NAD-preferring malic enzyme: localization, regulation and its potential role in herring (Clupea harengus) sperm cells.

Herring spermatozoa exhibit a high activity of NAD-preferring malic enzyme (NAD-ME). This enzyme is involved in the generation of NADH or NADPH in the decarboxylation of malate to form pyruvate and requires some divalent cations to express its activity. In order to confirm that NAD-ME isolated from herring sperm cells is localized in mitochondria, we performed immunofluorescent analysis and assayed spectrophotometrically the malic enzyme reaction. Production of polyclonal rabbit antibodies against NAD-ME from herring spermatozoa enabled identification of mitochondrial localization of this enzyme inside herring spermatozoa. The kinetic studies revealed that NAD-ME was competitively inhibited by ATP up to tenfold. Addition of fumarate reversed ATP-dependent inhibition of NAD-ME to 55 % of its maximum activity. The pH-dependent regulation of malic enzyme activity was also examined. Malic enzyme showed maximum activity at pH near 7.0 in all studied conditions. Finally, the role of malic enzyme activity regulation in mitochondria of herring sperm cells was discussed.

Purification and stability of octameric mitochondrial creatine kinase isoform from herring (Clupea harengus) organ of vision.

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.

Inhibition by tributyltin of herring skeletal muscle lactate dehydrogenase activity.

The activity of herring (Clupea harengus) skeletal muscle lactate dehydrogenase (EC 1.1.1.27) LDH-A4 isoenzyme was examined in the presence of tributyltin chloride (TBT). This paper reports the in vitro inhibition of LDH activity with increasing concentration of TBT. Bovine serum albumin (BSA) added to the LDH-A4 isoenzyme prior to the addition of TBT was able to protect enzyme activity against inhibition by this toxicant. The observed protection of LDH-A4 activity increased with increasing BSA concentration in the incubation medium. The results suggest that the presence of BSA could protect LDH activity from direct binding of TBT to LDH.

Enzyme activities in fish spermatozoa with focus on lactate dehydrogenase isoenzymes from herring Clupea harengus.

The activities of NAD- and NADP-dependent dehydrogenases and creatine kinase were compared in extracts of spermatozoa from herring (Clupea harengus), carp (Cyprinus carpio) and catfish (Clarias gariepinus). The activity of malic enzyme in herring spermatozoa was approximately 5 and 36 times higher than in carp and catfish spermatozoa. In contrast, lactate dehydrogenase activity in herring spermatozoa was very low. Herring spermatozoa possess two isoenzymes of lactate dehydrogenase: LDH-A(2)B(2) and LDH-B(4). Both herring spermatozoa isozymes were separated, partly purified and characterized by kinetic and physico-chemical properties. The pH optima and K(m) values for pyruvate reduction were 7.1, 7.25, 7.6 and 0.22, 0.07, 0.09 mM for LDH-A(4), LDH-A(2)B(2) and LDH-B(4), respectively. The isoenzymes also have different thermostabilities. High activity of malic enzyme in herring spermatozoa suggests adaptation to metabolism at high oxygen tension.