Figure 5. Estimation of specific activity of DProPHMs mutants by immunoblot
analysis. Aliquots of media collected during sequential 24 h periods (D2 and D3)
were assayed at low (0.5 mM) or high (100 mM) substrate concentration. (A)
Volumes of media containing 1000 pmol/h of PHM activity when assayed with 0.5 mM
a-N -acetyl-Tyr-Val-Gly were subjected to immunoblot analysis using Ab246. (B)
Volumes of media containing 9200 pmol/h of PHM activity when assayed with 100 mM
a-N -acetyl-Tyr-Val-Gly were subjected to immunoblot analysis. The volume of
medium analyzed varied from 0.05 to 0.005 mL; identical amounts of DProPHM-382s
medium were analyzed in both experiments. (C) Films were densitized, and data for
the two samples of each mutant were normalized to the average signal obtained for
DProPHM-382s. Ab246 recognizes a site distinct from that altered by mutation, and
the ratio plotted should reflect the amount of each protein needed to generate an
equivalent amount of PHM activity.
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