Abstract
Abstract Formyltetrahydrofolate synthetase has been purified to near homogeneity from the thermophile, Clostridium thermoaceticum, as judged from sedimentation velocity, amino-terminal analysis, and electrophoresis. The enzyme is exceptionally stable at high temperatures, especially at pH 7.0. Ammonium or potassium ions enhance its stability between pH 6 to 9. These ions are not required for activity, however. There is little or no carbohydrate or lipid present in the enzyme. The partial specific volume calculated from the amino acid composition is 0.747 cc per g. The thermostable formyltetrahydrofolate synthetase isolated from C. thermoaceticum is somewhat more hydrophobic than the similar enzyme isolated from two mesophilic Clostridia. Although the difference in hydrophobicity is small, it may explain the higher thermal stability of the enzyme from C. thermoaceticum.
Highlights
The partial specific volume calculated from the amino acid composition is 0.747 cc per g
The thermostable formyltetrahydrofolate synthetase isolated from C. thermoaceticum is somewhat more hydrophobic than the similar enzyme isolated from two mesophilic Clostridia
In this paper we report the purification, stability, and composition of formyltetrahydrofolate synthetase from C. thermoaceticum
Summary
C. thermoaceticum was grown on glucose in a fermenter containing 400 liters of medium with the composition as given by Lentz and Wood [28], but complemented with 45 ml of boiled and filtered tomato juice per liter. The only modification was that the assay was performed at 50” instead of at 37” In this assay the product of the reaction, IO-formyltetrahydrofolate, is determined spectrophotometrically after conversion to 5, lo-methenyltetrahydrofolate, which at 350 rnp has an E = 24.9. Protein was determined either by the biuret method [30] or with the phenol reagent method [31] with bovine serum albumin as standard Both methods give enzyme protein concentrations which are in agreement with concentrations calculated from the refractive index increment obtained in an ultracentrifuge with the use of interference optics [32]
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