ELSEVIER Biochimica et Biophysica Acta 1250 (1995) 76-82 Biochi~ic~a et Biophysica A~ta Glucose-6-phosphate dehydrogenase from the blood cells of two Antarctic teleosts: correlation with cold adaptation M. Antonietta Ciardiello, Laura Camardella, Guido di Prisco * Institute of Protein Biochemistry and Enzymology, C.N.R., Via Marconi 10, 1-80125 Naples, Italy Received 29 July 1994; revised 10 March 1995; accepted 10 March 1995 Abstract Glucose-6-phosphate dehydrogenase (G6PD) has been purified from the blood of two Antarctic teleost species, i.e., from the erythrocytes of Dissostichus mawsoni (family Nototheniidae), and from the plasma and cells of haemoglobinless Chionodraco hamatus (family Channichthyidae). The specific activities in haemolysates of Antarctic blood cells appear higher than that of a lysate of human erythrocytes. The two Antarctic enzymes have an apparent subunit molecular mass slightly higher than that of human G6PD; the electrophoretic behaviour on cellulose acetate is similar. Both Antarctic enzymes are irreversibly heat inactivated through a biphasic process. K m for glucose-6-phosphate (G6P) does not vary significantly with temperature, whereas K m for NADP increases at increasing temperature, kcat increases with temperature, with a break point at 35°C (in human G6PD, the break point is at 15 ° C). Thermodynamic and kinetic characterisation indicate that the catalytic performance of the enzyme of cold-adapted fish, at temperatures typical of their habitat, is more efficient than that displayed by G6PD from a temperate organism. Keywords: Glucose-6-phosphate dehydrogenase; Enzyme; Antarctic fish; Kinetic analysis; Thermodynamic analysis; Cold adaptation 1. Introduction During evolution, Antarctic fish developed special anatomical, physiological and biochemical characteristics. Assuming that cold adaptation is linked to modifications of the metabolic machinery, the study of structural and func- tional properties of enzymes with special metabolic roles, and in particular the regulation of catalytic activity by temperature, may provide clues to the understanding of the molecular bases of fish cold adaptation [1]. It was reported that at low temperature several enzymes from Antarctic fish are more efficient than their temperate counterparts, whereas others show little or no functional variations; these different behaviours may reflect the rela- tive importance of specific metabolic pathways [2]. We have focussed our attention on G6PD (EC 1.1.1.49), whose metabolic significance justifies the study of its properties in cold-adapted Antarctic fish. G6PD, localised in all cells, plays a key role in the metabolism of glucose. It catalyses the first reaction (oxidation of D-G6P to D-glu- Abbreviations: G6PD, Glucose-6-phosphate dehydrogenase; G6P, glucose 6-phosphate; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis. * Corresponding author. Fax: +39 81 593 6689. 0167-4838/95/$09.50 © 1995 Elsevier Science B.V. All rights reserved SSDI 0167-4838(95)00046-1 cono-A-lactone 6-phosphate and concomitant reduction of the coenzyme NADP) of the pentose-phosphate pathway which, with its close interaction with lipid biosynthesis, seems to play an important role in the metabolism of cold-adapted fish [3]. G6PD is considered to control the rate of this pathway, although the detailed mechanism of regulation has not yet been understood. G6PD-produced NADPH, used in lipid biosynthesis and in the protection of the cell from oxidative stress, is also the coenzyme of glutathione reductase and is required for the optimal func- tioning of catalase. Both reduced glutathione and catalase are essential for the detoxification from hydrogen perox- ide; therefore G6PD plays a main role in maintaining the physiological viability of erythrocytes, which are espe- cially sensitive to oxidative damage [4]. G6PD has been purified from the erythrocytes of the nototheniid Dissostichus mawsoni, and from the whole blood and cells of the channichthyid Chionodraco hama- ms. The purpose of this study was two-fold: (i) to begin an investigation on the catalytic behaviour of G6PD from cold-adapted fish, with special attention to the activity response to low temperatures; (ii) to examine G6PD in Channichthyidae, one of the five Antarctic families (Nototheniidae are grouped in another one) of the largely endemic suborder Notothenioidei. The pale-whitish blood