Sequence of pig lens aldose reductase and electrospray mass spectrometry of non‐covalent and covalent complexes

Abstract

The complete sequence of pig lens aldose reductase (EC 1.1.1.21), a member of the nicotinamide coenzyme-dependent aldo-keto reductase super family, was determined by the combined use of data obtained from Edman degradation, fast-atom-bombardment mass spectrometry and electrospray mass spectrometry. The N-terminal residue of human and pig aldose reductase was shown to be acetylated. The assignment of a disulfide bridge (Cys298 -Cys303) was obtained by mass spectrometry. Electrospray mass spectrometry has been used for molecular mass measurement of human muscle (35758 It7Da) and pig lens (35778 -C 3Da) aldose reductase; using mild ionization conditions, it has also been used to study the reversible interaction involved in a non-covalent complex with NADP' (36527 5 4Da). An alkylating analog of NADP' (3-chloroacetylpyridine-adenine dinucleotide phosphate) was used as an irreversible inhibitor to investigate the NADP binding site and the mass of the covalent complex was measured (36521 2 3Da).Aldose reductase (AR), a monomeric NADP-dependent aldo-keto reductase enzyme, has been isolated from various human and animal tissues [1][2][3][4][5]. It is implicated in the development of diabetic complications in non-insulin-dependent tissues and seems to be able to compete with hexokinase when the concentration of sugar is high. It catalyzes the reduction of various carbonyl substrates such as aldehydes (glucose) to the corresponding alcohols (sorbitol). Accumulation of these polyols generates a high osmotic pressure which causes cellular swelling. Under diabetic hyperglycemic conditions, accumulation of these compounds has been shown to be involved in physiological disorders [6] Abbreviations. ESMS, electrospray mass spectrometry ; AR, aldose reductase ; clac'PdADP, 3-chloroacetylpyridine -adenine dinucleotide phosphate; LCMS, on-line liquid chromatography mass spectrometry; rp-HPLC, reversed-phase high-performance liquid chromatography ; FABMS, fast-atom-bombardment mass spectrometry; Pth-Xaa, phenylthiohydantoin derivative of an amino acid.Enzymes. Aldose reductase (E. C. 1.1.1.21); trypsin (E. C. 3.4.21.4); chymotrypsin (E. C. 3.4.21.1); carboxypeptidase P (E. C. 3.4.16.1).Note. The novel amino acid sequence data published here have been deposited with the EMBL sequence data bank and are available under the accession number P80276 (pig AR) and P15121 (human AR).interpretation of these X-ray data [16] and to develop selective inhibitors, we have undertaken the determination of the complete primary structure, including post-translational modifications, and the location of cysteine bridges. In this sequence study, mass spectrometry has systematically supplemented Edman degradation. Indeed several mass spectrometry ionization techniques have shown their capacity not only to determine molecular masses but also to generate fragmentations that could help the peptide sequence determination [17, 181. Protein sequencing by mass spectrometry has been discussed and demonstrated [ 191. Electrospray mass spectrometry (ESMS) has clea...

Comparative anatomy of the aldo–keto reductase superfamily

“…A total of 42 oxidoreductases have been identified as members of the AKR superfamily (Table 1), including seven HSDs [five of which are also dihydrodiol dehydrogenases (DDs ; EC 1.3.1.20)] [20,25,[27][28][29][30][31][32][33], seven ADRs [5,[34][35][36][37][38][39][40][41], six ALRs [5,[42][43][44][45][46][47], two 3-oxo-5β-steroid 4-dehydrogenases (∆%-3-ketosteroid 5β-reductases) (ED 1.3.99.6) [48,49], three plant chalcone reductases [12,50,51], two 2,5-diketo-gulonate reductases from Corynebacterium [52,53], Pseudomonas morphine dehydrogenase (EC 1.1.1.218) [54], a putative reductase from Leishmania [55], human liver bile acid binding protein [29,31], bovine lung prostaglandin F synthase [17], bovine DD3 [56], frog lens ρ-crystallin [57], a mouse vas deferens protein [58], apple sorbitol-6-phosphate reductase [59], two xylose reductases from yeast [60,61],…”

Phosphorylation and O-Glycosylation Sites of Bovine Chromogranin A from Adrenal Medullary Chromaffin Granules and Their Relationship with Biological Activities

“…ES/MS-ES/MS analysis was done on a VG Bio-Q quadrupole mass spectrometer (Fisons Bio-Q; VG Bio-Tech) with a mass range of 4000 Da and operating in the positive ion mode (33). The peptide was dissolved in water/acetonitrile/acetic acid (49/50/1; v/v/v) at a concentration of about 2-5 pmol/l.…”

Ruminococcin A, a New Lantibiotic Produced by a Ruminococcus gnavu s Strain Isolated from Human Feces

“…Scanning was usually performed from m/z ϭ 500 to m/z ϭ 1,500 in 10 s with the resolution adjusted so that the peak at m/z ϭ 998 from horse heart myoglobin was 1.5 to 2 wide on the base. Calibration was performed by using the multiply charged ions produced by a separate introduction of horse heart myoglobin (16,950.4 Da) (10).…”