Further purification of epidermal growth factor by high-performance liquid chromatography

Matrisian, Lynn M.; Larsen, Brent; Finch, Joanne S.; Magun, Bruce E.

doi:10.1016/0003-2697(82)90015-x

Cited by 100 publications

(28 citation statements)

References 21 publications

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“…Routinely, the peptide isolated in our laboratory by this method yields a single band upon gel electrophoresis in sodium dodecyl sulphatecontaining polyacrylamide gels (15%) under reducing conditions and a single peak upon reversephase liquid chromatography (C-19 column) using an acetonitrile gradient in 0.1% trifluoroacetic acid. In a mitogenesis assay (human skin fibroblasts) EGF-URO prepared in our laboratory and stored frozen (0.2 to 1 mg ml-1 in 50 mM sodium bicarbonate) has a potency (EC50) of approximately 0.25ngmlP1 (Hollenberg & Gregory, 1980); this value is in accord with the potency reported for murine f-EGF-URO or for murine a-EGF-URO that has been stored frozen in buffer solutions (Matrisian et al, 1982 …”

Section: Reagentssupporting

confidence: 62%

Inhibition by anti‐inflammatory agents of contraction induced by epidermal growth factor‐urogastrone in isolated longitudinal smooth muscle strips from guinea‐pig stomach

Itoh

Muramatsu

Patel

et al. 1988

British J Pharmacology

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1 Epidermal growth factor-urogastrone (EGF-URO) caused a concentration-dependent contractile response of longitudinal muscle strips from the gastric body of the guinea-pig stomach. The contractile response to EGF-URO was monophasic, with tension returning rapidly to baseline. Desensitization was evident in that further addition of EGF-URO to the organ bath did not cause a second contraction. 2 Preincubation with indomethacin, ibuprofen, naproxen and aspirin markedly inhibited the contractions induced by EGF-URO with an order of potency (indomethacin > naproxen > ibuprofen > aspirin) that reflected the ability of these agents to inhibit cyclo-oxygenase. 3 The data indicate that prostanoids mediate the action of EGF-URO in the longitudinal muscle preparation.4 Auranofin (0.5 to 50juM), a chrysotherapeutic agent with antiproliferative properties used for treating rheumatoid arthritis, also markedly inhibited the EGF-URO response; however, other gold-containing compounds (aurothioglucose or gold sodium thiomalate at 30 to 100 pM) failed to cause significant inhibition. 5 Preincubation of preparations for 2 h with 1 JM hydrocortisone, prednisolone or dexamethasone caused an inhibition of EGF-URO-induced contraction of approximately 50%. However, steroids lacking either a 17x-hydroxyl (corticosterone) or an 1lI-hydroxyl (cortisone, deoxycorticosterone, prednisone) substituent did not inhibit the contraction caused by EGF-URO. For hydrocortisone, the inhibitory effect was half-maximal at 0.2 JM and was maximal at 1 pM. Cycloheximide (1O gM) blocked the inhibitory action of hydrocortisone and potentiated the contractile action of EGF-URO.6 The ability of a variety of steroidal and non-steroidal anti-inflammatory agents to interfere with the action of EGF-URO in a smooth muscle preparation suggests that these agents may also inhibit the action of EGF-URO mediated by prostanoids in other target tissues.7 The data also point to a potential role for EGF-URO in regulating gastric motility.

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Section: Reagentssupporting

confidence: 62%

Inhibition by anti‐inflammatory agents of contraction induced by epidermal growth factor‐urogastrone in isolated longitudinal smooth muscle strips from guinea‐pig stomach

Itoh

Muramatsu

Patel

et al. 1988

British J Pharmacology

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“…Biologically active EGF was prepared from mouse submaxillary glands by the method of Savage and Cohen (8). EGF was further purified by reversed-phase HPLC (9). The most abundant EGF species thus isolated, a-EGF, was iodinated by the chloramine-T method (10).…”

Section: Methodsmentioning

confidence: 99%

Recycling of epidermal growth factor in a human pancreatic carcinoma cell line.

Korc¹,

Magun²

1985

Proc. Natl. Acad. Sci. U.S.A.

