Metabolic control and structure of glycolytic enzymes. 17. Pig liver phosphofructokinase: asymmetry properties, proof of rapid association-dissociation equilibriums, and effect of temperature and protein concentration on the equilibriums

Abstract

Sedimentation coefficient determinations show that, under comparable conditions, pig liver phosphofructokinase (PL-PFK) is larger than any previously studied phosphofructokinase; the molecular weight of PL-PFK at 4 °C at a protein concentration of 5 mg/mL is estimated to be greater than 10 000 000. Furthermore, the enzyme is very asymmetric as indicated by a strong concentration dependence of the sedimentation coefficient data and by a minimum intrinsic viscosity value at 20.0 °C of 30.8 cm3/g. Kinetic and reversibility tests show that the enzyme exists as a temperaturedependent, concentration-dependent equilibrium mixture of polymeric forms. Dissociation (partial) is favored by either higher temperatures or lower protein concentrations. PL-PFK sediments as a single peak (s02o,w = 104 S) at both 4 and 23 °C, at concentrations above 3 and 9 mg/mL, respectively.An mammals, control of liver phosphofructokinase (PFK)1 is necessary for coordination of utilization of glucose through glycolysis and synthesis of glucose through gluconeogenesis.In addition, these reactions must be coordinated with the overall needs for, and supplies of, carbohydrates from all sources, including amino acids, Krebs cycle metabolites, fatty acids, etc. In this respect, the mammalian liver PFKs differ from their widely studied muscle counterparts, which, for the most part, are turned on or off only in response to needs for energy through degradation of glucose. This difference in complexity of functions raises the possibility of differences in structures and control properties between mammalian muscle and liver phosphofructokinases.Although procedures have recently been published for purification of phosphofructokinases from various liver sources,

Phosphofructokinase

“…Brand and Soling (52) found a molecular weight for the rat liver enzyme of 365,000 using a linear gradient polyacrylamide gel electrophoresis in the presence of ATP. Massey and Deal (87,88) observed that the pig liver phosphofructokinase sediments as a single boundary, but, depending on protein concentration and temperature, its sedimentation coefficient varies from 44 to 104 S. The 104 S protein is the largest phosphofructokinase thus far reported and may correspond to a molecular weight of at least 10 million.…”

“…Preparation. Phosphofructokinase from livers of chicken (85), rat (52,86), and pigs (87) and from pig kidney (88) has been isolated in pure form, with specific activities ranging from 86 to 134 units/ mg. The chicken liver phosphofructokinase has been crystallized (85).…”

“…Electrophoresis on cellulose acetate revealed that rat skeletal muscle and heart contain the single isozymic form A and liver contains isozyme A and isozyme B (108,109). By DEAE chromatography rat liver phosphofructokinase has been resolved into two isozymes (88). The major isozyme, which constitutes 85% of total phosphofructokinase activity, is B4 (L as defined by Dunaway and Weber) (86).…”

The Glycolytic Enzyme Phosphofructokinase-1 Assembles into Filaments

“…PFK1 was recently identified as a filament-forming enzyme in Saccharomyces cerevisiae (Shen et al, 2016). Filament formation by PFK1 may be evolutionarily conserved because purified liver PFK1 (PFKL) forms asymmetric oligomers in vitro, as determined by gel chromatography (Kemp, 1971), sedimentation with analytical ultracentrifugation (Trujillo and Deal, 1977), fluorescence polarization (Reinhart and Lardy, 1980), and fluorescence correlation spectroscopy (Ranjit et al, 2014). However, only one previous study showed filamentous structures by transmission EM (TEM; Foe Despite abundant knowledge of the regulation and biochemistry of glycolytic enzymes, we have limited understanding on how they are spatially organized in the cell.…”

Crystallization and preliminary crystallographic analysis of human muscle phosphofructokinase, the main regulator of glycolysis

“…Furthermore, binding of Pfk to intracellular proteins such as calmodulin (Marinho-Carvalho et al, 2006), actin (Liou & Anderson, 1980), microtubules (Vé rtessy et al, 1997) and caveolin 1 (Vallejo & Hardin, 2005) modulates the activity, oligomeric equilibrium and localization of Pfk within the cell. Moreover, the equilibrium between monomer, dimers, tetramers and higher oligomers affects Pfk activity (Trujillo & Deal, 1977;Luther et al, 1983). Mutations in the muscle Pfk gene can lead to type VII glycogen storage disease (Tarui disease; Toscano & Musumeci, 2007).…”