THE JOURNAL OF BIOLOGICAL CHEMlSTRY 0 1990 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 265, No. 7, Issue of March 5, pp. 3626-3635, 1990 Printed in U. S. A.

Sequence Analysis of Bovine Lens Aldose Reductase*

(Received for publication, December 19, 1988)

Sylvia Z. SchadeS, Sherrell L. Early+, T. Reginald William&, Ferenc J. Kezdys, Robert L. HeinriksonQ, Charles E. GrimshawIl, and Clyde C. Doughty+ From the *Department of Biological Chemistry, University of Illinois Health Sciences Center, Chicago, Illinois 60612, the §Biopolymer Chemistry Unit, The Upjohn Company, Kalamazoo, Michigan, 49001, and the Wepartment of Basic and Clinical Research, Research Znstitute of Scripps Clinic, Scripps Clinic and Research Foundation, La Jolla, California 92037

The covalent structure of bovine lens aldose reduc- tase (alditol-NADP+ oxidoreductase, EC 1.1.1.2 1) 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 3 15 amino acid residues. The enzyme is blocked at the amino terminus, and mass spectrom- etry was employed to identify the blocking acetyl group and to sequence the amino-terminal tryptic pep- tide. 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 ap- plication of algorithms for prediction of secondary structure and nucleotide binding domains are consist- ent with the view that aldose reductase is a double- domain protein with a &CY-@ secondary structural or- ganization. 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 contin- ues with a &a-@ fold characteristic of nucleotide bind- ing proteins.

Aldose reductase (alditol-NADP’ I-oxidoreductase, EC l.l.l.Zl), the first enzyme in the sorbitol pathway (Hers, 1960), catalyzes the following reaction.

D-Glucose + NADPH + H+ c) D-sorbitol + NADP’

The enzyme has a broad substrate specificity, ranging from aldoses to aromatic aldehydes, but the coenzyme requirement at pH 7.0 is specifically for NADPH (Hayman and Kinoshita, 1965). Aldose reductase has a key role in initiating the intra- cellular accumulation of polyob resulting in an alteration of the osmotic conditions. These osmotic changes may, in turn, lead to the formation of cataracts in diabetic and galactosemic animals (Kinoshita et al., 1981). In fact, aldose reductase is generally accepted to be the enzyme of prime importance in the etiology of diabetic complications, not only in lens but

* This work was supported by Grant EY 00449 (to C. C. D.) from the National Eye Institute, Grant DK 32218 from the National Institute for Diabetes and Digestive and Kidney Diseases, and by a grant from the Olive H. Whittier Fund (to C. E. G.). This is Publi- cation 5867 BCR from the Research Institute of Scripps Clinic, Scripps Clinic and Research Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 USC. Section 1734 solely to indicate this fact.

also in peripheral nerve and kidney (Kador and Kinoshita, 1985).

Sequences inferred from cDNA have been published for the aldose reductases from rat lens (Nishimura et al., 1989; Carper et al., 1987) and human placenta (Bohren et al., 1989; Chung and LaMendola, 1989). However, since enzymes catalyzing reactions at the branch points of metabolic cascades are usually subject to regulation by post-translational modifica- tions, it is important to elucidate the structure of an aldose reductase by methods of protein chemistry. In this paper we present a detailed account of the covalent structural analysis of bovine lens aldose reductase, an enzyme which has served as a paradigm for understanding the physical, chemical, and regulatory properties of this class of enzymes (Sheaff and Doughty, 1976). A preliminary report of this work has been presented in abstract form (Doughty et al., 1988).

EXPERIMENTAL PROCEDURES’

Enzyme Purification-Aldose reductase was purified to homoge- neity from bovine lens by the method of Sheaff and Doughty (1976), modified by elimination of the carboxymethylcellulose step. The final enzyme purity was greater than 98% as indicated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis under reducing conditions.

Predictiue Algorithms-Secondary structural predictions were based unon the methods of Chou and Fasman (1978) and Garnier et al. (1978). The algorithm of Wierenga et al. (1986) was used for identification of potential nucleotide binding regions.

RESULTS’

The sequence of the 315 amino acids in bovine lens aldose reductase is presented in Fig. 1. The structure was deduced from gas-phase sequencing of overlapping peptides generated from various S-alkylated derivatives of aldose reductase by CNBr treatment and by digestion with Stuphylococcus aureus V-8 protease, trypsin, chymotrypsin, metalloendopeptidase, and mouse submaxillary gland protease. Reverse-phase HPLC’ was used for initial fractionation of digests and also

i Parts of this paper (including portions of “Experimental Proce- dures” and “Results,” Figs. Ml, M9, M13, M16, and M18, as well as Tables Ml-M4) are presented in miniprint at the end of this paper. Miniprint is easily read with the aid of a standard magnifying glass. Full size photocopies are included in the microfilm edition of the Journal that is available from Waverly Press.

* The abbreviations used are: HPLC, high performance liquid chro- matoaranhv: AR. aldose reductase; nAR, native AR; CAMAR, S- - _ _. carboxamidomethyl-AR; PEAR, S-pyridylethyl-AR; SAR, succinyl- AR; AESAR, S-aminoethyl-SAR, BME, 2-mercaptoethanol; DMED, N,N’-dimethylethylenediamine; DTT, dithiothreitol; HEPES, N-2- hvdroxvethvlninerazine-N’-2-ethanesulfonic acid; HFA, hexafluo- &acetone trihh;drate; NEM, N-ethylmorpholine; NEMAc, NEM buffer (pH adjusted with sequence grade glacial HAc); PTC, phenyl- thiocarbamyl; SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel electrophoresis; TFA, trifluoroacetic acid; TPCK, N-tosyl-L-phen- ylalanine chloromethyl ketone.

