PRELIMINARY ESTIMATES OF THE DOPAMINE β‐HYDROXYLASE CONTENT AND ACTIVITY IN PURIFIED NORADRENERGIC VESICLES

R. L. Klein, D. F. Kirksey, R. A. Rush, M. Goldstein

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Abstract

The dopamine β‐hydroxylase (DβH) content and activity of large dense‐core noradrenergic vesicles purified from bovine splenic nerve were determined using two assay procedures : enzymic activity expressed in Units per mg protein and homospecific activity based on radioimmunoassay expressed in Units per mg DβH antigen. Approximately two‐thirds of the total enzyme activity is latent in these vesicles, even after various treatments designed to compromise vesicle integrity. DβH can be completely unmasked by brief treatment with 0.01‐0.05% Triton X‐100 and activity increases from 0.20 to 0.64 Units per mg vesicle protein. Calculations based on both assay methods suggested that an average of 7% (range 3‐15%) of the total vesicle protein was DβH and that the average vesicle contained about 4 molecules of enzyme (range 2‐9 molecules). The estimated homospecific activities indicated an average of 25 and 50% (range 18‐72%) of the vesicle enzyme was inactive in the various samples using the two antibodies. The vesicle can synthesize up to 30 molecules of noradrenaline/s per molecule of DβH at near optimal substrate concentration, and 60‐270 molecules of norepinephrine/s per vesicle. The assumptions used in the various calculations were critically analyzed and, based on the methods employed, it is tentatively considered to be unlikely that there could be more than 5‐12 molecules of DβH per vesicle. The possibility that circulating DβH originates primarily, if not exclusively, from the large dense‐core vesicle type is considered and the functional implications of the data support the concept of vesicle reuse during several cycles of exocytosis involving a quantal size equal to a fraction of the vesicle transmitter content.

Original languageEnglish
Pages (from-to)81-86
Number of pages6
JournalJournal of Neurochemistry
Volume28
Issue number1
DOIs
Publication statusPublished - Jan 1977

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