Chestkov V V, Laptev A V, Shishkin S S
Biokhimiia. 1988 Feb;53(2):322-7.
Phenylalanine hydroxylase was detected among human liver bioptats and autoptats extracted with 0.4% Triton X-100 from the 105,000 g homogenate fraction. In contrast to the membrane form of the rat liver enzyme, human liver phenylalanine hydroxylase is detected both by its enzymatic activity and immunochemically under non-denaturating conditions. The enzymatic activity of phenylalanine hydroxylase makes 5-15% of that of the cytoplasmic fraction and 20-30% of the amount of antigen in the cytoplasmic fraction and 20-30% of the amount of antigen in the cytoplasmic fraction as can be evidenced from rocket immunoelectrophoresis data. Immunoblotting of proteins performed after denaturating electrophoresis of the membrane and cytoplasmic fractions revealed an antigen band with a similar electrophoretic mobility. The subunit composition of the enzyme in both fractions was characterized by two-dimensional electrophoresis with subsequent immunoblotting. It was found that the membrane fraction of the enzyme is represented only by the L-subunit with Mr of 55 kD, whereas the cytoplasmic fraction, besides the predominant L-subunit, also contains 2H-subunits of the enzyme with Mr = 57 kD. These 2H-subunits differ between themselves as well as from the L-subunit by the pI value.
在用0.4% Triton X-100从105,000 g匀浆组分中提取的人肝脏活检组织和尸检组织中检测到了苯丙氨酸羟化酶。与大鼠肝脏酶的膜形式不同，人肝脏苯丙氨酸羟化酶在非变性条件下通过其酶活性和免疫化学方法均可检测到。从火箭免疫电泳数据可以看出，苯丙氨酸羟化酶的酶活性占细胞质组分酶活性的5 - 15%，占细胞质组分抗原量的20 - 30%。对膜组分和细胞质组分进行变性电泳后进行蛋白质免疫印迹分析，发现一条具有相似电泳迁移率的抗原带。通过二维电泳及随后的免疫印迹分析对两个组分中酶的亚基组成进行了表征。结果发现，酶的膜组分仅由分子量为55 kD的L亚基组成，而细胞质组分除了主要的L亚基外，还含有分子量为57 kD的该酶的2个H亚基。这2个H亚基在自身之间以及与L亚基之间在等电点值上存在差异。
