Alternate putrescine metabolites: quantitative analysis of delta'-pyrroline oxidation to 2-pyrrolidone in tissue homogenates by high-pressure liquid chromatography.
A sensitive analytical procedure for following the oxidation of delta'-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 delta'-pyrroline. Phenazine methosulfate and reduced glutathione 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 delta'-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 delta'-pyrroline to 2-pyrrolidone was localized in the cellular cytosol. These results demonstrate that the oxidation of delta'-pyrroline to 2-pyrrolidone is enzyme mediated and provide a useful method for further characterization of this metabolic step.
D000818:Animals; D002851:Chromatography, High Pressure Liquid; D005260:Female; D007700:Kinetics; D008099:Liver / Q000201:enzymology / Q000378:metabolism*; D009928:Organ Specificity; D010084:Oxidation-Reduction; D011758:Pyrroles / Q000378:metabolism*; D011760:Pyrrolidinones / Q000096:biosynthesis*; D051381:Rats; D011919:Rats, Inbred Strains; D013347:Subcellular Fractions / Q000201:enzymology
D011758:Pyrroles; D011760:Pyrrolidinones; C025565:delta(1)-pyrroline; C028537:2-pyrrolidone