Regulation of skeletal Muscle Glycolysis During Dehydration in the Aestivating African Clawed Frog, Xenopus Laevis

Abstract

ii The undersigned hereby recommend to the Faculty of Graduate Studies and Research acceptance of this thesis Regulation of skeletal muscle glycolysis during dehydration in the aestivating African clawed frog, Xenopus laevis. Abstract Seasonally arid conditions can trigger African clawed frogs (Xenopus laevis) to enter aestivation. This process includes whole body dehydration that at high levels can create hypoxic conditions due to impaired blood circulation and increase the need for glycolytic energy production. This thesis examines hexokinase (HK) and lactate dehydrogenase (LDH) purified from skeletal muscle of control versus dehydrated (~30% body water lost) frogs. Studies analyzed substrate affinities, urea effects, thermal stability and protein posttranslational modifications (PTM) to understand how enzyme properties are modified under dehydration stress. Muscle HK and LDH showed regulation by reversible protein phosphorylation and nitrosylation. These PTM's correlated with reduced affinities for glucose by HK and lactate by LDH, overall lower Vmax for LDH in both directions, and altered thermal stabilities. The two enzymes responded to the same PTMs, which suggests that coordinated controls over these first and last enzymes of anaerobic glycolysis contribute to dehydration responsive pathway regulation. 4.4. Future Directions 64 References 68 Appendix 1: Native Geographical Range of X. laevis in Africa 74 viii List of Abbreviations Acetyl-CoA -Acetyl coenzyme A AMP, ADP, ATP-Adenosine mono,-di,-or triphosphate DEAE-Diethylaminoethyl EDTA-Ethylenediamine tetraacetate EGTA-Ethyleneglycol bis tetracacetate G6P-Glucose-6-phosphate G6PDH-Glucose-6-phosphate dehydrogenase HK-Hexokinase I50-Inhibitor concentration reducing enzyme velocity by 50% Km-Substrate concentration producing half maximal enzyme activity LDH-Lactate dehydrogenase NAD, NADH-Oxidized and reduced nicotinamide adenine dinucleotide NaF-Sodium fluoride PAGE -Polyacrylamide gel electrophoresis PKA-cAMP-dependent protein kinase PKG-Protein kinase G PMSF-phenylmethysulfonyl fluoride PP2A-Protein phosphatase type-2A Pi -Inorganic phosphate PTM-Posttranslational modification PVDF-Polyvinylidene difluoride SDS-Sodium dodecyl sulfate Tm -Melting temperature TCA-Tricarboxylic acid cycle (citric acid cycle) TRIS -tris(hydroxymethyl)aminomethane Vmax -Maximal enzyme velocity ix List of Tables 55 1 Chapter 1:

The West African lungfish secretes a living cocoon during aestivation with uncertain antimicrobial function

“…This includes spadefoot toads (Zamora-Camacho et al 2019;Calabrese and Pfennig 2023;Chen et al 2023), African clawed frogs (Childers 2014), turtles (Ligon and Stone 2003), amphiumas (Smith and Secor 2017), and the Australian salamanderfish (Ogston et al 2016) among many other species known to aestivate. One of the most exceptional examples of aestivation are found in the South American (Lepidosiren paradoxa) and African lungfishes (Protopterus spp.…”