Abstract

Introduction

Materials & Methods

Results

Discussion & Conclusion

References

Discussion
Board

Chemicals and Animals

KT-5823 and H89 were obtained from Biomol (Plymouth Meeting, PA), PKA inhibitor peptide (5-24; PKA-I) was obtained from Sigma Chemical Co. (St. Louis, MO), [gamma³²P] ATP (3000 Cimmol^-1) was obtained from Mandel Scientific (Montreal, PQ), and phosphocellulose paper (P81) was obtained from Whatman (Canlab Corp., Mississauga, Ont.). All other chemicals and coupling enzymes were obtained from Sigma Chemical Co. (St. Louis, MO) or Boehringer Mannheim (Montreal, PQ). All buffers were adjusted to pH 6.8 at 21°C. Freshwater crayfish, Orconectes virilis, were purchased from Britannia Bait and Food Mart (Ottawa, Ont.) during the month of July, 1995. Animals were kept in an incubator at 15°C in plastic tubs containing their original water and bubbled with air. Control animals were sampled after 5-7 days under these conditions. Animals were killed by decapitation, and tissues (tail muscle, hepatopancreas) were immediately excised, frozen in liquid nitrogen, and then stored at –70°C. For subcellular fractionation studies, tissues were not frozen but were briefly held on ice in plastic tubes before homogenization. For anoxia exposure, water in a sealed container (except for a small hole to vent gas) was bubbled with nitrogen gas for 45 min (or until a lit match was extinguished immediately when held under the container lid). Crayfish were then quickly placed in the container. Bubbling with nitrogen gas was continued and animals were sampled at specified times, killed and dissected as above.

Protein Kinase A Assay

For in vivo studies, the activity of PKA was determined by a radioactive assay that measures the incorporation of radiolabeled ³²P-ATP onto the substrate kemptide (Jiang and Corbin 1991). Frozen samples of tail muscle or hepatopancreas were quickly weighed and then homogenized 1:10 w:v in PEM buffer (10 mM potassium phosphate, pH 6.8, 15 mM 2-mercaptoethanol, 2 mM EDTA, 20% v/v glycerol) with a few crystals of phenylmethylsulfonyl fluoride added immediately prior to homogenizing with a Polytron PT10 homogenizer. Homogenates were centrifuged for 5 min at 13,000 g in a Biofuge 15 microcentrifuge and then the supernatants were removed and used immediately for PKA assay. The degree of dilution used in this protocol ensured that endogenous cAMP in the tissues would not change the percentage of PKAc in vitro. Reactions were carried out in disposable glass tubes at 22°C and contained in a final 60 µl volume: 20 mM potassium phosphate (pH 6.8), 37.5 µM kemptide, 1 mM EDTA, 1 mM magnesium acetate, 0.245 mM cold ATP, 2.2 x 10⁶ dpm (1.0 µCi/assay) ³²P-ATP, and 10 µl of enzyme extract. Paired samples were run in the absence versus presence of 1 µM cAMP to assay the amount of free catalytic subunit versus total PKA, respectively; preliminary trials showed that this amount of cAMP was sufficient to fully dissociate all inactive PKA tetramers and release the catalytic subunits for assay. Other initial trials optimized time and protein amount to ensure that the standard reaction conditions used were not limited by enzyme concentration. Reactions were started with the addition of radioactive Mg-ATP and terminated after 10 min at 22°C by withdrawing a 40 µl aliquot and spotting this onto a 2 x 2 cm piece of Whatman P81 filter paper (Roskoski 1983). Papers were washed with four changes (50 ml each) of 75 mM H₃PO₄ for 5 min, then rinsed with distilled water and placed in 1.5 ml microcentrifuge tubes held in 20 ml scintillation vials. Radioactivity on papers was counted using Cerenkov methodology on a Packard Model 1900CA scintillation counter. One unit is defined as the amount of enzyme that transfers one nmol of phosphate to kemptide per minute at 22°C. The percentage of PKA that was present as the free catalytic subunit in tissues was calculated from the activities (U/gww) measured by assays run in the absence versus presence of cAMP.

