Purification from rat liver of a novel constitutively expressed member of the aldo-keto reductase 7 family that is widely distributed in extrahepatic tissues.
Antiserum raised against human aflatoxin B(1) aldehyde reductase 1 (hAFAR1) has been used to identify a previously unrecognized rat aldo-keto reductase (AKR). This novel enzyme is designated rat aflatoxin B(1) aldehyde reductase 2 (rAFAR2) and it characteristically migrates faster during SDS/PAGE than does the archetypal ethoxyquin-inducible rAFAR protein (now called rAFAR1). Significantly, rAFAR2 is essentially unreactive with polyclonal antibodies raised against rAFAR1. Besides its distinct electrophoretic and immunochemical properties, rAFAR2 appears to be regulated differently from rAFAR1 as it is expressed in most rat tissues and does not appear to be induced by ethoxyquin. Multiple forms of rAFAR2 have been identified. Anion-exchange chromatography on Q-Sepharose, followed by adsorption chromatography on columns of Matrex Orange A and Cibacron Blue, have been employed to purify rAFAR2 from rat liver cytosol. The Q-Sepharose chromatography step resulted in the resolution of rAFAR2 into three peaks of AKR activity, two of which were purified and shown to be capable of catalysing the reduction of 2-carboxybenzaldehyde, succinic semialdehyde, 4-nitrobenzaldehyde and 9,10-phenathrenequinone. The two most highly purified rAFAR2-containing preparations eluted from the Cibacron Blue column were 91 and 98% homogeneous. Analysis of these by SDS/PAGE indicated that the least anionic (peak CBA5) comprised a polypeptide of 37.0 kDa, whereas the most anionic (peak CBA6) contained two closely migrating polypeptides of 36.8 and 37.0 kDa; by contrast, in the present study, rAFAR1 was estimated by SDS/PAGE to be composed of 38.0 kDa subunits. Final purification of the 37 kDa polypeptide in CBA5 and CBA6 was accomplished by reversed-phase HPLC. Partial proteolysis of the two preparations of the 37 kDa polypeptide with Staphylococcus aureus V8 protease yielded fragments of identical size, suggesting that they represent the product of a single gene. Furthermore, the peptide maps from CBA5 and CBA6 differed substantially from that yielded by rAFAR1, indicating that they are genetically distinct from the inducible reductase. A peptide generated by CNBr digestion of the 37 kDa polypeptide from CBA6 was shown by Edman degradation to share 88% sequence identity with residues Tyr(168)-Leu(183) of rAFAR1. This provides evidence that the rat protein identified by its cross-reactivity with anti-hAFAR1 serum is an additional member of the AKR7 family.
Kelly
V P
VP
Biomedical Research Centre, Ninewells Hospital and Medical School, University of Dundee, Dundee DD1 9SY, Scotland, U.K.
Ireland
L S
LS
Ellis
E M
EM
Hayes
J D
JD
D000449:Aldehyde Reductase / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D003600:Cytosol / Q000201:enzymology; D005260:Female; D006801:Humans; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008114:Liver Neoplasms, Experimental / Q000201:enzymology; D008297:Male; D008969:Molecular Sequence Data; D009928:Organ Specificity; D010446:Peptide Fragments / Q000737:chemistry; D051381:Rats; D011916:Rats, Inbred F344; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D014407:Tumor Cells, Cultured
D007527:Isoenzymes; D010446:Peptide Fragments; D011994:Recombinant Proteins; D000449:Aldehyde Reductase