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Membrane-bound phenylalanine hydroxylase of human liver
V V Chestkov1, A V Laptev, S S Shishkin
1Institute of Medical Genetics, Academy of Medical Sciences, Moscow, Russia.
Abstract:
Phenylalanine hydroxylase (EC 1.14.16.1) antigen and activity have been identified among proteins extracted with a buffer containing 0.4% Triton X-100 from adult human liver bioptate fraction, which was sedimented at 105,000 x g (n = 4). This enzyme fraction was designated as a 'membrane-bound form of phenylalanine hydroxylase'. It amounted to 5-15% of phenylalanine hydroxylase activity and 15-25% of phenylalanine hydroxylase antigen content. After immunoblotting two-dimensional gels, the soluble (cytoplasmic) form of phenylalanine hydroxylase antigen displayed three spots: one spot corresponded to the L-subunit with a molecular weight of 55,000, the two other spots corresponded to the H-subunit with a molecular weight of 57,000. Only the L-subunit was revealed in the membrane-bound enzyme form. Both phenylalanine hydroxylase activity and antigen were also demonstrated in extracts from human embryonic livers (n = 7). However, in this case the membrane-bound phenylalanine hydroxylase amounted to 85% of the antigen content. Subunit compositions of the enzymes were similar in adult and embryonic livers. The differences in the subunit compositions and enzyme activities of membrane-bound and cytoplasmic forms of phenylalanine hydroxylase in adults and embryos may be due to other functions of this enzyme in the hepatocyte membrane.
Insights
A membrane-bound form of phenylalanine hydroxylase was identified in human liver, differing in subunit composition and activity between adults and embryos. This suggests distinct roles for the enzyme in different developmental stages.
Area of Science:
- Biochemistry
- Molecular Biology
- Cell Biology
Background:
- Phenylalanine hydroxylase (PAH) is crucial for phenylalanine metabolism.
- The enzyme's localization and forms in human liver, particularly during development, are not fully understood.
Purpose of the Study:
- To investigate the presence and characteristics of membrane-bound phenylalanine hydroxylase in adult and embryonic human liver.
- To compare the subunit composition and activity of soluble and membrane-bound PAH forms.
Main Methods:
- Proteins were extracted from human liver biopsies using Triton X-100 and ultracentrifugation.
- Enzyme activity and antigen content were quantified.
- Immunoblotting and two-dimensional gel electrophoresis were used to analyze subunit composition.
Main Results:
- A membrane-bound form of PAH was identified in adult human liver, constituting 5-15% of activity and 15-25% of antigen.
- The soluble PAH form showed L- and H-subunits (55,000 and 57,000 MW), while the membrane-bound form contained only the L-subunit.
- In embryonic livers, membrane-bound PAH was predominant (85% of antigen), with similar subunit compositions to adult livers.
- Differences in subunit composition and activity between membrane-bound and cytoplasmic forms were observed between adult and embryonic livers.
Conclusions:
- A distinct membrane-bound form of phenylalanine hydroxylase exists in human liver.
- Developmental differences in PAH localization and activity suggest alternative functions in the hepatocyte membrane.
- Further research is needed to elucidate the specific roles of membrane-bound PAH in adult and embryonic hepatocytes.