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D-Altronate: NAD-oxidoreductase of Escherichia coli K12 has been purified. The purification consists of four steps: nucleic acids precipitation with protamine sulfate; ammonium sulfate precipitation; ion exchange chromatography on DEAE-cellulose and Sephadex G-150 gel filtration. The overall procedure results in a 53-fold purification with a yield of 60%. Altronate oxidoreductase is an inducible enzyme: galacturonate, tagaturonate, glucuronate and fructuronate are good inducers. The activity remains stable at 4 °C or at — 20 °C for several months. Optimal activity is obtained at pH 6.3 for tagaturonate reduction, and at pH 8.9 for altronate oxidation. Altronate oxidoreductase does not require any cofactor, except NADH. The apparent Michaelis constants have been determined: for tagaturonate, the Km is 350 μM; for NADH, 60 μM; for altronate, 90 μM, and for NAD+, 110 μM. Moreover, altronate oxidoreductase is active with NADPH as coenzyme and the apparent Km value for NADPH is 400 μM. Other carbohydrates tested are unsuitable as substrates. Many other carbohydrates tested do not prove to be inhibitors. The value for the energy of activation is 38 kJ/mol (9 kcal/mol); the Q10 is 1.5. The results of heat inactivation of altronate oxidoreductase (58.5 °C) indicate homogeneous enzyme activity in crude extract. Thermal inactivations also indicate that the same enzyme, altronate oxidoreductase, is induced by galacturonate and glucuronate, and catalyzes the direct reactions with NADH or NADPH. Finally, it has been confirmed by heat inactivation that there is no interference between specific assays of altronate and mannonate oxidoreduetases. Compounds such as NADH, NADPH, altronate and mannonate protect enzymatic activity from degradation by heat, whereas NAD+, tagaturonate and fructuronate do not. NADH, in particular, is effective as a stabilizer; this ability is proportional to its concentration. Enzyme inhibition with para-chloromercuribenzoate is non-competitive and the apparent Ki is 1 mM. para-Chloromercuribenzoate also inactivates altronate oxidoreductase indicating homogeneous activity and specificity of the assay method in crude extracts. Chromatographic properties of the enzyme are identical whether the inducer is galacturonate or glucuronate. Using paper chromatography, the reaction product of enzyme action on D-tagaturonate is identified as D-altronate. [ABSTRACT FROM AUTHOR]

Human lactoferrin, the red iron-binding protein from milk, was prepared by chromatography on DEAE-cellulose of the milk proteins soluble in 2 M ammonium sulfate. Several preparations differing in iron content were obtained. Apolactoferrin was prepared by dialyzing the protein against 0.1 M citric acid. Saturation experiments with iron ascorbate indicated the iron : protein ratio at saturation to be 0.025 micromoles of metal per mg of protein. Assuming 80,000 to be the molecular weight of lactoferrin, this would correspond to a ratio of two iron atoms per molecule. With copper, a similar molar proportion at saturation was obtained. Gasometrie measurements were used to calculate the amount of carbon dioxide liberated upon dissociation of the metal-protein complexes by acidification. One mole of CO2 was freed per atom of iron or copper. [ABSTRACT FROM AUTHOR]

Collagenase was found to attack the collagen molecule at both ends at 10°. Six molecular fragments of different lengths were observed and could be correlated with the intact molecule as long-spacing segment fragments. It was possible to isolate the 2,600, 1,250 and 780 Å fragments by fractional ammonium sulphate precipitation. The sharp helix-coil transition on denaturation shows that the fragments have an intact triple helix. It was possible to separate the two shorter fragments into three components (the α1, α2 and α3 chain fragments) by chromatography on CM cellulose. The α1 and α3 chains have very similar amino acid compositions and peptide maps for both these fragments. This suggests that the differences between these two components are probably due to telopeptides and not to the amino acid sequence of the main chain. [ABSTRACT FROM AUTHOR]