Purification, molecular and kinetic characterization of phosphofructokinase-1 from the yeast Schizosaccharomyces pombe: evidence for an unusual subunit composition.
Phosphofructokinase-1 (Pfk-1) from Schizosaccharomyces pombe was purified by 54-fold enrichment to homogeneity elaborating the following steps: (a) Disruption 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 native enzyme exhibits a mass of 790+/-30 kDa, as detected by sedimentation equilibrium measurements. The apparent sedimentation coefficient was found to be s(20,c)=20.2+/-0.3 S. No significant dependence of the s-value on the protein concentration was observed in the range 0. 07-0.7 mg/ml. Polyacrylamide gel electrophoresis in presence of sodium dodecyl sulphate and MALDI-TOF spectra showed that the enzyme is composed of subunits of identical size of 100+/-5 kDa, forming an octameric structure. The N-terminus of the enzyme was found to be blocked. Sequences of tryptic and chymotryptic peptides of the subunit coincide with the proposed amino acid sequence as deduced from the gene from the EMBL library. The Pfk-1 coding sequence of S. pombe was transformed into a Pfk-1 double deletion mutants of Saccharomyces cerevisiae resulting in glucose-positive cells with enzyme activity in the crude cell extract. The kinetic analysis revealed less cooperativity to fructose 6-phosphate (n(H)=1.6) and less inhibition by ATP as compared to the enzyme from baker's yeast. Fructose 2,6-bisphosphate (in micromolar range) and AMP (in millimolar range) were found to overcome ATP inhibition and to increase the affinity to fructose 6-phosphate.
Reuter
R
R
Institut für Biochemie, Medizinische Fakultät, Universität Leipzig, Liebigstrasse 16, 04103 Leipzig, Germany.
Naumann
M
M
Bär
J
J
Haferburg
D
D
Kopperschläger
G
G
D000249:Adenosine Monophosphate / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000494:pharmacology; D000595:Amino Acid Sequence; D002499:Centrifugation, Density Gradient; D004591:Electrophoresis, Polyacrylamide Gel; D005636:Fructosephosphates / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010732:Phosphofructokinase-1 / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism; D021122:Protein Subunits; D012568:Schizosaccharomyces / Q000201:enzymology*; D016415:Sequence Alignment; D020539:Sequence Analysis, Protein; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D005636:Fructosephosphates; D021122:Protein Subunits; D000249:Adenosine Monophosphate; C027618:fructose-6-phosphate; D000255:Adenosine Triphosphate; D010732:Phosphofructokinase-1