Abstract
Abstract The effects of a series of univalent cation salts on certain properties of pyruvate kinase, including sedimentation behavior, perturbation of tryptophan residues, and stability of the enzyme, have been investigated. Ultraviolet difference spectra of pyruvate kinase in 0.1 m solutions of activator univalent cation chlorides (K+, NH4+, or Na+) in comparison with enzyme in 0.1 m nonactivator univalent cation chlorides (Li+, or trimethylammonium ion, or tris(hydroxymethyl)aminomethane cation) were determined. Tryptophanyl difference spectra with absorbance peaks at 286 mµ and 295 mµ were obtained under these conditions only when the nonactivator cation Tris+ was included in the experiment. Difference spectra of pyruvate kinase in 0.1 m KCl versus enzyme in 0.1 m LiCl with or without various substrates and Mg++ in the reaction showed no absorbance differences characteristic of the perturbation of tryptophan residues. In contrast, different comparisons of univalent cation chlorides at concentrations of 0.5 to 0.86 m showed capacities to perturb tryptophan residues of pyruvate kinase that were inversely correlated with the known capacities of univalent cations to activate the enzyme. A study of the stability of pyruvate kinase in a series of univalent cation salt solutions showed a positive correlation between the capacities of cations to protect against inactivation and known capacities to activate. In addition to the effect of univalent cations, different anion species influenced the extent of perturbation of tryptophan residues and the stability of the enzyme. The perturbation of the tryptophan residues in the presence of Tris+ (cation) was inhibited by the addition of multivalent anions, phosphate, arsenate, and sulfate. Also, the enzyme consistently showed greater stability in buffer solutions containing the multivalent anions phosphate, arsenate, or sulfate than in buffer solutions in which the predominant anions were acetate, chloride, or nitrate. The sedimentation behavior of pyruvate kinase in the ultracentrifuge was not greatly affected by type of univalent cation salt in the environment, with the exception that the s020,w value was lower in a solution of Tris-Cl than in other salts.
Highlights
KhaSe in 0.1 M solutions of activator univalent cation chlorides (K+, NH*+, or Na+) in comparison with enzyme in 0.1 M nonactivator univalent cation chlorides
Effect of increasing concentrations of KC1 and Tris-Cl on absorbance dijerence of pyruvate kinase at 695 rnp Initially each reaction mixture, in a volume of 3 ml, contained 0.33 M Tricine buffer, 6.7 X 1OP M mercaptoethanol, and 6 mg of pyruvate kinase prepared by the standard procedure (“Materials and Methods”)
No appreciable absorbance differences were observed at 286 and 295 rnp. These results provide no evidence, that activator cations as opposed to nonactivator cations at 0.1 M concentrations are responsible for perturbation of tryptophan residues
Summary
Source of Materials-Crystalline pyruvate kinase was obtained commercially (Calbiochem and Sigma) as a suspension in 2.1 M The salts used in all experiments were reagent grade. 3 and 4, stock salt solutions were utilized that had been extracted with diphenylthiocarbazone to remove trace metals [11]. Tricine was obtained from Calbiochem; EDTA, from Baker; mercaptoethanol, from Eastman Kodak; P-enolpyruvate, from Nutritional Biochemicals; and the barium salt of ADP, from Sigma. Tris-ADP was prepared from the barium salt of ADP as described by Miller and Evans [12].
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
