绵羊瘤胃细菌、原虫蛋白质分解代谢相关酶活力及谷氨酸脱氢酶体系的米氏常数值

Enzyme Activities Related to Protein Catabolism and KmValue of Glutamate Dehydrogenase System in Rumen Bacteria and Protozoa of Sheep

本试验旨在研究绵羊瘤胃细菌、原虫蛋白质分解代谢相关酶活力及谷氨酸脱氢酶体系的米氏常数(Km)值,为解释绵羊瘤胃细菌、原虫蛋白质分解代谢特征提供酶学依据。选用6只1岁左右安装永久性瘤胃瘘管的中国美利奴(新疆型)绵羊[平均体重为(32.00±1.36)kg],饲喂精粗比为30∶70的饲粮,依次采集饲喂前(0 h)和饲喂后1.5、3.0、6.0、9.0、12.0 h 6个时间点的瘤胃液,重复采集3次。分离和制备细菌、原虫破碎液,分别测定相关酶活力及谷氨酸脱氢酶体系的Km值。结果显示:1)绵羊瘤胃细菌、原虫破碎液中蛋白酶、谷丙转氨酶、谷草转氨酶和谷氨酸脱氢酶的活力随饲喂时间的延长均呈现先升高后降低的动态变化规律,总体在饲喂后1.5 h达到峰值;谷氨酸和氨含量也呈现相似的变化规律。原虫破碎液中参与蛋白质分解代谢的这4种酶的活力在各时间点均极显著高于细菌(P<0.01)。2)原虫破碎液谷氨酸含量极显著高于细菌(P<0.01);原虫破碎液氨含量在1.5、6.0、9.0和12.0 h显著或极显著高于细菌(P<0.05或P<0.01)。3)绵羊瘤胃细菌、原虫谷氨酸脱氢酶对烟酰胺腺嘌呤二核苷酸(NAD)的Km值分别为2.60×10^(-7)、1.48×10^(-7)mol/L;细菌、原虫谷氨酸脱氢酶对谷氨酸的Km值分别为8.41×10^(-6)、4.91×10^(-6)mol/L;细菌、原虫谷氨酸脱氢酶对还原型烟酰胺腺嘌呤二核苷酸(NADH)的Km值分别为3.80×10^(-8)、2.70×10^(-8)mol/L;细菌、原虫谷氨酸脱氢酶对α-酮戊二酸的Km值分别为1.16×10^(-6)、2.07×10^(-6)mol/L;细菌、原虫谷氨酸脱氢酶对氨的Km值分别为2.97×10^(-5)、1.40×10^(-5)mol/L。结果提示,总体上,绵羊瘤胃细菌、原虫中蛋白酶、谷氨酸脱氢酶、谷丙转氨酶、谷草转氨酶的活力在饲喂后1.5 h达到峰值,之后逐渐降低;绵羊瘤胃原虫中蛋白酶、谷丙转氨酶、谷草转氨酶和谷氨酸脱氢酶的活力均极显著高于细菌,原虫中蛋白质分解代谢更旺盛;瘤胃原虫中不仅存在谷氨酸转氨机制,还可能存在利用氨重新合成氨基酸的机制。

This study was to conducted to determine enzyme activities related to protein catabolism and Kmvalue of glutamate dehydrogenase（ GDH） system in rumen bacteria and protozoa of sheep,to provide enzymology reference to explain protein catabolism in rumen bacteria and protozoa of sheep. Six 1-year-old healthy Chinese merino sheep（ Xinjiang type） [average body weight was（ 32. 00 ± 1. 36） kg] were chosen. The sheep were installed with permanent fistula in rumen. Dietary forage to concentrate ratio was 30∶70. Rumen fluid was collected sequentially before feeding（ 0 h） and 1. 5,3. 0,6. 0,9. 0 and 12. 0 h after feeding,respectively,which were repeated 3 times. After centrifugation,crushing liquid of rumen bacteria and protozoa was prepared to determine the activities of enzymes and Km values of GDH system. The results showed as follows： 1） the activities of protease,glutamic-pyruvic transaminase（ GPT）,glutamic oxal（ o） acetic transaminase（ GOT）and GDH showed an initial increase and then a decrease as the feeding time passed by,and generally reached the maximum at 1.5 h after feeding in rumen bacteria and protozoa crushing liquid of sheep; the contents of glutamate and ammonia（ NH＋4） contents showed similar tendency. The activities of the four enzymes involved in protein metabolism in rumen protozoa crushing liquid were significantly higher than those in rumen bacteria（ P〈0.01）. 2） Glutamate content of rumen protozoa crushing liquid was significantly higher than that of bacteria（ P〈 0.01）. Ammonia content of rumen protozoa crushing liquid was significantly higher than that of bacteria at 1.5,6.0,9.0 and 12.0 h（ P〈 0.05 or P〈 0.01）. 3） Kmvalues of GDH to NAD in bacteria and protozoa crushing liquid were 2.60 × 10-7and 1.48 × 10-7mol/L,respectively; those to glutamate in bacteria and protozoa crushing liquid were 8.41 × 10-6and 4.91 × 10-6mol/L,respectively; those to NADH in bacteria and protozoa crushing liquid were 3. 80 × 10-8and 2. 70 × 10-8mol/L,respectively; those to α-oxoglutarate in bacteria and protozoa crushing liquid were 1.16 × 10-6and 2.07 × 10-6mol/L,respectively; those to ammonia in bacteria and protozoa crushing liquid were 2.97 × 10-5and 1.40 × 10-5mol/L,respectively. The results indicate that the activities of protease,GPT,GOT and GDH generally reach the maximum at 1.5 h after feeding,then gradually decrease in rumen bacteria and protozoa of sheep; meanwhile,the enzyme activities in protozoa are significantly higher than those in bacteria,and protein catabolism in protozoa is more active than in bacteria; there is not only a transamination mechanism of glutamate,but also a re-using ammonia mechanism for synthesis of amino acids in rumen protozoa.