Purification and characterization of two aldose reductase isoenzymes from rabbit muscle

Using a modification of the procedure of Kormann et al. (Kormann, A. W., Hurst, R. O., and Flynn, T. G. (1972) Biochim. Biophys. Acta 258, 40-55) for the purification of glycerol dehydrogenase, two enzymes have been purified from the skeletal muscle of male rabbits. From a consideration of their properties these enzymes have been named aldose reductase 1 and aldose reductase 2, respectively. Both enzymes are monomeric by the criteria of gel filtration and polyacrylamide gel electrophoresis in sodium dodecyl sulfate and both reductases are immunologically identical as shown by double immunodiffusion and rocket immunoelectrophoresis. Aldose reductases 1 and 2 have almost identical amino acid compositions, their NH2 termini are blocked and the COOH termini of both enzymes are apparently identical. The enzymes differ, however, in molecular weight with aldose reductase 2 having Mr = 41,500 and aldose reductase 1 Mr 40,200. Both enzymes have the broad substrate specificity typical of the aldehyde reductase family of enzymes; Km values of aldose reductase 1 for aldo sugars were similar to those reported for rabbit lens aldose reductase, and both aldose reductase 1 and 2 were inhibited by the commercial aldose reductase inhibitors Alrestatin and Sorbinil. Two aldose reductases, immunologically and electrophoretically identical to the muscle enzymes, were found in rabbit lens. Two aldose reductases were also detected in the skeletal muscle of male rats and pigs and in pig and bovine lens. The presence of relatively large amounts of aldose reductase in muscle identifies a new and rich source of the enzyme.     

The aldo-keto reductase superfamily. cDNAs and deduced amino acid sequences of human aldehyde and aldose reductases

Aldehyde reductase [EC 1.1.1.2] and aldose reductase [EC 1.1.1.21] are monomeric NADPH-dependent oxidoreductases having wide substrate specificities for carbonyl compounds. These enzymes are implicated in the development of diabetic complications by catalyzing the reduction of glucose to sorbitol. Enzyme inhibition as a direct pharmacokinetic approach to the prevention of diabetic complications resulting from the hyperglycemia of diabetes has not been effective because of nonspecificity of the inhibitors and some appreciable side effects. To understand the structural and evolutionary relationship of these enzymes, we cloned and sequenced cDNAs coding for aldose and aldehyde reductases from human liver and placental cDNA libraries. Human placental aldose reductase (open reading frame of 316 amino acids) has a 65% identity (identical plus conservative substitutions) to human liver and placental aldehyde reductase (open reading frame of 325 amino acids). The two sequences have significant identity to 2,5-diketogluconic acid reductase from corynebacterium, frog rho-crystallin, and bovine lung prostaglandin F synthase (reductase). Southern hybridization analysis of human genomic DNA indicates a multigene system for aldose reductase, suggesting the existence of additional proteins. Thus, the aldo-keto reductase superfamily of proteins may have a more significant and hitherto not fully appreciated role in general cellular metabolism.     

Aldose reductase and p-crystallin belong to the same protein superfamily as aldehyde reductase

Aldose reductase (EC 1.1.1.21) has been implicated in a variety of diabetic complications. Here we present the first primary sequence data for the rat lens enzyme, obtained by amino acid and cDNA analysis. We have found structural similarities with another NADPH-dependent oxidoreductase: human liver aldehyde reductase (EC 1.1.1.2). The identity between these two enzymes is 50%. Both enzymes share approx. 40-50% homology with p-crystallin, a major lens protein present only in the frog, Rana pipiens. We propose that aldose reductase, aldehyde reductase and p-crystallin are members of a superfamily of related proteins.     

The complete cDNA and amino acid sequence of bovine fetuin. Its homology with alpha 2HS glycoprotein and relation to other members of the cystatin superfamily.

