Composition of the aqueous phase of chromaffin granules

Abstract

Nuclear magnetic resonance spectroscopy has been used to determine the composition of the aqueous phase of bovine chromaffin granules. Relative concentrations of catecholamines (epinephrine plus norepinephrine), ATP and chromogranins have been measured from integrated intensities in the proton spectra using computer simulation techniques. Most or all of the catecholamines (97 +/- 8%) are present in the aqueous phase and contribute to the high resolution spectrum. The catecholamine:ATP molar ratio (4.41 +/- 0.45) determined by NMR is close to the value (4.45) derived from biochemical assay indicating that most or all of the ATP is present with catecholamine in the aqueous phase. Catecholamine:protein ratios show that approximately 45% of the soluble protein freed by lysis is not NMR visible. Intensity from this fraction does not appear under highly denaturing conditions (8 M urea) but reappears after hydrolysis. This behavior is similar to that of recently isolated soluble lipoprotein complexes. Variations in the NMR spectra associated with (1) different preparative procedures; (2) different suspension media, and (3) increasing osmolality are described. The fact that high concentrations of epinephrine and ATP (approximately 700 mM total) are dissolved in the aqueous phase implies that solution phase interactions at least partially ionic in nature are responsible for the low internal osmolality of chromaffin granules in vivo. Ordered phases containing a substantial fraction of the total catecholamine in an osmotically inactive form are not present.

Ultrastructural localization of chromogranin: a potential marker for the electron microscopical recognition of endocrine cell secretory granules

“…(1983aO'Connor et aI. ( ,b, 1984 in human tissues, and offers some support to the contention, based on biochemical evidence presented by Sen et al (1979), that one rfile for chromogranin (specifically chromogranin A) is to stabilize the stored components. containing cells.…”

Transport of Ca2+ and Na+ across the chromaffin-granule membrane

“…It has been suggested that its role is the stabilization of catecholamine-ATP complexes (Pletscher et al, 1974), but n.m.r. studies have provided no evidence that these complexes do in fact exist within the granules (Sharp & Richards, 1977;Sen et al, 1979). Another possibility is that the granules are a reservoir for Ca2+ that penetrates the plasma membrane during the secretory process (Serck-Hanssen & Christiansen, 1973); this is an attractive theory in view of the large number of Abbreviation used: Hepes, 4-(2-hydroxyethyl)-1piperazine-ethanesulphonic acid.…”

Phylogenetic considerations of neurosecretory granule contents: Role of nucleotides and basic hormone/transmitter packaging mechanisms.

“…The results of fluorescence spectrophotometry (STEFFEN et al, 1974) and NMR spectroscopy (GRANOT and ROSENHECK, 1978;SHARP and RICHARDS, 1977a, b;DANIELS et al, 1978;SHARP and SEN, 1978;SEN et al, 1979) all indicate that the chromaflin granule contents are in a "fluid" or mobile state. In NMR spectroscopy, broadening of absorption bands or decreases in the relaxation rate are associated with a decrease of molecular motion, a condition that does not exist within the chromaffin granule (SHARP and RICHARDS, 1977a, b;DANIELS et al, 1978;SHARP and SEN, 1978;SEN et al, 1979). At the high concentration of solutes within the granule interior, the contents of the chromaffin granule best represent a non-ideal but homogeneous viscous solution (NJUS et al, 1981) (Fig.…”