Purification and Characterization of Proinsulin mRNA from Rat B‐Cell Tumor

Itoh, Nobuyuki; Nose, Kiyoshi; Okamoto, Hiroshi

doi:10.1111/j.1432-1033.1979.tb13079.x

Cited by 86 publications

(27 citation statements)

References 25 publications

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“…Rat I and II preproinsulin mRNA levels have been measured in rat islets [31] and insulinoma ceils [30,32] and shown to be present in approximately equal amounts. It has, furthermore, been shown that both rat preproinsulin mRNAs appear to be regulated coordinately in vivo following fasting and refeeding of rats with sucrose [31].…”

Section: Discussionmentioning

confidence: 99%

Glucose stimulates the biosynthesis of rat I and II insulin to an equal extent in isolated pancreatic islets

1987

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The effects of glucose on insulin biosynthesis were studied by measuring the incorporation of radiolabelled amino acids into proinsulin/insulin in isolated rat islets. The islets were pulse labelled for 15 rain with [3H]ieucine (present in rat insulin I and II) or [35S]methionine (unique to rat insulin II) and then incubated for a 165 min post-label (chase) period during which the majority of labelled proinsulin was converted to insulin but under conditions whereby > 95% of radiolabelled proinsulin or insulin was retained in the islets. The newly synthesized, labelled, insulin was analyzed by high performance liquid chromatography. Rat I and II insulin biosynthesis was stimulated by 16.7 mM glucose to the same extent.

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Section: Discussionmentioning

confidence: 99%

Glucose stimulates the biosynthesis of rat I and II insulin to an equal extent in isolated pancreatic islets

1987

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“…Total RNA was extracted with phenol/chloroform at pH 9 [5] and purified by LiCl precipitation as previously described [9]. A 10% aliquot of total RNA was stored at -80°C for cDNA hybridisation.…”

Section: Methodsmentioning

confidence: 99%

Characterisation of rat 9‐kDa cholecalcin (CaBP) messenger RNA using a complementary DNA

Desplan

Brehier

Thomasset

1985

European Journal of Biochemistry

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The rat posesses two cholecalciferol-induced calcium-binding proteins, the cholecalcins (CaBP). The 9-kDa CaBP is mainly concentrated in the duodenum while 28-kDa CaBP is located in the kidney and cerebellum. The mRNA encoding 9-kDa CaBP has been characterised using the cloned cDNA, pC109, synthesised from rat duodenal 9-kDa CaBP mRNA J. Biol. Chem. 258, 13502-135051. Nucleotide sequence analysis of this cDNA shows the presence of two stop codons, TGA and TAG, at positions 207 and 271, respectively, of the 3' untranslated region. The cDNA-hybridised mRNA, isolated from rat duodenum, directs the cell-free synthesis of two proteins precipitable by antisera to 9-kDa intestinal CaBP. A major protein comigrates with 9-kDa CaBP whereas a minor product corresponds to a protein which is larger by 2000 Da. The minor protein appears to result from read-through of the 'leaky' UGA stop signal. No protein band which was immunoprecipitable with 28-kDa CaBP antiserum was detected when cDNA-hybridised mRNA from rat kidney and cerebellum was translated in a cell-free system. Northern blots show that the cDNA pC109 sequence hybridises to a homogeneous mRNA species 500-600 nucleotides long from rat duodenum. Larger mRNA species encoding 28-kDa CaBP are undetectable in rat kidney and cerebellum even under low stringency conditions. All these findings demonstrate that there is no cross-hybridisation between 9-kDa and 28-kDa CaBP mRNAs. Southern blot analysis of rat genomic DNA, that shows only one homologous 9-kDa gene, is consistent with these findings. Thus, all our data indicate that there are distinct genes coding for each rat cholecalcin.Cholecalcin, which is also known as calcium-binding protein (CaBP), is one of a group of intracellular proteins which bind calcium with high affinity ( K , = lo6 M-I) [l, 21 and its concentration is dependent upon 1,25-dihydroxycholeca1-ciferol (calcitriol) [3,4], the hormonal form ofvitamin D. Two forms have been identified in the rat [4]. A 28 000-M, molecule (28-kDa cholecalcin), found in kidney and cerebellum, has four calcium-binding sites, whereas a 9000-Mr protein (9-kDa cholecalcin) is mainly located in the duodenum and possesses two calcium-binding domains [l, 2, 41. The presence of two distinct mRNAs encoding either 9-kDa or 28-kDa cholecalcin was deduced from cell-free translation assays, since rat duodenal poly(A)-rich RNA directs the synthesis of 9-kDa cholecalcin whereas mRNAs isolated from kidney and cerebellum code for the 28-kDa cholecalcin [5]. We recently reported the molecular cloning of a cDNA fragment synthesised from rat duodenal mRNA encoding 9-kDa cholecalcin [6]. Nucleotide sequence analysis of a resulting 375-base-pair cDNA clone, pC109, allowed the assignment of 207 nucleotides in the coding region before the TGA stop codon [7] (Fig. 1). The deduced amino acid sequence for 9-kDa cholecdlcin contains the two 'EF hand' domains described by Moews and Kretsinger [8] corresponding to the calcium-binding sites I and I1 [7]. The homology observed between site I structure ...

