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Oxidative Stress Poster Session






Abstract

Introduction

Materials & Methods

Results

Discussion & Conclusion

References




Discussion
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Anoxia Effects On Enzymes Of Intermediary Metabolism In The Anoxia-Tolerant Freshwater Crayfish, Orconectes Virilis.


Contact Person: Dr. Kyra J. Cowan (kcowan@corr.com)


Introduction

Several aquatic invertebrates, such as the freshwater crayfish of eastern Canada, have had to adapt to rapid and dramatic fluctuations in PO2. Adaptation to anoxia is a key factor for the survival of Orconectes virilis, a small freshwater crayfish of eastern Canada, and they do so through several biochemical and physiological responses. A rapid increase in respiratory water flow, a reduction in ventilation volume, and the scope of energy produced during anaerobic metabolism could all contribute to survival under anoxic conditions. Adaptation to severe environmental conditions is often associated with metabolic rate depression (Storey and Storey 1990). The metabolic pathways of O. virilis are dramatically influenced by changes in available levels of oxygen. Two key metabolic enzymes involved in glycolysis, pyruvate kinase (PK) and phosphofructokinase (PFK), have been shown to be regulated by phosphorylation in several species (Storey 1984, Michaelidis and Storey 1990, Whitwam and Storey 1990, Fernandez et al. 1994, Benoit et al. 1994, Holwerda et al. 1983, Brooks and Storey 1990, Walsh et al. 1991). This is the case for this species, the regulation by reversible phosphorylation of which was found to be conducted by a cAMP-dependent protein kinase (PKA; Cowan and Storey 1999a). The purification and characterization of PKAc from O. virilis has also shown that this regulatory enzyme has very similar properties characteristic of PKAc found in both vertebrates and invertebrates (Cowan and Storey 1999b).

To further understand the metabolic responses to anoxia by a freshwater crayfish and to assess the connection between tolerance of this stress and changes to metabolic pathways, we focused on several key metabolic enzymes and their maximal activities in response to anoxic conditions. Nineteen regulatory enzymes were assayed in tail muscle and hepatopancreas of O. virilis, comparing enzyme activities under both control and anaerobic conditions.

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Materials and Methods

Chemicals and animals

Biochemicals were purchased from Sigma Chemical Co. (St. Louis, MO) or Boehringer-Mannheim Corp. (Montreal, Que.). Distilled, deionized water was used for the preparation of all solutions. Crayfish were obtained from the Ottawa region and arrived in plastic bags filled with their original freshwater. These animals averaged 7.1 +/- 0.3 cm in length and 16.3 +/- 1.6 g in weight. They were kept in an incubator at 10.5C in plastic tubs containing their original water and bubbled with oxygen. Anoxia was established by bubbling 50% original freshwater / 50% deionized distilled water with N2 (g) in a sealed container, containing enough bubbling water for the number of crayfish to be stressed. The water was gassed for 45 minutes until anoxic conditions were established. Crayfish to be stressed were sealed in the container for 20 hours, removed and immediately dissected. Tissue extraction involved decapitation and removal of the hepatopancreas and the tail muscle, which were then immediately frozen in liquid nitrogen and stored at 70C.

Enzyme analysis

Samples of frozen tissues were quickly weighed and then homogenized 1:5 w/v in 20 mM imidazole, pH 7.0, 15 mM -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. The homogenate was centrifuged at 14000 x g for 25 minutes at 4C in a Baxter Canlab Biofuge 15 and the supernatant was removed and stored on ice to be used for enzyme analysis.

Maximal activities of metabolic enzymes were assayed at 25C in a final volume of 250 l (plus extract volume) using a Dynatech MR5000 microplate reader at an absorbance of 340 nm (unless otherwise noted). Assay conditions were optimized for all enzymes using extracts of O. virilis tail muscle and hepatopancreas. Blanks (minus the most specific substrate) were subtracted. Optimal assay conditions were as follows:

Hexokinase (HK; E.C. 2.7.1.1): 100 mM Tris buffer (pH 8.0), 1 mM EDTA, 2 mM MgCl, 20 mM glucose, 0.2 mM NADP+, 1 mM ATP and 1 unit (U) glucose-6-phosphate dehydrogenase.

Glucokinase (GK; E.C. 2.7.1.2): 100 mM Tris buffer (pH 8.0), 1 mM EDTA, 2 mM MgCl2, 100 mM glucose, 0.2 mM NADP+, 1 mM ATP and 1 unit (U) glucose-6-phosphate dehydrogenase.

