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Yeast-derived recombinant human insulin-like growth factor I: Production, purification, and structural characterization (1990)

Elliott, Steve ; Fagin, Katherine D. ; Narhi, Linda O. ; [et al.]

Springer

The protein journal 9 (1990), S. 95-104

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ISSN: 1573-4943

Keywords: Insulin-like growth factor ; glycosylation ; disulfide pairing ; circular dichroism ; mutation

Source: Springer Online Journal Archives 1860-2000

Topics: Chemistry and Pharmacology

Notes: Abstract Recombinant human insulin-like growth factor I (IGF-I) is efficiently expressed and secreted fromSaccharomyces cerevisiae using a yeast α-factor leader to direct secretion. However, approximately 10–20% of the IGF-I was in a monomeric form, the remaining materials being disulfide-linked aggregates. When the purified material was subjected to reverse-phase high-performance liquid chromatography (rp-HPLC), it gave two doublet peaks, I and II. Upon reduction, doublet peaks I and II converged to one doublet peak. This suggests that peaks I and II result from different disulfide structures, and the doublet feature of each peak results from other causes. Different disulfide structures between peaks I and II were also suggested from the near UV circular dichroism of these proteins. Only the peak II was biologically active, indicating that peak II has the correct disulfide structure. Concanavalin A affinity chromatography of the purified peak II doublet showed binding of the subpeak with an earlier rp-HPLC retention time, indicating that it was glycosylated. Sequence analysis of tryptic peptides suggested that Thr29 was the site of glycosylation. Site-directed mutagenesis was used to convert Thr29 to Asn29. This substitution reduced, but did not eliminate IGF-I glycosylation, suggesting additional glycosylation sites. The site of carbohydrate addition was consistent with the model that O-glycosylations occur on hydroxyl amino acids near proline residues in β-turns.

Type of Medium: Electronic Resource

URL: http://dx.doi.org/10.1007/BF01024990

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The in vivo formation and repair of DNA adducts from 1′-hydroxysafrole (1981)

Phillips, David H. ; Hanawalt, Philip C. ; Miller, James A. ; [et al.]

New York, NY : Wiley-Blackwell

Journal of Supramolecular Structure and Cellular Biochemistry 16 (1981), S. 83-90

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ISSN: 0275-3723

Keywords: safrole ; 1′-hydroxysafrole ; 1′-acetoxysafrole ; carcinogen-DNA adducts ; DNA repair ; 5-bromodeoxyuridine density labelling ; repair patch size ; Chemistry ; Molecular Cell Biology

Source: Wiley InterScience Backfile Collection 1832-2000

Topics: Biology , Chemistry and Pharmacology , Medicine

Notes: 1′-Hydroxysafrole is a proximate carcinogenic metabolite of the naturally occurring hepatocarcinogen safrole. Comparison by high-performance liquid chromatography of the nucleoside adducts obtained from hepatic DNA of adult female mice treated with [2′,3′-3H]1′-hydroxysafrolc with those formed by reaction of deoxyribonucleo-sides with electrophilic derivatives of 1′-hydroxysafrole indicated that the four in vivo adducts studied were derived from an ester of 1′-hydroxysafrole. Three of the four adducts comigrated with products of the reaction of 1′-acetoxysafrole with deoxyguanosine, whereas the fourth adduct comigrated with the major reaction product of the ester with deoxyadenosine. Analysis of the three deoxyguanosine ad-ducts indicated that all three involve substitution on the 2-amino group of guanine. A sample of the major adduct prepared from deoxyguanylic acid has been charac-terized from its NMR spectrum as N2-(trans-isosafrol-3′-Y1)-deoxyguanosine, and the deoxyadenosine adduct has been similarly characterized as N6-(trans-isosafrol-3′-yl)-deoxyadenosine.Repair replication was measured in cultured human T98G cells exposed to 1′-acetoxysafrole using the combined 5-bromodcoxyuridine density label and radioiso-topic label metnod. At a concentration of 1 mM 1′-acetoxysafrole, the amount of repair synthesis approached maximum values only about 15% of those obtained af-ter saturating doses of ultraviolet light. Repair patch size distribution was found to be similar in cells treated with ultraviolet light or 1′-acetoxysafrole as determined by the density of repair-labeled DNA relative to that of parental DNA.

Additional Material: 5 Ill.