M. Kaloyianni et al., PURIFICATION AND KINETIC-PROPERTIES OF PHOSPHOFRUCTOKINASE FROM RANA-RIDIBUNDA ERYTHROCYTES, Comparative biochemistry and physiology. B. Comparative biochemistry, 107(3), 1994, pp. 479-487
Phosphofructokinase (PFK) from Rana ridibunda erythrocytes was purifie
d about 570-fold by column chromatography on Cibacron Blue Sepharose.
The resulting enzyme preparation had a specific activity of 1.94 U/mg
protein and a pH maximum of 7.6. The molecular weight as determined by
HPLC chromatography was 330,000 Da. The S-0.5 value for fructose-6-ph
osphate (F6P) was 5.6 mM and the K-m for ATP 0.87 mM. The enzyme was s
ensitive to inhibition by ATP which was increased with lower F6P conce
ntrations. At physiological levels of 2,3-diphosphoglycerate (0.35 mu
mol/ml RBC), 20% of PFK activity was inhibited. Significant activation
s under cellular conditions were exercised by AMP and, to a lesser ext
ent, by P-i. Micromolar concentrations of fructose-2,6-bisphosphate an
d glucose-1,6-bisphosphate were also potent activators of the erythroc
yte enzyme. Fructose-1,6-bisphosphate (10-50) mu M activated the enzym
e to a limited extent. With respect to these effects, it is suggested
that PFK is a significant enzyme in regulating the glycolytic flux of
Rana ridibunda red blood cells. The existence of a regulatory mechanis
m controlled by the energy status of the red cell, as well as the stat
e of oxygenation of haemoglobin, is discussed, in which PFK occupies a
central role.