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PANC-1 human pancreatic carcinoma cells readily bound and internalized 12I-labeled epidermal growth factor (EGF). Bound 125I-labeled EGF was then partially processed to a number of high molecular weight acidic species. (5), and the gastrointestinal hormone cholecystokinin (6). Further, high concentrations of EGF decrease the number of EGF receptors on the cell surface through a process termed down-regulation (7). We now report an additional mechanism by which some cells may modulate EGF receptor function. In PANC-1 human pancreatic carcinoma cells, internalized EGF is processed mainly to a number of high molecular weight products. Two major products of processed EGF, along with intact EGF, are released into the culture medium. One of these products as well as intact EGF can rebind to the cell surface. These data suggest that in certain cells, recirculation of bindable EGF products may be important in the intracellular disposition of cell-surface EGF receptors. MATERIALS AND METHODSCell Culture. PANC-1 human pancreatic carcinoma cells (CRL 1469) Internalization, Processing, and Degradation Studies. To monitor 125I-labeled EGF internalization, cultured cells were washed as for binding studies and then were incubated for 4 min at 40C with 500 mM NaCl, titrated to pH 2.5 with acetic acid (12). Radioactivity removed by acid treatment (surfacebound) and acid-resistant (internalized) radioactivity were then determined separately. Internalized ligand was localized with the aid of Percoll gradient fractionation of cell homogenates (11). Cell disruption was carried out as described (11), except that homogenization buffer contained 1 mM EDTA, 10 mM acetic acid, and 10 mM triethanolamine at pH 7.4, and was supplemented with 1 mM benzamidine and 0.25 mM phenylmethylsulfonyl fluoride (PMSF) to prevent in vitro proteolysis. Acid phosphatase, UDP-galactosyl transferase, and NADPH-cytochrome c reductase were assayed as reported (11) and served as markers for lysosomes, Golgi apparatus, and endoplasmic reticulum, respectively. To monitor 251I-labeled EGF processing, cell-bound and dissociated radioactivity were analyzed by IEF on horizontal agarose gels using internal standards generated with the aid of pI marker proteins (13). The locations of 125I-labeled proteins were determined by autoradiography of analytical gels after drying (13). Degradation of 125I-labeled EGF in incubation medium was monitored by analyzing supernatant radioactivity by Sephadex G-25 column chromatography. RESULTSStudies with a highly purified species of EGF (pI 4.55) indicate that, after binding to target cells, the peptide is shortened by the sequential removal of carboxyl-terminal Abbreviations: EGF, epidermal growth factor; IEF, isoelectric focusing. 6172The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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“…EGF was prepared as described (29,30) and iodinated by using the immobilized lactoperoxidase methAbbreviations: EGF, epidermal growth factor; PDGF, plateletderived growth factor; PMA, 4B-phorbol 12,8-myristate 13a-acetate; TPCK-treated trypsin, tosylphenylalanyl chloromethyl ketonetreated trypsin; kinase C, protein kinase C.…”

Section: Introductionmentioning

confidence: 99%

Platelet-derived growth factor mimics phorbol diester action on epidermal growth factor receptor phosphorylation at threonine-654.

Davis¹,

Czech²

1985

Proc. Natl. Acad. Sci. U.S.A.

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Addition of platelet-derived growth factor (PDGF) to quiescent WI-38 human fetal lung fibroblasts mimics the effect of tumor-promoting phorbol diesters to inhibit the high-affinity binding of '25I-labeled epidermal growth factor (125I-EGF). PDGF, like phorbol.diesters, was found to increase the phosphorylation state of EGF receptors immunoprecipitated from intact fibroblasts that were labeled to equilibrium with (32liphosphate. Phosphoamino acid analysis of the EGF receptors indicated that both PDGF and phorbol diesters increased the level of [32P~phosphoserine and [32P~phosphothreonine. Phosphopeptide mapping of the EGF receptor demonstrated that PDGF increased the phosphorylation of several sites and induced the phosphorylation of a site that was not observed to be phosphorylated on EGF receptors isolated from control cells. This latter phosphorylation site on the EGF receptor was identified as threonine-654, previously shown to be phosphorylated in response to phorbol diesters in intact cells or by purified protein kinase C in vitro. Further, it was observed that PDGF mimicked the action of phorbol diesters to inhibit the EGF-dependent tyrosine phosphorylation of the EGF receptor in [32Pjphosphate-labeled fibroblasts.These results are consistent with the hypothesis that increases in diacylglycerol and Ca2' levels caused by addition of PDGF to fibroblasts activate protein kinase C and that this kinase, at least in part, mediates the effect of PDGF on the phosphorylation of the EGF receptor. The data further suggest that protein kinase C may play an important role in the regulation of cellular metabolism and proliferation by PDGF.Platelet-derived growth factor (PDGF) is a potent polypeptide mitogen that is released into the blood during platelet lysis after tissue injury. The full mitogenic effect of PDGF is only expressed in the presence of other growth factors such as epidermal growth factor (EGF) and insulin (1). Interestingly, this effect is also characteristic of tumor-promoting phorbol diesters such as 4p-phorbol 12/3-myristate 13a-acetate (PMA), which stimulate the growth of quiescent fibroblasts synergistically with EGF and insulin (2, 3). An-other striking similarity between the actions of PDGF and phorbol diesters is a rapid inhibition of 125I-labeled EGF (125I-EGF) binding to the receptors ofcultured cells (4-12). In view of the similarity of these actions of PDGF and PMA on the EGF receptor, we considered the possibility that these two agents may have a similar mechanism of action.It has been reported that PMA causes the phosphorylation of the EGF receptor on senine and threonine residues, and it has been suggested that this phosphorylation is causally related to the effect of PMA on the EGF receptor (13)(14)(15). Phosphopeptide mapping of the EGF receptor has revealed that the phosphorylation state of several sites is increased after treatment of cells with phorbol diester (13,15,16). One of these sites was observed to be uniquely phosphorylated on the EGF receptor isolated from PMA-trea...

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Further purification of epidermal growth factor by high-performance liquid chromatography

Cited by 100 publications

References 21 publications

Inhibition by anti‐inflammatory agents of contraction induced by epidermal growth factor‐urogastrone in isolated longitudinal smooth muscle strips from guinea‐pig stomach

Inhibition by anti‐inflammatory agents of contraction induced by epidermal growth factor‐urogastrone in isolated longitudinal smooth muscle strips from guinea‐pig stomach

Recycling of epidermal growth factor in a human pancreatic carcinoma cell line.

Platelet-derived growth factor mimics phorbol diester action on epidermal growth factor receptor phosphorylation at threonine-654.

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