The cDNA sequence encoding the bovine fetal protein fetuin is reported. The deduced amino acid sequence is identical with that obtained from amino acid sequencing. The protein is a single chain preceded by a signal sequence. The three N-linked glycosylation sites have been determined. The sequence of fetuin shows over 70% similarity to human alpha 2HS glycoprotein. All of the cysteine residues are conserved in both proteins, suggesting that fetuin has the same arrangement of disulfide loops as alpha 2HS glycoprotein and may also be a member of the cystatin family. Southern blot analysis indicates that a single gene codes for fetuin. No evidence for a separate gene for a bovine alpha 2HS glycoprotein was obtained; thus, fetuin in cattle and alpha 2HS glycoprotein in the human are equivalent proteins.     

Differential expression of the "B" subunit of the vacuolar H(+)-ATPase in bovine tissues.

The B subunit is one of two nucleotide-binding polypeptides found in all members of the vacuolar class of H(+)-translocating ATPases. We have isolated aDNA clone encoding the bovine brain B (58 kDa) subunit and have deduced its amino acid sequence. The bovine brain amino acid sequence is 99% identical to a partial cDNA reported from human brain. Northern blot analysis of RNA isolated from bovine tissues and a bovine kidney cell line reveals that two messages of approximately 3.2 and 2.0 kilobases (kb) are expressed in all tissues examined except brain, where only the 3.2-kb message can be detected. Northern blotting of RNA isolated from human fibroblast and human lung tumor cell lines reveals that three messages of approximately 6.0, 3.2, and 2.0 kb are expressed, whereas only the 3.2-kb message is expressed in a human brain tumor cell line. This is the first demonstration of tissue-specific expression of multiple forms of a vacuolar H(+)-ATPase subunit. We have also isolated a partial cDNA clone from bovine brain which appears to encode an isoform of the B subunit. The deduced amino acid sequence is 82% identical to the major bovine brain B subunit sequence; it does not hybridize with either the 3.2- or 2.0-kb message on Northern blot. Southern blot analysis of bovine genomic DNA with probes derived from both isolated cDNAs indicates that the bovine B subunit is encoded by a multigene family.     

Purification and characterization of aldose reductase and aldehyde reductase from human kidney.

Aldose reductase and aldehyde reductases have been purified to homogeneity from human kidney and have molecular weights of 32,000 and 40,000 and isoelectric pH 5.8 and 5.3, respectively. Aldose reductase, beside catalyzing the reduction of various aldehydes, reduces aldo-sugars, whereas aldehyde reductase, does not reduce aldo-sugars. Aldose reductase activity is expressed with either NADH or NADPH as cofactor, whereas aldehyde reductase utilizes only NADPH. Both enzymes are inhibited to varying degrees by aldose reductase inhibitors. Antibodies against bovine lens aldose reductase precipitated aldose reductase but not aldehyde reductase. The sequence of addition of the substrates to aldehyde reductase is ordered and to aldose reductase is random, whereas for both the enzymes the release of product is ordered with NADP released last.     

Sequence analysis of bovine lens aldose reductase

The covalent structure of bovine lens aldose reductase (alditol-NADP+ oxidoreductase, EC 1.1.1.21) was determined by sequence analysis of peptides generated by specific and chemical cleavage of the homogeneous apoenzyme. Peptides, purified by reverse-phase high performance liquid chromatography were subjected to compositional analysis and sequencing by gas-phase automated Edman degradation. Aldose reductase was found to contain 315 amino acid residues. The enzyme is blocked at the amino terminus, and mass spectrometry was employed to identify the blocking acetyl group and to sequence the amino-terminal tryptic peptide. The aldose reductase was shown to contain no carbohydrate despite the fact that the enzyme contains the consensus sequence -Asn-Lys-Thr- for N-linked glycosylation. Comparative sequence analysis and application of algorithms for prediction of secondary structure and nucleotide binding domains are consistent with the view that aldose reductase is a double-domain protein with a beta-alpha-beta secondary structural organization. The NADPH binding site appears to be associated with the amino-terminal half of the enzyme. Modeling studies based on the tertiary structures of dihydrofolate and glutathione reductases indicate that the NADPH binding site begins at Lys-11 and continues with a beta-alpha-beta fold characteristic of nucleotide binding proteins.     

