Electrophoretic analysis of proteinases in sodium dodecyl sulfate-polyacrylamide gels containing copolymerized radiolabeled protein substrates: Application to proenkephalin processing enzymes

Journal Article · Mon Oct 01 00:00:00 EDT 1990 · Analytical Biochemistry; (USA)

DOI:https://doi.org/10.1016/0003-2697(90)90147-2· OSTI ID:6011248

Irvine, J W; Roberts, S F; Lindberg, I ^[1]

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1. Louisiana State Univ. Medical Center, New Orleans (USA)

A novel method is described for the zymographic analysis of proteinases in sodium dodecyl sulfate-polyacrylamide gels containing copolymerized radiolabeled protein substrates such as ({sup 35}S)methionine-labeled proenkephalin or {sup 125}I-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 ({sup 35}S)proenkephalin. Soluble- and membrane-bound adrenal trypsin-like enzyme were isolated from bovine adrenal chromaffin granules. Both proteinases cleaved ({sup 35}S)methionine-labeled proenkephalin but not {sup 125}I-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.

• OSTI ID: 6011248
• Journal Information: Analytical Biochemistry; (USA), Journal Name: Analytical Biochemistry; (USA) Vol. 190:1; ISSN ANBCA; ISSN 0003-2697
• Country of Publication: United States
• Language: English

Related Subjects

550201* -- Biochemistry-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
AMINO ACIDS
AUTONOMIC NERVOUS SYSTEM AGENTS
AUTORADIOGRAPHY
BETA DECAY RADIOISOTOPES
BETA-MINUS DECAY RADIOISOTOPES
CARBOXYLIC ACIDS
CHEMICAL COMPOSITION
DAYS LIVING RADIOISOTOPES
DRUGS
ELECTRON CAPTURE RADIOISOTOPES
ELECTROPHORESIS
ENDORPHINS
ENKEPHALINS
ENZYMES
EVEN-ODD NUCLEI
HORMONES
HYDROLASES
INSULIN
INTERMEDIATE MASS NUCLEI
IODINE 125
IODINE ISOTOPES
ISOTOPES
LIGHT NUCLEI
LIPOTROPIC FACTORS
METABOLISM
METHIONINE
NEUROREGULATORS
NUCLEI
ODD-EVEN NUCLEI
ORGANIC ACIDS
ORGANIC COMPOUNDS
ORGANIC SULFUR COMPOUNDS
PEPTIDE HORMONES
PEPTIDE HYDROLASES
PEPTIDES
POLYPEPTIDES
PROTEINS
RADIOISOTOPES
SUBSTRATES
SULFUR 35
SULFUR ISOTOPES