Purified human erythrocyte pyrroline-5-carboxylate reductase. Preferential oxidation of NADPH.
Pyrroline-5-carboxylate reductase catalyzes the final step in proline synthesis by NAD(P)H-dependent reduction of pyrroline-5-carboxylate. We have purified and characterized this enzyme from human erythrocytes. Purification to homogeneity (approximately 600,000-fold) was accomplished by sonication, ultracentrifugation, 2',5'-ADP-Sepharose affinity chromatography, and DEAE-Sephacel ion exchange chromatography. The enzyme runs as a single band of 30,000 Mr on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Sizing chromatography under nondenaturating conditions demonstrates activity in the 300,000-350,000 Mr range, suggesting that the native enzyme exists as a 10- to 12-mer. The purified enzyme exhibits kinetic characteristics similar to those previously described for whole red cell homogenates. The Vmax is 10-fold higher and the Km for pyrroline-5-carboxylate is 7-fold higher with NADH versus NADPH as cofactor. The affinity for NADPH is 15-fold higher than that for NADH. Erythrocyte pyrroline-5-carboxylate reductase is competitively inhibited by NADP+. Unlike the enzyme from some other sources, erythrocyte pyrroline-5-carboxylate reductase is not inhibited by proline or ATP. Double label studies using [14C]pyrroline-5-carboxylate and [3H]exNADPH in the presence of both NADH and NADPH were performed to determine the preferred source of reducing equivalents. In the presence of physiologic concentrations of pyrroline-5-carboxylate and both pyridine nucleotides, all of the reducing equivalents came from NADPH. We suggest that, in some cell types including human erythrocytes, a physiologic function of pyrroline-5-carboxylate reductase is the generation of NADP+.
Merrill
M J
MJ
Endocrinology Section, National Cancer Institute, Bethesda, Maryland 20892.
Yeh
G C
GC
Phang
J M
JM
D050842:1-Pyrroline-5-Carboxylate Dehydrogenase; D004789:Enzyme Activation; D004912:Erythrocytes / Q000201:enzymology*; D006801:Humans; D007700:Kinetics; D008970:Molecular Weight; D009243:NAD / Q000378:metabolism / Q000502:physiology; D009249:NADP / Q000378:metabolism* / Q000502:physiology; D010084:Oxidation-Reduction; D000587:Oxidoreductases Acting on CH-NH Group Donors / Q000097:blood* / Q000302:isolation & purification / Q000502:physiology; D011392:Proline / Q000096:biosynthesis / Q000302:isolation & purification
D009243:NAD; D009249:NADP; D011392:Proline; D050842:1-Pyrroline-5-Carboxylate Dehydrogenase; C494240:ALDH4A1 protein, human; D000587:Oxidoreductases Acting on CH-NH Group Donors