Pre-proparathyroid Hormone: A Direct Translation Product of Parathyroid Messenger RNA

Abstract

An 8-15S RNA fraction from calf parathyroid glands stimulated the incorporation of radioactive lysine and methionine into protein by 15-to 30-fold in a wheat germ extract. The major product, representing 25% of the total protein synthesized, could be bound to an antiserum to parathyroid hormone and binding was inhibited by parathyroid hormone. The chromatographic mobilities of the two tryptic peptides of the cell-free product that contained methionine were identical to the corresponding peptides of parathyroid hormone. Upon electrophoresis in acidic or sodium dodecyl sulfate-acrylamide gels, the cell-free product migrated Parathyroid hormone (PTH) is initially synthesized as a precursor, proparathyroid hormone (ProPTH) (1,5,6). In studies in intact cells, it is not possible to demonstrate that ProPTH is the initial, direct product of the translation of messenger RNA for PTH. To determine whether the parathyroid messenger RNA might contain more information than is represented in the structure of ProPTH, we have begun studies to isolate the messenger RNA for PTH and to translate it in a heterologous cell-free system. We now Abbreviations: PTH, parathyroid hormone; ProPTH, proparathyroid hormone; pre-ProPTH, pre-proparathyroid hormone; TMV, tobacco mosaic virus; TSSE buffer, 0.01 TM Tris HCl, pH 7.4, 0.5% sodium dodecyl sulfate, 0.1 MI NaCl and 1 m1M ethylenediaminetetraacetate. 3731report that RNA isolated from parathyroid glands can be translated in cell-free extracts of wheat germ and that the major product synthesized is slightly larger than ProPTH. The results indicate that the product, termed pre-ProPTH, may be a precursor to ProPTH. METHODSIsolation of RNA. Calf parathyroid glands, obtained from a local abattoir at the time of slaughter, were immediately placed ini cold Earle's balanced salt solution and used within 2 hr. Approximately 1 g (wet weight) of glands was minced and homogenized with a motor-driven Teflon-glass homogenizer at 00 in 5 ml of 0.25 M sucrose, 0.01 M Tris-HCl, pH 7.4 (25°), containing 100 ,g/ml of heparin. The homogenate was centrifuged at 1000 X g for 5 min. The supernatant was adjusted to 1.0% sodium dodecyl sulfate (Na DodSO4) and 0.04 M Na ethylenediaminetetraacetate (EDTA), pH 7.4, and deproteinized by shaking three times with two volumes of phenol-chloroform-isoamyl alcohol (1: 1: 0.04) and finally three times with chloroform-isoamyl alcohol (1:0.04) (7).The final chloroform phase was re-extracted with 5 ml of 0.01 M Tris * HCl, pH 7.4,0.5% Na DodSO4, 0.1 M NaCl, and 1 mM EDTA (TSSE buffer). RNA was precipitated overnight at -20°by the addition of 2 volumes of ethanol to the combined aqueous phases. The RNA was dissolved in 0.4 ml of TSSE buffer and was layered over a 5-20% sucrose

Proinsulin biosynthesis in broken-cell preparations of islets of Langerhans

“…The single peak of proinsulin obtained on gel electrophoresis contrasts with the predominant labelling in insulin that takes place when whole rabbit islets are incubated with labelled amino acids for extended periods (Parry & Taylor, 1974). Other studies (Permutt & Boime, 1975;Weber, 1975) Broken-cell 2mM-Glucose (10) 4.04+0.57 preparation 16mM-Glucose (10) 7.63±1.00* 16mM-Galactose (6) 5.31 + 1.81 16mM-Sucrose (6) 5.43 + 0.94 Crude cell2mM-Glucose (10) 0.37+0.12 free system * Value significantly different from control with P<0.01. precursor of proinsulin in a similar way to the production of a parathyrin precursor (Kemper et al, 1974). However, in the present experiments there is no evidence that significant amounts of material closely related to proinsulin, but of higher molecular weight, are being labelled.…”

Mechanisms for the incorporation of proteins in membranes and organelles.

“…It was observed (16,17,47,109,138,194,223) that primary translation products synthesized in the absence of microsomal membranes (presecretory proteins) contain amino-terminal segments (referred to as signal segments), which are not present in the final secretory products or in products found within the microsomal lumen (prosecretory and secretory proteins). Indeed, Milstein et al (138) produced an independent formulation of the signal hypothesis based on their observation that immunoglobulin light chains synthesized in vitro by microsomes, or in extracts that contained membranes, were processed by removal of an amino-terminal segment to a polypeptide with the same electrophoretic mobility as the mature light chain.…”

Cell-free synthesis of rat preproinsulins: characterization and partial amino acid sequence determination.

“…Automated sequence determination of labeled cell-free product purified by immunoprecipitation discloses the presence of 23 additional amino acids NH-terminal to the B chain sequence of proinsulin. The partial amino acid sequence of this extension is as follows: NH2-XLeu (Lys) Met-X-Phe-Leu-Phe-Leu-Leu (Lysy Leu-Leu-X-Leu-X-X-X-X-X-X-X-X-proinsulin.…”