Isolation of peptides from porcine intestinal tissue that induce extracellular acidification in CHO cells: identification of the active peptide as IGF-I and characterization of a fragment of calponin H1 processed at a dibasic site.
Chinese hamster ovary (CHO) cells are widely used as hosts for receptor expression and pharmacological studies. However, several endogenous receptor populations are present on these cells. Intestinal tissue extracts were found to induce strong extracellular acidification responses (ECAR) in CHO cells, yet several pure hormonal peptides, such as VIP, secretin, CCK, GIP, and galanin were ineffective. It is not known, which are the active compounds in the extracts that can stimulate the extracellular acidification in CHO cells. These active substances may be ligands for yet unknown receptors that are present natively in this cell type. We therefore decided to identify the active compound(s) by isolation from intestinal extract and structural characterization. Using chromatographic separations in combination with microphysiometry we have purified and characterized one such bioactive ligand. Structural analysis indicated that the isolated peptide was identical to insulin-like growth factor I (IGF-I). In the intestine, IGF-I is present in low amounts and has previously been detected only with radioimmunoassays. The results indicate that CHO cells express functional receptors for IGF-I. Among the peptides extracted from the intestine IGF-I is probably the strongest stimulator of ECAR in CHO cells. Moreover, IGF-I acts synergistically with other factors present in the crude tissue extract. Additionally, a fragment of calponin H1 (residues 1-43), previously not described at the protein level, was identified in the IGF-I containing fractions. The fragment was characterized by mass spectrometry and found to be N-terminally modified by acetylation suggesting that the whole protein bears the same posttranslational modification.
Bonetto
V
V
Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden.
Eriste
E
E
Jonsson
A P
AP
Efendic
S
S
Jörnvall
H
H
Sillard
R
R
D000107:Acetylation; D000818:Animals; D016466:CHO Cells; D002135:Calcium-Binding Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D002457:Cell Extracts / Q000737:chemistry / Q000494:pharmacology; D006224:Cricetinae; D004357:Drug Synergism; D006863:Hydrogen-Ion Concentration; D007334:Insulin-Like Growth Factor I / Q000737:chemistry / Q000302:isolation & purification* / Q000494:pharmacology*; D007422:Intestines / Q000737:chemistry*; D008840:Microfilament Proteins; D010455:Peptides / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013552:Swine; D000096985:Calponins
D002135:Calcium-Binding Proteins; D002457:Cell Extracts; D007334:Insulin-Like Growth Factor I; D008840:Microfilament Proteins; D010455:Peptides; D000096985:Calponins