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“…Total RNA was extracted with phenol-chloroform according to Itoh et al (18). Poly A+ RNA was separated out by chromatography on oligo(dT)-cellulose T7 as described by Aviv and Leder (19).…”

Section: Ilmentioning

confidence: 99%

Vitamin D-Dependent Calcium-Binding Proteins (CaBPs) in Human Fetuses: Comparative Distribution of 9K CaBP mRNA and 28K CaBP during Development

et al. 1987

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ABSTRACT. The vitamin D-dependent calcium-binding protein (CaBP) cholecalcin or calbindin, has been used as a molecular marker of 1,25-dihydroxyvitamin D3 action. Mammals possess two CaBPs: a 9,000 mol wt (9K CaBP) and a 28,000 mol wt (28K CaBP). The distinct localization of each protein in the rat has been previously described with the aid of specific radioimmunoassays developed for each CaBP. Antibodies raised against the rat 28K CaBP can be used to detect this protein in a number of mammalian species including humans. In contrast, antibodies against rat 9K CaBP do not cross react with human 9K CaBP, but human 9K CaBP mRNA can be analyzed using a cDNA probe for rat 9K CaBP mRNA. Such a cross-hybridization between the rat cDNA probe and human CaBP mRNA was demonstrated by Northern analysis. We have documented the distribution and evolution of 28K CaBP and 9K CaBP mRNA in human tissues during fetal development from 14 to 32 wk of gestation. 28K CaBP was only present in kidney and cerebellum, and not detectable in duodenum. There was a 2-fold increase of 28K CaBP in the cerebellum between 14 and 24 wk of gestation. The 9K CaBP mRNA was unevenly distributed in human fetal tissues. 9K CaBP mRNA was present in classical vitamin D target tissues such as duodenum and placenta; high levels of 9K CaBP mRNA also were found in thymus and lung. These findings suggest a role for the hormone not only in the duodenum, placenta and kidney, but during the development of specific organs such as cerebellum, lung and thymus, and in agreement with many recent studies, point to a role of vitamin D in cellular differentiation and growth. (Pediatr Res 21: 362-367, 1987) Abbreviations 1,25(OH)2D3, 1,25-dihydroxyvitamin D3 CaBP, calcium-binding protein 9K CaBP, 9,000 molecular weight calcium-binding protein 28K CaBP, 28,000 molecular weight calcium-binding protein PGFa, prostaglandin DEPC, diethylpyrocarbonate SDS, sodium dodecyl sulfateThe vitamin D-dependent CaBP, cholecalcin (1) or calbindin, is one of a group of intracellular proteins that bind calcium with an affinity constant Ka = lo6 M-' (2, 3). These proteins are the best known molecular markers of vitamin D action on target cells and have been characterized for a number of species (3). Birds have only one CaBP of 28,000 molecular weight, while two CaBPs have been described in mammals (3, 4). A small 9K CaBP having two calcium-binding sites was first located in the absorptive cells of the duodenum (5) but is also found in the placenta (6, 7). A larger 28K CaBP, similar to the chick 28K CaBP (3), possesses four calcium-binding sites and is concentrated in the distal tubules of the kidney (8) and the Purkinje cells of cerebellum (9). Although their exact molecular functions remain unclear, 9K CaBP in the duodenum and the placenta as well as 28K CaBP in the kidney are probably involved in calcium transfer. 28K CaBP has been proposed as a marker of Purkinje cell differentiation and development of the cerebellum (10). Vitamin D-dependence of rat intestinal 9K CaBP gene expression ...

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Purification and Characterization of Proinsulin mRNA from Rat B‐Cell Tumor

Cited by 86 publications

References 25 publications

Glucose stimulates the biosynthesis of rat I and II insulin to an equal extent in isolated pancreatic islets

Glucose stimulates the biosynthesis of rat I and II insulin to an equal extent in isolated pancreatic islets

Characterisation of rat 9‐kDa cholecalcin (CaBP) messenger RNA using a complementary DNA

Vitamin D-Dependent Calcium-Binding Proteins (CaBPs) in Human Fetuses: Comparative Distribution of 9K CaBP mRNA and 28K CaBP during Development

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