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Herring (Clupea harengus) shows the unique behavior of reproductive biology in which spermatozoa remains in the surrounding media for extended periods. It is an excellent model for studying the malic enzyme (ME) and creatine kinase (CK) biochemical properties because of their high activity and variability of molecular isoforms. The specific activity of NAD-preferring ME in herring spermatozoa is the highest among other fish spermatozoa and is localized in its large mitochondrion. Two different CK isoforms, dimer and octamer, were detected in herring spermatozoa. It has already been shown that CK isoforms play an important role in energy homeostasis by catalyzing a reversible transfer of the phosphate of ATP to creatine to yield ADP and creatine phosphate (CP) (creatine/CP circuit). Two lactate dehydrogenase (LDH) isoenzymes were also shown in herring spermatozoa, LDH-B4 and LDH-A2B2. In this mini-review, the role of ME and energy transport system with easily diffusible creatine and CP in herring spermatozoa is discussed.

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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.

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Two forms of NADP-dependent malic enzyme (ME, EC 1.1.1.40) were purified from the abdomen muscle of the crayfish Orconectes limosus (Rafinesque, 1817) and the shrimp Crangon crangon L., 1758 by affinity chromatography on 2′,5′-ADP-Sepharose 4B, with good qualitative recovery in a single step, using a substrate activation method with a malate–manganese chloride pair. The enzymes were identified by native polyacrylamide gel electrophoresis stained for protein and enzyme activity. The faster migrating mitochondrial enzyme from crayfish is inhibited by sulfhydryl reagent and loses its activity. Ellman’s Reagent, 5,5′-Dithiobis-(2-nitrobenzoic acid) (DTNB), can be used for the differentiation and measurement of cytoplasmic and mitochondrial malic enzyme in decapod crustacean tissue.