Sedimentation study of a catalytically active form of rabbit muscle Phosphofructokinase at pH 8.55

Lyndal K. Hesterberg, James Lee

Research output: Contribution to journalArticle

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Abstract

The enzymatic active form of rabbit muscle phosphofructokinase (PFK) was observed directly by using the method of reacting or active enzyme centrifugation (AEC). These studies were performed in two assay systems: a coupled enzyme and a pH-dependent dye-linked system in glycylglycine buffer at pH 8.55 and 23 ± 1°C. The sedimenting band of PFK was stabilized by three solvent systems: 50% (v/v) D2O, 10% (w/v) sucrose, and 4% (v/v) or 10% (v/v) glycerol. The active PFK species sediments as a single component with a sedimentation coefficient of 12.4 ± 0.5 S, after correcting for protein-solvent interactions. Although PFK may undergo association-dissociation, there is no observable change in the value of s20,w over a 57-fold range of protein concentration. Throughout this range only a single active species of PFK was observed, and within an experimental uncertainty of ±10%, the enzymatic activity observed in the sedimentation studies accounts for the total enzymatic activity observed in the steady-state kinetics. Partially purified PFK was subjected to AEC analysis. Results reveal the presence of again a single active form sedimenting at the same rate as the purified enzyme. Results from sedimentation velocity studies indicate that the stabilizing solvents employed in AEC enhance the self-association of PFK. However, such an enhancement alone cannot account for the observation of a single active species with a sedimentation coefficient of 12.4 S. The interactions between solvent additives and PFK were studied by density measurements and by the application of multicomponent theory. Results from such a preferential solvent interaction study indicate that PFK is preferentially hydrated in the presence of sucrose or glycerol. The enhancement of PFK self-association is most likely due to a nonspecific solvent-protein interaction.

Original languageEnglish (US)
Pages (from-to)2030-2039
Number of pages10
JournalBiochemistry
Volume19
Issue number10
StatePublished - 1980
Externally publishedYes

Fingerprint

Phosphofructokinases
Sedimentation
Muscle
Rabbits
Muscles
Centrifugation
Enzymes
Association reactions
Glycerol
Sucrose
Glycylglycine
Proteins
Uncertainty
Assays
Buffers
Sediments
Coloring Agents
Observation

ASJC Scopus subject areas

  • Biochemistry

Cite this

Sedimentation study of a catalytically active form of rabbit muscle Phosphofructokinase at pH 8.55. / Hesterberg, Lyndal K.; Lee, James.

In: Biochemistry, Vol. 19, No. 10, 1980, p. 2030-2039.

Research output: Contribution to journalArticle

Hesterberg, Lyndal K. ; Lee, James. / Sedimentation study of a catalytically active form of rabbit muscle Phosphofructokinase at pH 8.55. In: Biochemistry. 1980 ; Vol. 19, No. 10. pp. 2030-2039.
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