The Significance of Vitamin C in Norepinephrine Synthesis in Isolated Bovine Adrenal Chromaffin Cell Granules
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Authors
Sinda, Richard
Issue Date
1987
Type
Thesis
Language
en_US
Keywords
Alternative Title
Abstract
Ascorbic acid (vitamin C) is a vitamin/cofactor of great
importance in the functioning of the human body. Although most
mammals can synthesize ascorbic acid from glucose derivatives,
humans cannot. Lack of sufficient ascorbic acid in the diet can
result in the deficiency disease, scurvy. The current.
recommended daily allowance in the United states is 60 mg., but
this amount is based on the prevention of scurvy. The optimal
amount of a vitamin may, in principle, be determined by
determining a concentration that produces maximal activity for
all of the enzymes that vitamin influences. Dopamine
beta-hydroxylase (DBH) in the chromaffin cells of the adrenal
medulla was the enzyme studied. DBH is located in organelles
called chromaffin granules and is thought to be dependent of
ascorbic acid for maximal activity. The currently accepted
hypothesis is that cytosolic ascorbic acid passes electrons via
an electron shuttle system to intragranular ascorbic acid which
subsequently passes them to DBH. Thus, the behavior of the
isolated enzyme in the presence of ascorbic acid is not
representative of its behavior in situ (in cells and tissues).
The role(s) of ascorbic acid in the production of norepinephrine
from dopamine (which DBH catalyzes) was investigated using both
lysed and intact chromaffin granules from bovine adrenal medulla.
DBH activity was assessed by measuring the rate of conversion of
tyramine to octopamine. Fer intact granules, the Km for ascorbic
acid was 0.085 mM and the specific activity Vrnax was 392 nmol
octopamine/A540 granules/30 minutes. For lysed granules, the Km
for ascorbic acid was 3.33 mM and the specific activity Vmax was
2500 nmol octcpamine/A540 granules/30 minutes. It was found that
electrons are passed directly from intragranular ascorbic acid to
DBH rather than via an intermediate. The intragranular
inhibitor(s) of DBH could be inactivated by dilution using lysed
granule preparations of 0.02 A540 granule concentration and
lower. The activity of DBH was still increasing at an
intragranular concentration equivalent of 10 mM which, in nature,
would cause osmotic lysis of the granule. This shows that DBH is
not as easily activated by ascorbic acid as are the enzymes which
are used as standards to determine the recommended daily
allowance.
Description
iv, 43 p.
Citation
Publisher
Kalamazoo College
License
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