Pyrroline 5-carboxylate dehydrogenase of the mitochondrial matrix of rat liver. Purification, physical and kinetic characteristics.
The oxidation of proline to glutamate in mitochondria requires two enzymes, proline oxidase and pyrroline 5-carboxylate (P5C) dehydrogenase. In this paper we report an 800-fold purification P5C dehydrogenase from rat liver mitochondria to yield an essentially homogenous protein. The protein, whose Mr is 59,000, is an alpha 2 dimer (Mr = 115,000) in solution with an isoionic point at pH 5.7. The substrates P5C and NAD+ have apparent dissociation constants of 0.16 and 1.0 mM, respectively. Studies have been conducted to see if the conversion of glutamate and NADH to P5C and NAD+ is catalyzed by this enzyme. These studies have established that if the reverse reaction occurs the rate is 1/15,000th of the rate at which P5C is oxidized to glutamate. The concentration of the substrates needed in the assay results in a high background that interferes with accurate spectrophotometric analysis of the rate of NADH production; therefore a radiochemical (2) or a new colorimetric (3) assay was used here. A number of aldehydes were tested as substrates. It was found that the rat and human enzymes (4) have similar requirements for an aldehyde to be a substrate. Both of these proteins interacted with a polyclonal rabbit anti-rat P5C dehydrogenase serum.
Small
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Department of Biochemistry and Nutrition, School of Medicine, University of North Carolina, Chapel Hill 27599-7260.
Jones
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ME
D050842:1-Pyrroline-5-Carboxylate Dehydrogenase; D000818:Animals; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008930:Mitochondria, Liver / Q000201:enzymology*; D008970:Molecular Weight; D000587:Oxidoreductases Acting on CH-NH Group Donors / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism; D051381:Rats; D011919:Rats, Inbred Strains; D013057:Spectrum Analysis; D013379:Substrate Specificity; D014018:Tissue Distribution
D050842:1-Pyrroline-5-Carboxylate Dehydrogenase; D000587:Oxidoreductases Acting on CH-NH Group Donors