Isolation, Purification, Cloning, Kinetic Properties Of Enzymes Related To Lipid Metabolism And Response To Copper And Zinc In Ctenopharyngodon Idella

Grass carp（Ctenopharyngodon idella） is one of the largest-scaled fish species for aquaculture in China. In 2011, the production of grass carp in China reached 4.2 million tons（FAO, 2012）. The study was conducted to determine the enzymatic characterization,kinetics, molecular characterization of key enzymes（G6PD and CPT I） related to lipid metabolism of grass carp. Moreover, we investigated the effects of waterborne Cu and Zn on the lipid metabolism-related enzyme’s activity and its gene expression level.1 Purification and characterization of glucose-6-phosphate dehydrogenase（G6PD）from grass carp（C. idella）In the present study, G6 PD was purified from grass carp（C. idella） hepatopancreas using the methods of 2’,5’-ADP-Sepharose 4B affinity chromatography followed by DEAE Sepharose Fast Flow ion exchange chromatography. The characterization of G6 PD and inhibition effects of several metal ions on G6 PD activity in vitro were also determined. Grass carp hepatopancreas G6 PD, with a specific activity of 18 U/mg protein,was purified 1066–fold with a yield of 19.5% and Mr of 71.85 k Da. The enzyme had a temperature optimum of 42 o C, p H optimum of 7.5 and 9.0. The Km values for G6-P and NADP+were determined to be 0.026, 0.0068 m M, respectively. The Vmax values for G6-P and NADP+ were 2.20 and 2.27 μM min^-1 mg protein^-1, respectively. The catalytic efficiency for G6-P and NADP as substrates were 0.085 and 0.334 × 10-6min^-1mg protein^-1, respectively. Inhibition effects of metal ions on the purified G6 PD activity indicated that IC50 values of Zn²⁺, Mn²⁺, Al³⁺, Cu²⁺and Cd²⁺were 0.42, 0.54, 0.94, 1.20 and 4.17 m M, respectively. The Ki constants of Zn²⁺, Al³⁺, Cu²⁺ and Cd²⁺ were 0.52, 1.12,0.26 and 4.8 m M, respectively. Zn²⁺, Al³⁺ and Cd²⁺ showed competitive inhibition while Cu²⁺inhibited the G6 PD in a noncompetitive inhibition manner. Of the metal ions tested,Zn2+ exhibited much higher inhibitory effect at lower concentrations, so it was a more potent inhibitor than others. Our study provided important information about the control of the grass carp liver PPP, the biosynthesis of several important related biomolecules and the status of detoxification systems in grass carp liver in relation to metabolism.2 Cloning and expression of CPT I from grass carp（C. idella）In the present study, we successfully cloned the complete c DNA sequences of three CPT Iα genes（CPT Iα1a, CPT Iα1b and CPT Iα2a） and one CPT Iβ gene isoforms fromgrass carp. We analyzed the molecular structure and function of four isoforms, and detected their tissue expression patterns. In the present study, alignment of CPT I amino acid sequences from grass carp to those from other fishes and mammals suggested some important substitutions occurring at Val19, Leu23 and Ser24in grass carp. Multiple protein post-translational modification sites included a potential asparagine glycosylation sites at residues Asn312, an acetylation site at residues Ala2, as well as a potential tyrosine phosphorylation site at residue Thr313, Thr603, Tyr720, serine phosphorylation site at residue Ser24, Ser38, Ser740, Ser748. Most of these residues are conserved in the proteins from other species. Furthermore, the four CPT I isoforms transmembrane domains were predicted by TMpred analysis, indicating that there are two different domains. Generally speaking,amino acid substitutions or/and mutations and difference transmembrane domain may partially explain the CPT I sensitivity to malonyl-Co A and/or catalytic activity in grass carp compared to mammals.The tissue-specific expression of four CPT I isoforms was determined via real-time q PCR in grass carp across liver, brain, gill, heart, white muscle, red muscle, kidney,spleen and intestine. Four isoforms were expressed in all the tested tissues, but at varying levels. CPT Iα1a and CPT Iβ m RNA were expressed preferentially in heart. CPT Iα2a and CPT Iα1b expression was the highest in liver.3 Study on kinetic properties of CPT I from grass carp（C. idella）In order to get knowledge of kinetic parameters of CPT I from grass carp,mitochondria from different tissues（liver, heart, muscle, spleen and intestine, respectively）were isolated and their integrity was detected by determination of citrate synthase activity.For CPT I activity, optimum temperature, p H, incubation time and protein concentration were 37 o C, p H 7.5, 3 to 25 min and 10-150 μg/ml, respectively. Under optimum conditions, we analyzed the changes of CPT I activity in different substrates at different concentrations in different tissues and found they all meet the saturation curve（Michaelis-Menten）. The reaction followed a Michaelis-Menten. Lineweaver–Burk plots were drawn by using 1/V versus 1/S values. The Km for carnitine was highest in the intestine. The Km for palmitoyl-Co A was highest in the muscle and lowest in the heart.The Vmax for carnitne followed the order: heart > intestine > while muscle > spleen >liver. The Vmax for palmitoyl-Co A in different tissues followed the order: heart >intestine = liver > spleen > white muscle. Catalytic efficiency for L-carnitne was higher in heart and lower in intestine, whereas higher and lower catalytic efficiency forpalmitoyl-Co A was observed in heart and white muscle, respectively. Thus, our results suggested the differences in CPT I kinetics among different tissues, which may be a reflection of different capacities