3628

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FIG. 1. The alignment of the pep- tides used for determining the amino acid sequence of bovine lens aldose reductase. Only the portions of each peptide which were sequenced are included, and the shaded areas represent residues identified by Edman degrada- tion. The designations 7’, CT, RC, CB, E, SA, and DP represent peptides de- rived by digestion with trypsin, chymo- trypsin, mouse submaxillary gland pro- tease (endoproteinase Arg C), CNBr, S. aureus V-8 protease, metalloendopepti- dase, and by acid cleavage of Asp-Pro bonds, respectively.

Sequence Analysis of Aldose Reductase 3629 10 20 30 40

A~~~-AHN %v ‘~YTGAKMPILGLGTWKSPPGKVTEAVKVAIDLGYRHIDCAHVY

50 60 70 80 90 100 ONENEVGLALC~AKLC~EKVVKREDLF~VSKLWCTYHDKDLVKGAC~KTLSDLKL

I

110 120 130 140 150

DYLDLYLIHWPTGFKPGKDFFPLDEDGNVIPSEKDFVDTWTAMEELVDEGLVKA

160 170 180 190 200

,G”SNFNHLOVEK,LNKPGLKYKPAVNOlECnPYLTOEKLlaYCNSK~,“VTAY

210 220 230 240 250 260

SPLGSPDRPWAKPEDPSILEDPRIKAIADKYNKTTADVLIRFPI~~NLIVIPKS

I RC-2

270 280 290 300 310

VTPERIAENFC1VFDFELDKEDMNTLLSYNRDWRACALVSCASHRDYPFHEEF

for final purification of select fractions. Representative HPLC separations are described in the Miniprint Section.3 Amino acid compositional data for peptides obtained by digestion with trypsin (Table Ml), CNBr (Table M2), S. aureu~ V-8 protease (Table M3), metalloendopeptidase (Table M4), and mouse submaxillary gland protease (endoproteinase Arg C) (Table M2) are consistent with the sequence given in Fig. 1. Results from automated Edman degradation are listed in Table M5.3 All peptides were sequenced after purification to homogeneity except for tryptic peptides T-1OA and T-lOB, which were sequences as a mixture, the V-8 protease peptide E-5, which contained peptides beginning at residues 65 and 71, and one CNBr peptide, DP, which was sequenced as a mixture with CB-3. Note that peptide CB-3A is a fragment of CB-3 obtained by prolonged incubation with CNBr, thereby resulting in an additional cleavage between Asp-224 and Pro- 225, one of two Asp-Pro bonds in aldose reductase.

Only two of the four potential cyanogen bromide fragments proved to be useful for sequence analysis, the amino-terminal dodecapeptide, CB-1, and the carboxyl-terminal tricosapep- tide, CB-4. CB-2 and CB-3,132 and 141 amino acids in length, respectively, could not be resolved by HPLC under a variety of conditions. Therefore, other strategies were used to gener- ate large fragments for overlapping peptides in this central region of the sequence. Particularly helpful was the fact that mouse submaxillary gland protease resulted in a single cleav- age between residues Tyr-209 and Ser-210, producing peptide RC-2. Peptides derived from V-8 protease and tryptic digests provided the majority of the confirming sequence data, and

3Experimental details are available from the authors for these data: Figs. M2-M8, MlO-M12, M14, M15, Ml7 and M19, and Table M5.

peptide SA-4 from a metalloendopeptidase digest provided two necessary overlaps.

Aldose reductase has a blocked amino terminus, as indi- cated by the fact that no phenylthiohydantoin-derivatives were generated during Edman degradation of the reduced and pyridylethylated intact protein (S-pyridylethyl-aldose reduc- tase) and of the amino-terminal peptides nTR-1, RC-1, and CB-1. Mass spectrometric analysis of nTR-1 and CB-1 re- vealed that the amino-terminal Ala was acetylated. The mass spectrometric sequencing further identified residues 4 and 6 as either Leu or Ile,4 residues indistinguishable because of identical masses. Amino acid analysis of CB-1 shows the presence of both Leu and Ile in equimolar amounts (Table M2). In the cDNA sequence of human placental aldose reduc- tase, residue 4 is Ile and 6 is Leu (Bohren et aZ., 1989; Chung and LaMendola, 1989), and the assignments shown in Fig. 1 are based upon homology. The amino acid composition ob- tained from sequence analysis matched the total amino acid composition of aldose reductase determined by both Pica-Tag and Durrum ion exchange analysis (Table I). The Pica-Tag analysis indicated there were 7 cysteine residues, in agreement with the analysis from the sequence data.

In view of the fact that the protein contains the sequence -Asn-Lys-Thr-, a consensus sequence for N-linked glycosyl- ation, it should be noted that we did not observe any carbo- hydrate at Asn-241 during the course of our sequence analysis. Moreover, direct microanalysis of aldose reductase for carbo- hydrate proved that the enzyme is not glycosylated to any significant extent.

DISCUSSION

The amino acid sequence of bovine lens aldose reductase, shown in Fig. 1, is the first for an aldose reductase to be

’ K. Rinehart, personal communication.

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3630 Sequence Analysis of Aldose Reductase

TABLE I Amino acid composition of bovine lens aldose reductase

The amino acid composition determined by Pica-Tag analysis is the mean of four determinations on a recent preparation of bovine lens aldose reductase. The data were calculated on the basis of Phe = 12. Trp was not determined (ND). The Durrum analysis is the mean of duplicate determinations reported previously (Doughty et al., 1982). The earlier reported values, except for Trp and Cys, have been recalculated based on a total of 315 amino acids.