Comparative studies on aldose reductase from bovine, rat and human lens

A purification scheme for aldose reductase (alditol: NADP+ 1-oxidoreductase, EC 1.1.1.21) developed using bovine lens tissue including an affinity chromatographic step is presented which is particularly suited for small quantities of lenses. Using the affinity chromatographic method as a key step also makes it possible to obtain preparations of rat lens aldose reductase which are homogeneous. The behavior of crude preparations of aldose reductase from human lens on both ion-exchange and affinity chromatography was similar to the chromatographic behavior of the enzyme from rat and bovine lens. Comparative studies of aldose reductase obtained from the lenses of the three species demonstrate the similarity of the enzymes. These comparisons were based on molecular weights, isoelectric points, chromatographic behavior and kinetic data. Homotropic cooperativity for both NADPH and glyceraldehyde, as evidenced by a downward curvature in the Lineweaver-Burk double-reciprocal plots, had been demonstrated for aldose reductase obtained from bovine lens (Sheaff, C.M. and Doughty, C.C. (1976) J. Biol. Chem. 251, 2696-2702). Similarly, cooperativity was observed with the enzyme from both rat and human lenses and the apparent Km values at both high and low concentrations of substrate are comparable for the lens aldose reductases from all three species for both substrates.     

The complete amino acid sequence of a human folate binding protein from KB cells determined from the cDNA

Two species of folate binding protein (FBP), an integral membrane-associated form and a soluble secreted form, have been previously purified from cultured human KB cells. The complete nucleotide sequence of the complementary DNA (cDNA) clone for the coding region of the mature membrane-associated FBP has now been determined, and the deduced amino acid sequence has been computer-analyzed for a prediction of the secondary structure of the protein. The clone has 857 nucleotides of which 678 comprise the coding region for 226 amino acids. The deduced amino sequence contains the identical sequence of the published 18 NH2-terminal amino acids of the purified FBP from KB cells and the published partial amino acid sequence of the human milk FBP except for 1 residue. There was also over 90% homology with the published amino acid sequence of the bovine milk FBP. A total of 16 cysteine residues has been conserved in the three proteins indicating that this amino acid may provide a tertiary structure which is required for its ligand binding function. Northern blot analysis using the cDNA probe identified a single band of 1.28-kilobase pair mRNA in KB cells which was 4.7-fold more intense in folate-depleted cells than in normal cells. These results indicate that the membrane FBP and the soluble FBP in the medium are translation products of the same gene. Computer analysis of the deduced amino acid sequence indicates that there is only one stretch of amino acids of sufficient hydrophobicity and length to span the lipid bilayer of the plasma membrane, but it lacked a predictable helical structure. Those regions of the sequence which did have a predictable helical structure lacked sufficient hydrophobicity required for a membrane anchor. Thus, it is likely that the fatty acids previously reported to be present in the membrane-associated FBP from these cells rather than a peptide sequence provide an important membrane anchoring function.     

Aldose reductase from human psoas muscle. Affinity labeling of an active site lysine by pyridoxal 5'-phosphate and pyridoxal 5'-diphospho-5'-adenosine

