Abstract

Introduction

Materials & Methods

Results

Discussion & Conclusion

References

Discussion
Board

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 22°C. 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 15°C. 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 -70°C.

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 4°C, 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 25°C 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 25°C. 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. 2.7.1.1), 6-phosphofructo-1-kinase (PFK; E.C. 2.7.1.11), aldolase (ALD; E.C. 4.1.2.13), pyruvate kinase (PK; E.C. 2.7.1.40), lactate dehydrogenase (LDH; E.C. 1.1.1.27), carnitine octanoyltransferase (COT; E.C. 2.3.1.21), carnitine palmitoyltransferase (CPT; E.C. 2.3.1.21), 3-hydroxyacyl-CoA dehydrogenase (HOAD; E.C. 1.1.1.35), beta-hydroxybutyrate dehydrogenase (BDH; E.C. 1.1.1.30), ATP-citrate lyase (CL; E.C. 4.1.3.8), fatty acid synthetase (FAS), isocitrate dehydrogenase, NADP-dependent (IDH; E.C. 1.1.1.42), glucose-6-phosphate dehydrogenase (G6PDH; E.C. 1.1.1.49), 6-phosphogluconate dehydrogenase (6PGDH; E.C. 1.1.1.43), malic enzyme (ME; E.C. 1.1.1.40), fructose-1,6-bisphosphatase (FBPase; E.C. 3.1.3.11), phosphoenolpyruvate carboxykinase (PEPCK; E.C. 4.1.1.32), glutamate dehydrogenase (GDH; E.C. 1.4.1.2), serine dehydratase (SDH; E.C. 4.2.1.13), glutamate-oxaloacetate transaminase (GOT; E.C. 2.6.1.1), glutamate-pyruvate transaminase (GPT; E.C. 2.6.1.2), 5'-nucleotidase (5NT; E.C. 3.1.3.5), adenylate kinase (AK; E.C. 2.7.4.3), creatine kinase (CK; E.C. 2.7.3.2), malate dehydrogenase, NAD-dependent (MDH-1; E.C. 1.1.1.38) or NADP-dependent (MDH-2; E.C. 1.1.1.40), citrate synthase (CS; E.C. 4.1.3.7).

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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