Subcellular fractionation

Fresh tissue samples (tail muscle and hepatopancreas) were removed from control and 20 h anoxic crayfish and immediately homogenized 1:4 w/v in TE buffer (20 mM Tris-HCl, pH 7.5, 5 mM MgCl₂, 10 mM EDTA) containing 0.25 M sucrose and kept on ice. The crude fraction was centrifuged at 200 g for 10 min to pellet cell debris. The resulting supernatant was layered over an equal volume of TE buffer containing 0.34 M sucrose and centrifuged at 1000 g for 10 min. The supernatant was removed and held on ice while the pellet was resuspended in TE/0.25 M sucrose buffer containing 1 % v/v Triton X-100 and recentrifuged at 1000 g. The pellet from this centrifugation was the plasma membrane fraction (PM) and was resuspended in a minimum amount of TE/0.25 M sucrose buffer. The supernatant from the first 1000 g spin was recentrifuged, this time at 17,000 g for 10 min, and the pellet was resuspended in a minimum amount of TE/0.25 M sucrose; this represents the mitochondrial fraction (MT). The supernatant from t his last centrifugation contained the microsomal and cytosolic fractions (MC/CY). All fractions were stored on ice until assayed for the distribution of enzymes.

Assay of marker enzymes and protein

To determine the purity of subcellular fractions, the activities of selected marker enzymes were assayed using optimal assay conditions as follows:

5'-Nucleotidase (5NT; E.C. 3.1.3.5): 20 mM imidazole buffer (pH 7.0), 500 mM MgSO₄, 12 mM AMP, 0.15 mM NADH, 2.0 mM 2-oxoglutarate and 1 U each of glutamate dehydrogenase and adenosine deaminase.

Citrate synthase (CS; E.C. 4.1.3.7): 50mM imidazole buffer (pH 8.1), 0.2% (v/v) Triton X-100, 2 mM oxaloacetate, 0.1mM acetyl-CoA and 0.1 mM 5,5’-dithiobis-(2-nitrobenzoic acid) (DTNB). Enzyme activity was monitored at 410 nm.

Lactate dehydrogenase (LDH; E.C. 1.1.1.27): 50 mM imidazole buffer (pH 7.0), 2 mM pyruvate, 0.15 mM NADH.

Soluble Protein

Protein concentration in enzyme extracts was determined by the Coomassie blue G-250 binding method (Bradford, 1976) using the Bio-Rad commercial kit and bovine serum albumin as the standard. Assays were conducted using the Dynatech MR-5000 microplate reader at 595 nm, with a total well volume of 310 µl.

Purification of tail muscle PKAc

Tissue homogenization (at a 1:5 ratio w:v and in the presence of 1 µM cAMP) and centrifugation was as described above for the PKA assay. Samples of supernatant were loaded onto a Matrix Red column (2.5 x 5 cm) equilibrated in PEM buffer. The column was washed with 10 ml of PEM buffer to flush out nonspecific protein, then eluted with 5 ml of the same buffer to remove the catalytic subunit of PKA, which has partial specificity for this column. The eluted 1 ml fractions were assayed for PKA activity. Peak fractions were pooled, loaded onto a hydroxylapatite column (2.5 x 3 cm) equilibrated in PEM buffer, flushed with 5 ml of PEM buffer and then eluted with 10 ml of the buffer. Fractions of 1 ml were again collected and assayed for PKA activity. Pooled peak fractions were then loaded onto a protamine agarose column (1 x 3 cm) equilibrated in PEM buffer which was then washed with 10 ml PEM buffer. The column was then eluted with 10 ml 250 mM KCl in PEM buffer, including 20 % glycerol. Fractions of 1 ml were c ollected and assayed for PKA activity.

Gel filtration and SDS-PAGE

The molecular weight of the catalytic subunit was determined by a Sephacryl S-300 gel filtration column (2.5 x 75 cm) equilibrated in PEM buffer, including 20 % glycerol. The flow rate was approximately 5-6 ml/h and 1 ml fractions were collected as assayed for PKA activity in the absence of added cAMP. Calibration standards were cytochrome c (14.5 kDa), triosephosphate isomerase (43 kDa), hexokinase (100 kDa), lactate dehydrogenase (140 kDa), and pyruvate kinase (280 kDa). SDS-polyacrylamide gel electrophoresis was performed according to Laemmli (1970) using a 10 % acrylamide separating gel using Coomassie Brilliant Blue as a stain. Protein standards were: A) rabbit muscle myosin (205 kDa), B) E. coli ß-galactosidase (116 kDa), C) rabbit muscle phosphorylase b (97 kDa), D) bovine albumin (66 kDa), E) egg albumin (45 kDa), and F) carbonic anhydrase (29 kDa).

Data analysis

Statistical analysis was performed by using the two-tailed Student-Newman-Keuls test, enzyme kinetics software written by Brooks (1992).

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