The reaction of aldose reductase from human psoas muscle with either pyridoxal 5'-phosphate (PLP) or pyridoxal 5'-diphospho-5'-adenosine (PLP-AMP) results in a pseudo first-order 2-fold activation of the enzyme with the stoichiometric incorporation of 1 mol of either reagent per mol of enzyme. However, in addition to an increase in Vmax there was also an increase in Km for both substrate, DL-glyceraldehyde, and coenzyme, NADPH. This resulted in an overall decrease in catalytic efficiency (kcat/Km). Spectral analysis indicated that activation by both PLP and PLP-AMP was accompanied by Schiff's base formation and epsilon-pyridoxyllysine was identified in hydrolysates of the reduced enzyme PLP-complex. Digestion of either PLP-modified or PLP-AMP-modified aldose reductase with endoproteinase Lys-C followed by high performance liquid chromatography purification and amino acid sequencing of the pyridoxyllated peptide revealed that PLP and PLP-AMP had modified the same lysine residue. A 32-residue peptide containing the essential lysine was found to be highly homologous with a segment of the sequence of both human liver aldehyde reductase and rat lens aldose reductase. A tetrapeptide (Ile-Pro-Lys-Ser) containing the essential lysine was identical in all three enzymes. These results highlight the close structural similarity between members of the aldehyde reductase family.     

Deduced amino acid sequence of mature chicken testis ferredoxin

The cDNA sequence encoding the complete mature form of the steroidogenic ferredoxin from chicken testis has been determined and the amino acid sequence deduced therefrom has been compared with the sequences of bovine, human and porcine steroidogenic ferredoxins. The chicken sequence is between 84% and 88% identical with those of the other mitochondrial iron-sulfur proteins. Thus, the amino acid structure of steroidogenic ferredoxins which transfer electrons to mitochondrial forms of cytochrome P-450 has been very highly conserved over evolutionary time.     

Purification and properties of aldose reductase and aldehyde reductase II from human erythrocyte

Aldose reductase (EC 1.1.1.21) and aldehyde reductase II (L-hexonate dehydrogenase, EC 1.1.1.2) have been purified to homogeneity from human erythrocytes by using ion-exchange chromatography, chromatofocusing, affinity chromatography, and Sephadex gel filtration. Both enzymes are monomeric, Mr 32,500, by the criteria of the Sephadex gel filtration and polyacrylamide slab gel electrophoresis under denaturing conditions. The isoelectric pH's for aldose reductase and aldehyde reductase II were determined to be 5.47 and 5.06, respectively. Substrate specificity studies showed that aldose reductase, besides catalyzing the reduction of various aldehydes such as propionaldehyde, pyridine-3-aldehyde and glyceraldehyde, utilizes aldo-sugars such as glucose and galactose. Aldehyde reductase II, however, did not use aldo-sugars as substrate. Aldose reductase activity is expressed with either NADH or NADPH as cofactors, whereas aldehyde reductase II can utilize only NADPH. The pH optima for aldose reductase and aldehyde reductase II are 6.2 and 7.0, respectively. Both enzymes are susceptible to the inhibition by p-hydroxymercuribenzoate and N-ethylmaleimide. They are also inhibited to varying degrees by aldose reductase inhibitors such as sorbinil, alrestatin, quercetrin, tetramethylene glutaric acid, and sodium phenobarbital. The presence of 0.4 M lithium sulfate in the assay mixture is essential for the full expression of aldose reductase activity whereas it completely inhibits aldehyde reductase II. Amino acid compositions and immunological studies further show that erythrocyte aldose reductase is similar to human and bovine lens aldose reductase, and that aldehyde reductase II is similar to human liver and brain aldehyde reductase II.     

Cloning and expression of human aldose reductase

The complete amino acid sequence of human retina and muscle aldose reductase was determined by nucleotide analysis of cDNA clones isolated using synthetic oligonucleotide probes based on partial amino acid sequences of purified human psoas muscle aldose reductase. The cDNA sequence differs substantially in the noncoding and coding regions of recently published sequences of this enzyme. The mRNA for aldose reductase was abundantly expressed in HeLa cells, but only scarcely in a neuroblastoma cell line. Recombinant baculovirus containing one of the muscle cDNA clones was constructed and used to infect Spodoptera frugiperda (SF9) cells. A prominent protein with an apparent molecular size of 36 kDa was identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the culture medium as well as in the homogenate of SF9 cells after 2 days of infection. Culture medium or the supernatant fraction of cell homogenates containing this protein had high aldose reductase activity which showed characteristics of the reported human enzyme. These findings indicate that the amino acid sequence reported in this paper represents human retina and muscle aldose reductase and that functional human aldose reductase can be expressed in large amounts in a baculovirus expression system. The result should facilitate refined structural analysis and the development of new specific aldose reductase inhibitors for the treatment of diabetic complications.     