Number in 8Npe”Ce

Methods

Pica-Tag DllrlUm

Nonpolar Ala Val Leu Ile Pro Met Phe TTp

Polar GUY Ser Thr CYS Trr Asn Gln

Acidic

20 19.3 24 20.6 32 31.7 20 17.8 20 21.5

3 2.1” 12 12

Asp Glu

Composition analysis AspfAsn Glu/Gln

Basic LYE Arg His

6 ND

5 14.8 3 11.4 4 14.1 I 6.5’ 3 12.2 4 1

23 21

35.6 32.3

28 21.9 10 10.7

9 9.2

20.3 21.8 33.0 17.9 22.0

3.1 11.9

6.4”

16.3 13.2 13.9

5.ad 10.9

37.8 34.4

28.1 10.2

8.9

u Determined as the phenylthiocarbamyl derivative of homoserine. *Determined by the spectrophotometric method of Edelhoch

(1967). ’ Determined as the phenylthiocarbamyl derivative of pyridyl-

ethylcysteine. d Determined by 5,5’-dithiobis-(2-nitrobenzoic acid) titration.

derived by protein sequencing. Critical overlaps of peptides were established by analysis of peptides generated by a variety of cleavage procedures. The NH* terminus of the enzyme has been shown to be acetylated, and therefore in the sequence analysis of the NH*-terminal peptides by mass spectrometry assignments of Ile-4 and Leu-6 are based upon composition of the peptide and reference to cDNA-derived sequences (Fig. 2). Sequence analysis of peptides containing the potential site for N-linked glycosylation (Asn-241) showed good yields of phenylthiohydantoin-Asn, thereby indicating the absence of any significant level of carbohydrate. This conclusion has been confirmed by microanalysis of the intact aldose reduc- tase for carbohydrate. Indeed, the consensus sequence, Asn- Lys-Thr, is conserved among all aldose reductase sequences determined thus far (Fig. 2), and it might come as a surprise that the protein is not glycosylated. Perhaps the location of the sequence near the COOH terminus of the molecule pre- vents post-translational modification due to folding of the growing polypeptide chain. Finally, validating the results of cDNA sequencing (Carper et al., 1987; Nishimura et al., 1989; Bohren et al., 1989; Chung and LaMendola, 1989), the present work establishes that the natural protein is a single polypep- tide of 315 amino acids and is not processed proteolytically to multichain forms.

Not surprisingly, comparison of the bovine lens aldose reductase sequence with those of related enzymes from rat

3 14 ANb -AH---NIVL YTGMnPIW LGTwI[S---P PXYTBAVKY AIDLGYRHID CAAYYQNENE ANh -AS---BILL NNGAMPILG LGTt,KS---P PGQVTBAYK” AIDYGYNHID CANVYPNBNB ANr NAS---HLBL NNGTINPTLG LGTWKS---P PGQYTBAVIY AIDIIGYNHID CAWYPNBKB ALh AAS---CYLL BTGQKEPLIG LGTWS---II PGQ"KAAYI(Y ALSVGYPHID CAAIYGNKPK PSI, MDPKS4R"KL NDGHPIPYLG FGTYAPKKVP KSRALKATKF AIWGPRHVD SAHLYQNBBQ DG “(TVPS--I”L NDGNSIPPLG YGVPK---VP PAMQRAYKK ALWGYRI(ID TAAIYGNSEG C*f ~..----_-- ---~~~~~~- ~-- - - - - - - - - - - - - - - - - - -----___.- .---------

.:; , , .:;, >:g :i, :;;c: /i , i/i liii’b:. ,. .:i,ym;;, , ;;;I m ii;

80 92 ARb “GLALQAKL- QKKWKRKDL FIVSKLWCTY HDKDLVKGAC QKTLSDLKLD yLDLyLIN"P ARh "GYAIQKKL- RKQVYKRBKL FIVSKLYCTY HKKGLYKGAC QKTLSDLKLD YLDLYLINWP ARr "GVALQKKL- KKPVYKRQDL PIYSKLWXP HDQSWKGAC PKTLSDLPLD YLDLYLIHHP ALh IGKALKKDYG PGKAYPRKKL FYTSKLWKTK HSPKDYKPAL RKTLADLQLK YLDLYLMHWP Psb VGQAIRSKIA -MjT"KRKDI FYTSKLWCNS LQPKLYWAL KKSLQNLQLD YVDLYIIHSP DG "GAAIAA--- --SGIARDDL PITTKLWNDR SEDKPAAAI ARSLAKLALD QVDLYLVHWP CRf ~...__^^-_ ---------- ---------- ---------L gNSLRD"G"D YLDLFL"H"P

:ii, I;; :i; g ji:: I;; !;:I :. i i i ,s, ,jK c ;;~il'xJ!, ,

131 138 ARb TGFKPGKDFC OLDKDGNVIP SKKDFVDTWI MKKLVDKGL YKAIGVSNFN HLQVRKILNK ARh TGFKPGKKFF PLDKSGWWP SDTWILDTWA A"KKL"DRGL "KAIGISNFN HIAVEHILNK ARr TGFKPGPDYB PLDASGNVIP SDTDPVDTHT MRQLYDKGL MAIGVSNFN PLQIERILNK ALh YAPRRCDNPE' PKNADGTICY DSTHYKKWK ALBALVAKGL VPALGLSNPN SRQIDDILSV PSb “SLKPGNKPY PKDKSGKLIF DSVDLCRTWK ALKK--DAGL TKSIGVSNFN RKQLEKILN- DC T-------.- ---------p AADNYVRAWK KLIIKLRAAGL TRSIGVSNHL VPHLBRIVAA CRf “SLKPSGAS!, PSDKDKPFIY DNVDLCATWK ALBARKDAGL VESLGVSNPN RRQLERILNK

/;/ii i i i ... "i, ; m :i/i ;iii'jl;"'lii iim igp$ 111";

181 188 199 ARK PGLKYKPAVN eIscRpyLTe KKLIQYCNSK GIVVTAYSPL GSPD-RPWAK PRDPSILKDP ARh FGLKYKPAYN QIBCtIPYLTP IWILIPYCQSK GIWTAYSPL GSPD-BPWAK PRDPSLLEDP ARr PGLKYKPAYN PIBCHPYLYQ SKLIKYCHCK GIWTAYSPL GSPD-ROWAK PEDPSLLKDP ALL ASVA--PAVL ewmpyLAe NBLIAHCOAR GLEVTAYSPL GSSD-RAWRD PDKPvLLBEp PS~ PGLKYKPV~N emxtpyLNe SKLLKFCKSH DIVLVAYAAL GAQLLSKWVN SNI(~VLLRDP Lx TGYV--PA"N eIRLHpAYB4 RRITDWAAA" DYKIBSWPL GQGKYD---- -----LPGAB CRf PGLKYKPYCN eYBCH"YLNQ NKLHSYCKSK DIVLVTYSVL GSHRDRNWYD LSLPVLLDDP

iii ,i; g$,, , i i i i i i II ::::::.,:.:: :::::::.. .:

259 ARb RIKAIADKYN KTTAQPYLIRF PIQRNLIVIP KSVTPERIAE NFQVFDPRLD KBDMNTLLSY ARh RIKAIAMHN KTTAQVLIRF PIIQRNLVYIP KSVTPERIAK NFKVPDFELS SQDHTTLLSY ARr RIKKIAAKYN KTTAQVLIRP PIQRNLVVIP KSVTPARIAI NFKVPDFELS NBDliATLLSY ALh YVLALARKYG RSPAQILLRW QYQNKVICIP KSITPSRILQ NIKVPDPTFS PBKNKQLNAL PSb "LCAIAKKHK QTPALYALRY QYQRGVWLA KSFNKKRIKK NMeVFDFBLT PBDHKAIDGL cc PVTAAAAAHG KTPAQAVLRH ELQKGFVYFP KSVRRKRLKB NLDVFDFDLT DTEIAAIDAl CRf ILNKVAAKYN RTSAKIAIRP ILQKGIWLA KSFTPANIKP NLGVFKFELK PKDIIKSLRSL

. . , i; I;-;;;;:, i';l'i :;'li' /:. ::li/j m 1;; / II; ~ilill";i ./::/ E :j/ ::: ..: .:.

298 303 ARb NRDWR-ACAL EC------- -ASHRDyPPs RKF ARh NRHWR-"CAL LSC------m ~TSAKDYPFH RKF ARr NRWR-"CAL WC------- ~AKSKDYPPH AR" AL,, NFCNIIBYIVPN LTVDGKRYPR DAGHPLYPFN DPY PSb NRNIRYY--- ------DFQK GIGHPKYPFS SKY DG DP-------- ----GDGSGR "SARPD---- RYD CRf DRNLHYG--- ------PFRI "KQOWHYPFN Dsy

j: g y:

FIG. 2. Comparison of the amino acid sequence of bovine lens aldose reductase with those of analogous proteins. The following symbols are used: ARb, bovine lens aldose reductase (this paper); ARh, human placental aldose reductase (Chung and La- Mendola, 1989); ARr, rat lens aldose reductase (Nishimura et al., 1989); ALh, human liver aldehyde reductase (Bohren et al., 1989); PSb, bovine prostaglandin synthase F (Watanabe et aZ., 1988); DG, Corynebacterium 2,5-diketo-o-gluconate reductase (Anderson et al. 1985); CRf, frog lens e-crystallin (Tomarev et al., 1984). The sequences were aligned using the CLUSTAL program (Higgins and Sharp, 1988).

lens and human placenta shows a high degree of identity over the entire length of the molecules (Fig. 2). This sequence homology extends to include other keto and aldose reductases (Nishimura et al., 1989) such as human liver aldehyde reduc- tase, bovine lung prostaglandin F synthase, and Corynebac- terium 2,5-diketo-D-gluconate reductase (Fig. 2). Less ex- pected is the homology seen with frog lens t-crystalline (To- marev et al., 1984), which aligns with the COOH-terminal region of the reductases (Fig. 2). Lens crystallins are generally thought to be structural proteins, and yet, the duck lens t- crystallin is virtually identical to duck lactate dehydrogenase B4 (Wistow et al., 1987).

The extensive sequence homology among these functionally related enzymes allows one to speculate about potential active site regions and structure-function relationships in the aldose reductases that are predictable from the amino acid sequence alone. The favorable alignment of the lens protein in the COOH-terminal segment suggests that aldose reductases may be double-domain proteins. This inference is supported by their well documented allosteric kinetic behavior (Sheaff and Doughty, 1976) characteristic of multidomain enzymes. From the composition alone, one would infer that the aldose reduc-

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Sequence Analysis of Aldose Reductase 3631

tases are a/p proteins (Nakashima et al., 1986), and, indeed, application of algorithms for prediction of secondary structure (Chou and Fasman, 1978; Garnier et al., 1978) indicates a regular pattern of alternating a-helices and P-strands punc- tuated by turns (data not shown). More importantly, in the region near residue 90-100 both algorithms gave ambiguous predictions with no clearly defined secondary structural ele- ments. Such predictions are hallmarks of interdomain con- necting segments.

The enzymes compared in Fig. 2 all employ nicotinamide adenosine dinucleotides as cofactor, and it has been exten- sively documented that mononucleotide binding sites have a characteristic /3-a-P supersecondary structural organization, the so-called Rossmann fold (Rossmann and Argos, 1977; Ohlsson et al., 1974). Recently, Wierenga et al. (1986) have proposed an algorithm for identifying probable protein seg- ments constituting nucleotide binding domains. Application of this method to bovine lens aldose reductase revealed that one, and only one, region of the molecule, that beginning at Lys-11, is a probable site for binding of the NADPH cofactor. Most characteristic of the beginning P-segment is the occur- rence of Gly residues in the 6- and 8-positions (residues 16 and 18), and these are conserved throughout all enzymes listed in Fig. 2. If the NADP binding site is, indeed, in this region, then it is possible to predict further the general fea- tures of the tertiary structure of the site by comparison of the aldose reductase sequence with those of two NADP-dependent enzymes, dihydrofolate reductase (Freisheim and Matthews, 1984) and glutathione reductase (Thieme et al., 1981) for which the tertiary structures are known. In the case of the dihydrofolate reductases, Gly and Thr residues have been shown to interact specifically with the cofactor (Freisheim and Matthews, 1984). By our alignment we find that these residues correspond to Gly-25 and Thr-28 in the bovine lens enzyme, and these residues are, indeed, conserved in all al- dehyde and aldose reductases (Fig. 2). Illustrated in Fig. 3 is a representation of the nucleotide binding fold in aldose reductase predicted from these structural comparisons.

