Effect Of Amino Acids Pattern And Methionylmethinoine On The Synthesis Of α_s-casein In Cultured Bovine Mammary Tissue

This study was conducted to establish an in vitro model for culture of lactating bovine mammary tissue and evaluate the effect of different amino acids patterns on synthesis of as-casein in cultured mammary tissue. The following series of experiments were performed:(1) Establishment of Western-blotting method for detection of as-casein (Expt 1); (2) Effect of lysine(Lys), methionine(Met), threonine(Thr) and phenylalanine (Phe) on synthesis ofαs-casein in cultured mammary tissue (Expt 2); (3) Effect of proportion of Met and methionylmethionine(Met-Met) on synthesis of as-casein in cultured mammary tissue (Expt 3); and (4) Effect of pattern of amino acids on synthesis of as-casein in cultured mammary tissue (Expt 4).In Expt 1, mammary tissue from dairy cows was cultured in stationary culture (24 well plastic culture plate). Explants were incubated at 37℃(95% air; 5% CO2) in DMEM/F12 medium containing 2μg/ml hydrocortisone,5μg/ml transferrin, 10μg/ml insulin,5μg/ml prolactin and 10% FCS. The mammary tissue did not attach entirely to the bottom of culture plate until 1d. Based on the MTT value, it was indicated that the proliferated ability of cultured mammary tissue was stable after 4d.The method to detect as-casein in the tissue culture was established. The bovine as-casein was used to immunize the New Zealand Rabbits, and the antibody of as-casein was obtained. It was observed that the efficiency of membrance transfer was higher by wet method than by semi-dry method.Expt 2 was carried out to study the effects of different concentration of Lys, Met, Thr and Phe on synthesis of as-casein in cultured mammary tissue. The medium modified by Lys, Met, Thr and Phe consisted of Lys at 120,145,170,195,220,245 and 270μg/ml, Met at 50,60,70,80,90,100 and 110μg/ml, The at 73,90,105,120, 135,150 and 165μg/ml and Phe at 75,90,105,120,135,150 and 165μg/ml, respectively. The synthesis of as-casein was significantly high with adding level of Lys at 195μg/ml (P＜0.01), Met from 80 to 100μg/ml (P＜0.01), Thr at 118 and 133μg/ml (P＜0.01), and Phe from 135 to 165μg/ml (P＜0.01), respectively. The optimal adding level of Lys, Met, Thr and Phe was estimated to be at 203,81,118 and 137μg/ml, respectively.Expt 3 was conducted to investigate the effect of proportion of Met and Met-Met on synthesis ofαs-casein in cultured mammary tissue. The modified medium consisted of 50,67.5,85,102.5 and 120μg/ml of Met, accordingly supplemented with Met-Met at levels:70,52.5,35,17.5 and 0μg/ml, respectively. The synthesis ofαs-casein was significantly high with adding level of Met and Met-Met at 85 and 35μg/ml (P＜0.01). The optimal adding level of Met and Met-Met was estimated to be at 81 and 38μg/ml, respectively. It is inferred that the optimal proportion of Met and Met-Met in medium was at 2:1.In Expt 4, the different proportation of Lys, Met, Thr and Phe was designed according to the pattern of amino acids of milk protein, Western-blotting detection and abundance ofαs-casein mRNA, respectively. In Trial 1,the modified medium consisted of proportion of Lys (200μg/ml), Met, Thr and Phe at 100:34:56:63, 100:40:58:68 and 100:54:62:64, respectively. In Trial 2, replacing Met by Met-Met at the optimal proportion (2:1) made the modified medium consist of proportion of Lys, Met, Met-Met, Thr and Phe at 100:22.5:11.5:56:63,100:26.5:13.5:58:68 and 100:34.5:17.5:62:64. The synthesis ofαs-casein was significantly increased when proportion of Lys, Met, Thr and Phe was at 100:40:58:68(P＜0.01), suggesting that casein-based amino acid pattern resulted in optimalαs-casein synthesis. The synthesis ofαs-casein was significantly higher when Met was replaced by Met-Met(P＜0.01), and was highest when proportion of Lys, Met, Met-Met, Thr and Phe was at 100:22.5:11.5:56:63. From these results, it is indicated that mammary tissue can utilize Met-Met for milk protein synthesis with a higher efficiency than Met.In summary, mammary tissue cultured in stationary culture can grow and proliferate normally. The established Western-blotting method can be used for detection ofαs-casein in the medium. The optimal concentration of Lys, Met, Thr and Phe in medium was at 203,81,118 and 137μg/ml. Different proportion of Met and Met-Met can affect the synthesis of as-casein with the optimal proportion at 2:1. The optimal proportion of Lys, Met, Thr and Phe was at 100:40:58:68 and the optimal proportion of Lys, Met, Met-Met, Thr and Phe was at 100:22.5:11.5:56:63.