Edward F. Skorkowski

Effect of tributyltin on adenylate content and enzyme activities of teleost sperm: a biochemical approach to study the mechanisms of toxicant reduced spermatozoa motility.

The effects of tributyltin (TBT) on the energy metabolism and motility of fish spermatozoa were investigated in vitro in African catfish and common carp. A significant (P<0.05) decrease of the duration and the intensity of motility was observed in catfish spermatozoa exposed to 0.27 microg/l TBT for 24 h. Exposure of catfish spermatozoa to 2.7-27 microg/l TBT caused an instant decrease in ATP content. In the presence of 27 microg/l TBT approximately 55% of the initial ATP concentration in catfish semen was lost after 60 min incubation while AMP concentrations increased and the total adenine nucleotide (TAN) pool remained unchanged. The reduction in sperm ATP levels could not be attributed to cell death since viability decreased only slightly over the period of exposure. In carp by contrast, none of the adenylates concentrations studied (ATP, ADP and AMP) were affected by TBT exposure at any experimental condition. However, carp sperm motility was significantly reduced by exposure to 2.7 microg/l TBT. Among the enzymes investigated only lactate dehydrogenase (LDH) in catfish sperm was significantly (P<0.01) affected by 27 microg/l TBT treatment with a reduction in activity of approximately 75%. Compared with carp sperm before TBT exposure, that of catfish had lower adenylate contents and overall lower enzymatic activities; this explains its slower sperm velocity and shorter duration of movement as measured by computer assisted sperm analysis (CASA). The present in vitro study shows that catfish spermatozoa are more sensitive to TBT exposure (and probably to other toxicants) than those of carp.

Quantitative determination of creatine kinase release from herring (Clupea harengus) spermatozoa induced by tributyltin

Creatine kinase (CK, ATP creatine phosphotransferase, EC 2.7.3.2) is an enzyme participating in ATP regeneration, which is the primary source of energy in living organisms. We demonstrated that CK from herring spermatozoa has high activity ( approximately 452 micromol/min/g of fresh semen) and has a different electrophoretic mobility from isoenzymes present in skeletal muscle. In our study, we investigated toxic effect of tributyltin (TBT) on herring spermatozoa using a specific sperm viability kit to observe live and dead sperm cells with a confocal microscope. Treatment of herring spermatozoa with TBT caused a time-dependent decrease of viability: 35% nonviable cells with 5 microM TBT and more than 90% nonviable cells with 10 microM TBT after 6 h exposure. We also monitored CK release from damaged spermatozoa into surrounding medium containing different concentrations of TBT. The higher concentration of TBT was used the more CK release from spermatozoa was observed. We suggest that CK could be a good biomarker of sperm cell membranes degradation in the case when lactate dehydrogenase release from permeabilized cells is not possible for rapid determination of the effect of TBT.

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.

Mitochondrial malic enzyme from crustacean and fish muscle

1. In contrast to mammalian skeletal muscle mitochondria, the only substrate that crustacean and fish mitochondria oxidize at a high rate is malate. 2. The mitochondria isolated from muscles of fish and crayfish exhibit a high activity of malic enzyme. 3. Assuming that malic enzyme is responsible for the conversion of malate to pyruvate in animal muscle, it could be expected that the mitochondria which possess high activity of this enzyme should oxidize malate very rapidly when oxygen is available. 4. Some properties of different molecular forms of malic enzyme are reviewed.

Evidence for the role of malic enzyme in the rapid oxidation of malate by cod heart mitochondria

Mitochondria isolated from the heart of cod (Gadus morrhua callarias) oxidized malate as the only exogenous substrate very rapidly. Pyruvate only slightly increased malate oxidation by these mitochondria. This is in contrast with the mitochondria isolated from rat and rabbit heart which oxidized malate very slowly unless pyruvate was added. Arsenite and hydroxymalonate (an inhibitor of malic enzyme) inhibited the respiration rate of mitochondria isolated from cod heart, when malate was the only exogenous substrate. Inhibition caused by hydroxymalonate was reversed by the addition of pyruvate. In the presence of arsenite, malate was converted to pyruvate by cod heart mitochondria. Cod heart mitochondria incubated in the medium containing Triton X-100 catalyzed the reduction of NADP+ in the presence of L-malate and Mn2+ at relatively high rate (about 160 nmoles NADPH formed/min/mg mitochondrial protein). The oxidative decarboxylation of malate was also taking place when NADP+ was replaced by NAD+ (about 25 nmol NADH formed per min per mg mitochondrial protein). These results suggest that the mitochondria contain both NAD+- and NADP+-linked malic enzymes. These two activities were eluted from DEAE-Sephacel as two independent peaks. It is concluded that malic enzyme activity (presumably both NAD+- and NADP+-linked) is responsible for the rapid oxidation of malate (as the only external substrate) by cod heart mitochondria.

