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Helen Thao:
Synthesis and Molecular Analysis of
Apo-Alkaline Phosphatase; Putative Subatrate for
Metallothionein.
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SYNTHESIS AND MOLECULAR
ANALYSIS OF APO-ALKALINE PHOSPHATASE; PUTATIVE SUBSTRATE FOR METALLOTHIONEIN.
H. Thao and A. Z. Mason, Ph.D.
Department of Biological Sciences, California State
University, Long Beach, CA.
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Chemical reactions in living organisms
occur rapidly at moderate temperature and under mild conditions primarily
because of the catalytic action of specialized proteins called enzymes.
Each step in a chain of biochemical reactions is usually catalyzed by a
specific enzyme. The lack of even one enzyme can prove to be fatal
to the organism. Many enzymes require metal ions in their structures
in order for them to be active and functional. In most instances,
the specificity of the enzyme for a particular metal is absolute, and the
addition of the incorrect metal to the enzyme can impair its functioning,
sometimes irreversibly. Although the importance of metals in metallo-enzyme
biochemistry is well known, the cellular mechanisms which ensure the addition
of the correct metal to the correct protein at the appropriate time and
place are not fully understood. It is generally hypothesized that
a number of transport proteins capable of metal transference are involved
in the regulation of metal donation to these metallo-enzymes. In
order to test this hypothesis, a study has been initiated to monitor and
quantify the kinetic transfer of zinc from the transport protein metallothionein
to the zinc requiring enzyme alkaline phosphatase.
Alkaline phosphatase hydrolyzes
phosphate esters of primary and secondary alcohols, cyclic aliphathic alcohols,
sugar alcohols, phenols, and amines. It also hydrolyzes inorganic pyrophosphate and 5’-terminal phosphates of single and double-stranded
DNA or RNA. In its active form the enzyme occurs as a dimer, in which
each of its monomer contains two atoms of Zn2+ and one molecule of Mg2+.
One of the zinc atom is tightly bound to the enzyme and it is necessary
for the structural stability of the enzyme. The other zinc atom is
loosely bound and it is required for the catalytic activity of the enzyme.
The Mg2+ molecule acts as an allosteric activator and binds to a site that
is different from the motif binding site.
To study the role of metallothionein
in the transfer of zinc to alkaline phosphatase, it is first necessary
to remove the metal from the enzyme to form apo (non-metal) alkaline phosphatase.
This poster discusses the procedures necessary for apo-alkaline phosphatase
synthesis and the biochemical and analytical procedures necessary to show
enzymatic impairment accompanying metal removal. Ultimately, the
demonstration of bi-directional transfer of Zinc between these two proteins,
metallothionein and alkaline phosphatase, will provide important information
of how metals are incorporated into metallo-enzymes.
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