title: Purification and stability of octameric mitochondrial creatine kinase isoform from herring (Clupea harengus) organ of vision.
abstract: Creatine kinases (CKs) constitute a large family of isoenzymes that are involved in intracellular energy homeostasis. In cells with high and fluctuating energy requirements ATP level is maintained via phosphocreatine hydrolysis catalyzed by creatine kinase. In contrast to invertebrates and higher vertebrates, in poikilothermic vertebrates the adaptations for the regulation of energy metabolism by changes in the oligomeric state of CK isoforms are not well known. The present study aimed at identification of herring eye CK isoforms and focuses on factors affecting the CK-octamer stability. In addition to the CK octamer, three different dimeric isoforms of CK were detected by cellulose acetate native electrophoresis. Destabilization of octamer was studied in the presence of TSAC substrates and about 50% of octamers dissociated into dimers within 24h. Moreover, we found that the increase of temperature from 4 °C to 30 °C caused rapid inactivation of dimers in TSAC-treated samples but did not affect octameric structures. In a thermostability assay we demonstrated that octamers retain their activity even at 50 °C. Our results indicate that destabilization of the octameric structure can lead to loss of enzyme activity at higher temperatures (above 30 °C). Furthermore, our results based on N-terminal sequence analysis suggest that probably the mitochondrial s-type CK, rather than u-type, is predominantly expressed in herring eye. In conclusion the existence of four various CK isoforms in one organ may reflect complex regulation of energy metabolism in the phototransduction process in teleost fishes.
authors.affiliation: Department of Molecular Evolution, Faculty of Biology, University of Gdańsk, 80-308 Gdańsk, Poland.
authors.forename: Natalia
authors.initials: N
authors.lastname: Niedźwiecka
authors.affiliation: Department of Molecular Evolution, Faculty of Biology, University of Gdańsk, 80-308 Gdańsk, Poland; Department of Virus Molecular Biology, Intercollegiate Faculty of Biotechnology UG-GUM, University of Gdańsk, 80-822 Gdańsk, Poland. Electronic address: katarzyna.grzyb@biotech.ug.edu.pl.
authors.forename: Katarzyna
authors.initials: K
authors.lastname: Grzyb
authors.affiliation: Department of Molecular Evolution, Faculty of Biology, University of Gdańsk, 80-308 Gdańsk, Poland.
authors.forename: Agnieszka
authors.initials: A
authors.lastname: Nona-Mołdawa
authors.affiliation: Department of Molecular Evolution, Faculty of Biology, University of Gdańsk, 80-308 Gdańsk, Poland.
authors.forename: Jadwiga
authors.initials: J
authors.lastname: Gronczewska
authors.affiliation: Department of Molecular Evolution, Faculty of Biology, University of Gdańsk, 80-308 Gdańsk, Poland.
authors.forename: Edward F
authors.initials: EF
authors.lastname: Skorkowski
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D052281:Creatine Kinase, Mitochondrial Form / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism; D004795:Enzyme Stability; D005123:Eye / Q000201:enzymology*; D005399:Fishes; D015971:Gene Expression Regulation, Enzymologic; D007527:Isoenzymes / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D008969:Molecular Sequence Data; D009928:Organ Specificity; D055503:Protein Multimerization; D020836:Protein Structure, Quaternary; D013696:Temperature
chemical_list: D007527:Isoenzymes; D052281:Creatine Kinase, Mitochondrial Form
keywords: Herring; MtCK; Octamer; Stability; TSAC