Phosphofructokinase (PFK; E.C. 2.7.1.11): 20 mM imidazole (pH 7.1), 10 mM MgCl2, 50 mM KCl, 2.5 mM ATP, 0.15 mM NADH, 12.5 mM fructose-6-phosphate (F6P), and 1 U each of aldolase, glycerol-3-phosphate dehydrogenase and triosephosphate isomerase.

Aldolase (ALD; E.C. 4.1.2.13): 50 mM imidazole buffer (pH 7.0), 2 mM MgCl2, 0.2 mM fructose-1,6-bisphosphate (FBP), 0.15 mM NADH, 1 U triosephosphate isomerase and 2 U glycerol-3-phosphate dehydrogenase.

Glyceraldehydephosphate dehydrogenase (GAPDH; E.C. 1.2.1.12): 50 mM imidazole (pH 7.2), 20 mM 3-phosphoglycerate, 5 mM MgSO4, 1 mM ATP, 0.15 mM NADH, and 1 unit (U) PGK.

Pyruvate kinase (PK; E.C. 2.7.1.40): 50 mM imidazole (pH 7.0), 50 mM KCl, 5 mM MgCl2, 0.4 mM phosphoenolpyruvate (PEP), 2 mM ADP, 0.15 mM NADH, 0.2% v/v rotenone saturated ethanol and 2 U lactate dehydrogenase.

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

ATP-citrate lyase (CL; E.C. 4.1.3.8): 200 mM Tris-HCl buffer (pH 8.4), 10 mM -mercaptoethanol, 0.2 mM coenzyme A (CoA), 0.15 mM NADH, 10 mM MgCl2, 20 mM potassium citrate, 10 mM ATP, 10 mM MgCl2, and 1 U malate dehydrogenase.

Fatty acid synthetase (FAS): 100 mM potassium phosphate buffer (pH 7.0), 3 mM EDTA, 5 mM dithiothreitol, 0.05 mM malonyl-CoA, 0.032 mM acetyl-CoA, 0.3 mM NADPH and 0.2% v/v rotenone saturated ethanol.

Isocitrate dehydrogenase, NADP+-dependent (IDH; E.C. 1.1.1.42): 50 mM imidazole buffer (pH 7.5), 2 mM MgCl2, 6 mM DL-isocitrate and 0.4 mM NADP+.

Glucose-6-phosphate dehydrogenase (G6PDH; E.C. 1.1.1.49): 60 mM Tris-HCl buffer (pH 7.5), 3.3 mM glucose-6-phosphate, 0.4 mM NADP+ and 6 mM 1 MgCl2.

Malic enzyme (ME; E.C. 1.1.1.40): 75 mM Triethanolamine buffer (pH 7.4), 4 mM MnCl2, 2.5 mM L-malate, 0.2 mM NADP+.

Fructose-1,6-bisphosphatase (FBPase; E.C. 3.1.3.11): 25 mM imidazole buffer (pH 8.0), 5 mM MgSO4, 0.2 mM NADP+, 6 mM FBP and 1 U each of glucose-6-phosphate dehydrogenase and phosphoglucose isomerase.

Phosphoenolpyruvate carboxykinase (PEPCK; E.C. 4.1.1.32): 100 mM imidazole buffer (pH 6.6), 6 mM -mercaptoethanol, 50 mM sodium bicarbonate, 1.2 mM PEP, 1.25 mM inosine diphosphate, 1 mM MnCl2, 0.15 mM NADH and 2.5 U malate dehydrogenase. All solutions were degassed prior to assay.

Glutamate dehydrogenase (GDH; E.C. 1.4.1.2): 100 mM Triethnolamine buffer (pH 8.0), 5 mM EDTA, 200 mM ammonium carbonate, 7.5 mM 2-oxoglutarate, 0.15 mM NADH, and 1 mM ADP.

Serine dehydratase (SDH; E.C. 4.2.1.13): 200 mM potassium phosphate buffer (pH 8.0), 2 mM EDTA, 100 mM L-serine, 0.11 mM pyridoxal phosphate, 0.15 mM NADH, 0.2% v/v rotenone saturated ethanol, and 1 U lactate dehydrogenase.

Glutamate-oxaloacetate transaminase (GOT; E.C. 2.6.1.1): 500 mM imidazole buffer (pH 7.8), 30 mM -mercaptoethanol, 15 mM 2-oxoglutarate, 250 mM L-aspartate, 0.11 mM pyridoxal phosphate, 0.15 mM NADH and 1 U malate dehydrogenase (NAD+ dependent).

