Gel Electrophoretic Profiles of Proteinases in Dark-Germinated Flax Seeds

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Abstract
The proteinases present in dark-germinated flax seeds (L. usitatissimum) were studied as a function of germination at 25.degree. C. A majority of activity was present in basic proteinases with an acidic pH optimum and a temperature optimum of 45.degree. C in the digestion of hemoglobin. Electrophoresis in a sodium dodecyl sulfate-polyacrylamide mixture which had been polymerized with gelatin was used to separate proteins in extracts of seedings. Subsequent activation of proteinases with Triton X-100 and resultant digestion of gelatin proved reproducible and afforded detection and good quantification of various proteinase zones. An EDTA-sensitive proteinase zone, P4 (about 60,000 daltons), appeared at day 3 after imbibition and attained maximum activity at day 4. This correlates with a rapid loss in vivo of the glyoxysomal enzyme, isocitrate lyase (EC 4.1.3.1). EDTA also slowed the loss of isocitrate lyase activity in extracts of 4-day seedlings in a dose-dependent manner. The addition of leupeptin, .alpha.-tolylsulfonyl fluoride, Pepstatin A, p-chloromercuribenzoate or 1,10-phenanthroline prior to, during, or after exchange of triton X-100 for sodium dodecyl sulfate had almost no inhibitory effect upon proteinases in 4-day seedlings.