The sequence of human retinal S-antigen reveals similarities with alpha-transducin

The complete amino acid sequence of human retinal S-antigen (48 kDa protein), a retinal protein involved in the visual process has been determined by cDNA sequencing. The largest cDNA was 1590 base pairs (bp) and it contained an entire coding sequence. The similarity of nucleotide sequence between the human and bovine is approximately 80%. The predicted amino acid sequence indicates that human S-antigen has 405 residues and its molecular mass is 45050 Da. The amino acid sequence homology between human and bovine is 81%. There is no overall sequence similarity between S-antigen and other proteins listed in the National Biomedical Research Foundation (NBRF) protein data base. However, local regions of sequence homology with alpha-transducin (T alpha) are apparent including the putative rhodopsin binding and phosphoryl binding sites. In addition, human S-antigen has sequences identical to bovine uveitopathogenic sites, indicating that some types of human uveitis may in part be related to the animal model of experimental autoimmune uveitis (EAU).     

Formation of sorbitol 6-phosphate by bovine and human lens aldose reductase, sorbitol dehydrogenase and sorbitol kinase

Formation of sorbitol 6-phosphate by bovine and human lens aldose reductase and sorbitol dehydrogenase by the reduction of glucose 6-phosphate and fructose 6-phosphate, respectively, has been demonstrated. The reaction product has been identified by Dowex-formate column chromatography, gas chromatography and mass spectrometry. Sorbitol 6-phosphate can also be formed by the phosphorylation of sorbitol by lens sorbitol kinase in the presence of ATP.     

Purification and characterization of the recombinant human aldose reductase expressed in baculovirus system

Large quantities of recombinant human aldose reductase were produced using Spodoptera frugiperda cells and properties of the enzyme were characterized. Direct purification of the recombinant aldose reductase by affinity column chromatography using Matrex gel orange A yielded a single 36 kDa band, similar in size to the purified human muscle aldose reductase, on a sodium dodecyl sulfate-polyacrylamide gel after silver staining. The isoelectric point of the recombinant enzyme was 5.85 which is identical to the human muscle aldose reductase. Following the treatment with an acylamino-acid releasing enzyme, the blocked NH2-terminal amino acid was identified to be acetylalanine. The successive NH2-terminal sequence and that of the COOH-terminal peptide concurred with the expected translated sequence. Kinetic analyses of the recombinant enzyme activity for various substrates and the cofactor, NADPH, demonstrated a good agreement with the previously reported kinetic data on the purified human aldose reductase. A high concentration of (NH4)2SO4 elicited a significant increase in both Km and Kcat for DL-glyceraldehyde as well as D-glucose. Although IC50 values for most of the aldose reductase inhibitors with recombinant enzyme were found to fall within the comparable range of those obtained with nonhuman mammalian enzymes, the IC50 value for epalrestat was more than 10-fold higher in the recombinant enzyme. These results indicate that the recombinant human aldose reductase expressed in the baculovirus system possesses structurally and enzymatically similar properties as those reported for the native human enzyme and should serve as a superior enzyme preparation to nonhuman mammalian enzymes for the screening of the efficacy and potency of newly developed aldose reductase inhibitors.     

