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Regulation of Canine Skeletal Muscle Phosphofructokinase-1 by Adenine Nucleotides Shuichiro Kanai Takuro Shimada Takanori Narita Ken Okabayashi*

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Regulation of Canine Skeletal Muscle Phosphofructokinase-1 by Adenine Nucleotides Shuichiro Kanai Takuro Shimada Takanori Narita Ken Okabayashi* Regulation of Canine Skeletal Muscle Phosphofructokinase-1 by Adenine Nucleotides Shuichiro Kanai Takuro Shimada Takanori Narita Ken Okabayashi* Department of Veterinary Medicine, College of Bioresource Sciences, Nihon University, 1866 Kameino, Fujisawa, Kanagawa, 252-0880, Japan *Corresponding author. E-mail: [email protected] KEY WORDS: Canine skeletal muscle, by ATP, while UTP and cAMP regulated phosphofructokinase-1, adenine nucleotides PFK-1 activity. The results also suggested that ADP binds to the allosteric sites (for ABSTRACT ATP and AMP) of PFK-1. Each adenine Phosphofructokinase-1 (PFK-1) is the most nucleotide functions as either an activating important rate-controlling enzyme for gly- or inhibitory effector of PFK-1 reaction in colysis in both prokaryotes and eukaryotes. canine skeletal muscle. Therefore, intracel- PFK-1 activity is regulated by multiple cel- lular nucleotides may play an important role lular metabolites, including nucleotides. In- in regulating glucose metabolism by binding tracellular nucleotides are produced through to the allosteric site of the enzyme. various metabolic processes, including en- ergy metabolism and intracellular signaling INTRODUCTION pathways. The activity of PFK-1, purified Glycolysis plays a central role in eukaryotic from canine skeletal muscle, was evaluated energy metabolism by producing NADH in the presence of various concentrations of and ATP, along with pyruvate, which is adenine nucleotides to examine the regula- converted to acetyl-coenzyme A for subse- tion of glucose catabolism in canine skeletal quent utilization in the tricarboxylic acid muscle. Although UTP did not inhibit PFK-1 cycle. Phosphofructokinase-1 (EC: 2.7.1.11, activity to the same extent as ATP, it substi- PFK-1), the most important rate-controlling tuted for ATP as a phosphate donor. cAMP enzyme in glycolysis, catalyzes the phos- functioned in a similar manner as AMP, as phorylation of fructose 6-phosphate (F-6-P) an activating effector of the PFK-1 reaction. by ATP to form fructose 1,6-bisphosphate. ADP activated PFK-1 at low concentra- Three subunits of mammalian PFK-1 (M, L, tions, but slightly inhibited PFK-1 at higher and P) are present in various proportions in concentrations. The results suggested that different tissues.1 However, skeletal muscle canine PFK-1 had three different bind- is an exception, as it expresses only the M- ing sites for adenine nucleotides acting as type, specifically a homotetramer of M-type, phosphate donors, activating effectors, and while all other tissues express distinct levels inhibitory effectors. Moreover, PFK-1 was of all three subunits.2 shown to be activated by AMP and inhibited Recent studies reported the crystal struc- 10 Vol. 17, No.1, 2019 • Intern J Appl Res Vet Med. tures of eukaryotic PFK-1 from yeast,3, 4 rab- was purchased from GE Healthcare (Little bit muscle,4 and human muscle,5 which were Chalfont, UK). F-2,6-P2 was synthesized generally based on crystallographic studies from D-fructose-1,2-cyclic-6-diphosphate as of PFK-1 purified from various eukaryotes. described previously.20 The molar concen- PFK-1 is regulated by adenine nucleo- tration of F-2,6-P2 was measured based on tides; the enzyme is inhibited by ATP and the correlated measurement of phosphorus activated by AMP.2, 6–11 Mammalian PFK-1 concentration.21 contains catalytic sites for binding to ATP Animals and F-6-P, inhibitory sites for ATP and The beagles were housed in appropriate citrate, and activator sites for AMP/ADP and facilities to ensure animal welfare. They 12 fructose-2,6-bisphosphate (F-2,6-P2). were fed commercial dry food and had free In humans, muscle PFK deficiency, access to water. The beagles were housed known as Tarui’s disease, is an autosomal in adequately sized cages under a 12-h recessive disorder characterized by exercise- dark–light cycle (light from 06:00 to 18:00) induced muscle weakness, pain, cramping, in an air-conditioned environment. This myoglobinuria, and hemolysis.13–15 In ani- study protocol was approved by the Nihon mals, hereditary muscle PFK deficiency with University Animal Care and Use Committee chronic hemolysis and exertional myopathy, (permission number: AP13B074-1). Sodium due to a single nonsense mutation in PFK, pentobarbital (150 mg/kg body weight) was has been reported in English springer span- administered intravenously for sacrifice. iels, whippets, and wachtelhunds.16–19 Purification of PFK-1 Very few physiological studies have After sacrifice, canine skeletal muscle PFK- been conducted on canine PFK-1, and thus 1 was purified as described previously with