1.25 microCi) of [35S]proenkephalin. Soluble- and membrane-bound adrenal trypsin-like enzyme were isolated from bovine adrenal chromaffin granules. Both proteinases cleaved [35S]methionine-labeled proenkephalin but not 125I-labeled proinsulin. Moreover, both had a Mr of approximately 30,000. The potential of this technique for general use is discussed. An additional method using the synthetic fluorogenic substrate t-butoxycarbonyl Glu-Lys-Lys aminomethylcoumarin is also described. SN - 0003-2697 UR - https://www.unboundmedicine.com/medline/citation/2285141/Electrophoretic_analysis_of_proteinases_in_sodium_dodecyl_sulfate_polyacrylamide_gels_containing_copolymerized_radiolabeled_protein_substrates:_application_to_proenkephalin_processing_enzymes_ DB - PRIME DP - Unbound Medicine ER -