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

Journal Article · Wed Aug 05 00:00:00 EDT 1987 · J. Biol. Chem.; (United States)

OSTI ID:6127750

Rhodes, C J; Lucas, C A; Mutkoski, R L; Orci, L; Halban, P A

Isolated rat pancreatic islets were pulse-labeled for 5 min with (/sup 3/H)leucine then chased for 25 min, during which time endogenously labeled (/sup 3/H)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 (/sup 3/H)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 had no effect on the ATP stimulation of proinsulin conversion. When granules were incubated in a more acidic buffer, 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.

• Research Organization: Brigham and Women's Hospital, Boston, MA
• OSTI ID: 6127750
• Journal Information: J. Biol. Chem.; (United States), Vol. 262:22
• Country of Publication: United States
• Language: English

Related Subjects

59 BASIC BIOLOGICAL SCIENCES
ATP
BIOLOGICAL FUNCTIONS
INSULIN
BIOSYNTHESIS
SECRETION
ATP-ASE
CENTRIFUGATION
ENZYME INHIBITORS
LEUCINE
LIQUID COLUMN CHROMATOGRAPHY
LYSOSOMES
MITOCHONDRIA
PANCREAS
PROTONS
RATS
TRACER TECHNIQUES
TRITIUM COMPOUNDS
ACID ANHYDRASES
AMINO ACIDS
ANIMALS
BARYONS
BODY
CARBOXYLIC ACIDS
CELL CONSTITUENTS
CHROMATOGRAPHY
DIGESTIVE SYSTEM
ELEMENTARY PARTICLES
ENDOCRINE GLANDS
ENZYMES
FERMIONS
FUNCTIONS
GLANDS
HADRONS
HORMONES
HYDROLASES
ISOTOPE APPLICATIONS
LABELLED COMPOUNDS
MAMMALS
NUCLEONS
NUCLEOTIDES
ORGANIC ACIDS
ORGANIC COMPOUNDS
ORGANOIDS
ORGANS
PEPTIDE HORMONES
PHOSPHOHYDROLASES
RODENTS
SEPARATION PROCESSES
SYNTHESIS
VERTEBRATES
550201* - Biochemistry- Tracer Techniques