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Helen Thao:
Synthesis and Molecular Analysis of
Apo-Alkaline Phosphatase; Putative Subatrate for
Metallothionein.
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Although metal ions are known to play an essential
role in the activity of metalloenzymes, very little is known about the
intra-cellular processes that ensure that the correct metal is transported and associated with the appropriate enzyme. In order to study these
transfer processes, it is necessary to first generate an apo-enzyme that
can act as a recipient substrate for metal transfer. In this study,
the apo form of the zinc requiring enzyme alkaline phosphatase was prepared
by the use of a chelating resin. The degree of sequestration was
monitored both enzymatically and by coupled HPLC-ICPMS. Elemental
profiles indicated that the zinc in both the apo and native forms of the
protein was associated with two major peaks with retention times of 10.733
and 15.5 minutes and approximate molecular weights of 150 kDa and 40 kDa
respectively. Of these two peaks, the former contained the majority
of the enzymatic activity. Comparative HPLC-ICPMS analyses of the
native and apo-protein showed an approximate 70% reduction in zinc in the
first peak in the apo-alkaline phosphatase sample. This coincided
with a similar 73% reduction in p-nitro phenyl phosphate dephosphorylation.
Alternative chelating procedures are currently being developed to increase
the efficacy of the sequestration process.
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