Restricted diffusion of tyrosine hydroxylase and phenylethanolamine N-methyltransferase from digitonin-permeabilized adrenal chromaffin cells.
Tyrosine hydroxylase [TyrOHase; tyrosine 3-monooxygenase; L-tyrosine,tetrahydropteridine:oxygen oxidoreductase (3-hydroxylating), EC 1.14.16.2] and phenylethanolamine N-methyltransferase, EC 2.1.1.28) are involved in catecholamine biosynthesis and are considered soluble proteins. However, they may actually be localized on the surface of the chromaffin granule. We have used the detergent digitonin to permeabilize the plasma membrane of cultured adrenal chromaffin cells to investigate the subcellular localization of TyrOHase and PMTase. A digitonin titration of the release of proteins and catecholamines revealed the existence of at least three subcellular compartments that are distinguished by their digitonin sensitivity: (i) soluble proteins, which were released upon treatment of the cells with low digitonin concentrations (5 microM), (ii) a "digitonin-sensitive" cytoplasmic protein pool, which required higher concentrations of digitonin for release (10 microM) and included TyrOHase and PMTase, and (iii) the chromaffin granule, which was insensitive to digitonin. Analysis of the rates of release of all of these proteins revealed that the rate of TyrOHase and PMTase release was slower at 10 microM than at 40 microM digitonin, while the rates of release of the other proteins were similar at both concentrations and varied in proportion to their respective sizes. Treatment with cytoskeletal disrupting agents had no effect on TyrOHase or PMTase efflux. These data suggest that TyrOHase and PMTase are in a detergent-labile association in the cell. This is consistent with the concept that TyrOHase and PMTase may be localized on the surface of the chromaffin granule.
Kelner
K L
KL
Morita
K
K
Rossen
J S
JS
Pollard
H B
HB
D000818:Animals; D002395:Catecholamines / Q000096:biosynthesis / Q000378:metabolism; D002417:Cattle; D002837:Chromaffin Granules / Q000187:drug effects / Q000201:enzymology*; D002838:Chromaffin System / Q000201:enzymology*; D003593:Cytoplasm / Q000201:enzymology; D004058:Diffusion; D004072:Digitonin / Q000494:pharmacology*; D004305:Dose-Response Relationship, Drug; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D008970:Molecular Weight; D010625:Phenylethanolamine N-Methyltransferase / Q000378:metabolism*; D014446:Tyrosine 3-Monooxygenase / Q000378:metabolism*
D002395:Catecholamines; D007770:L-Lactate Dehydrogenase; D014446:Tyrosine 3-Monooxygenase; D010625:Phenylethanolamine N-Methyltransferase; D004072:Digitonin