Purification and properties of malic enzyme from herring Clupea harengus spermatozoa Natalia Niedźwiecka, Edward F. Skorkowski ⁎ Gdańsk University Biological Station, Department of Molecular Evolution, 80-308 Gdańsk, Poland abstract article info Article history: Received 30 August 2012 Received in revised form 5 January 2013 Accepted 6 January 2013 Available online 11 January 2013 Keywords: Fish Herring Clupea harengus Spermatozoa Malic enzyme Herring spermatozoa exhibit higher activity of malic enzyme (ME) than Atlantic salmon (Salmo salar), brown trout (Salmo trutta), carp (Cyprinus carpio) and African catfish (Clarias gariepinus) spermatozoa. Two molecular forms of ME are present in herring spermatozoa: an NAD-preferring malic enzyme with very high activity and an NADP-specific malic enzyme with much lower activity (ratio about 33:1). NAD-preferring ME was purified by chromatography on DEAE-Sepharose, Red Agarose and Sephadex G-200 to a specific activity of 36 μmol/min/mg protein and NADP-specific ME on DEAE-Sepharose and 2′5′-ADP Sepharose. The molecular mass for NAD-preferring and NADP-specific ME determined by SDS-PAGE was equal to 61 and 64 kDa, respectively. High activity of ME suggests adaptation of herring spermatozoa to metabolism at high oxygen tension for herring spawn. © 2013 Published by Elsevier Inc. 1. Introduction It was shown earlier that activities of enzymes involved in the total generation of NADPH e.g. isocitrate dehydrogenase, glucose-6- phosphate dehydrogenase and malic enzyme are higher in herring spermatozoa than in carp and catfish spermatozoa (Gronczewska et al., 2003). On the other hand the activity of lactate dehydrogenase in herring spermatozoa is very low, about 7 times lower than that of malic enzyme (Gronczewska et al., 2003). Both these enzymes could compete for the same substrate, pyruvate formed during glycolysis and catabolism of some amino acids. Malic enzyme catalyses the reversible decarboxylation of malate to form pyruvate in the presence of NAD or NADP and a divalent cation (Mn 2+ or Mg 2+ ). It has been shown that pyruvate could stimulate spermatozoa motility and viability when added as a substrate to the incubation medium (Lahnsteiner et al., 1999; Ziętara et al., 2009). In fish ME exists as a homotetramer. Each ME form has different ki- netic and physico-chemical properties and exhibits distinct tissue- specific expression (Skorkowski, 1988). Various molecular forms of ME are classified based on their relative affinity for coenzyme. Three forms of ME coded by three independent loci occur in salmon trout heart: cytosol NADP-dependent, and two mitochondrial forms one spe- cific for NADP (NADP-dependent) and the other preferring the NAD coenzyme (NAD(P)-dependent) (Skorkowski, 1988). Both ME forms exhibit differences in Michaelis constant for malate and pyruvate and also in the relative rates of malate decarboxylation versus pyruvate carboxylation. According to our knowledge properties of ME from vertebrate sper- matozoa have not been previously described. The aims of the study were (1) to estimate some of important enzymes that enable fish sper- matozoa to fulfil their requirement for NADPH; (2) to demonstrate ME form in herring spermatozoa and (3) to determine some properties of herring spermatozoa NAD-prefering malic enzyme which dominates in the cell. 2. Materials and methods 2.1. Sperm collection Spermatozoa were collected from mature male herring (Clupea harengus and Clupeidae) from the Baltic Sea (fresh mass 109–132 g), Atlantic salmon (Salmo salar; fresh mass 4800–5500 g), and brown trout (Salmo trutta; fresh mass 4000–4500 g). Herring were purchased from a local fisherman at Swibno in the spring and salmon and trout in the autumn while fish enter the mouth of the Vistula River to spawn. Spermatozoa were collected from sexually mature males of African cat- fish (Clarias gariepinus; fresh mass 1400–1600 g). Six males of carp (Cyprinus carpio; fresh mass 1600-1800 g) were injected with 2 mg dry carp pituitary extract per kg of carp wet weigh in 0.6% saline and stripped after 24 h. Intratesticular sperm from herring, catfish and sal- monids were squeezed out after decapitation of the fish and removal of the testis. The sperm was diluted 1:10 in cold 30 mM Tris–HCl buffer, pH 8.2 containing 80 mM NaCl, 40 mM KCl and 0.1 mM CaCl 2 . Diluted spermatozoa were centrifuged at 800 g for 10 min and frozen in dry Comparative Biochemistry and Physiology, Part B 164 (2013) 216–220 ⁎ Corresponding author. Tel.: +48 58 523 6049. E-mail address: edward.skorkowski@biol.ug.edu.pl (E.F. Skorkowski). 1096-4959/$ – see front matter © 2013 Published by Elsevier Inc. http://dx.doi.org/10.1016/j.cbpb.2013.01.001 Contents lists available at SciVerse ScienceDirect Comparative Biochemistry and Physiology, Part B journal homepage: www.elsevier.com/locate/cbpb