Glutamate-pyruvate transaminase (GPT; E.C. 2.6.1.2): 500 mM imidazole buffer (pH 7.3), 30 mM -mercaptoethanol, 15 mM 2-oxoglutarate, 500 mM L-alanine, 0.11 mM pyridoxal phosphate, 0.15 mM NADH and 1 U lactate dehydrogenase.

Branched chain alpha-ketoacid dehydrogenase (KADH, E.C.): 30 mM potassium phosphate buffer (pH 7.5), 1 mM dithiothreitol, 2 mM MgCl2, 0.1 % Triton X-100, 10 U LADH, 0.4 mM CoA, 0.4 mM TPP, 0.2 mM NAD+, 2 mM alpha-ketoisovalerate

Data analysis

Statistical analysis used the Students t-test.

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Results

Table 1 shows the significant changes to maximal activities of metabolic enzymes assessed in two tissues (tail muscle and hepatopancreas) of control and 20 hours anoxic O. virilis. Activities are presented as milliunits per gram wet weight. Table 2 shows enzyme maximal activities in tissues from control animals or, if no effect of anoxia occurred, shows combined values for tissues from control and anoxic animals.

Table 1.

Significant changes to maximal activities of enzymes in tail muscle and hepatopancreas of O. virilis.

                                        Control                           Anoxic
                                        mU/gww                         mU/gww
Tail Muscle
ALD                            30645 +/- 3630 (6)             20400 +/- 1690 (6) *
CL                               457 +/- 42.7 (7)               186 +/- 27.3 (5) **
ME                               496 +/- 50.4 (4)               390 +/- 10.9 (6) *
GPT                             27.2 +/- 5.87 (5)              47.2 +/- 6.19 (6) * 
HK                              20.1 +/- 3.86 (4)              55.1 +/- 6.92 (4) *
GK                              31.6 +/- 6.14 (8)            0.513 +/- 0.086 (8) **
KADH                            67.5 +/- 5.55 (7)              9.03 +/- 2.36 (5) **
SDH                             7 92 +/- 47.6 (6)               327 +/- 40.4 (6) **
PFK                              9312 +/- 543 (7)               6551 +/- 180 (7) **

Hepatopancreas
ALD                              265 +/- 10.2 (7)               429 +/- 43.0 (6) **
LDH                            19146 +/- 1630 (7)              24067 +/- 805 (6) *
FBP                             45.3 +/- 6.17 (6)              65.0 +/- 3.94 (7) *
PK                               6325 +/- 518 (6)               4523 +/- 411 (6) *
G6PDH                           93.2 +/- 17.4 (5)               304 +/- 38.9 (7) **
GPT                              171 +/- 16.1 (7)               320 +/- 30.9 (8) **
GK                              32.5 +/- 4.35 (6)              75.6 +/- 8.79 (5) **
KADH                            21.5 +/- 2.87 (6)              39.9 +/- 5.14 (5) *
SDH                              624 +/- 63.0 (6)               953 +/- 38.6 (6) **
PFK                              246 +/- 29.3 (6)               116 +/- 6.23 (6) **
Data are mU per gram wet weight, means +/- SEM, n = 4-8. *Significantly different from corresponding control values, P<0.05. **Significantly different from corresponding control values, P<0.005.

Table 2.

Maximal activities of enzymes in tail muscle and hepatopancreas of O. virilis.

                                 Tail Muscle                    Hepatopancreas
                                 mU/gram wet weight             mU/gram wet weight

HK                               20.1 +/- 3.86 (4)              5.43 +/- 0.719 (8) a
GK                               31.6 +/- 6.14 (8)               32.5 +/- 4.35 (6)
PFK                               9312 +/- 543 (7)                246 +/- 29.3 (6)
ALD                             30645 +/- 3630 (6)                265 +/- 10.2 (7)
GAPDH                            7788 +/- 544 (15) a            8777 +/- 1055 (16) a
PK                             58014 +/- 6057 (10) a              6325 +/- 518 (6)
LDH                           144101 +/- 8992 (14) a            19146 +/- 1630 (7)

GDH                            21788 +/-  1498(14) a             3743 +/- 180 (15) a
GPT                              27.2 +/- 5.87 (5)               171 +/- 16.1 (7)
GOT                            63083 +/- 1475 (16) a            18031 +/- 654 (16) a
SDH                               792 +/- 47.6 (6)a              624 +/- 63.0 (6)
KADH                             67.5 +/- 5.55 (7)              21.5 +/- 2.87 (6)

G6PDH                           62.7 +/- 10.4 (13) a            93.2 +/- 17.4 (5)

FBPASE                          11.4 +/- 0.78 (13) a           45.3 +/-  6.17 (6)
PEPCK                            796 +/- 36.8 (13) a            229 +/- 25.7 (12) a
ME                                496 +/- 50.4 (4)              359 +/- 24.2 (14) a

CL                                457 +/- 42.7 (7)              126 +/- 8.92 (14) a
FAS                             83.8 +/- 5.80 (13) a            121 +/- 16.8 (13) a
IDH                            16900 +/- 1217 (14) a             929 +/- 108 (10) a

Values are mU/gram wet weight, means +/- SEM, with n in brackets and represent data for control crayfish alone or where indicated by (a) data for control and anoxic crayfish are combined. Data were combined when no significant difference was found between the values from the two states.

