Regulation of phosphofructokinase and the control of cryoprotectant synthesis in a freeze-avoiding insect CLARKP. HOLDENAND KENNETHB. STOREY Departments of Biology and Chemistry, Carleton Universiry, Ottawa, ON KIS 5B6, Canada Received February 23, 1993 Accepted June 23, 1993 HOLDEN,C.P., and STOREY,K.B. 1993. Regulation of phosphofructokinase and the control of cryoprotectant synthesis in a freeze-avoiding insect. Can. J. Zool. 71: 1895 - 1899. Phosphofructokinase (PFK) from larvae of the freeze-avoiding gall moth Epiblema scudderiana was purified 7 1 1-fold using ATP-agarose affinity chromatography to a final specific activity of 23 Ulmg protein. The native molecular mass of the enzyme was 420 000 + 20 000 Da. The enzyme showed an optimum pH of 8.13 + 0.2 1 at 22°C and 8.19 + 0.1 1 at 5°C. Arrhenius plots of PFK activity showed a sharp break at 9°C. So,, values for fructose 6-phosphate showed positive thermal modifica- tion, decreasing with decreasing assay temperature; the opposite was true for ATP-Mg2+. PFK was activated by fructose 2,6-bisphosphate, AMP, and inorganic phosphate; activator effects were temperature-dependent. The enzyme was inhibited by ATP-M~~+,citrate-Mg2+, and phosphoenolpyruvate. The positive effects of low temperature on enzyme kinetic proper- ties would promote PFK activity to channel glycolytic carbon flow into the production of glycerol during cold-stimulated cryoprotectant synthesis. HOLDEN,C.P., et STOREY,K.B. 1993. Regulation of phosphofructokinase and the control of cryoprotectant synthesis in a freeze-avoiding insect. Can. J. Zool. 71 : 1895 - 1899. De la phosphofructokinase (PFK) prklevke chez des larves d'Epiblema scudderiana, un papillon gallicole rkfractaire au gel, a kt6 rendue 7 1 1 fois plus concentrke par chromatographie d'affinitk a 1'ATP-agarose lui confkrant une activitk spkcifique finale de 23 Ulmg protkine. La masse molkculaire originale de l'enzyme ktait de 420 000 + 20 000 Da. L'enzyme avait un pH optimal de 8,13 + 0,21 a 22°C et de 8,19 f O,11 a 5°C. La courbe d'Arrhenius de l'activitk de la phosphofructokinase a dkmontrC qu'il y avait une coupure brusque a 9°C. La valeur So,, pour le fructose-6 phosphate a mis en lumikre une modification thermique positive, dkcroissant parallklement au dkcroissement de la tempkrature de I'expkrience; l'inverse se produisait dans le cas de 1'ATP-Mg2+. La PFK est activke par le fructose 2,6-biphosphate, 1'AMP et le phosphate inorga- nique; l'effet des activateurs depend de la tempkrature. L'enzyme est inhibke par 1'ATP-Mg2+, le citrate-Mg2+ et le phos- phoknolpyruvate. Les effets positifs d'une tempkrature faible sur les propriktks cinktiques de l'enzyme peuvent favoriser l'activitk de la PFK de fa~ona orienter l'utilisation du carbone glycolytique vers la production de glyckrol au cours de la synthkse de la substance cryoprotectrice stimulke par le froid. [Traduit par la rkdaction] For personal use only. Introduction regulatory controls applied at key loci of glycolysis carbon is Many insects endure winter exposure to deep subzero tem- channeled into the accumulation of glycerol (in most species) peratures without freezing. To do so they use a variety of bio- or other polyhydric alcohols (Storey and Storey 1991). chemical adaptations including the addition of antifreeze proteins 6-Phosphofructo- 1-kinase (PFK) is an important regulatory, to body fluids and a buildup of extremely high concentrations and often rate-limiting, enzyme of the glycolytic pathway and of low molecular weight carbohydrates (sugars or polyols) that gates the commitment of sugar phosphates (derived from glyco- act as cryoprotectants (Storey and Storey 1992). Larvae of gen or glucose) into the triose phosphate portion of the path- the gall moth Epiblema scudderiana (Clemens) (Lepidoptera, way. The enzyme is subject to a wide variety of regulatory Olethreutidae) accumulate glycerol as their cryoprotectant in mechanisms including allosteric controls by powerful activa- concentrations of 2 M or more, representing about 19% of the tors and inhibitors, post-translational modification by protein total body mass of the insect (Rickards et al. 1987). As a phosphorylation and dephosphorylation, enzyme aggregation, result, the supercooling point of the larvae is lowered to and enzyme binding subcellular macromolecules (e.g., F-actin) - 38°C in midwinter compared with - 12°C in the absence of (Hue and Rider 1987; Pilkis et al. 1987; Storey 1988). Insects Can. J. Zool. Downloaded from www.nrcresearchpress.com by CARLETON UNIV on 03/22/16 cryoprotectant, allowing the species to overwinter without that produce glycerol as their cryoprotectant require a cold- freezing in exposed galls on the woody stems of goldenrod active PFK that facilitates triose phosphate formation. The plants (Rickards et al. 1987). importance of control at the PFK locus