大鼠肝脏组氨酸-丙酮酸转氨酶同工酶的纯化、表征及鉴定

Purification, characterization and identification of rat liver histidine-pyruvate aminotransferase isoenzymes.

作者信息

Noguchi T, Okuno E, Minatogawa Y, Kido R

出版信息

Biochem J. 1976 Apr 1;155(1):107-15. doi: 10.1042/bj1550107.

DOI:10.1042/bj1550107

PMID:938469

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC1172807/

Abstract

Histidine-pyruvate aminotransferase (isoenzyme 1) was purified to homogeneity from the mitochondrial and supernatant fractions of rat liver, as judged by polyacrylamide-gel electrophoresis and isolectric focusing. Both enzyme preparations were remarkably similar in physical and enzymic properties. Isoenzyme 1 had pI8.0 and a pH optimum of 9.0. The enzyme was active with pyruvate as amino acceptor but not with 2-oxoglutarate, and utilized various aromatic amino acids as amino donors in the following order of activity: phenylalanine greater than tyrosine greater than histidine. Very little activity was found with tryptophan and 5-hydroxytryptophan. The apparent Km values were about 2.6mM for histidine and 2.7 mM for phenylalanine. Km values for pyruvate were about 5.2mM with phenylalanine as amino donor and 1.1mM with histidine. The aminotransferase activity of the enzyme towards phenylalanine was inhibited by the addition of histidine. The mol.wt. determined by gel filtration and sucrose-density-gradient centrifugation was approx. 70000. The mitochondrial and supernatant isoenzyme 1 activities increased approximately 25-fold and 3.2-fold respectively in rats repeatedly injected with glucagon for 2 days. 2. An additional histidine-pyruvate aminotransferase (isoenzyme 2) was partially purified from both the mitochondrial and supernatant fractions of rat liver. Nearly identical properties were observed with both preparations. Isoenzyme 2 had pI5.2 and a pH optimum of 9.3. The enzyme was specific for pyruvate and did not function with 2-oxoglutarate. The order of effectiveness of amino donors was tyrosine = phenylalanine greater than histidine greater than tryptophan greater than 5-hydroxytryptophan. The apparent Km values for histidine and phenylalanine were about 0.51 and 1.8 mM respectively. Km values for pyruvate were about 3.5mM with phenylalanine and 4.7mM with histidine as amino donors. Histidine inhibited phenylalanine aminotransferase activity of the enzyme. Gel filtration and sucrose-density-gradient centrifugation yielded a mol.wt. of approx. 90000. Neither the mitochondrial nor the supernatant isoenzyme 2 activity was elevated by glucagon injection.

摘要

通过聚丙烯酰胺凝胶电泳和等电聚焦判断，从大鼠肝脏的线粒体和上清液部分将组氨酸 - 丙酮酸氨基转移酶（同工酶1）纯化至同质。两种酶制剂在物理和酶学性质上非常相似。同工酶1的pI为8.0，最适pH为9.0。该酶以丙酮酸作为氨基受体时有活性，但以2-氧代戊二酸为底物时无活性，并且利用各种芳香族氨基酸作为氨基供体，其活性顺序为：苯丙氨酸＞酪氨酸＞组氨酸。色氨酸和5-羟色氨酸的活性极低。组氨酸的表观Km值约为2.6mM，苯丙氨酸约为2.7mM。以苯丙氨酸作为氨基供体时，丙酮酸的Km值约为5.2mM；以组氨酸作为氨基供体时，丙酮酸的Km值约为1.1mM。添加组氨酸会抑制该酶对苯丙氨酸的氨基转移酶活性。通过凝胶过滤和蔗糖密度梯度离心测定的分子量约为70000。在反复注射胰高血糖素2天的大鼠中，线粒体和上清液中的同工酶1活性分别增加了约25倍和3.2倍。2. 从大鼠肝脏的线粒体和上清液部分又部分纯化了另一种组氨酸 - 丙酮酸氨基转移酶（同工酶2）。两种制剂观察到几乎相同的性质。同工酶2的pI为5.2，最适pH为9.3。该酶对丙酮酸具有特异性，对2-氧代戊二酸不起作用。氨基供体的有效性顺序为：酪氨酸 = 苯丙氨酸＞组氨酸＞色氨酸＞5-羟色氨酸。组氨酸和苯丙氨酸的表观Km值分别约为0.51和1.8mM。以苯丙氨酸作为氨基供体时，丙酮酸的Km值约为3.5mM；以组氨酸作为氨基供体时,丙酮酸的Km值约为4.7mM。组氨酸抑制该酶的苯丙氨酸氨基转移酶活性。凝胶过滤和蔗糖密度梯度离心得到的分子量约为90000。注射胰高血糖素后，线粒体和上清液中的同工酶2活性均未升高。