A rapid and sensitive detection of proteolytic enzymes after electrophoresis

Dahlmann, Burkhardt; Jany, Klaus-Dieter

doi:10.1016/s0021-9673(00)91224-x

Cited by 19 publications

(2 citation statements)

References 5 publications

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“…Disc polyacrylamide-gel electrophoresis at pH4.3 was done in capillaries as described by Dahlmann & Jany (1975) at an acrylamide concentration of 7.5 % (w/v). Proteolytic activity was detected by photopaper test as described by Jany (1976).…”

Section: Disc Electrophoresismentioning

confidence: 99%

Purification and some properties of an alkaline proteinase from rat skeletal muscle

Dahlmann¹,

Reinauer²

1978

Biochemical Journal

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1. Rat skeletal muscle was homogenized in 0.05M-Tris/HCl, pH 8.5, containing 1M-KCl. Myofibrillar proteins were precipitated by addition of (NH4)2SO4 (33% saturation). 2. The alkaline proteolytic activity that was precipitated with the myofibrillar proteins was solubilized with trypsin (conjugated to Sepharose) and further purified by affinity chromatography, ion-exchange chromatography and gel filtration. 3. The purified enzyme migrates as a single band in polyacrylamide-disc electrophoresis, and has optimum hydrolytic activity with azocasein and [14C]haemoglobin as substrates at pH 9.4 and 9.6 respectively. Its apparent molecular weight, as determined by gel filtration on Sephadex G-75, is 30800. 4. The purified alkaline proteinase is strongly inhibited by equimolar amounts of soya-bean trypsin inhibitor and ovomucoid, whereas di-isopropyl phosphorofluoidate and alpha-toluenesulphonyl fluoride have no effect. On the other hand N-ethylmaleimide and p-chloromercuribenzoate have inhibitory effects on the enzyme activity. 5. Bivalent metal ions (Fe2+, Co2+, Zn2+, Mg2+, Mn2+) diminish the proteolytic activity, at 1mM concentrations. Ca2+ ions and the metal-ion-chelating agent EDTA are without effect on enzyme activity. 6. The enzyme is part of the alkaline proteolytic activity that appears to be associated with myofibrillar proteins.

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Section: Disc Electrophoresismentioning

confidence: 99%

Purification and some properties of an alkaline proteinase from rat skeletal muscle

Dahlmann¹,

Reinauer²

1978

Biochemical Journal

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“…Polyacrylamide Gel Electrophoresis. Gel electrophoresis at pH 4.3 (Reisfeld et al, 1962) was carried out on a micro scale for which protein staining, as well as enzyme specific staining, was performed and described previously (Dahlmann & Jany, 1975). Determination of the molecular weight by gel electrophoresis without sodium dodecyl sulfate was performed on gels of increasing acrylamide concentrations (12.5-25%) according to Hedrick and Smith (1968) using bovine trypsin (Mt 23 500), myoglobin (17 200), ribonuclease A (13 400), and cytochrome c (12 400) as standards.…”

Section: Jany E Imentioning

confidence: 99%

Enzymic and chemical properties of an endopeptidase from the larva of the hornet Vespa crabro

Jany¹,

Haug²,

Pfleiderer³

et al. 1978

Biochemistry

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An endopeptidase from the larvae of the hornet Vespa crabro has been purified to homogeneity. The enzyme has been characterized with respect to molecular weight, amino acid compositon, and amino- and carboxyl-terminal sequences. The catalytic properties of the hornet protease are similar to those of bovine chymotrypsin with respect to inactivation by phenylmethanesulfonyl fluoride and carbobenzoxyphenylalanine chloro ketone and preferential peptide bond cleavage at aromatic amino acid residues. In contrast to bovine chymotrypsin, the hornet protease is not inhibited by the basic pancreatic Kunitz inhibitor, soybean inhibitor, or chicken ovomucoid. The molecular weight, as determined by several independent methods, was found to be 14 500. The protease is a single-chain protein containing two disulfide bonds. The terminal sequences are: NH2-Ile-Val-Gly-Gly-Ile-Asp.....Gly-Lys-Tyr-Pro-Tyr-Gln-Val-Ser-Leu-Arg-COOH.

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A simple and rapid technique for postelectrophoretic detection of proteases using azocasein

Peyronel¹,

Cantera²

1995

Electrophoresis

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Azocasein, diffused into gels after polyacrylamide gel electrophoresis, provides a useful substrate for simple and rapid protease detection. After electrophoresis, the gels are incubated in an azocasein solution, immersed in trichloroacetic acid and then treated with NaOH. Proteolytic zones appear as uncolored bands on an orange background. The gels can be stored in trichloroacetic acid for months. This technique gives good results with serine proteases, acid proteases and metalloproteases. The method can be used under nondenaturing conditions and in the presence of sodium dodecyl sulfate. The sensitivity of this method is at the submicrogram protein level for trypsin.

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A rapid and sensitive detection of proteolytic enzymes after electrophoresis

Cited by 19 publications

References 5 publications

Purification and some properties of an alkaline proteinase from rat skeletal muscle

Purification and some properties of an alkaline proteinase from rat skeletal muscle

Enzymic and chemical properties of an endopeptidase from the larva of the hornet Vespa crabro

A simple and rapid technique for postelectrophoretic detection of proteases using azocasein

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