The usefulness of the analytical electrofocusing in a thin-layer polyacrylamide gel (PAG) in the histochemistry of enzymes cleaving peptide bonds.

Abstract

The usefulness of the analytical electrofocusing in a thin-layer polyacrylamide (PAG) plate is shown on the basis of experiments with 10%–20% homogenates of various rat, rabbit and human organs as well as in lysates of isolated human lymphocytes and leucocytes in 2% Triton X100. 0.1–0.3 μl of 12000 g supernatants were applied on LKB Ampholine PAG plates pH range 3.5–9.5 and subjected to electrofocusing. Afterwards portions of PAG plates were processed in optimized histochemical media for the demonstration of enzymes cleaving peptide bonds using various substrates. The same media were used in the histochemical detection of enzymes in sections on slides or semipermeable membranes. Electrofocused zymograms display species and organ differences. Ala-MNA, Leu-MNA and Met-MNA furnish similar zymograms. Bands obtained with Ala-MNA are most intense. Zymograms with Gly-Pro-MNA and Lys-Pro-MNA at pH 7.2 are not entirely identical. The majority of bands is more intense when Gly-Pro-MNA is used as the substrate and is due to the activity of DAP IV. The anodal band(s) focusing around pH 4.9 (rat) or 5.5 (man) is (are) much stronger with Lys-Pro-MNA and DAP II is responsible for it (them). Zymograms with His-Ser-MNA and Lys-Ser-MNA are similar. However, they differ form those revealed with Gly-Pro-MNA and Lys-Pro-MNA. Zymograms of lysates of leucocytes obtained with naphthol AS-D-chloroacetate and Ac-Ala-l-naphthyl ester are not identical showing that more than one enzyme is responsible for the bands. Results obtained with closely related substrates such as Ac-Ala-l-naphthyl ester and Ac-Met-l-naphtyl ester are not identical either. Zymograms of lysates of human lymphocytes revealed with Gly-Pro-MNA at pH 7.2 and Lys-Ala-MNA or Lys-Pro-MNA at pH 5.5 or 5.3 respectively show clearly the presence of DAP IV and DAP II in these cells. The analytical electrofocusing in PAG plates is a very useful tool in the histochemistry (and biochemistry) of enzymes cleaving peptide bonds. It helps very much in the evaluation of the substrate specificity, choosing of the discriminating substrate and enables a quick and reliable testing of the quality of various batches of commercially supplied substrates, diazonium salts and other reagents. The correlation of zymograms with the “in situ” pattern helps in the elucidation of the origin of individual bands in zymograms and suggests different molecular forms of peptidases in different localizations.