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Pyrroline-5-carboxylate synthesis from glutamate by rat intestinal mucosa
Abstract:
The mitochondria of rat intestinal mucosa were found to have an enzymatic activity that converts radioactive glutamate to pyrroline-5-carboxylate (P5C) in the presence of ATP, NADPH, and MgCl2. The product of this enzyme was identified as P5C by the fact that it was converted to proline by chemical reduction with NaBH4 or by enzymatic reduction with NADH in the presence of purified yeast P5C reductase. The product was demonstrated to be P5C rather than pyrroline-2-carboxylate by thin layer chromatography. The presence of the activity in mitochondria prepared from intestinal mucosa of germ-free rats proved that this activity is of mammalian origin. Omission of either ATP, NADPH, or MgCl2 from the reaction mixture resulted in little or no activity. The optimal pH appeared to be about 7.0 under the conditions used. Substrate saturation curves in the presence of an ATP and an NADPH regeneration system gave apparent Km values of 2.5 mM for glutamate, 0.19 mM for ATP, and 6.5 microM for NADPH in the presence of 20 mM MgCl2. The mitochondrial preparation usually produced P5C at a rate of 1.2 to 1.6 nmol/mg/min at 20 degrees C when incubated with 1 mM glutamate, 3 mM ATP, 0.2 mM NADPH, and 20 mM MgCl2.
Insights
Mitochondria in rat intestinal lining possess an enzyme that converts glutamate to pyrroline-5-carboxylate (P5C). This discovery confirms mammalian origin for this crucial metabolic pathway.
Area of Science:
- Biochemistry
- Cell Biology
- Metabolic Pathways
Background:
- Mitochondria are vital organelles involved in cellular metabolism.
- The conversion of glutamate to proline is a key metabolic process.
- Understanding the cellular location and origin of enzymes is crucial for metabolic research.
Purpose of the Study:
- To identify and characterize enzymatic activity in rat intestinal mucosa mitochondria.
- To determine the substrate and product of this mitochondrial enzymatic activity.
- To confirm the mammalian origin of the observed enzymatic activity.
Main Methods:
- Incubation of rat intestinal mucosa mitochondria with radioactive glutamate, ATP, NADPH, and MgCl2.
- Identification of the product (pyrroline-5-carboxylate, P5C) via chemical and enzymatic reduction to proline.
- Thin-layer chromatography to distinguish P5C from pyrroline-2-carboxylate.
- Enzyme assays using mitochondria from germ-free rats to establish mammalian origin.
- Determination of optimal pH, substrate saturation kinetics (Km values), and reaction rates.
Main Results:
- Mitochondria from rat intestinal mucosa exhibit enzymatic activity converting glutamate to P5C.
- The reaction requires ATP, NADPH, and MgCl2, with optimal activity around pH 7.0.
- Apparent Km values were determined for glutamate (2.5 mM), ATP (0.19 mM), and NADPH (6.5 microM).
- Activity was confirmed in germ-free rat mitochondria, indicating mammalian origin.
- The mitochondrial preparation produced P5C at rates of 1.2–1.6 nmol/mg/min.
Conclusions:
- Rat intestinal mucosa mitochondria possess a distinct enzymatic activity responsible for P5C synthesis from glutamate.
- This pathway is of mammalian origin and requires specific cofactors (ATP, NADPH, MgCl2).
- The characterized kinetic parameters provide insights into the enzyme's function and regulation within intestinal mitochondria.