Human muscle aldose reductase reacts with pyridoxal phos- phate to form a Schiff s base at a lysine residue contained in the fragment -Ile-Pro-Lys-Ser- (Morjana et al., 1989). Our laboratory has shown that lysine residue 262 of bovine lens

FIG. 3. Ribbon representation of the proposed secondary structure of the potential nucleotide binding site of bovine lens aldose reductase (segment 1 l-38), as deduced by analogy with the structure of the nucleotide binding site of Lactoba- cillus casei dihydrofolate reductase (segment 37-64) (Freish- eim and Matthews, 1984).

aldose reductase can be similarly modified with pyridoxal phosphate (Doughty et al., 1982; Schade et al., 1983; Schade et al., 1984; Schade et al., 1987). Complete protection against pyridoxal phosphate modification of the bovine lens enzyme was afforded by 2’-AMP but only slight protection by either 3’- or 5’-AMP, suggesting that Lys-262 is interacting with the 2’-phosphate moiety of NADP(H). Experiments with the human muscle enzyme lead to a similar conclusion (Morjana et al., 1989). Lys-262 and Ser-263 are conserved in all of the proteins shown in Fig. 2, and amino acids surrounding this invariable pair are similar. One might, then, conclude that Lys-262 is in close proximity to the nucleotide binding region of the first domain of these enzymes and that it participates in cofactor binding. A more direct role for Lys-262 as part of the catalytic apparatus cannot be ruled out at present.

Finally, the location of cysteinyl or half-cystine residues in the aldose reductases and homologous proteins listed in Fig. 2 leads us to speculate with regard to possible disulfide bond pairings in these molecules. In general, cysteine residues are not necessarily conserved among homologous proteins, and they are readily replaceable by hydrophilic polar amino acids, most often Ser or Thr. In contrast, half-cystine residues are highly conserved as pairs. Moreover, since half-cystines are generally found in the interior of the molecule, they are replaced most often by hydrophobic aliphatic amino acids, such as Val and Met. Two Cys residues are conserved through- out the proteins in Fig. 2 with the exception of diketogluconate reductase, namely, 186 and 199 (bovine enzyme numbering). We would predict that these are linked half-cystines. The frog lens crystallin has only two more Cys residues; these corre- spond to Cys residues in prostaglandin synthase but to valines or leucine at 181 and 138 in all of the aldose reductases. We predict another disulfide bond linking these half-cystines in prostaglandin synthase and frog lens crystallin. Again, the aldose reductases all have a unique pair of Cys residues at positions 298 and 303 corresponding to valines in the aldehyde reductase. We infer that half-cystines 298 and 303 form a disulfide pair. We also predict disulfide pairing of the unique pair of Cys residues in aldehyde reductase, which correspond to Ile-131 and Val-259 in bovine lens aldose reductase. The remaining Cys residues, 44, 80, and 92 in the aldose reduc- tases, and the unique Cys in the rat lens enzyme correspond- ing to Ser-201 are all predicted to be present as the free thiol. Indeed, Cys-80 can be replaced by Asn, and Cys-44 in Ser in the homologous proteins. The only dubious assignment is that of Cys at 92, which corresponds to Leu in three homologous proteins. We feel that this Cys can be predicted as the sulfhy- dry1 form because of the absence of a corresponding hydro- phobic Cys partner. Further experimentation will be neces- sary to clarify this issue.

At the beginning of the work presented in this paper, no information was available concerning the amino acid sequence of any aldose reductase. With the help of our partial sequence, it became possible to isolate and sequence the cDNA corre- sponding to aldose reductase, and at the time of this writing, several such sequences have been so derived. We felt, however, that it was important to continue our sequence analysis of the protein and to determine the complete structure of the native enzyme, since post-translational modifications were expected to occur with an enzyme of this complexity and this allosteric kinetic behavior. In fact, we were able to establish that the amino terminus is acetylated and that the enzyme is a single peptide chain. We also showed that no carbohydrates or cofactors are bound covalently to the functional enzyme. The amino acid sequences obtained by cDNA and amino acid sequencing methods are in full agreement. Perhaps equally

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3632 Sequence Analysis

important is the fact that the chemical sequencing did not reveal any variants in any of the amino acid positions, thereby showing that bovine lens aldose reductase is a single enzyme and not a mixture of closely related isozymes. Finally, we feel that the techniques developed in this work for generating, purifying, and identifying all peptide fragments will play an essential role in all future work concerning the identification of functionally important residues and the delineation of the active site by chemical derivatizations.

Acknowledgments-We thank Dr. Kenneth Rinehart for helpful discussions and determination of the sequence of the blocked amino- terminal peptide by mass spectrometry, Saw Kyin for gas-phase sequence determinations, Dr. Stephen Chung for sharing unpublished data on the cDNA sequence of aldose reductase, Scott McKee for some of the enzyme preparations, Dr. Roger A. Poorman for help in writing programs for predictive algorithms, Dr. Jean C. S. Chang for help in preparation of some of the tables and figures and some HPLC analyses, Ilene M. Reardon for her help in the graphic displays, and Susan K. Lanting for typing the manuscript.

