Evidence for isoaspartyl (deamidated) forms of mouse epidermal growth factor.
A variant form of mouse submaxillary gland epidermal growth factor ( EGF) was identified by isocratic reversed-phase HPLC of EGF obtained by Bio-Gel P-10 column chromatography ("culture grade"). The variant form was essentially absent in preparations of EGF further purified by chromatography on DEAE-cellulose ("receptor-grade" EGF). The spectral properties and amino acid composition of the variant form (EGF-I) could not be distinguished from those of the intact polypeptide isolated by HPLC (alpha- EGF). Receptor-binding and mitogenic properties of EGF-I were also equivalent to those of alpha- EGF. These data suggested that EGF-I was structurally very similar to EGF. However, the very low yield (less than 4%) obtained by Edman degradation indicated that the N-terminal (Asn1) of the polypeptide was modified. Isoelectric focusing of EGF-I revealed two major immunoreactive bands: one with a pI equivalent to that of alpha- EGF (pI 4.6) and another at pI 4. 1. Alkaline treatment of alpha- EGF (0.1 M NH4OH) yielded peak material by HPLC that coeluted with EGF-I; the alkaline-generated EGF-I yielded bands that also focused at pH 4.6 and 4. 1. Ammonium hydroxide treatment of [des-Asn1]-EGF (beta- EGF) did not produce conversion to EGF-I. On the basis of these data, we propose that EGF-I was formed by selective deamidation of the N-terminal Asn of intact EGF. This notion is also supported by liquid secondary ion mass spectrometry, which showed that EGF-I was approximately 1.5 mass units greater than alpha- EGF. The heterogeneity observed by isoelectric focusing supports previous studies which have shown that, following deamidation of N-terminal asparagine, a beta-aspartyl shift can occur, which in the present study might yield succinimido-aspartyl1- EGF and beta-aspartyl1- EGF. Low yields observed during Edman degradation indicate that negligible amounts occur as the alpha-aspartyl1- EGF isomer.[1]References
- Evidence for isoaspartyl (deamidated) forms of mouse epidermal growth factor. DiAugustine, R.P., Gibson, B.W., Aberth, W., Kelly, M., Ferrua, C.M., Tomooka, Y., Brown, C.F., Walker, M. Anal. Biochem. (1987) [Pubmed]
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