In general, anoxia had no effect on enzyme maximal activities of GAPDH, PEPCK, IDH, FAS, GOT, or GDH in either tissue, and out of the 19 activities detected GK, ALD, PFK, AND GPT were changed significantly between control and anoxic animals in both tissues. In liver 9 out of 19 enzymes were affected during anoxia exposure, as was the case for tail muscle. Hence, anoxic conditions seemed to cause a specific and important reorganization of enzyme activities in both hepatopancreas and tail muscle.

Tail Muscle

The effect of anoxia on the maximal activities of the 9 enzymes in crayfish tail muscle which were significantly affected by anaerobic conditions are depicted in Table 1. Activity of the rate-limiting enzyme of glycolysis, PFK, decreased by 30 % during anoxia and that of the initial step of glycolysis, GK, decreased by 98 %. The maximal activity of ALD also decreased by 33 % during anoxia. The activity of one other enzyme of glycolysis, HK, was most strongly affected, increasing during anoxia by 2.7-fold. CL activity fell, down 59 % compared to controls, as did ME, down 21 %. Tail muscle GPT activities represented the only other muscle enzyme activity that rose during anoxia, up 74 %. Both KADH and SDH activities dropped with respect to controls, down 87 and 59 %, respectively.

Hepatopancreas

Anoxia induced significant changes in the activities of ten enzymes in hepatopancreas (Table 1). Of these, only the activity of the glycolytic enzymes, PFK and PK, decreased during anoxia, dropping by 53 and 28 %, respectively. However, activity of three other glycolytic enzymes, GK, ALD, and LDH, increased by 132, 62, and 26 %. Activities of a gluconeogenic enzyme, FBP, also increased significantly during anoxia, up 43 % from control values. In addition, G6PDH activities increased 3.3-fold, KADH activities by 1.9-fold and GPT and SDH rose 87 and 53 % from their corresponding control values.

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Discussion and Conclusion

After twenty hours of anoxia, the metabolic make-up of crayfish organs had changed considerably. Organ-specific patterns were apparent in the responses of metabolic enzymes in both tissues. In tail muscle, exposure to anoxia altered the maximal activities of almost half of the metabolic enzymes assayed, as was the case in hepatopancreas, indicating that both tissues undergo a profound metabolic reorganization that impact on numerous pathways of intermediary metabolism. There could be several influences at work during anoxia exposure that could differentially affect the metabolic make-up of different organs. The first priority is to maintain glucose supply and this is done by depleting glycogen reserves, increasing gluconeogenesis from glycerol or from amino acids supplied by the breakdown of muscle protein, and switching most organs to a primary dependence on lipid oxidation. Thus, during anoxia it is expected that the crayfish tissues adapt to suppress glycolysis and fatty acid synthesis and to increase protein catabolism, gluconeogenesis, and lipid oxidation. Another influence on metabolic reorganization is metabolic arrest. Overall suppression of metabolic rate has been linked with reversible phosphorylation controls on key enzymes (Storey and Storey, 1990; Cowan and Storey 1998) but may also involve specific changes in the maximal activities of selected enzymes to suppress nonessential metabolic functions during long term dormancy. These factors can impact on the metabolic make-up of crayfish organs to optimize enzymatic pathways to meet the challenges of the dormant state.

During anoxia the maximal activities of nine metabolic enzymes changed significantly in tail muscle. Activities of three enzymes of glycolysis, GK, ALD, and PFK, were reduced; notably PFK is the rate-limiting enzyme of the pathway so this suggests that glycolytic rate was suppressed. PFK activities are much higher in tail muscle than HK activities. Reduced CL activity in tail muscle during anoxia may be linked with low rates of fatty acid synthesis in the anaerobic state. GPT activity was also greatly enhanced in tail muscle (by 74 %). This indicates that some protein catabolism must have taken place, and that anaerobic crustaceans must rely on the fermentation of glutamate during periods of anoxia exposure.

