Cell Biology Poster Session
Materials and Methods
Adult golden mantled ground squirrels, Spermophilus lateralis, were obtained in the Crooked Creek area of the White Mountains of California. All animals were individually housed in rat cages at the animal care facility of the University of California at Irvine, and maintained at 22°C on a fall (10L:14D) photoperiod. The squirrels were maintained on a diet high in polyunsaturated fats (15% sunflower oil providing linoleic acid, 18:2) which results in a depot fat composition very close to that seen in the wild and leads to a normal pattern of hibernation by the animals (5). The animals were allowed to feed and fatten for the next 7 weeks. At the end of this time (September), several squirrels were sacrificed by decapitation while euthermic. Other squirrels were induced to hibernate at an ambient temp of 3.5°C. After 30 days (October) of hibernation, these were sacrificed while torpid. Samples of hind leg skeletal muscle were quickly excised and frozen in liquid nitrogen. Tissues were air-freighted on dry ice to Carleton University, and were then stored at -80°C until use.
Muscle Adenylate Levels
Adenylates and IMP were determined in protein free perchloric acid extracts using an ion-exchange HPLC method (6). The system consisted of a Beckman 110B solvent delivery system with a Bio-Rad C18 HL 90-5S column (250 x 4.6 mm) and a photodiode array detector set at 254 nm. Elution was by binary gradient using mobile phases of 30 mM KH2PO4 (pH 5.45), 7.5 mM tetrabutylammonium phosphate (TBAP) and 30 mM KH2PO4 (pH 7.0), 7.5 mM TBAP and 50% v/v acetonitrile.
Measurement of Na+K+-ATPase Activity
Frozen tissue samples (0.2 g) from euthermic or hibernating S. lateralis were homogenized 1:5 w/v in homogenization buffer (25 mM imidazole, pH 7.4, 25 mM NaF, 2 mM EDTA, 2 mM EGTA, 250 mM sucrose, 0.2% w/v deoxycholate, 1 mM dithiothreitol) with 1 mM phenylmethylsulphonyl fluoride (PMSF) added at the time of homogenization. The homogenate was allowed to sit on ice for 30 min prior to centrifugation at 10,000g for 10 min at 5°C. Endogenous phosphate was removed from a 0.5 mL aliquot of supernatant by centrifugation through a 10 ml column of Sephadex G-25 equilibrated in homogenization buffer(7).
The assay method was based on that described by Crombie et al. (8). Each extract was assayed in triplicate in a control buffer lacking K+ (120 mM NaCl; 5 mM MgCl2, 25 mM imidazole-HCl; 1 mM ouabain, pH 7.4) to measure non-specific ATPase activity and in an experimental buffer containing K+ (100 mM NaCl; 20 mM KCl; 5 mM MgCl2, 25 mM imidazole-HCl; pH 7.4) to measure total ATPase activity; the difference in reaction rates between the two gave the activity due to Na+K+-ATPase. An aliquot of enzyme extract was assayed at 25°C in a final volume of 0.25 mL, and the assay started by the addition of ATP to give a final concentration of 3 mM. Initial experiments determined that at 25°C production of phosphate was linear with respect to time and the volume of homogenate assayed. Aliquots of the assay mixture were taken for inorganic phosphate analysis by the malachite green-molybdate dye binding method (9). One unit is defined as the amount of enzyme that releases 1 µmol of inorganic phosphate per hour.
Phosphorylation and dephosphorylation studies on Na+K+-ATPase
Tissue samples from euthermic or hibernating animals were homogenized and desalted by centrifugation through a Sephadex G-25 column pre-equilibrated in 50 mM imidazole, pH 7.0, 25% (v/v) glycerol, 0.2% (w/v) deoxycholate, 10 mM 2-mercaptoethanol and 0.1 mM EDTA (Incubation Buffer). The stimulation of endogenous protein kinases and phosphatases to alter the Na+K+-ATPase activity was through the addition of appropriate effectors to give a final total volume of 0.15 mL. All incubations for kinase stimulation contained 30 mM NaF to inhibit protein phosphatases, 10 mM Mg.ATP, and other effectors as noted in the figure legends. Endogenous phosphatases were stimulated with 15 mM MgCl2 and 1.3 mM CaCl2 in the absence of ATP and NaF (+Mg2+/+Ca2+/-NaF). Reactions were incubated for 5 h at 30°C and then Na+K+-ATPase activity was measured.
In order to further assess dephosphorylation, enzyme in muscle extracts from euthermic and hibernating animals was first experimentally phosphorylated by incubating the desalted supernatant with 30 mM NaF, 15 mM MgCl2, 10 mM ATP, and 1 µM cAMP for 2 h at 25°C. Samples were desalted by passage through Sephadex G-25 columns equilibrated in Incubation buffer and then dephosphorylation was initiated by adding 10 U of alkaline phosphatase. After 2 h incubation at 25°C, samples were assayed for Na+K+-ATPase activity. By decreasing the incubation time spent at higher temperature, these conditions attempted to maximize the recovery of enzyme activity by dephosphorylation.
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|MacDonald, J.A.; Storey, K.B.; (1998). Regulation of ground squirrel Na+K+-ATPase activity by reversible phosphorylation during hibernation.. Presented at INABIS '98 - 5th Internet World Congress on Biomedical Sciences at McMaster University, Canada, Dec 7-16th. Available at URL http://www.mcmaster.ca/inabis98/cellbio/macdonald0174/index.html|
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