Translation of messenger ribonucleic acid from isolated pancreatic islets and human insulinomas.

Abstract

RNA preparations from isolated rat pancreatic islets and from human insulinomas were injected into oocytes of Xenopus laevis which were then incubated with[3H]leucine. Acid-ethanol extracts of the oocytes were immunoprecipitated with anti-insulin serum using the double antibody technique. Sodium dodecyl sulfate disc gel electrophoresis of the immunoprecipitates showed the presence of an insulin-displaceable immunoreactive material with a molecular weight of about 18,000 in extracts from oocytes injected with RNA of 9-11 S. This immunoreactive product was not detected in extracts from oocytes injected with buffer or 4-8S RNA or RNA heavier than 11 S. These observations suggest the involvement of a precursor larger than proinsulin in the biosynthesis of insulin.It is now well established that the biosynthesis of insulin in the pancreatic beta cell proceeds via a single-chain polypeptide precursor which has been identified and characterized as proinsulin (1-5). Based on a molecular weight for proinsulin of approximately 10,000, it can be speculated that the translation of the messenger RNA for proinsulin would require the participation of three ribosomes. Studies by Permutt and Kipnis (6) on the stimulatory effect of glucose on protein synthesis in isolated rat pancreatic islets provided indirect support for this speculation. Tjioe and Kroon (7) immunoassayed for insulin in polysome preparation from rat islets and concluded that trisomes participated in the biosynthesis of insulin. However, we (8) have observed that immunoassayable insulin was mainly associated with tetrasomes and pentasomes obtained from fetal bovine pancreas, when the breakdown of polysomes by RNase was completely prevented by antibody against RNase. Our observation suggests that polysomes larger than trisomes may be involved in the biosynthesis of insulin. The participation of polysomes larger than expected could be due to either the presence of untranslatable regions of nucleotides in the messenger RNA or the synthesis of a protein larger than anticipated. In order to establish if a protein larger than proinsulin is synthesized, we have extracted and fractionated RNA from isolated rat pancreatic islets and from human insulinomas and have used the RNA fractions as templates for protein synthesis in the oocytes of Xenopus laevis.MATERIALS AND METHODS Preparation of RNA. RNA was extracted from the tissue homogenate using phenol according to the method of Palmiter (9). Five thousand rat islets isolated by the collagenase method of Lacy and Kostianovsky (10) and two human insulinomas were extracted. The RNA preparation was fractionated by centrifugation through a 10-20% sucrose gradient in 0.01 M tris(hydroxymethyl)aminomethane (Tris)-HCI, pH (500 ,Ci/ml) for 40 hr followed by three successive changes of medium containing nonradioactive leucine (1 mg/ml) at 30 min intervals. The oocytes were then homogenized in 0.5 ml of acid-ethanol (75 ml of absolute ethanol, 1.5 ml of concentrated HC1, and water to 100 ml). The homogenate was centrifuged ...

Translation and stability of human globin mRNA in Xenopus oocytes.

“…The oocytes of the African frog Xenopus laevis are capable of translating exogenous, injected mRNAs with high efficiency and for several days (3). A variety of heterologous mRNAs have been translated in the frog oocytes including rabbit (4,5) and mouse globin mRNA (6)(7)(8), human proinsulin mRNA (9), immunoglobulin mRNA (10), and ovalbumin mRNA (11).…”