Evolution of the Aldose Reductase-Related Gecko Eye Lens Protein ρB-Crystallin: A Sheep in Wolf's Clothing

ρB-crystallin (AJ245805) is a major protein component (20%) in the eye lens of the gecko Lepidodactylus lugubris. Limited peptide sequence analysis earlier revealed that it belongs to the aldo-keto reductase superfamily, as does the frog lens ρ-crystallin. We have now determined the complete cDNA sequence of ρB-crystallin and established that it is more closely related to the aldose reductase branch of the superfamily than to frog ρ-crystallin. These gecko and frog lens proteins have thus independently been recruited from the same enzyme superfamily. Aldose reductase is implicated in the development of diabetic cataract in mammals, and, if active, ρB-crystallin might be a potential risk for the gecko lens. Apart from a replacement 298 Cys → Tyr, ρB-crystallin possesses all amino acid residues thought to be required for catalytic activity of the aldose reductases. However, modeling studies of the ρB-crystallin structure indicate that substrate specificity and nicotinamide cofactor affinity might be affected. Indeed, neither recombinant ρB-crystallin nor the reverse mutant 298 Tyr → Cys showed noticeable activity toward aliphatic and aromatic substrates, although cofactor binding was retained. Various other oxidoreductases are known to be recruited as abundant lens proteins in many vertebrate species; ρB-crystallin demonstrates that an aldose reductase-related enzyme also can be modified to this end. Received: 18 July 2000 / Accepted: 3 November 2000     

Primary structure of human plasma fibronectin--characterization of the 6,000 dalton C-terminal fragment containing the interchain disulfide bridges

The carboxy terminal fragment of human plasma fibronectin has been isolated after tryptic digestion and separation by DEAE-cellulose chromatography and gel filtration on Sephadex G-50. It has a molecular weight of 6,000 which changes to 3,000 after reduction indicating that the fragment is a dimer. We have determined the amino acid sequence of the 6kDa fragment and showed that it contains 26 residues including two half-cystines which form two interchain disulfide bridges. The 6kDa fragment is not phosphorylated as in bovine fibronectin although its amino acid sequence is identical to that reported for bovine plasma fibronectin. When compared to the sequence deduced from a rat cDNA, one amino acid substitution can be found. It appears that the carboxyl end of fibronectin is highly conserved among species.     

Molecular cloning and expression of rat liver 3 alpha-hydroxysteroid dehydrogenase

Complementary DNA clones encoding 3 alpha-hydroxysteroid dehydrogenase (3 alpha HSD) were isolated from a rat liver cDNA lambda gt11 expression library using monoclonal antibodies as probes. The sizes of the cDNA inserts ranged from 1.3-2.3 kilobases. Sequence analysis indicated that variation in the DNA size was due to heterogeneity in the length of 3' noncoding sequences. A full-length cDNA clone of 1286 basepairs contained an open reading frame encoding a protein of 322 amino acids with an estimated mol wt of 37 kDa. When expressed in E. coli, the encoded protein migrated to the same position on sodium dodecyl sulfate-polyacrylamide gels as the enzyme purified from rat liver cytosols. The protein expressed in bacteria was highly active in androsterone reduction in the presence of NAD as cofactor, and this activity was inhibited by indomethacin, a potent inhibitor of 3 alpha HSD. The predicted amino acid sequence of 3 alpha HSD was related to sequences of several other enzymes, including bovine prostaglandin F synthase, human chlordecone reductase, human aldose reductase, human aldehyde reductase, and frog lens epsilon-crystalline, suggesting that these proteins belong to the same gene family.     

cDNA cloning of Gb, the substrate for botulinum ADP-ribosyltransferase from bovine adrenal gland and its identification as a rho gene product

A 1.5 kilobase cDNA coding for the complete amino acid sequence of Gb, the substrate for ADP-ribosyltransferase in C1 and D botulinum toxins from bovine adrenal gland, has been isolated from a cDNA library of bovine adrenal gland. This cDNA encodes a polypeptide of 21,770 Da consisting of 193 amino acid residues, and the deduced amino acid sequence contains all the partial amino acid sequences reported previously (Narumiya, S., Sekine, A., and Fujiwara, M. (1988) J. Biol. Chem., 263, 17255-17257). Sequence comparison revealed that Gb is identical with the product of human rho clone 12 (rho A). The present results also confirmed our suggestion that the ADP-ribosylation occurs at Asn41 in the putative effector domain of the rho gene product.