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กก Principal
Areas of Research Metallobiochemistry The CBBSM
has long been recognized as one of the leading contributors to present day understanding
of the role of zinc in biological systems, particularly zinc metalloenzymes. Starting with
the discovery of zinc in carboxypeptidase in 1954, Professor Vallee and his colleagues
have made a continuous series of important findings that have become the foundations of
the field of metallobiochemistry. Today more
than 300 enzymes have been found to contain zinc and many of them were identified in the
CBBSM. Fundamental contributions to
instrumental analysis of zinc in biological material and techniques to avoid metal
contamination were crucial for these findings and have become the hallmarks of the
laboratory. Because the physicochemical
properties of zinc are not amenable to spectral investigation, the laboratory developed
the highly successful and widely emulated approach of metal substitution for studying the
coordination characteristics of metals in catalytic active sites. A significant consequence of this was the
recognition that active site metals have highly unusual spectral properties indicative of
an "entatic" environment in which the metal is poised for catalysis. When coupled with rapid kinetic methods spectrally
active metals can be used to measure events that occur during the catalytic process. In
addition to carboxypeptidase, two other early zinc metalloenzyme discoveries made in the
laboratory and subsequently studied in great detail were horse liver alcohol dehydrogenase
and E. coli alkaline phosphatase. Many
pioneering advances were made with these systems such as the recognition that zinc can
have a structural as well as catalytic role in enzymes, and that two (or more) zinc ions
can cooperate to form an active site - later termed a co-catalytic site. The work on carboxypeptidase was extended to
additional metallopeptidases starting with the bacterial neutral protease, thermolysin,
and later to angiotensin converting enzyme, astacin, and the matrix metalloproteinases. As a result of this work researchers in the
laboratory were the first to recognize a unique, characteristic sequence of amino acids, a
signature sequence, that signalled a catalytic zinc binding site. When structures of these proteins began to be
reported, the generality of this sequence analysis was extended to include all amino acids
involved in active site metal coordination and the significance of amino acid spacing was
appreciated. Later this analysis was applied
to structural zinc sites in gene regulatory proteins adding zinc clusters and zinc twists
to the previously recognized zinc fingers. The
work on alcohol dehydrogenase led to studies of the enzyme in human liver, which unlike
the horse enzyme, was found to catalyze the oxidation of ethylene glycol. This led to the use of ethanol for the treatment
of ethylene glycol poisoning in humans, a life-saving therapeutic intervention. Further
studies, based on a novel application of affinity chromatography, led to the discovery of
isozymes of the human enzyme which in turn allowed identification of genetic variations in
different populations, and eventually to work on the active principles in Chinese herbal
medicines that deter alcohol consumption. Another
early discovery was metallothionein, a cysteine-rich molecule with the highest metal
content of any known protein. Based on the
rationale that the chemical properties of cadmium Angiogenin and Angiogenesis Angiogenin
is a 14kDa protein that was first identified in a tumor cell conditioned medium on the
basis of its ability to induce blood vessel formation.
It is a member of the ribonuclease superfamily of proteins with close
structural similarity to pancreatic RNase A. It
has extremely low catalytic activity which is nevertheless essential for its biological
activity. During 1998-1999, research focused
on the potential of anti-angiogenin antibodies and other antagonists to inhibit the growth
of human tumors in nude mice both to demonstrate the importance of angiogenin in the early
stages of tumor growth and to assess the effectiveness of these antagonists as therapeutic
agents. We also continued to explore the
mechanism of action of angiogenin at the molecular and cell biological level, and to
characterize the structure/function relationships of angiogenin, the protein ribonuclease
inhibitor and other types of inhibitors of angiogenin. Alcohol Metabolism and Daidzin Daidzin, a
constituent of an ancient Chinese herbal treatment (Radix puerariae) for alcohol
abuse, selectively suppresses home-cage ethanol intake by Syrian golden hamsters under a
two-bottle, free choice (ethanol/water) condition. The
ethanol intake suppressive The
mechanism by which daidzin selectively suppresses ethanol intake in laboratory animals is
unknown at this time. We have shown that
daidzin is a selective and potent inhibitor of mitochondrial aldehyde dehydrogenase
(ALDH-2). ALDH-2 catalyzes the detoxification
of acetaldehyde, an intermediate of ethanol metabolism.
Some humans inherit an inactive variant form of ALDH-2, and in these
individuals alcohol abuse is rare. Based on
these findings, we postulated that daidzin may act by mimicking the consequences of the
apparently harmless natural mutation of the ALDH-2 gene.
To evaluate this hypothesis, we synthesized a series of structural analogs
of daidzin and tested and compared their ALDH-2 inhibitory activity with their
antidipsotropic activity. The results
demonstrated a direct correlation between the two and raised the possibility that daidzin
may, in fact, suppress ethanol intake by inhibiting ALDH-2. Developmental
Biology and Cellular Differentiation
In the
course of studies of the changes in cellular composition that occur during development
after the fertilization of Xenopus laevis oocytes, it became apparent that the
major events that occur during this process are the result of cellular programming by
critical biomolecules. Thus, the frog
requires two to three years to produce mature eggs capable of being fertilized. In marked contrast, and evidently as a direct
consequence, once fertilization takes place, a full tadpole with a complement of organs
derived from all three germ lines is formed in less than 30 hours. This means that it takes nearly 900-fold more time
to mature a single-celled egg than to make an entire tadpole. To achieve this biological feat, X. laevis oocytes
must produce and store chemical signals required for differentiation and organogenesis and
use them later during the period of rapid embryogenesis.
These embryo-derived differentiation factors, which we will call differins,
would comprise a class of compounds that, acting singly or in combination, induce
commitment of primordial or stem cells and direct them along specific differentiation
paths. X. laevis oocytes could serve
as one of the systems with which to identify and isolate such substances while the embryo
itself can provide the means to test their function(s).
Bioactive lipids were selected as the first class of compounds to study. |