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Sequence Analysis of Aldose Reductase

Sequence Analysis of Bovine Lens Aldore Reductare’

6XPERfMENTALPROCIDUREI

Materials Pept,des were purlfled wng a Waters HPLC system conr,r,,ng of two Mode, 510 pumpr,K injector, and an automated gradbent controller A Waters .,go mu,t,-wavelength detector was “ red to mon,tor the eluate dwnc, Dec.t,de ou,,f,<at,on and dur,no am,no ac,d anelyw Module

The data from amono ad analysr (Pit&iag) v& analyzed by a wat;rr 730 Data The HPLC 10Ivents and chem,calr were arqwed from ather Aldr,rh. 5,

Fher Chem,cal Hexaffuoroacetme tr,h drate and N,N’-d,methylethy,ene d

3 ma. ~,er<e. o, ,am,ne were

purchased from Aldnrh Ethylen,m,ne (a~ ,“e, W&I obtaned from Serva The enz mes wed ,n thlr study we,e TPCK-t,eated trypun and ch protexe from Mtler Sc,ent,f,r, tr,fola fro” t

motrypr~n from Cooper Bwmed,ce.l. r aureus “ -8 01s metalloendopept~dare (E c. 3 4 2m ,CN

Immunob~olog~calr, and rn~u~e rubmar+w, gland protease type XX (endop,oteInare A,g C) ‘a,“. ‘,““,> “-ik%i~phere dumnrfor peptkde purlfsatmn were obtamed from AIITech (Synchro ak RP- PC% 25 cm x 4 1 mm ID. 300 Al and Vydac (C-4.5 “ucro”. 25 cm x 4 6 mm ID. 300 A. pH rta g le C-8. 10 “vtron. 25 cm x 4 6 mm ID. 300 A,. A Water, P,co-Tag R cd”mn wes used to separate the phenylth,o~arbamylder,vat,verofthehyd,oly2ed ammoac,dr

Reverse-phere dumnr for pept,de pur~fsatmn were obtamed from AIITech (Synchro ak RP- PC% 25 cm x 4 1 mm ID. 300 Al and Vydac (C-4.5 mwo”. 25 cm x 4 6 mm ID. 300 A. pH rta g le C-8. 10 mtron. 25 cm x 4 6 mnl ID. 300 A,. A weten Plco-Tag R col”mn wes “ red to separate the phenylth,o~arbamylder,vat,verofthehyd,oly2ed ammoac,dr

Pe tide Purlhcatlon b HPLC Each dlgert was lnltlally frxtmnated on one Of three reverse- Phase y at c 8 PH rtable. or synctlropat C-8) the peptider we,e eluted by ~nc,eas,n

After In,e<tlon of the do est.

90 m,“, armd,cated ,n the md~wdual 9 the concentratwx of eluent B. usually over a per,od o 30. ‘I

of 6 M ,gu,er lfan eluted peat warto be further pu,,‘,ed. 100 PI

? uanidme-“Cl was added t., the eluate. followed by lyophnlolat~on of the ~oIut,on to a

~“a,, “0 “me to wkh a” equal volume 0, more of appropriate el”e”t war added Ths pept,de mxture WeI the” fraalonated on one Of the tame three rewrre phare L0,“rn.I often urmg a shallower g,ad,e”t than was “ red durmg chmmatography of the o,rgr”al d, ert part~cufarly w”h the t

,n ,,,a” cater. pt~ peptlder. seve,al roundr of pur~f,cat~on were Per armed 9 6 W,t some

d,geru. IDS-PAGE ana ys,t followed by protem detectvan ~4th ~,lver star” war used to check the 7 p”,,ty Of each Of the HPLC f,act,onr

Pe t,de Pur,fncat,on b HPLC Each dlgert was m,t,ally fractmnated on one O‘ three reverse- Phase y at C 8 pH rtable. or lynchropat C-8) the peptider we,e eluted by ~ncreas,”

After ~n,e<t~on of the do est.

90 m,“, armd,cated ,n the n”d,wdual 9 the concentratwx of eluent B. usually over a per,od o 30. ‘I

of 6 M ,gu,er lfan eluted peat warto be further pu,,‘,ed. 100 PI

? uanidme-“Cl was added t., the eluate. followed by lyophnlolat~on of the ~oIut,on to a

~“a,, “0 “me to wkh a” equal volume 0, more of appropriate el”e”t war added Ths pept,de mxture WeI the” fraalonated on one Of the tame three rewrre phare L0,“rn.I often urmg a shallower g,ad,e”t than was “ red durmg chmmatography of the o,rgr”al d, ert ,n ,,,a” cater. part~cufarlv w,th the twm~ pept,des. seve,al roundr of our,f,cat,on were cser armed 9 6 w,, s,me d,g< L”,,.,“, FokIIVI,

Amino Acid A Amino Acid Analwr Protem and peptnde sampler (0 5 2 nmolj were hydrolyzed ~4th 6 N HCI kontaining t HCI kontalnlng cl 1% phenol) at 105’c ‘0, X-24 h. 0, preferably at 1so’c for 1 h. Fewer problemswere entouniered wh fomat~on of colored by-brod&r’and droplet condenkon in prablemrwere encountered wth fo,mat~on of colored by-products and droplet condenrat,on I,, the Pco-Tag tuber when the rhoner tnrubatmn time at higher temperature war “red. although the Pco-Tag tuber when the rhoner tnrubatmn time at higher temperature war “red. although the extent of hydro,yr,r of the extent of hydro,yr,r of pept,de pept,de reduced under those cond,t,onr reduced under those cond,t,onr

bonds between paws of very hydrophob,c am,“., ac,dr was bonds between paws of very hydrophob,c am,“., ac,dr was In general, the longer mcubatlo” time war “ red for large, In general, the longer mcubatlo” time war “ red for large,

peptlder. The arn,n~ arods were convetted to then, re~pect,ve pheny,thioca,bamy, (WC, peptlder. The arn,n~ arods were convetted to then, re~pect,ve phe”y,thioca,bamy, (WC, dewaWe and guant,tated by HPLC “rmg Water’s P,co-Tag I dewaWe and guant,tated by HPLC “rmg Water’s P,co-Tag I

7 7 stem stem Cyltelne war determmed as Cyltelne war determmed as

the PTC derwatwe of I wboxam~domethyl 0, S-pyrldy ethyl cyrte~ne Meth,on,ne war the PTC derwatwe of I wboxam~domethyl 0, S-pyrldy ethyl cyrte~ne Meth,on,ne war detemvned at the PTC derwatlve of homorerlne detemvned atthe PTC derwatlve ofhomorer~ne

