Dopamine beta-hydroxylase distribution in density gradients: physiological and artefactual implications.
Knowledge of the vesicular origin of circulating dopamine beta-hydroxylase (DbetaH) is indispensable for any attempts to explain the parallelism or lack of it between circulating enzyme and catecholamines as they may relate to physiological stress, forms of hypertension, neurological disorders, and the response to pharmacological agents. The present study represents an effort to evaluate and to place in proper perspective data based on the DbetaH activity found in the region of the light vesicle peak of noradrenaline (NA), which is used as a quantitative measure of a population of small terminal vesicles. Distributions of vesicles and subvesicular components are compared with DbetaH and NA in sucrose-D2O density gradients used to prepare relatively pure fractions of large dense cored vesicles (LDV) from bovine splenic nerve. Although NA in sedimentable particles of the light vesicle peak is likely to be a valid measure of a small vesicle population, the following is demonstrated: (1) A substantial fraction (25%-37%) of the total sedimentable DbetaH activity can be proven to distribute in the region of the light vesicle peak from a tissue with an insignificant small vesicle population. Based on studies of vesicles from sequential nerve segments, this enzyme activity probably corresponds to a population of "immature" LDV which are undergoing axoplasmic transport and have not synthesized their full complement of transmitter. (2) Physical lysis which depletes the matrix of LDV causes redistribution of DbetaH activity from the heavy vesicle peak into the region of the light vesicle peak. Analogously, DbetaH associated with exocytosed LDV and retrograde transport particles is also likely to contaminate the region of the light vesicle peak. (3) Based on available data, it can be calculated that each small dense cored vesicle could contain only 0.1-0.5 molecules of DbetaH and that a contamination of only 0.016% LDV can account for all of the DbetaH reported to occur in the light vesicle peak of normal rat vas deferens preparations.
D000818:Animals; D002417:Cattle; D002458:Cell Fractionation / Q000379:methods; D002499:Centrifugation, Density Gradient; D004299:Dopamine beta-Hydroxylase / Q000032:analysis*; D009419:Nerve Tissue Proteins / Q000032:analysis; D009638:Norepinephrine / Q000032:analysis; D010525:Peripheral Nerves / Q000201:enzymology / Q000648:ultrastructure*; D013572:Synaptic Vesicles / Q000032:analysis / Q000201:enzymology*
D009419:Nerve Tissue Proteins; D004299:Dopamine beta-Hydroxylase; D009638:Norepinephrine