Use of gel electrophoresis for the study of enzymatic activities of cold-adapted bacteria.
Glutamate dehydrogenase (GDH) and lactate dehydrogenase (LDH) activity of 13 cold-adapted strains, isolated from cold soils and showing GDH and/or LDH activity in spectrophotometric assays, were revealed by the use of electrophoresis on a nondenaturing acrylamide gel (zymogram). Psychrophilic strains were grown at 4 degrees C and 10 degrees C and the psychrotolerant strains at 4 degrees, 20 degrees and 28 degrees C. Incubation with the specific substrate and staining were done at 4, 28 or 37 degrees C. In the most cold-adapted strains, LDH and GDH production was high at 4 degrees C. In psychrotrophic strains, enzyme production and activity were greater at 20 or 28 degrees C than at lower temperatures. LDH remained active up to 37 degrees C while GDH activity was more thermolabile. GDH activity was NAD-dependent in some psychrophilic strains. In other strains, it was dependent on NAD(P) only or on both NAD and NAD(P). Two bands were seen for GDH or LDH activity in some strains. This method, which does not require a dialysis step, can be used to study the influence of temperature on enzyme production and activity, and the co-factor dependence. It detects phenotypic differences between isozymes, providing data for systematics.
Berchet
V
V
Laboratoire d'Ecologie Microbieunne (UMR-CNRS 5557). Université Claude Bernard Lyon 1, Villeurbanne, France.
Boulanger
D
D
Gounot
A M
AM
D000222:Adaptation, Physiological; D001419:Bacteria / Q000145:classification / Q000201:enzymology* / Q000254:growth & development; D003080:Cold Temperature; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D004783:Environmental Microbiology; D004798:Enzymes / Q000032:analysis*; D014644:Genetic Variation; D005969:Glutamate Dehydrogenase / Q000378:metabolism; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D009243:NAD / Q000378:metabolism; D009249:NADP / Q000378:metabolism
D004798:Enzymes; D009243:NAD; D009249:NADP; D007770:L-Lactate Dehydrogenase; D005969:Glutamate Dehydrogenase