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The African clawed frog (Xenopus laevis) withstands prolonged periods of extreme whole-body dehydration that lead to impaired blood flow, global hypoxia, and ischemic stress. During dehydration, these frogs shift from oxidative metabolism to a reliance on anaerobic glycolysis. In this study, we purified the central glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) to electrophoretic homogeneity and investigated structural, kinetic, subcellular localization, and post-translational modification properties between control and 30% dehydrated X. laevis liver. GAPDH from dehydrated liver displayed a 25.4% reduction in maximal velocity and a 55.7% increase in its affinity for GAP, as compared to enzyme from hydrated frogs. Under dehydration mimicking conditions (150 mM urea and 1% PEG), GAP affinity was reduced with a K m value 53.8% higher than controls. Frog dehydration also induced a significant increase in serine phosphorylation, methylation, acetylation, beta-N-acetylglucosamination, and cysteine nitrosylation, post-translational modifications (PTMs). These modifications were bioinformatically predicted and experimentally validated to govern protein stability, enzymatic activity, and nuclear translocation, which increased during dehydration. These dehydration-responsive protein modifications, however, did not appear to affect enzymatic thermostability as GAPDH melting temperatures remained unchanged when tested with differential scanning fluorimetry. PTMs could promote extreme urea resistance in dehydrated GAPDH since the enzyme from dehydrated animals had a urea I 50 of 7.3 M, while the I 50 from the hydrated enzyme was 5.3 M. The physiological consequences of these dehydration-induced molecular modifications of GAPDH likely suppress GADPH glycolytic functions during the reduced circulation and global hypoxia experienced in dehydrated X. laevis.

Titel:	Mind the GAP: Purification and characterization of urea resistant GAPDH during extreme dehydration.
Autor/in / Beteiligte Person:	Hadj-Moussa, H ; Wade, SC ; Childers, CL ; Storey, KB
Link:	Zum Volltext
Zeitschrift:	Proteins, Jg. 89 (2021-05-01), Heft 5, S. 544-557
Veröffentlichung:	New York, NY : Wiley-Liss ; <i>Original Publication</i>: New York : Alan R. Liss, c1986-, 2021
Medientyp:	academicJournal
ISSN:	1097-0134 (electronic)
DOI:	10.1002/prot.26038
Schlagwort:	Acetylation Amphibian Proteins isolation & purification Amphibian Proteins metabolism Animals Binding Sites Dehydration physiopathology Droughts Glyceraldehyde 3-Phosphate metabolism Glyceraldehyde-3-Phosphate Dehydrogenases isolation & purification Glyceraldehyde-3-Phosphate Dehydrogenases metabolism Glycolysis physiology Kinetics Liver chemistry Male Methylation Models, Biological Models, Molecular Nitroso Compounds chemistry Nitroso Compounds metabolism Phosphorylation Polyethylene Glycols chemistry Protein Binding Protein Conformation, alpha-Helical Protein Conformation, beta-Strand Structural Homology, Protein Substrate Specificity Thermodynamics Urea chemistry Amphibian Proteins chemistry Dehydration metabolism Glyceraldehyde 3-Phosphate chemistry Glyceraldehyde-3-Phosphate Dehydrogenases chemistry Liver enzymology Protein Processing, Post-Translational Xenopus laevis metabolism
Sonstiges:	Nachgewiesen in: MEDLINE Sprachen: English Publication Type: Journal Article; Research Support, Non-U.S. Gov't Language: English [Proteins] 2021 May; Vol. 89 (5), pp. 544-557. <i>Date of Electronic Publication: </i>2021 Jan 06. MeSH Terms: Protein Processing, Post-Translational* ; Amphibian Proteins / chemistry ; Dehydration / metabolism ; Glyceraldehyde 3-Phosphate / chemistry ; Glyceraldehyde-3-Phosphate Dehydrogenases / chemistry ; Liver / enzymology ; Xenopus laevis / metabolism ; Acetylation ; Amphibian Proteins / isolation & purification ; Amphibian Proteins / metabolism ; Animals ; Binding Sites ; Dehydration / physiopathology ; Droughts ; Glyceraldehyde 3-Phosphate / metabolism ; Glyceraldehyde-3-Phosphate Dehydrogenases / isolation & purification ; Glyceraldehyde-3-Phosphate Dehydrogenases / metabolism ; Glycolysis / physiology ; Kinetics ; Liver / chemistry ; Male ; Methylation ; Models, Biological ; Models, Molecular ; Nitroso Compounds / chemistry ; Nitroso Compounds / metabolism ; Phosphorylation ; Polyethylene Glycols / chemistry ; Protein Binding ; Protein Conformation, alpha-Helical ; Protein Conformation, beta-Strand ; Structural Homology, Protein ; Substrate Specificity ; Thermodynamics ; Urea / chemistry References: Hillman SS. 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Mind the GAP: Purification and characterization of urea resistant GAPDH during extreme dehydration.