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Intracellular pteroylpolyglutamate hydrolase from human jejunal mucosa. Isolation and characterization
Abstract:
Human jejunal intracellular pteroylpolyglutamate hydrolase was purified 30-fold from intestinal mucosa. The apparent molecular weight of the enzyme was 75,000 by Sephadex G-200 gel filtration, and the isoelectric point was at pH 8.0. The enzyme was maximally active at pH 4.5 and was unstable at increasing temperatures. Intracellular pteroylpolyglutamate hydrolase cleaved both terminal and internal gamma-glutamate linkages. In contrast, brush-border pteroylpolyglutamate hydrolase catalyzed the hydrolysis of only terminal gamma-glutamate linkages. The intracellular enzyme showed greatest affinity for the complete folic acid molecule with longer glutamate chains. Subcellular fractionation studies showed the intracellular enzyme was localized in lysosomes. These data show that the properties of human jejunal intracellular pteroylpolyglutamate hydrolase are distinct from those of the brush-border enzyme but are similar to the properties of intracellular pteroylpolyglutamate hydrolase described in other tissues.
Insights
Human jejunal intracellular pteroylpolyglutamate hydrolase, an enzyme found in lysosomes, differs from the brush-border enzyme. It cleaves both internal and terminal glutamate linkages in folate polyglutamates.
Area of Science:
- Biochemistry
- Cell Biology
- Gastroenterology
Background:
- Folate metabolism is crucial for cellular functions.
- Pteroylpolyglutamate hydrolase enzymes play a key role in folate processing.
- Distinct intracellular and brush-border forms of this enzyme exist in the jejunum.
Purpose of the Study:
- To purify and characterize human jejunal intracellular pteroylpolyglutamate hydrolase.
- To compare its properties with the brush-border enzyme.
- To determine its subcellular localization.
Main Methods:
- Enzyme purification from intestinal mucosa.
- Biochemical assays for enzyme activity and substrate specificity.
- Sephadex G-200 gel filtration for molecular weight determination.
- Isoelectric focusing for isoelectric point determination.
- Subcellular fractionation.
Main Results:
- The intracellular enzyme was purified 30-fold.
- Apparent molecular weight: 75,000 Da; isoelectric point: pH 8.0.
- Optimal activity at pH 4.5; enzyme unstable at higher temperatures.
- Cleaved both terminal and internal gamma-glutamate linkages.
- Localized to lysosomes.
- Showed highest affinity for longer-chain folate polyglutamates.
Conclusions:
- Human jejunal intracellular pteroylpolyglutamate hydrolase has distinct biochemical properties compared to the brush-border enzyme.
- Its substrate specificity and cleavage pattern suggest a role in intracellular folate processing.
- Lysosomal localization supports its involvement in folate degradation or recycling pathways.