Summary
A new, abnormal erythrocyte G-6-PD variant, Gd Frankfurt, was first purified chromatographically on DEAE cellulose. Further purification was performed by fractionate precipitation with ammonium sulfate and by double gel filtration on Sephadex G-200. By these procedures a nearly 14000-fold concentration was obtained. In this preparation as well as in a control preparation of the normal enzyme variant Gd B the molecular weight, the content of sulfhydryl groups and the inhibition of enzyme activity by N-ethylmaleimide was determined. The content of sulfhydryl groups was found to be the same as in the normal enzyme variant.
The molecular weights are slightly different: for the abnormal enzyme variant the value obtained was 223 000, and 243 500 for the normal enzyme. The inhibition by N-ethyl-maleimide showed to be somewhat stronger in the abnormal variant than in the normal enzyme. Peptide analysis after tryptic digestion was performed by fingerprint technique on silica gel-starch thin layers. 39 peptides of the abnormal variant showed the same pattern as in the normal enzyme whereas one peptide was found in a differing localization. Amino acid analysis' of the differing peptides revealed a substitution of glutamic acid for lysine in the abnormal enzyme variant.
Zusammenfassung
Eine neue, anomale erythrocytäre G-6-PD-Variante, Gd Frankfurt, wurde nach chromatographischer Reingung auf DEAE-Cellulose, durch fraktionierte Ammoniumsulfat-Fällung und zweimalige Gelfiltration auf das rund 14000fache angereichert. Mittels Gelfiltration wurde eine Molekulargewichtsbestimmung durchgeführt, ferner wurde der SH-Gruppen-Gehalt und die Hemmung der Aktivität durch N-Äthylmaleimid bestimmt. Im Vergleich zum Normalenzym, Gd B, fand sich — bei gleichem SH-Gruppen-Gehalt — ein von der Norm nur geringfügig abweichendes Molekulargewicht von 223 000 gegenüber 243 500 beim Normalenzym und eine etwas stärkere Hemmung durch N-Äthylmaleimid. Nach tryptischer Verdauung ergab die Peptidanalyse mittels Fingerprint-Verfahrens auf Kieselgel-S-Dünnschichtplatten ein abweichendes Verhalten für 1 Peptid, während die restlichen 39 Peptide die gleiche Lokalisation aufwiesen. Die Aminosäuren-Analyse dieser differienden Peptide deckte für die neue Variante einen Austausch von Lysin gegen Glutaminsäure auf.
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Nowicki, L., Strobel, S. & Martin, H. Über eine neue erythrocytäre Glucose-6-Phosphatdehydrogenase-Variante, ‚Typ Frankfurt‘. Klin Wochenschr 52, 485–492 (1974). https://doi.org/10.1007/BF01468537
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DOI: https://doi.org/10.1007/BF01468537
Key words
- New erythrocyte G-6-PD variant
- Gd Frankfurt
- Structural abnormality
- Molecular weight
- Peptide analysis
- Amino acid substitution
- Lys → Glu