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A cholecystokinin molecule larger than any previously chemically characterized was purified from canine proximal small intestine mucosa. The purification procedure consisted of sequential steps of affinity chromatography, gel filtration, and high pressure liquid chromatography. Activity was detected and quantitated by radioimmunoassay with an antibody that recognized the carboxyl terminal sequence of porcine cholecystokinin. Microsequencing of the purified peptide revealed an amino terminal nonadecapeptide sequence (AQKVNSGEPRAHLGALLAR) not present in known cholecystokinin molecules followed by a nonadecapeptide sequence (YIQQARKAPSGRMSVIKNL) that corresponds exactly to the amino terminal sequence of porcine cholecystokinin 39 except for reversed positions of a Met and a Val residue. Based on the sequence analysis, immunoreactivity, and presence of biological activity in two bioassay systems, this peptide, tentatively named cholecystokinin 58, may be a biosynthetic precursor of the smaller forms previously characterized in gastrointestinal and brain tissues. 相似文献
163.
The two dimorphic forms of chloroplast isolated from maize leaves utilized acetate for fatty acid biosynthesis and had similar requirements for cofactors. The oleate:palmitate ratio of the fatty acid products was lower for bundle sheath chloroplasts as was acetate incorporation into total fatty acids. Galactose from UDP-galactose was incorporated into galactolipids by both morphological forms to give monogalactosyl diacylglycerol and digalactosyl diacylglycerol in the ratio of 4:1. 相似文献
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Heinz Nika Edward Nieves David H. Hawke Ruth Hogue Angeletti 《Journal of biomolecular techniques》2013,24(3):132-153
We previously adapted the β-elimination/Michael addition chemistry to solid-phase derivatization on reversed-phase supports, and demonstrated the utility of this reaction format to prepare phosphoseryl peptides in unfractionated protein digests for mass spectrometric identification and facile phosphorylation-site determination. Here, we have expanded the use of this technique to β-N-acetylglucosamine peptides, modified at serine/threonine, phosphothreonyl peptides, and phosphoseryl/phosphothreonyl peptides, followed in sequence by proline. The consecutive β-elimination with Michael addition was adapted to optimize the solid-phase reaction conditions for throughput and completeness of derivatization. The analyte remained intact during derivatization and was recovered efficiently from the silica-based, reversed-phase support with minimal sample loss. The general use of the solid-phase approach for enzymatic dephosphorylation was demonstrated with phosphoseryl and phosphothreonyl peptides and was used as an orthogonal method to confirm the identity of phosphopeptides in proteolytic mixtures. The solid-phase approach proved highly suitable to prepare substrates from low-level amounts of protein digests for phosphorylation-site determination by chemical-targeted proteolysis. The solid-phase protocol provides for a simple, robust, and efficient tool to prepare samples for phosphopeptide identification in MALDI mass maps of unfractionated protein digests, using standard equipment available in most biological laboratories. The use of a solid-phase analytical platform is expected to be readily expanded to prepare digest from O-glycosylated- and O-sulfonated proteins for mass spectrometry-based structural characterization. 相似文献
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D H Hawke P M Yuan K J Wilson M W Hunkapiller 《Biochemical and biophysical research communications》1987,145(3):1248-1253
Microbore HPLC methodology permits rapid and sensitive mapping of human saliva proteins. Saliva is sampled and processed in less than one hour, greatly reducing the likelihood of artifactual protein degradation. As little as 50 microliters of saliva yields proteins in sufficient quantities and purity to obtain amino terminal sequences directly. By this route we have discovered a 14 kDa protein extremely homologous to Cystatin S, but amino-terminally extended by eight amino acids. 相似文献
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