Preliminary result of respiratory kinetics in zooplankton samples: MALASPINA 2010

Aknowledgements This research, is funded by the Formation and Perfection of the Researcher Personal Program of the Basque Country Government, the EXZOME project (CTM 2008 – 01616/MAR) and the CAMVALEX (CTM 10-09515-F) from the Spanish Science and Education Ministry. References Lowry, O. H., N. J. Rosebrough, et al. (1951). "Protein measurement with the folin phenol reagent." Journal of Biological Chemistry 193: 265 - 275. Packard, T. T., E. Berdalet, et al. (1996). "Oxygen consumption in the marine bacterium Pseudomonas nautica predicted from ETS activity and bisubstrate enzyme kinetics." Journal of Plankton Research 18(10): 1819 – 1835. Packard and Gomez (2008). "Exploring a first-principles-based model for zooplankton respiration." ICES Journal of Marine Science 65(3): 371 - 378. Wagner, T.C., Scott, M.D. (1994). “Single extraction method for the spectrophotometric quantification of oxidized and reduced pyridine nucleotides in eritrocytes”. Analytical Biochemistry 222: 417-426 Respiration Oxygen electrodes Protein (Lowry et al., 1951) ETS activity (Packard et al.,1996) Pyridine nucleotides (Wagner and Scott, 1994) Preliminary result of respiratory kinetics in zooplankton samples: MALASPINA 2010 N. Osma a*, T. Packard a, M. Gómez a a Institute of Oceanography and Global Change, University of Canary Islands. Canary Islands, Spain. * E–mail: [email protected] Fig 4. Analytical methods Fig 5. Lineweaver-Burk reciprocal plot for calculating the kinetic parameters in bisubstrate reactions. B/O Hespérides Fig 3. 100 µm mesh size WP-2 net used for sampling. Fig 1. Cruise track of the R/V Hesperides during the LEG 3, 4 and 7. Fig 2. Size fractionation and on board incubation system. Pyridine Nucleotides Fig 6. Protein specific NADH and NADPH levels in well-fed and starved zooplankton. The specific concentration of both NADH and NADPH were not significantly different in well-fed zooplankton (p>0.001) of the three size fractions. Levels of NADH were only significantly different between well-fed and starved conditions in 100500 µm size fraction. 100-500 µm 500-1000 µm > 1000 µm Table II. Kinetic constants of NADH and NADPH in two size fractions of zooplankton, There are no significative differences between the equilibrium constant (Km) and the apparent dissociation constant (Kia) for NADH and NADPH when well fed and starved organisms are compared. The maximum velocity (Vmax) of the reaction decreases in starved zooplankton. Data are given as mean ± SD. Numbers in parentheses represent the number of data used to calculate the average values. The respiratory oxygen consumption (R) is a physiological process that occurs wherever the oxygen and the organic matter are present. It is catalyzed by the enzymatic activity of the respiratory electron transfer system (ETS). This activity, in turn, is controlled by the availability of substrates, i.e., nicotinamide adenine dinucleotide (NADH) and nicotinamide adenine dinucleotide phosphate (NADPH). The Enzyme Kinetic Model (EKM) argues that, according to the theory of MichaelisMenten, the maximum activity of these enzymes and the availability of their substrates control the in vivo activity of the ETS enzymes at constant temperatures (Packard and Gomez, 2008). Up to now, no study has measured the NADH and NADPH intracellular levels and applied them to the EKM in the zooplankton. In the present work, we have measured the intracellular level of substrates and calculated the kinetics constants. Field samples were collected during three legs of the Malaspina 2010 oceanographic cruise (Fig 1). The physiological measurement of respiration were accomplished on board in well fed and in 24h starved organisms. The enzyme activities and kinetics and intracellular substrates levels were determined back at laboratory. INTRODUCTION I. NADH levels remained nearly constant from well-fed to starvation conditions in 500-1000 µm and >1000 µm size fractions. However, they decreased in 100-500 µm size fraction. Highest size classes might have a larger reserve and 24 h of starvation might not challenge them. II. Specific respiratory oxygen consumption decreases with size fraction, both in well-fed and starvation conditions. III. There are no significant differences in the kinetics constants between well-fed and starved organisms. CONCLUSSIONS Table I. Protein specific rates of respiratory oxygen consumption (R) and potential respiration (Φ) both in well-fed and in starved zooplankton. Data are given as means ± SD. ENZYME KINETIC MODEL Ro = Vmax: Maximum velocity of the reaction S1, S2: Substrate concentration of NADH and NADPH K1, K2: Michaelis constant of NADH and NADPH Kia: Apparent dissociation constant 𝑉𝑉𝑉𝑉 · [𝑆𝑆 ·𝑆𝑆] 𝐾𝑆 · 𝐾𝐾𝑉 + 𝐾𝑆 · [𝑆𝑆] + 𝐾𝑆 · [𝑆𝑆] + [𝑆𝑆 ·𝑆𝑆]