Purification, molecular and kinetic characterization of phosphofructokinase-I from the yeast Schizosaccharomyces pombe: evidence for an unusual subunit composition
R. Reuter et al., Purification, molecular and kinetic characterization of phosphofructokinase-I from the yeast Schizosaccharomyces pombe: evidence for an unusual subunit composition, YEAST, 16(14), 2000, pp. 1273-1285
Phosphofructokinase-1 (Pfk-1) from Schizosaccharomyces pombe was purified b
y 54-fold enrichment to homogeneity elaborating the following steps: (a) Di
sruption of the cells with glass beads; (b) fractionated precipitation with
polyethylene glycol 6000; (c) affinity chromatography on Cibacron-Blue F3G
-A-Sephadex G 100; (d) ion exchange chromatography on Resource Q, The nativ
e enzyme exhibits a mass of 790+/-30 kDa, as detected by sedimentation equi
librium measurements. The apparent sedimentation coefficient was found to b
e s(20,c)=20.2+/-0.3 S. No significant dependence of the s-value on the pro
tein concentration was observed in the range 0.07-0.7mg/ml. Polyacrylamide
gel electrophoresis in presence of sodium dodecyl sulphate and MALDI-TOF sp
ectra showed that the enzyme is composed of subunits of identical size of 1
00+/-5 kDa, forming an octameric structure, The N-terminus of the enzyme wa
s found to be blocked, Sequences of tryptic and chymotryptic peptides of th
e subunit coincide with the proposed amino acid sequence as deduced from th
e gene from the EMBL library. The Pfk-1 coding sequence of S, pombe was tra
nsformed into a Pfk-1 double deletion mutants of Saccharomyces cerevisiae r
esulting in glucose-positive cells with enzyme activity in the crude cell e
xtract. The kinetic analysis revealed less cooperativity to fructose 6-phos
phate (n(H) = 1.6) and less inhibition by ATP as compared to the enzyme fro
m baker's yeast. Fructose 2,6-bisphosphate (in micromolar range) and AMP (i
n millimolar range) were found to overcome ATP inhibition and to increase t
he affinity to fructose 6-phosphate. Copyright (C) 2000 John Wiley & Sons,
Ltd.