The Significance of Vitamin C in Norepinephrine Synthesis in Isolated Bovine Adrenal Chromaffin Cell Granules
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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.