Stimulation by ATP of proinsulin to insulin conversion in isolated rat pancreatic islet secretory granules. Association with the ATP-dependent proton pump

Rhodes, C. J.; Lucas, C. A.; Mutkoski, R. L.; Orci, Lelio; Halban, Philippe A.

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Title		Stimulation by ATP of proinsulin to insulin conversion in isolated rat pancreatic islet secretory granules. Association with the ATP-dependent proton pump
Authors		Rhodes, C. J. Lucas, C. A. Mutkoski, R. L. Orci, Lelio Halban, Philippe A.
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
Full text		Article - Limited access to UNIGE Other version: http://www.jbc.org/content/262/22/10712.full.pdf
Structures		Faculté de médecine / Section de médecine clinique / Département de médecine
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