RESUMEN
A heat- and acid-stable protein fraction that inhibited peptide chain initiation in rabbit reticulocyte lysates was extracted from frozen, powdered rat skeletal muscles by stepwise trichloroacetic acid precipitation. Streptozotocin-induced diabetes increased the inhibitory activity; this was prevented by insulin therapy. Size-exclusion high-performance liquid chromatography resolved four inhibitory fractions; only one was consistently increased (approximately 2-fold) in muscle extracts from diabetic rats. Polysome profiles of lysates incubated with this fraction indicated peptide chain initiation inhibition. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis the purified inhibitory fraction migrated with apparent Mr 30 and 32 kDa, which on Western blot immunostained with antisera against histone H1/H1(0). Perchloric acid extraction of muscle homogenates yielded approximately twofold more H1 from diabetic than from control rats; yield from diabetics decreased to control values 5 h after subcutaneous insulin injection. Inclusion of detergent during homogenization increased H1 yield more from muscles of control than from diabetic rats and abolished the difference between them. Because H1 affects several biochemical reactions, its facilitated extraction from insulin-deprived tissues can bias interpretation of studies of insulin action.
Asunto(s)
Diabetes Mellitus Experimental/metabolismo , Histonas/farmacología , Músculos/metabolismo , Biosíntesis de Proteínas/efectos de los fármacos , Animales , Fraccionamiento Químico , Cromatografía Líquida de Alta Presión , Diabetes Mellitus Experimental/tratamiento farmacológico , Electroforesis en Gel de Poliacrilamida , Histonas/aislamiento & purificación , Histonas/metabolismo , Insulina/uso terapéutico , Focalización Isoeléctrica , Masculino , Músculos/análisis , Ratas , Ratas Endogámicas , Ribonucleasas/metabolismo , EstreptozocinaRESUMEN
Histone-H1 purified from rat skeletal muscle is a relatively potent inhibitor of peptide chain initiation in a cell free system, the rabbit reticulocyte lysate (50% inhibition at approximately 0.4 microM). H1 does not inhibit formation of the ternary complex nor its attachment to 40S ribosomes; the data are compatible with H1 binding to mRNA. The inhibition shows mRNA selectivity: translation of beta-globin mRNA is more affected than that of alpha-globin mRNA and hepatic albumin mRNA more than total hepatic mRNA. Whether or not histone-H1 plays a role in translational regulation in intact cells is conjectural, it may serve as a useful model for protein-mRNA interactions.