Cell Biology Poster Session



Materials & Methods


Discussion & Conclusion



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Reorganization of organ metabolic potential and signal transduction capacity during estivation in spadefoot toads, Scaphiopus couchii.

Contact Person: Kenneth B. Storey (kenneth_storey@carleton.ca)

Materials and Methods

Spadefoot toads were obtained from the Tucson, Arizona area in July and air freighted to the Carleton lab where they were held for 2 days at 22C. Control animals were sampled and remaining toads were induced to estivate by placing them in plastic tubs containing 6-8 inches of damp soil at 15C. Animals burrowed into the soil within 24 h and did not emerge again until gently dug out two months later. Mean body masses were 27.42 0.72 g (n=40) for control and 19.37 0.97 g (n=35) for estivated toads. Toads were killed by pithing and tissue samples were excised, frozen in liquid nitrogen and stored at -70C.

Samples of frozen tissues were homogenized 1:5 w/v in 20 mM imidazole, pH 7.0, 15 mM 2-mercaptoethanol, 50 mM NaF, 2 mM EDTA, 2 mM EGTA and 20% v/v glycerol with 0.1 mM phenylmethylsulfonyl fluoride added immediately prior to homogenizing. After centrifugation at 14,000 x g for 25 minutes at 4C, the supernatant was removed and stored on ice.

Glycogen phosphorylase was measured as in (5). Maximal activities of all other metabolic enzymes were assayed spectrophotometrically at 25C in a volume of 250 ul using a Dynatech MR5000 microplate reader. One unit is defined as the amount of enzyme that uses 1 umol of substrate per minute at 25C. Assays were optimized using extracts of toad liver and optimal assay conditions were as in Cowan (6) (available from K.B. Storey on request). Enzymes assayed were: hexokinase (HK; E.C., 6-phosphofructo-1-kinase (PFK; E.C., aldolase (ALD; E.C., pyruvate kinase (PK; E.C., lactate dehydrogenase (LDH; E.C., carnitine octanoyltransferase (COT; E.C., carnitine palmitoyltransferase (CPT; E.C., 3-hydroxyacyl-CoA dehydrogenase (HOAD; E.C., beta-hydroxybutyrate dehydrogenase (BDH; E.C., ATP-citrate lyase (CL; E.C., fatty acid synthetase (FAS), isocitrate dehydrogenase, NADP-dependent (IDH; E.C., glucose-6-phosphate dehydrogenase (G6PDH; E.C., 6-phosphogluconate dehydrogenase (6PGDH; E.C., malic enzyme (ME; E.C., fructose-1,6-bisphosphatase (FBPase; E.C., phosphoenolpyruvate carboxykinase (PEPCK; E.C., glutamate dehydrogenase (GDH; E.C., serine dehydratase (SDH; E.C., glutamate-oxaloacetate transaminase (GOT; E.C., glutamate-pyruvate transaminase (GPT; E.C., 5'-nucleotidase (5NT; E.C., adenylate kinase (AK; E.C., creatine kinase (CK; E.C., malate dehydrogenase, NAD-dependent (MDH-1; E.C. or NADP-dependent (MDH-2; E.C., citrate synthase (CS; E.C.

cAMP-Dependent protein kinase (PKA) and calcium/phospholipid-dependent protein kinase C (PKC) were determined by radioactive assay as in (7,8). The % PKA present as the active catalytic subunit was determined from assays in the absence versus presence of 1 micromolar cAMP. Protein phosphatase type-1 (PP-1) activity was measured as the ability to dephosphorylate 32P-labeled glycogen phosphorylase a (9) comparing untreated (active) versus trypsinated (total) extracts. Protein phosphatase types-2A, -2B, and -2C activities were determined as in (10) using a molybdate / malachite green dye solution to measure phosphate release from specific phosphopeptide substrates: for PP-2A, RRApTVA in the absence versus presence of 2.5 nM okadaic acid; for PP-2B, DLDVPIPGRFDRRVpSVAAE with 1 nM okadaic acid; for PP-2C, RRApTVA with 1 micromolar okadaic acid in the presence/absence of 10 mM Mg2+. One unit of phosphatase activity is defined as the amount of enzyme that catalyzes the release of 1umol of phosphate per minute.

Protein was determined by the Coomassie blue G-250 binding method. Statistical analysis used the Student's t-test.

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Storey, KB; Cowan, KJ; MacDonald, JA; Storey, JM; (1998). Reorganization of organ metabolic potential and signal transduction capacity during estivation in spadefoot toads, Scaphiopus couchii.. 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/storey0151/index.html
© 1998 Author(s) Hold Copyright