Comparative studies on NADP-linked dehydrogenases in some tissues of fish and crustaceans

Abstract 1. 1. The activities of isocitrate dehydrogenase (EC 1.1.1.42), malic enzyme (EC 1.1.1.40) and glucose-6-phosphate dehydrogenase (EC 1.1.1.49) were measured in extracts of various tissues obtained from three species of fish and three crustaceans. 2. 2. The activity of isocitrate dehydrogenase greatly exceeded that of malic enzyme in fish muscle and was only slightly higher in crustaceans muscle. 3. 3. Very low activity of glucose-6-phosphate dehydrogenase was detected in all somatic muscle investigated. 4. 4. In the heart muscle of fish the activities of the three enzymes studied were greater than those in somatic muscle. 5. 5. However the isocitrate dehydrogenase/malic enzyme and isocitrate dehydrogenase/glucose-6-phosphate dehydrogenase activities ratios were generally similar in somatic and heart muscle of fishes. 6. 6. Insignificant differences were observed between the livers and gills of fish with respect to the three enzyme activities tested. 7. 7. Fish livers and gills displayed significantly higher glucose-6-phosphate dehydrogenase activity levels than somatic muscle of fish. 8. 8. Slight differences were observed in isocitrate dehydrogenase and matic enzyme activities between somatic muscle and livers or gills.

Short term cadmium intoxication of the shrimp Palaemon serratus: Effect on adenylate metabolism

Abstract 1. 1. Shrimps were exposed for 96 hr to various concentrations of cadmium under laboratory conditions. The LC 50 was around 4 ppm Cd in water, which corresponded to 0.180 μg/g wet weight of cadmium in tail muscles. 2. 2. The effect of various concentrations of cadmium on adenylates was analyzed in tail muscles: At subletal cadmium concentrations, no variation of ATP, ADP and of the adenylate sum occurred, while the AMP concentration began to decrease from 0.06 ppm. At the LC 50 , the ATP, ADP and AMP concentrations dropped acutely, the ATP/ADP ratio increased acutely. The apparent equilibrium constant of the adenylate kinase reaction increased significantly from 2 ppm Cd, indicating an impairment in energetic metabolism. Cadmium intoxication did not influence the value of the adenylate energy charge.

High activity of NADP-dependent malic enzyme in mitochondria from abdomen muscle of the crayfish Orconectes limosus.

1. Mitochondria isolated from abdomen muscle of crayfish Orconectes limosus exhibit malic enzyme activity in the presence of L-malate, NADP and Mn2+ ions after addition of Triton X-100. Under optimal conditions about 230 nmole of reduced NADP and an equivalent amount of pyruvate are produced per min per mg of mitochondrial protein. 2. The pH optimum for decarboxylation of L-malate is about 7.5. 3. The apparent Km for L-malate, NADP and Mn2+ ions was found to be 0.66, 0.012, and 0.0025 mM, respectively. 4. The requirement for Mn2+ can be replaced by Mg2+, Co2+ and Ni2+ ions; however, higher concentrations of these ions than Mn2+ are required for a full stimulation of malic enzyme activity. 5. Oxaloacetate and pyruvate inhibited the enzyme activity in a competitive manner with apparent Ki values of 0.05 mM and 5.4 mM, respectively.

Purification and properties of the heart type lactate dehydrogenase of the cod (Gadus morhua) from the baltic sea: Comparison with LDH-A4 and LDH-C4

Abstract 1. 1. Cod from the Baltic Sea possesses polymorphic Ldh-B locus accounted for by two alleles, b and b′, with frequencies of 0.87 and 0.13, respectively. 2. 2. Both LDH-B alloenzymes, LDH-B4 and LDH-B′4, were purified to specific activities of 553 and 416 μmol NADH min−1 mg−1 respectively. 3. 3. Cod LDH-B4 and LDH-B′4 alloenzymes possess the same kinetic and physicochemical properties. The pH optima for pyruvate reduction and lactate oxidation are 6.9 and 10.5, respectively. Both alloenzymes have similar heat stability. 4. 4. Some kinetic properties of all cod homotetramers are compared.

Unusual expression of the threespine stickleback (Gasterosteus aculeatus) lactate dehydrogenase isoenzymes and partial characterization of purified LDH-A4

1. 1. The threespine stickleback possesses the same LDH isoenzyme in heart and skeletal muscles. 2. 2. The Ldh-B locus reduced its expression to eye and brain tissues in the threespine stickleback. 3. 3. The threespine stickleback LDH-A4 was purified to homogeneity from skeletal muscle to a specific activity of 384 μmol NADH min−1 mg−1 and some kinetic properties are reported. 4. 4. The Km values for pyruvate reduction (0.30 mM) and lactate oxidation (50 mM) or NAD+ inhibition ratio (1.1) confirm that the most common isoenzyme of the threespine stickleback tissues is LDH-A4.