of fatty acid oxidation in different tissues grass carp.4 The composition of carnitine and kinetic of CPT I in grass carp（C. idella） at different developmental stagesThe ontogeny and kinetics of CPT I was investigated in hepatopancreas and muscle throughout four developmental stages（newly-hatched larvae, one-month-old juvenile,three-month-old, and six-month-old, respectively） of grass carp C. idella. In the present study, TC contents in hepatopancreas were the highest at hatching and one-month-old grass carp, and the lowest at three-month-old grass carp. TC content varied with developmental stages. The present study also indicated that the lowest FC concentration in hepatopancreas was found at three-month-old grass carp, inferring that most of FC was acylated at the stage. In muscle, TC content declined from hatching to three-month old grass carp and then increased at six-month-old grass carp. The ratio of AC/FC provides a useful marker of changes in carnitine metabolism. The highest ratio of AC/FC in hepatopancreas was found in 1-month-old grass carp, and the lowest ration of AC/FC was found in hatching. In contrast, in muscle, the ratio of AC/FC was the highest for3-month-old grass carp, and the lowest for 1-month-old grass carp. Different profiles of the ratio of AC/TC were observed in tested tissues and also in different developmental stages with the same tissue, which indicated different physiological state of metabolism and energy demands. In hepatopancreas, the maximal velocity（Vmax） significantly increased from hatching to one-month-old grass carp and then gradually declined at six-month-old grass carp. In muscle, CPT I activity was the highest at one-month-old grass carp, nearly two-fold higher than that at hatching（P < 0.05）. The Michaelis constant（Km） value was also the highest for one-month-old in both tested tissues. Carnitine concentrations（FC, AC and TC） were the lowest for three-month-old grass carp, and remained relatively constant in both tissues from fish under the other developmental stages. The FC concentration in hepatopancreas and muscle at four developmental stages were less than the respective Km, indicating that grass carp required supplemental carnitine in their food to ensure that CPT I activity was not constrained by carnitine availability.5 Effect of waterborne copper on the lipid metabolism in hepatopancreas and muscle of grass carp（C. idella）The present study was performed to determine the effect of waterborne copper（Cu）exposure on lipid metabolism in hepatopancreas and muscle of grass carp C. idella. Grass carp were exposed to four waterborne Cu levels（control, 0.058, 0.150, 0.199 mg/l） for 14 days. The lipid content, Cu accumulation, the activities of several enzymes and expression levels of genes involved in lipid metabolism were determined in hepatopancreas and muscle. Waterborne Cu exposure reduced lipid content, growth performance, and increased Cu accumulation in both tested tissues. In hepatopancreas and muscle, the activities and the m RNA expression of lipogenic enzymes（G6PD, 6PGD,ICDH, ME and FAS） decreased with increasing waterborne Cu levels. Furthermore, the m RNA expressions of lipolytic enzymes（HSL and ATGL） and transcription factor（SREBP-1c and PPARα） were down-regulated by Cu exposure. Waterborne Cu exposure induced the down-regulation of activities of enzymes and the m RNA expression of both lipogenesis and lipolysis, resulting in reduced lipid contents mainly through inhibition of lipogenesis prevailing over reduced lipolysis. To our knowledge, the present study provided experimental evidence that waterborne Cu exposure can perturb the normal processes of lipid metabolism, which provided new insight into the relationship between Cu and lipid deposition in fish.6 Effect of waterborne zinc on the lipid metabolism in hepatopancreas and muscle of grass carp（C. idella）The present study was performed to determine the effect of waterborne zinc（Zn）exposure on lipid metabolism in hepatopancreas and muscle of grass carp C. idella. Grass carp were exposed to four waterborne Zn levels（control, 0.99, 2.50, 3.30 mg/l） for 14 days. The lipid content, Zn accumulation, the activities of several enzymes and expression levels of genes involved in lipid metabolism were determined in hepatopancreas and muscle. In hepatopancreas, lipid content, G6 PD, 6PGD, ICDH, ME and FAS as well as m RNA levels of G6 PD, 6PGD, ME, FAS and SREBP-1c increased with increasing Zn concentrations. But, activity and m RNA level of CPT I genes and, PPARα, CPT Iα1a,CPT Iα2a and HSL genes decreased with increasing Zn concentrations. In muscle, lipid content, activities of G6 PD, ICDH, ME and FAS as well as m RNA expression of G6 PD,6PGD, ME, FAS and SREBP-1c decreased with increasing Zn concentrations, however,the activity of CPT I and m RNA expression of PPARα, HSL and ATGL gene increased with increasing Zn concentrations. The differential Pearson correlations between transcription factors（SREBP-1c and PPARα） the activities and m RNA expression oflipogenic enzymes and their genes were observed between hepatopancreas and muscle.Different response patterns of several tested enzymes and genes to waterborne Zn exposure indicated the tissue-specific regulatory effect of lipid metabolism following waterborne Zn exposure. To our knowledge, the present study provided experimental evidence that waterborne Zn exposure can perturb the normal processes of lipid metabolism, which provided new insight into the relationship between Zn and lipid deposition in fish.