Sylvia J. Downing

Enzymatic synthesis and purification of L-pyrroline-5-carboxylic acid.

Abstract Δ 1 -Pyrroline-5-carboxylic acid (P5C) is an intermediate in the metabolism of proline, ornithine, and glutamic acid. It has been obtainable by chemical synthesis only as a mixture of the d - and l -stereoisomers. We report a method for the enzymatic synthesis of P5C on a preparative scale. The P5C is formed from l -ornithine by purified ornithine aminotransferase and then isolated by Dowex-50W cation-exchange resin chromatography. The purified compound exists entirely as the biologically active l -stereoisomer. With [ 14 C]ornithine as precursor, high specific activity uniformly labeled [ 14 C]P5C can be obtained.

Stimulation of the hexose-monophosphate pentose pathway by Δ1-pyrroline-5-carboxylic acid in human fibroblasts

Abstract L-pyrroline-5-carboxylic acid, an intermediate in the interconversions of glutamic acid, ornithine and proline, is a potent stimulator of the hexose-monophosphate pentose pathway in cultured human fibroblasts. These studies suggest that pyrroline-5-carboxylate reductase, which catalyzes the conversion of pyrroline-5-carboxylate to proline coupled with the oxidation of NADPH, provides the NADP for the observed activation of the hexose-monophosphate pentose pathway.

A radioisotopic assay for ornithine-δ-transaminase

Abstract We describe a radioisotopic assay for ornithine-δ-transaminase using precursor ornithine-U-14C. We quantitate the product Δ1-pyrroline-5-carboxylate-14C by cation-exchange chromatography. The sensitivity of the method allows for measurement of enzyme activity in extracts prepared from 105 mammalian cells.

Linkage of the HMP pathway to ATP generation by the proline cycle

Abstract The reactions catalyzed by proline oxidase and pyrroline-5-carboxylate reductase form a catalytic cycle linking the hexose-monophosphate pentose (HMP) pathway to mitochondrial ATP generation. The cycling of proline and pyrroline-5-carboxylate couples glucose oxidation to ATP generation by a mechanism independent of the Embden-Meyerhof pathway and the tricarboxylic acid cycle.

A radioisotopic assay for Δ1-pyrroline-5-carboxylate reductase

Abstract We describe a radioisotopic assay for Δ 1 -pyrroline-5-carboxylate reductase. In this assay we use Δ 1 -pyrroline-5-carboxylate[U- 14 C] and isolate product l -[U- 14 C]proline by cation-exchange column chromatography.

Proline inhibition of pyrroline-5-carboxylate reductase: differences in enzymes obtained from animal and tissue culture sources.

Abstract Pyrroline-5-carboxylate reductase obtained from several cell lines in culture has been shown to be approximately 50X more sensitive to proline inhibition than enzyme obtained from animal tissue sources. The description of this inhibition and possible causes for the difference between animal and tissue culture pyrroline-5-carboxylate reductase are presented.

Proline biosynthesis: Multiple defects in Chinese hamster ovary cells

Abstract The biochemical basis for the proline requirement of Chinese hamster ovary cells has been investigated. The results indicate an absence of conversion of glutamate to proline and a 90% reduction in ornithine transaminase, the first enzyme in the pathway converting ornithine to proline. Possible explanations for this double defect are discussed.

Amino acid transport in adult diaphyseal bone contrast with amino acid transport mechanisms in fetal membranous bone

Abstract 1. 1. This report describes a system for in vitro study of amino acid transport in intact sections of adult rat diaphyseal bone. 2. 2. Kinetic analysis of amino acid transport indicates a common transport mechanism for neutral amino and imino acids in adult diaphyseal bone. This is in contrast to evidence for separate transport mechanisms for neutral amino and imino acids in fetal rat calvaria. 3. 3. Removal of Na + from the incubation medium decreases but does not abolish transport of both neutral amino and imino acids in adult rat diaphysis. 4. 4. The implications of differences in transport mechanisms between fetal membranous and adult diaphyseal bone are discussed.