Abstract
On the basis of structural homology and similar biological activity, gastrin-releasing peptide (GRP) has been considered the mammalian equivalent of amphibian bombesin. In this paper we now show this to be incorrect. Chromatography of frog (Bombina orientalis) gut extracts demonstrated two peaks of bombesin-like immunoreactivity (BLI), one similar in size to GRP and one similar in size to amphibian bombesin. These peaks were purified by high pressure liquid chromatography then subjected to mass spectrometric analyses to determine molecular weights and amino acid sequence. Based on the amino acid sequence of the lower molecular weight BLI species, a mixed oligonucleotide probe was prepared and used to screen a B. orientalis stomach cDNA library. Sequence analysis showed that all hybridizing clones encoded a 155-amino acid protein homologous to the mammalian GRP precursor. The mass spectra of the high and low molecular weight peaks of frog gut BLI were consistent with their origin from the processing of the frog GRP (fGRP) precursor into GRP-29 and GRP-10, just like the processing of the rat GRP precursor. Sequence homology showed that the fGRP precursor is more homology showed that the fGRP precursor is more closely related to the mammalian GRP precursors than to either the frog bombesin or frog ranatensin precursors. Northern blot analysis showed that fGRP is encoded by a mRNA of 980 bases, clearly different from the 750-base mRNA which encodes frog bombesin. Northern blot analysis and in situ hybridization showed fGRP mRNA in frog brain and stomach and bombesin mRNA in frog skin, brain, and stomach. That frogs have independent genes for both GRP and bombesin raises the possibility that mammals have an as yet uncharacterized gene encoding a true mammalian bombesin.
Highlights
Lower molecular weight BLI species, a mixed oligonu- isolated from porcine stomach a 27-amino acid peptide hocleotide probe was prepared and used to screen a B. mologous to thecarboxyl terminus of bombesin and named it orientalis stomach cDNA library
Sequence GRPand gastrin-releasing peptide (GRP)-10 have been considered the mammalian homology showed that the fGRP precursor is more equivalents of amphibian bombesin
In this paper we report that frog besin raises the possibility that mammals have an as (B.orientalis) gut BLI does not derive primarily from the frog yet uncharacterized gene encodinga true mammalian bombesin precursor, but rather like mammalian BLI, is prebombesin
Summary
Frog Gut Extraction and Chromatography-Frog gut tissue contained approximately 150pmol/g of BLI. LSIMS analysis before and aftersuch treatment showed a mass shift in only one of the molecular ion components (Fig.[3]) In these spectra (onlypartial spectra arsehown) the MH’ ion at m/z 1093of the untreatedfraction is virtually absent in the oxidized fraction, and two new peaks appear at m/z MH+ 1109 (+16 Da)and MH’ 1125 (+32 Da). This suggests the presence of two oxidizable residues, and is consistent with the presence of one methionine and one tryptophan typical of most bombesin-like peptides. (yea3)and 225 (y8bs)and for His-Trp at m/z 296 (y8a3)and 324 (~sb[3])
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