Sequence A”a,ys,s Automated ammo aced lequen<,n of seelected Pept,des and pept,de mrture~ was ccnduned by ur,ng an ABI model 47OA Gas P are Sequencer at the “mverrrt of % lll,n.m B,otech”alogy Center I” champa~gnlubana The PTH derrvat8ver were analyze d manual qeam” I” some cases. and by on-lme HPLC I” others

by Manual requenc,ng of the ‘8~

few am,no arrd rer,duer was used 8” some cares to select larger peptnder ‘or further gas-phase sequenmg Iran. 1386,. In all cares, the peptIde subjected to gas-phase Iequencmg were I” the n.nomolar roncentratmn range At least FWO ddferent. ovetlapplng peptldes were used to de‘me each POIW,” on the complete requence (deWed I” Table MW. and a, least tw, repl,cate *-*--,-=*--‘-ere performed for each Peptode

ylatmn wth 4-Vmvlpvndlne and lodaa‘etamlde Preparat,on of ose reductare (PEAR) by reductmn and alkylatron of tFie cyrtemer ,n aldore “g I-v,“y,pyr,d,ne was conducted urmg a method wpphed by the ,,n,verrny of low Center TO 1 0 ml 0‘0 5 M N.ethvlmoroholine acetate buffer INFMA~I OH

at 22-C ‘~4 h. d~“,nylpyr,dl”e (8 PI) was added a;d the lght

,e.;t,,,ar c&t,,& at The rexbon war rtopped bv the addltlon of 24 uI of 2. mertaotoethano, ,BME,

2.Am,;d;thv!atwn ofS4R To 1.5 mg (38 “mater) lyoph,t,red IAR was added 0 5 ml of 0.5 M NEMAc (pH 8 3,. 0 5 g of guamdme-HCI. and 2 mg of DU h&urn atmocphere and Incubated overmght at 22’c

The ,ea<tw” was placed under a Ethylenm~n

and the rextoo” was rtrrred ‘0, a” add,tianal 2 h under the he,,, was quenched of 88% ‘orm~ sod and d,,uted

3633

RESULTS

dvomatogiam ire rubre&ently p&f#ed) iFe amlno acld-comporltion;dete,~ned for ih; derlgnated peptlderare ,ncluded I” Table M 1 Bared on data farreveral long pept,der obta,ned from other dogertr. it war realized that the very early poRton of the dwxwogram rho,.,” I” F,g MI r”“~t conts~” te~eral hyd,oph,,,c pept,der For this reason. rubrequent frypt,‘ d,gel,l of CAMAR were frad~onated by HPLC wng a ddferent gradlent and deteCtv,n at 220 nm ,n order to rubdlvnde there h”drophWc pept,der #“to a fracw” nA’ ofverv earlu-elut,“. wec,es m,ddle fractmnr dengnated’l to 5. and a tmal fiact,on “B” conrirlmg o‘tl;e mom hy&phob,;l;ept,der and other longer peptsder (F,g M2)) The “KC rechromato ,433, and a‘ fract,on ‘8” m F,g M43 2

raphy of ‘radio” “A- $3 shown I” F,g

to those rnde vzted I” F,g Ml Purr‘rcatmn of the ml die ‘<actronr yrelded peptrder swm,a,

The peptr s et I” Table Ml account for alI of the fmal sequence esept for four short tryPt,< peptldescompwng a total of 18 am,no acrdr The peptnder ,n Table Ml are dewed for the most part from trypt,(d,geltlofCAMAR. aithoughthree peptlderpresented (7.5. T lO.a”d~-2d)were dewed from a ,,ypt,c dlgert of PEAR v,h,ch had bee” labeled ,n the a‘t~ue we ,wth pyr,doxal 5’.phosphate. and one peptlde CT 16, from a tryptac digest of natwe aldore redunare wh,ch had not been rub,ected to redurtlo” and alt~labon ofthe wste,ne reroduer

lnrompleie cleavage was observed b&ween Lys-t7kPro-179. Lyt- 135.Asp-136. Lyr 85.A\rp-86. and Arg-69.au-70 Such mcomplete deavager have been observed to occur between tmxla ,enduer I” other proterns &Wlro” eta!, 1982, 1986, The peptrdel a‘CAMAR were much thoner and thur easer to quanitty than the 9

enerated by trypsm drgertmn anger peptlder generated by

the other d,gertr. yet the longer peptlder were better w,ted lo, automated gar phase SW”e”Cl”g

Trypw Dqert a‘ nTR-I and “TR-2 Peptldes “n-1 and nTR-2. dewed from d hm,, d,gert of nAR. were reparately digested wth tryps” atter the ~ylte,nes had been reduced and alt lated w,th 4-vmylpyndme The dlgertr were fractmnated by reverre-phase HPLC &gr M5-!,W and r the

P eptoder ,wlated from these digests (TRTR14 and “TW.TR,~. tee Fogs MS-M‘S, we,e used to

con ,rrn the armno aud sequence already detemxned by gas-phase requenc~ng of PeptIdes Dbta,nedfromtheotherd,Oertr

5. aweus” Proteale Dv-x! cleavage at glutamate rer,dur

d Dlgermn of PEAR with 5 aureut “ -8 p,~teaw was complete for ‘I #‘the rerndue war not p,eGFG&, ‘allowed b” another charoed

rendue. The length of the peptlder coWRed from HPLC fract,o”atmn of the &gest vaned f& 1 t-80 + am,no at!dr ,F,gs Mg-Ml1 and Table ,451, Most afthe ‘ollected Peaks were requenced wthout further p”r,f~ca,,o” and leveral peats were p”r,f,ed tr, obtain am,“0 ac,d LW,,POI,,,D” dara

Mo”se Submax*llarv Gland Proteare Dwest PEIR dIgested wrth Morse rubmaxrllary gland protease produced two malo, pepodes CRC-’ and Rc-2. Fig Ml231 wh,th were rechromatoa,aohed and rublected to oarahare Cdman d.nnrlar~o” Peptlde Rc-2 gave one