the characteristics of canine PFK-1 remain some modifications.7 Briefly, fresh skeletal unclear. Understanding the physiological muscle was resected from sacrificed beagles regulation of canine skeletal muscle PFK- and stored at -80°C until PFK-1 purifica- 1 by adenine nucleotides is important for tion. All purification steps were carried clarifying energy metabolism in canine out below 4°C, except for heat treatment. skeletal muscle and the effects of abundant Fifteen grams of skeletal muscles, minced intracellular adenine nucleotides on glucose with scissors, were dissolved in homogeni- metabolism. In this study, regulation of zation buffer (50 mM Tris-phosphate buffer canine muscle PFK-1 activity by adenine (pH 8), 1 mM PMSF, and 10 mM DTT), and nucleotides was examined using partially homogenized 5 times for 1 min each with a purified PFK-1 from canine skeletal muscle. Teflon homogenizer at 500 rpm. The homog- MATERIALS AND METHODS enate was ultracentrifuged at 100,000 ×g for Materials 30 min. The supernatant was heat-treated at 52°C for 3 min, and then centrifuged The chemicals and reagents used in this at 10,000 ×g for 10 min to separate the dena- study were obtained from Wako Pure Chem- tured protein. The heat-treated supernatant icals (Osaka, Japan) and Sigma-Aldrich (St. was loaded onto a Blue Sepharose 6 Fast Louis, MO, USA). ATP, ADP, AMP, UTP, Flow (Cibacron Blue) column (1 × 5 cm) cAMP, citrate, aldolase (EC: 4.1.2.13), and equilibrated with buffer A (50 mM Tris- glycerol 3-phosphate dehydrogenase (EC: phosphate buffer (pH 8), 0.1 mM EDTA (pH 1.1.1.8) were purchased from Wako Pure 8), 0.05 mM fructose 1,6-bisphosphate, 10 Chemicals. F-6-P, fructose 1,6-bisphosphate, mM DTT). After washing the column with D-fructose-1,2-cyclic-6-diphosphate, NAD, 50 ml buffer A containing 0.15 mM ADP, and triosephosphate isomerase (EC: 5.3.1.1) PFK-1 was eluted with buffer B (50 mM were purchased from Sigma-Aldrich. Blue Tris-phosphate buffer (pH 8), 0.1 mM EDTA Sepharose 6 Fast Flow as Cibacron Blue (pH 8), 0.05 mM fructose 1,6-bisphosphate, Intern J Appl Res Vet Med • Vol. 17, No. 1, 2019. 11 Table 1. Purification of PFK-1 from canine skeletal muscle Step Total activity Total protein Specific activity Yield Purification (units) (mg) (U/mg protein) (%) (fold) Ultracentrifugation 38.03 1313.4 0.029 100.0 1.00 Heat treatment 28.80 421.47 0.068 75.7 2.36 Cibacron Blue 20.74 15.03 1.380 54.5 47.66 affinity column Dialysis 12.03 2.61 4.609 31.6 159.18 5 mM ATP, 2 mM F-6-P, 10 mM DTT) at a µmol of F-6-P per min at 25°C. flow rate of 1 ml/min. Fractions with PFK-1 The allosteric-kinetic properties of PFK- activity were collected, precipitated with 1 were determined in a reaction mixture con- 50% ammonium sulfate, and centrifuged at taining the following components in a final 10,000 ×g for 30 min. The precipitate was volume of 1 ml: 50 mM HEPES buffer (pH dissolved in 50 mM Tris-phosphate buffer 7.3), 100 mM KCl, 6.5 mM MgCl2, 1 mM with 10 mM DTT and dialyzed overnight to NH4Cl, 5 mM KH2PO4, 0.3 mM NADH, eliminate ammonium sulfate. The dialyzed aldolase (0.5 U), glycerol 3-phosphate solution was used as purified PFK-1. dehydrogenase (0.5 U), triosephosphate Protein Estimation isomerase (5 U), and varying concentrations Protein concentration after each purification of F-6-P, ATP, and/or AMP. step and that of purified PFK-1 were de- The PFK-1 activity of each enzyme 22 termined using the Bradford method with solution was expressed as v/Vmax for nor- bovine serum albumin as the standard. malization, where v is the activity at pH 7.3 PFK-1 Assay (intracellular physiological condition), and V is the maximum activity determined at The PFK-1 assay was conducted as reported max previously.7 The enzymatic reaction was pH 8.2 for excluding the allosteric effects of initiated by adding enzyme solution at 25°C regulatory factors. and monitoring the rate of NADH oxidation at 340 nm. Figure 1. pH-dependent activity profile of The pH-dependent activity profile was canine muscle PFK-1. PFK-1 activity was shown by the value of decreasing absor- assayed using 5 µl of purified PFK-1 solu- bance. PFK-1 activity regulation was deter- tion in a total volume of 1 ml containing 1 mined by velocity standardized by maxi- mM F-6-P, 5 mM ATP, and 0.1 mM AMP at mum velocity (v) calculated based on the various pH levels. Values show the decrease in absorbance at 340 nm in 1 min. The optimal condition (Vmax) in each experiment, in order to eliminate interindividual differ- results are shown as the mean ± SEM of 3 ences or effects of the freeze-thaw process.7 independent experiments. Optimal PFK-1 activity was determined in a reaction mixture containing the follow- ing components in a final volume of 1 ml: 50 mM HEPES buffer (pH 8.2), 100 mM KCl, 6.5 mM MgCl2, 1 mM NH4Cl, 5 mM KH2PO4, 0.3 mM NADH, aldolase (0.5 U), glycerol 3-phosphate dehydrogenase (0.5 U), triosephosphate isomerase (5 U), 1 mM F-6-P, 5 mM ATP, and 0.1 mM AMP.
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