has been illustrated by The synthesis of glycerol as a cryoprotectant by this and the much greater activities of the enzyme in insects that pro- other insect species is fueled from massive stores of glycogen duce glycerol than in trehalose-producing species (Hayakawa that are accumulated by the fat body during summer feeding and Chino 1982) and by a sharp increase in the product of PFK, (Miller 1976; Storey and Storey 1991). During the autumn the fructose 1,6-bisphosphate, upon cold exposure, indicating an activities and ratios of glycogenolytic and hexose monophos- activation of flux through the enzyme (Churchill and Storey phate cycle enzymes are optimized for the efficient and quan- 1989). The importance of PFK control is further illustrated in titative conversion of stored glycogen into glycerol as the dual-polyol-producing species by the strong temperature- weather cools. Most cold-hardy insects reshnd to a tempera- dependent controls on PFK that facilitate glycerol synthesis at ture trigger in the range 0 -5 "C, with maximal rates of glyco- moderate temperatures (1 0 - 15"C) but block enzyme activity gen to cryoprotectant conversion occurring between 0 and at lower temperatures to divert carbon flow into the synthesis -5°C (Storey and Storey 1991, 1992). Cold activation of of sorbitol instead (Storey 1982; Storey and Storey 1991). glycogen phosphorylase initiates glycogen breakdown, and via The present study examines the kinetic and regulatory prop- Printed In Canada / ImprimC au Canada CAN. J. ZOOL. VOL. 71, 1993 TABLE1. Purification of PFK from Epiblema scudderiana Total Total Specific protein activity Fold activity 6%) (u) % yield purification (U/mg) Crude homogenate 192 6.62 - - 0.033 10% PEG 75 .O 3.85 58.0 1.60 0.05 1 ATP-agarose 0.044 1.01 15.0 71 1 23.2 erties of partially purified PFK from E. scudderiana and ana- (1 x 2 cm) equilibrated with HB. The column was washed in lyzes the interactions of enzyme properties with temperature sequence with 1 mL of HB, 1 mL of 1 M KC1 in HB, and 5 mL of and high glycerol concentrations to determine the factors that HB. PFK was then eluted in a single peak with a linear gradient of are important in enzyme control during cryoprotectant syn- 0-5 mM each of F6P + ADP in HB containing 500 mM KCl. One- millilitre fractions were collected and assayed for PFK activity. Peak thesis. tubes were pooled, stored in 40% glycerol, and used as the source of PFK for all kinetic studies. Materials and methods Molecular mass determination Animals and chemicals The native molecular mass of PFK was determined by Sephacryl Galls containing E. scudderiana caterpillars were collected in the S-300 gel filtration chromatography. The column buffer consisted of autumn of 1989. In the laboratory the galls were placed at either 15 50 mM KH2P0,, 15 mM 2-mercaptoethanol, 0.1 % w/v NaN, and or -4°C; these acclimation temperatures were chosen because at 10% V/Vglycerol, pH 7.2. A 100-pL aliquot of crude supernatant was 15°C the insects do not synthesize glycerol, whereas at -4°C loaded onto the column and fractions of 0.5 mL were collected. Stan- glycerol production occurs at a high rate (Rickards et al. 1987). After dards were run in the same manner and detected by activity assays acclimation for 3 weeks, galls were opened and the larvae were at 340 nm for rabbit muscle PFK (360000), rabbit muscle aldolase removed, frozen in liquid nitrogen, and then transferred to -80°C (160000), and rabbit liver fructose 1,6-bisphosphatase (140000) or until use. Biochemicals were purchased from Boehringer-Mannheim by absorbance at 280 nm for bovine blood hemoglobin (64 500) and Corp., Montreal, Quebec, or Sigma Chemical Co., St. Louis, bovine heart cytochrome c (13 370). The molecular weight of PFK Missouri. Sephacryl S-300 was obtained from Pharmacia Fine was determined from a plot of Ka versus log molecular mass for the Chemicals, Uppsala, Sweden. Protein was determined by the method protein standards. of Bradford (1976) using the Bio-Rad Laboratories prepared reagent and a standard of bovine gamma globulin. Isoelectrofocusing Column isoelectrofocusing was performed by the method of Enzyme assay and kinetic studies Vesterberg (1971), using an LKB Products 8101 (1 10 mL) column PFK activity was monitored by following NADH oxidation at and pH 3.5 to 10 LKB ampholines in a sucrose density gradient with 340 nm using a Gilford 240 recording spectrophotometer with a column development at 500 V for 14 h. Samples of crude supernatant LaudaFor personal use only. K-2/R water bath attached for temperature control of the of larvae acclimated to both -4 and 15°C were analyzed. One- cuvettes; kinetic properties were assessed at both 22 and 5°C. millilitre fractions were collected and assayed for PFK activity as Optimal assay conditions were 50 mM imidazole-HC1 buffer (pH 7.2 described above.