In hepatopancreas, changes in the activities of metabolic enzymes were also consistent with changes in fuel use patterns in the anaerobic state. The strong trend among those enzymes that changed was for elevated enzyme activities (8 out of 10 cases) (Figure 2). Activities of two glycolytic enzymes were reduced in hepatopancreas of anoxic crayfish, consistent with carbohydrate sparing. PFK and PK activities were reduced or inhibited whereas gluconeogenesis was promoted through FBPase activity. In crayfish hepatopancreas, activities clearly favour gluconeogenesis during anoxia. Significantly increased levels of G6PDH during anoxia may be linked to the enhanced GK activities found in hepatopancreas under the same conditions. Amino acid oxidation also appears to be facilitated in hepatopancreas of anoxic crayfish with elevated levels of SDH and GPT.

Several changes to metabolic pathway potential occur in order to optimize the crayfish ability to survive conditions without environmental oxygen. Anoxia also affects the activities or protein levels of several key regulatory enzymes including protein kinase A, protein phosphatase types 1 and 2, numerous tyrosine kinase and phosphatases, MAPKs, and a variety of transcription factors involved in regulating induced gene espression (Cowan and Storey 1999a, 1999b, 1999c). The future directions of studies into anoxia-tolerance in the freshwater crayfish will lead us into completing the picture of what actually occurs during metabolic rate depression in O. virilis, including an analysis of amino acid levels and glycogen storage in tissues, assays for glycogen phosphorylase, and levels of phosphagen arginine phosphate.

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References

Benoit, M A, Debauche, P, Devos, P (1994) Phosphofructokinase from the posterior gills of the euryhaline crab, Eriocheir sinensis: evidence for its regulation by phosphorylation. J. Comp. Physiol. B 164: 165-171

Brooks, S.P.J. (1992) A simple computer program with statistical tests for the analysis of enzyme kinetics. Bio-Techniques. 13: 906-911.

Brooks, S P J, Storey, K B (1990) cGMP-stimulated protein kinase phosphorylates pyruvate kinase in an anoxia-tolerant marine mollusc. J Comp Physiol B 160: 309-316

Cowan, K J, Storey, K B (1999a) Reversible phosphorylation control of glycolysis in the anoxia tolerant crayfish, Orconectes virilis, submitted.

Cowan, K J, Storey, K B (1999b) Purification and characterization of a c-AMP-dependent protein kinase from the tail muscle of the crayfish. Orconectes virilis, submitted.

Cowan, K J, Storey, K B (1999c) Signal transduction in the anoxia-tolerant crayfish, Orconectes virilis, submitted.

Fernandez, M, Cao, J, Dolores, M, Ramos-Martinez, J I, Villamarin, J A (1994) Phosphofructokinase from mantle tissue of Mytilus galloprovincialis: purification and effects of phosphorylation on the enzymatic activity. Biochem. Mol. Biol. Int. 33: 355-364

Hochachka, P W, Somero, G N (1984) Biochemical adaptation. Princeton University Press, Princeton, N J.

Holwerda, D A, Veenhof, P R, van Heugten, H A A, Zandee, D I (1983) Regulation of mussel pyruvate kinase during anaerobiosis and in temperature acclimation by covalent modification. Mol. Physiol 3: 225-234

Michaelidis, B, Storey, K B (1990) Phosphofructokinase from the anterior byssus retractor muscle of Mytilus edulis: modification of the enzyme in anoxia and by endogenous protein kinases. Int. J. Biochem. 22:759-765

Storey, K B (1984) Phosphofructokinase from the foot muscle of the whelk, Busycotypus canaliculatum: evidence for covalent modification of the enzyme during anaerobiosis. Arch. Biochem. Biophys. 235: 665-672

Storey, K B, Storey, J M (1990) Facultative metabolic rate depression: molecular regulation and biochemical adaptation in anaerobiosis, hibernation, and estivation. Quart Rev Biol 65: 145-174.

Walsh, D A, Newsholme, P, Cawley, K C, van Patten, S M, Angelos, K L (1991) Motifs of protein phosphorylation and mechanisms of reversible covalent regulation. Physiol. Rev. 71:285

Walsh, D A, Van Patten, S M (1994) Multiple pathway signal transduction by the cAMP-dependent protein kinase. FASEB J. 8: 1227-1236

Whitwam, R E, Storey, K B (1990) Regulation of phosphofructokinase during estivation and anoxia in the land snail, Otala lactea. Physiol. Zool. 64: 595-610

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Cowan, K.J.; Storey, K.B.; (1998). Anoxia Effects On Enzymes Of Intermediary Metabolism In The Anoxia-Tolerant Freshwater Crayfish, Orconectes Virilis.. 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/oxidative/cowan0410/index.html
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