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Changes of the quaternary structure of microvillus aminopeptidase in the membrane
Abstract:
Microvillus aminopeptidase (EC 3.4.11.2) is an enzyme with a molecular weight around 300 000. Normal preparations contain three different subunits (subunit A, Mr 162 000; subunit B, Mr 123 000; subunit C, Mr 61 000). The relationship between the three subunits was studied by immunoelectrophoresis using specific antibodies against individual denatured subunits and by densitometric scanning of polyacrylamide gels after separation of the three subunits. The results suggest that microvillus aminopeptidase initially appears in the membrane as a symmetric molecule built up to two identical A subunits. These subunits are then split into equimolar amounts of subunit B and subunit C by trypsin. Subunit B cannot generate subunit C but may be further degraded. The reaction sequence described is one which occurs in vivo. Treatment of purified aminopeptidase with trypsin increases the specific activity twofold. This phenomenon does not seem to be correlated to the generation of subunit B and subunit C or to the transformation of amphiphilic form into hydrophilic form.
Insights
Microvillus aminopeptidase, an enzyme, is initially a dimer of subunit A. Trypsin cleaves subunit A into subunits B and C in vivo, affecting enzyme activity and structure.
Area of Science:
- Biochemistry
- Enzymology
- Molecular Biology
Background:
- Microvillus aminopeptidase (EC 3.4.11.2) is a large enzyme (approx. 300,000 MW) found in normal preparations.
- It consists of three distinct subunits: A (162,000 MW), B (123,000 MW), and C (61,000 MW).
Purpose of the Study:
- To elucidate the structural relationship between the three subunits of microvillus aminopeptidase.
- To understand the in vivo processing and activation of this enzyme.
Main Methods:
- Immunoelectrophoresis with specific antibodies against denatured subunits.
- Densitometric scanning of polyacrylamide gel electrophoresis (PAGE) separations.
Main Results:
- Microvillus aminopeptidase initially exists as a symmetric dimer of two identical subunit A molecules.
- Trypsin cleaves subunit A into equimolar amounts of subunit B and subunit C.
- Subunit B can be further degraded but does not generate subunit C.
- This cleavage process occurs in vivo.
- Trypsin treatment doubles the specific activity of purified aminopeptidase, independent of subunit generation or amphiphilic/hydrophilic transformation.
Conclusions:
- The study proposes a sequential model for microvillus aminopeptidase processing, starting from a subunit A dimer.
- Trypsin-mediated cleavage is a key step in generating active enzyme forms in vivo.
- The observed increase in specific activity upon trypsin treatment requires further investigation beyond subunit composition changes.