Stimulation by ATP of proinsulin to insulin conversion in isolated rat pancreatic islet secretory granules. Association with the ATP-dependent proton pump.
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.
D000251:Adenosine Triphosphatases / Q000037:antagonists & inhibitors; D000255:Adenosine Triphosphate / Q000494:pharmacology*; D000818:Animals; D002458:Cell Fractionation; D002499:Centrifugation, Density Gradient; D002851:Chromatography, High Pressure Liquid; D003594:Cytoplasmic Granules / Q000187:drug effects / Q000378:metabolism*; D004024:Dicyclohexylcarbodiimide / Q000494:pharmacology; D007328:Insulin / Q000378:metabolism*; D007473:Ion Channels / Q000378:metabolism; D007515:Islets of Langerhans / Q000648:ultrastructure*; D008297:Male; D011384:Proinsulin / Q000378:metabolism*; D011522:Protons; D051381:Rats; D011919:Rats, Inbred Strains; D014220:Trialkyltin Compounds / Q000494:pharmacology
D007328:Insulin; D007473:Ion Channels; D011522:Protons; D014220:Trialkyltin Compounds; C011559:tributyltin; D004024:Dicyclohexylcarbodiimide; D000255:Adenosine Triphosphate; D011384:Proinsulin; D000251:Adenosine Triphosphatases