

Other version: http://www.jbc.org/content/262/22/10712.full.pdf
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Stimulation by ATP of proinsulin to insulin conversion in isolated rat pancreatic islet secretory granules. Association with the ATP-dependent proton pump |
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Published in | The Journal of biological chemistry. 1987, vol. 262, no. 22, p. 10712-10717 | |
Abstract | Isolated rat pancreatic islets were pulse-labeled for 5 min with [3H]leucine then chased for 25 min, during which time endogenously labeled [3H]proinsulin becomes predominantly compartmented in immature secretory granules. The islets were then homogenized in isotonic sucrose (pH 7.4) and a beta-granule preparation obtained by differential centrifugation and discontinuous sucrose gradient ultracentrifugation. This preparation was enriched 8-fold in beta-granules. Aside from contamination with mitochondria and a limited number of lysosomes, the beta-granule preparation was essentially free of any other organelles involved in proinsulin synthesis and packaging (i.e. microsomal elements and, more particularly, Golgi complex). Conversion of endogenously labeled [3H]proinsulin was followed in this beta-granule fraction for up to 2 h at 37 degrees C in a buffer (pH 7.3) that mimicked the cationic constituents of B-cell cytosol, during which time 92% of the beta-granules remained intact. Proinsulin conversion was analyzed by high performance liquid chromatography. The rate of proinsulin conversion to insulin was stimulated by 2.2 +/- 0.1-fold (n = 6) (at a 60-min incubation) in the presence of ATP (2 mM) and an ATP regenerating system compared to beta-granule preparations incubated without ATP. This ATP stimulation was abolished in the presence of beta-granule proton pump ATPase inhibitors (tributyltin, 2.5 microM, or 1,3-dicyclohexylcarbodiimide, 50 microM). Inhibitors of mitochondrial proton pump ATPases (sodium azide, 20 mM, or oligomycin, 10 micrograms/ml) had no effect on the ATP stimulation of proinsulin conversion. When granules were incubated in a more acidic buffer (pH 5.5), proinsulin conversion was increased relative to that at pH 7.3. At pH 5.5, ATP no longer stimulated conversion, and tributyltin and 1,3-dicyclohexylcarbodiimide had no effect. Disrupted granules only converted proinsulin to a limited extent, and neither ATP nor the inhibitors affected conversion. It is therefore suggested that ATP stimulation of proinsulin conversion in isolated, intact, beta-granules is secondary to intragranular acidification by an ATP-dependent proton pump (reflecting the low pH optimum for proinsulin conversion), rather than ATP dependence of converting activity per se. | |
Keywords | Adenosine Triphosphatases/antagonists & inhibitors — Adenosine Triphosphate/ pharmacology — Animals — Cell Fractionation — Centrifugation, Density Gradient — Chromatography, High Pressure Liquid — Cytoplasmic Granules/drug effects/ metabolism — Dicyclohexylcarbodiimide/pharmacology — Insulin/ metabolism — Ion Channels/metabolism — Islets of Langerhans/ ultrastructure — Male — Proinsulin/ metabolism — Protons — Rats — Rats, Inbred Strains — Trialkyltin Compounds/pharmacology | |
Identifiers | PMID: 2440873 | |
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Citation (ISO format) | RHODES, C. J. et al. Stimulation by ATP of proinsulin to insulin conversion in isolated rat pancreatic islet secretory granules. Association with the ATP-dependent proton pump. In: The Journal of biological chemistry, 1987, vol. 262, n° 22, p. 10712-10717. https://archive-ouverte.unige.ch/unige:8993 |