Abstract
Membranes were isolated from mitochondria and chromaffin granules of bovine adrenal medullae. The cross-contamination between the two membranes was examined by comparing the radioactive bands on autoradiograms of gels after phosphorylation of the membranes with [γ-32P]-ATP and decoration with [125I]concanavalin A and [125I]protein A with antibody that was raised against chromaffin-granule membranes. It was found that the membranes cross-contaminated each other by less than 10%. The technique of immunodecoration with antibodies against β subunits of proton-ATPases from yeast mitochondria, spinach chloroplasts, andE. coli membranes was used for quantitative estimation of proton-ATPase complexes in chromaffin granules and mitochondrial membranes. It was found that chromaffin-granule membranes contain less than 10% of the amount of proton-ATPase complex in mitochondrial membranes. The specific ATPase activity of chromaffin-granule membranes was on the order of 30 to 50% of the mitochondrial membranes. The ATPase activity of the chromaffin-granule membranes was more sensitive to 4-acetamido-4′-isothiocyano-2,2′-disulfonic acid stilbene and 4-chloro-7-nitrobenzofurazan. It was much less sensitive than the mitochondrial membranes to antibody against β subunit of proton-ATPase fromE. coli membranes. After solubilization of chromaffin-granule membranes by octyglucoside and cholate and subsequent centrifugation on sucrose gradient, two different ATPase enzymes were separated. The heavier enzyme was identical to the mitochondrial-ATPase complex, while the lighter enzyme was identified as a novel ATPase, which might be responsible for the special properties of the ATPase activity of chromaffin-granule membranes.
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Abbreviations
- DCCD:
-
dicyclohoxylcarbodiimide
- NBD-Cl:
-
4-chloro-7-nitrobenzofurazan
- SITS:
-
4-acetamido-4′-isothiocyano-2,2′-disulfonic acid stilbene
- SDS:
-
sodium dodecyl sulfate
- MES:
-
2-(N-morpholino)ethane sulfonic acid
- FITC:
-
fluorescein isothiocyanate
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Cidon, S., Nelson, N. Properties of a novel ATPase enzyme in chromaffin granules. J Bioenerg Biomembr 14, 499–512 (1982). https://doi.org/10.1007/BF00743074
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DOI: https://doi.org/10.1007/BF00743074