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Two feeding trials were conducted to study the requirements of juvenile cobia for dietary methionine and lysine. Experiment I compared both fishmeal-based (fish meal and soybean meal as protein sources) and casein-based (casein and gelatin as protein sources) diets. All diets were isonitrogenous and isoenergetic. Crystalline amino acids was pre-coated with carboxymethylcellulose (CMC) and supplemented to simulate the amino acid pattern of juvenile cobia muscle protein except methionine. Five methionine levels of 0.45, 0.70, 0.95, 1.20 and 1.45% (1.07, 1.66, 2.26, 2.86 and 3.45% of protein) were studied. The test diets contained 0.16% cystine. Juvenile cobia (initial weight 35g) were fed test diets for 8 weeks. The results showed that the cobia used fish meal more effectively than casein. Final weight, percent weight gain (PWG), feed conversion ratio(FCR), hepatosomatic index (HSI), condition factor (CF), protein efficiency ratio (PER), protein retention (PR), carcass proximate composition and free methionine concentration in the serum were significantly enhanced (p<0.05) by increasing dietary methionine level. But profile of essential amino acids in the muscle and serum superoxidase and lysozyme activity were not significantly affected. Weight gain of the cobia fed the fishmeal-based diet containing 1.2% methionine was significantly higher than the other dietary groups. Methionine content of 0.45% resulted in reduced growth. No significant difference in weight gain was found in the casein-containing diets when methionine was 0.7% and over. The cobia fed diet containing 0.45% methionine started show apparent skin lesions in their heads on the sixth week of the growth trial and significant mortalities were observed since the seventh week (p<0.05). The optimum dietary level of methionine for cobia, estimated by the broken-line regression analysis on weight gain, was 1.10% (2.62% of protein) based on the fishmeal-based diet results and was 0.70% (1.67% of protein) based on the casein-based diet results. Experiment II used fish meal and wheat gluten as the protein sources. Pre-coated crystalline amino acids were supplemented to simulate the amino acid pattern of juvenile cobia muscle protein except lysine. Lysine levels including 1.49, 1.75, 2.00, 2.25, 2.50 and 2.75% (3.54, 4.16, 4.76, 5.35, 5.95 and 6.54%) were studied. Juvenile cobia (initially 12g) were fed test diets for 8 weeks. The results showed that the final weight, PWG, FCR, CF, PER, PR, carcass proximate composition and free lysine concentration in the serum were significantly enhanced (p<0.05) by increasing lysine levels. But HSI and essential amino acid profile of fish in the muscle were not significantly affected. Final weight and percent weight gain of the cobia fed diets containing 1.75% lysine or over were significantly higher than fish fed diet contained 1.49% lysine. FCR was lower for cobia fed diets containing 1.75% lysine or over than the 1.49% lysine group. The optimum dietary level of lysine, estimated by the broken-line regression analysis on weight gain was 1.76% (4.19% of protein), and was 1.95% (4.64% of protein) on serum free lysine concentration. There was no overt deficiency symptom, such as caudal fin rot in rainbow trout, in the cobia during the growth trial.

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ODC activity of the altered proteins was measured and compared to that of the full length 461 amino acid containing ODC. Mouse ODC cDNA sequences were deleted from either 5' or 3' ends using exonuclease III and Mung Bean nuclease treatments. An internal deletion was obtained by Hinc II and Bcl I restriction endonuclease digestion of the full length ODC cDNA. Capped mRNAs were synthesized in vitro using the resulting deleted DNA as templates, and the protein was translated in vitro. The results indicate that the protein in which translation initiates at internal AUG start codons does not have any activity. The protein with 39 amino acids deleted from carboxy-terminus maintains 12% of the activity, while deletion of greater than 79 amino acids have no activity. An internal deletion from amino acid 290 to 331 and which may contain the suspected ornithine binding site has no activity. These results suggest that the entire amino acid sequence of mouse ODC is required for full activity of the enzyme.

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