Alternate putrescine metabolites: Quantitative analysis of Δ′-pyrroline oxidation to 2-pyrrolidone in tissue homogenates by high-pressure liquid chromatography
✍ Scribed by David W. Lundgren; Holly Tevesz; Elisa A. Krill
- Book ID
- 102988346
- Publisher
- Elsevier Science
- Year
- 1985
- Tongue
- English
- Weight
- 846 KB
- Volume
- 148
- Category
- Article
- ISSN
- 0003-2697
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✦ Synopsis
A sensitive analytical procedure for following the oxidation of A'-pyrroline to 2-pyrrolidone in tissue homogenates is described. Homogenates are extracted with chloroform/acetonitrile and fractionated by high-performance liquid chromatography, and 2-pyrrolidone is quantitated by monitoring the column effluent at 200 nm. The lower limit of 2-pyrrolidone that can be accurately (+5%) quantitated is approximately 100 pmol. Phenazine methosulfate significantly enhances the rate of 2-pyrrolidone biosynthesis from A'-pyrroline. Phenazine methosulfate and reduced gfutathione are required to obtain proportionality between 2-pyrrolidone formation and incubation time. Formation of 2-pyrrolidone as a function of protein concentration is linear and 2-pyrrolidone biosynthesis as a function of A'-pyrroline concentration is characterized by hyperbolic kinetics. Based on analysis of enzyme activity in different tissues, liver appears to play the dominant role in 2-pyrrolidone biosynthesis. The metabolic step from A'-pyrroline to 2-pyrrolidone was localized in the cellular cytosol. These results demonstrate that the oxidation of A'-pyrroline to 2-pyrrolidone is enzyme mediated and provide a useful method for further characterization of this metabolic step. o 1985 Academic PI~SS, h.