Reinheitskriterien für das kristallisierbare Isoenzym der D-Glycerinaldehyd-3-phosphat-Dehydrogenase aus Bäckerhefe

Abstract

Zusammenfassung: Es wird eine verbesserte Methode zur Darstellung von großen Mengen reiner, kristallisierter Glycerinaldehyd-3-phosphat-Dehydrogenase aus Bäckerhefe beschrieben. Die Verbesserungen bestehen in der Verwendung von 5mM EDTA in allen Lösungen und von schwermetallfreiem Ammoniumsulfat für die Kristallisation. Nucleinsäuren werden mit Protaminsulfat und gebundene Nucleotide mit Aktivkohle entfernt. Das Präparat zeichnet sich durch bisher unerreichte Werte der spezifischen Aktivität sowie des Verhältnisses £280/^260 aus, die durch weitere Reinigungsoperationen nicht mehr verändert werden. Die elektrophoretische und histochemische Analyse Summary: An improved method is reported for the preparation of large quantities of pure, crystalline glyceraldehyde-3-phosphate dehydrogenase from baker's yeast. The improvements consist of 1) inclusion of 5mM EDTA in all solutions, 2) crystallization with ammonium sulfate of low heavy metal content, 3) removal of nucleic acids by precipitation with protamine sulfate, and 4) removal of bound nucleotides with washed charcoal. The specific activity and the ratio of £280/^260 of the preparation exceed the values previously reported. They did not change subsequently in further attempts at purification. Electrophoresis and histochemical analysis of the In vorangegangenen Arbeiten 1 » 2 wurde der Mechanismus

Aspartate‐β‐Semialdehyde Dehydrogenase from Escherichia coli

“…The absorption spectrum ( A : : $?&: , , , = 1 ; AZ8,,/ A260 = 1.8) agrees with the absence of any bound nucleotide [18]. Charcoal treatment, used for the removal of bound nucleotides from glyceraldehyde-3-phosphate dehydrogenase [l] did not change the absorption spectrum.…”

Purification and characterization of aspartic β-semialdehyde dehydrogenase from yeast and purification of an isoenzyme of glyceraldehyde 3-phosphate dehydrogenase

“…Glyceraldehyde-3-Phosphate Dehydrogenase Isozyme. The highest activity reported for this enzyme is 145-158 jumol of NADH/minute per milligram at pH 8.5 (Kirschner and Voigt, 1968), while the values more usually reported for pure enzyme range from 60 to 90 /xmol of NADH per minute per milligram at 30°. Our preparations routinely show a specific activity of about 120 yumol per minute per milligram at pH 7.5.…”

“…Enzyme samples were desalted on Sephadex G-25 before protein determinations in order to remove dithiothreitol which causes abnormally high Lowry determinations. The concentration of purified glyceraldehyde-3-phosphate dehydrogenase was measured by the absorbance at 280 nm, using t280% 0.89 (Kirschner and Voigt, 1968).…”

“…Yeast glyceraldehyde-3-phosphate dehydrogenase, present to the extent of about 5 % of the dry weight of yeast (Krebs et al, 1953), has been purified and studied extensively. Because of the current interest in this enzyme and the puzzling nature of its subunit interactions (Kirschner and Voigt, 1968;Kirschner et al, 1966;Trentham, 1968; Stancel and Deal, 1969), we also give a purification procedure for one isozyme which we obtained as a by-product of our efforts to purify the aspartic /3-semialdehyde dehydrogenase.…”