Native agarose-polyacrylamide gel electrophoresis allowing the detection of aminopeptidase, dehydrogenase, and esterase activities at the nanogram level: enzymatic patterns in some Frankia strains.
Nanogram amounts of soluble aminopeptidases, dehydrogenases, and esterases were detected by nondenaturing ultralow gelling point agarose-polyacrylamide gel electrophoresis (ULGA-PAGE). Cytosolic fractions from Frankia sp. were electrophoresed at 4 degrees C in the presence of Co2+, Zn2+, or Mg2+ ions. Then, aminopeptidases and esterases were revealed by simultaneous capture staining by using fast garnet GBC diazonium salt as the chromogenic coupling compound. Dehydrogenases were revealed by using nitro blue tetrazolium salt as electron acceptor. A variety of aminopeptidases, dehydrogenases, and esterases could be identified by their migration in ULGA-PAGE and by their sensitivities to NaCl, CoSO4, ZnSO4, and MgCl2 when assayed "ingel." The presence of agarose was essential for the detection of the complex enzyme patterns. The patterns were remarkably similar for the five Frankia strains isolated from Allocasuarina and Casuarina host plants and differed from those of Frankia strains isolated from Comptonia and Hippophaë host plants. A nomenclature is proposed for aminopeptidases and other Frankia enzymes. The richness of the Frankia amino-peptidases and esterases zymograms makes them adequate marker enzymes for taxonomical, genetic, or biochemical studies. Dehydrogenases might also be useful, although a more restricted number of bands were found with L-lactic and L-malic acid as substrates.
Benoist
P
P
Laboratoire d'Enzymologie, CNRS, Gif-sur-Yvette, France.
Schwencke
J
J
D000190:Actinomyces / Q000201:enzymology* / Q000254:growth & development; D000626:Aminopeptidases / Q000032:analysis*; D003600:Cytosol / Q000201:enzymology; D004587:Electrophoresis, Agar Gel; D004591:Electrophoresis, Polyacrylamide Gel; D004950:Esterases / Q000032:analysis*; D010088:Oxidoreductases / Q000032:analysis*; D012680:Sensitivity and Specificity; D013379:Substrate Specificity; D009626:Terminology as Topic
D010088:Oxidoreductases; D004950:Esterases; D000626:Aminopeptidases