However. the c,eaua.ae Ilte

amount of another proteare w,,h chymotryps~nlste spe<,f,c,ty. t,nce lays to reach complet,on. Funhe, d,oertion of RC-I wth CNB, ,ree

contaminated woth a small the dlpcrtnon reqwed 5 c below yneldedth,ee pept>desRg M13andTableM3,

Metalloendaeept,dare Dwertlo” of Am T,eatme”t o‘ AESAR wth metallc.endooet,,~dare orodured several neotzdes two of whoth were rub,ected to gas-phase Ldman aegrad~tlbn ,Frg

and SA.4 Fable MS, were CYI 44 and Cvr-02. ,et~e<twel~ Although the drgestuon went to rompletron. a~ md,cated b low Whnle the N-ruwnylated aldote reductare was I& %

reverse phase HkC. the ;,eld w& le 8” the NEMAc buffer used far

aml”oethylatmn of the cyrtelner. AEIAR appeared to be poorly wluble I” th,r buffer. wh,ch m,ght erp,a,n the low y,eld of the d, err. have been cross-INnted by the

Another explanation 15 that the AEIAR molecules may ethy ensm~ne used to alkylate the cylteme rerlduer. thereby P

preventmg d,ger,,on bythe “tetalloendopept,dare Cle.vaww,th CW-Cleavage atthethree meth~on~nesin aldore redunare should y,e,d four

PeptIdes. barr,ng any ac,dolytlc cleavages When PEAR war treated ~8th CNB, I” 70% fomw a<,d. SIX 0, seven pepndes were formed. ar determ,“ed by “WC @g MIS’) and SDS-PAGE analyr~r The four expected major pept,der were 12.W 132. and 141 amino wdr ,n length. and sIaned w,th Ala1 KB-11. A~286 (CB-4). Pro-13 KB-2). and Glu-145 ICB-3). rerpect,vely nc,dolyt,c cleavage occurred at the two Asp&o bonds found ,n the pw,e,n (ASP 224.pro-225 and Alp-230.Pro-231). and with longer mcubatmn tmws, add,t,o”al cleavager occurred at ASP. 2%Trp-J-35 and at Asp-308-Tyr-309 resolved bv HPLC

The iwo larger peptcdes KB-2 and CB-3) could not be Fdman deqradatton v‘th,r rombmed peat gave 3 armno termma, ~equen<es.

and Pro-231. ,ex,ect,“ely Edman dewadatvan of De&de CB-3A

I a”re”s V-8 Preteare Dvx~tron PEAR ,I 5 mg, 67 ““I.,,) ,.,=I upended I” 2 5 ml of 0 1 M NEBA< buffermntanng 2% Wv,SDSand 2 mM EOTA(pH 8 1, V-8pmtease waraddedtogwe a “ -8 pmteare/PEAR rat10 a‘ l’E.O,wtiwt,and rnwbated ford h at 37-C After4 h, addmona, V-8 pwteare war added to gwe a 1 30 &v”wt) rat80 and the d,gestmn war cawnued for 17 hat WC The reactlo” was rtopged by map-freenng the rample at -WC The dagert war rub,ected to erhaurt~ve dlslyslt at 5 C aga,“rtwate, to remove SDS p,,ort., HPLC analyr,r

Moure Submax,Ilarv Gland Pmteare Dagelt~on -TO a ~wpens,~n of PEAR (2.5 mg. 67 nmol) ,n 0.4 M amm.,n,um bicarbonate buffer (PH 8 0) <anta,mng 0 14 M NEMk. 0 I mM EDTA. 0 I ,,a, glycme. and 0 2% hv/vl 505. war added mouse rub max,l!ary protease kndoprote,nare erg C, at 0 h, 4 h, and 20 h to give a P,oteare,PEAR rat10 of 1 100. I 50. and 1 40 (w”,.,,). rerpett,~e,~ The dl

B ertmn. conducted for a total of 26 h at 37’C. was smpped by map- ‘reez,ng the sample at

-7 Y Metalloendopept~dare Cleavarse at Armnoeth”lcyr,eme TO 24 nmol of AEIAR ,n 100 ,, 0‘0

I ~ed,g~~t:il~~nHla~ftOPPed after 6 h mcvbatro” at47’c by map-‘reezmg the~amplea, -78-C

1 8 91 at UC was added 2 5 umtr meWloendopeptw3are m 100 PI of NEMA~ buffer

Chvmotw.rm Dvzertlo” of Am,“0 Terminal Pec,t,der T-1 and E-1 -To 3 nmol .,f ,yoph,,,led peptrde T-7. the purlfled amino-terminal Peptlde from a reverred-phase HPLC separat,on (~,g Ml81 of a trypl,” dogert of nAR conducted 8” 0 1 M amm~n,um borbonate buffer. war added 10 ng of a-chymatryprln m 20 ~1 of 0 05 M dmmon~um formate buffer (PH 6 41 to glue a chymotrypnnlpept~de ram of I 360 (wUw1, and the mixture was mrubated at 37~ far 4 h Ammonium formate was used ar the ,eactlon buffe, wIthout adjurtment of the PH. in order to

El re~entany~o”tam~“at~on oftheramplewhlch mnght mterferew~th therubre y marrrpectramet

de,on,red water ear z Samplerwere lyophiked fourt~merwnh the addltlon o 100 PI ofi-,,lla-0 9

uentrequewng

time Chymotrypr,n was added to 1 nmo, of am,no.,e,m,na, pep,,de CB~, the purlfled armno-termnal pept,de obtamed from a CNB, d,gert of DMED-PEAR at d <hymotlplln/DMED.PEAiR rat,0 of 1 100 ~wvwt) I” 20 p, 0‘0 1 M ammq”i,,m b,<arbonaie and the d,ge,tmn m,“t”re was mwbated at 37-C for 4 h peak CB IST-I (tee F’gfM 13, II presented I” Table MI

The ammo d‘rd vx,,p~~,t,on of the early The ammo acld <ompo~~t~on of the larger

peak~hawedthatth~r ract,on <onta,ned anotherPept,deofuntnownar,g,n

700TGGiGkwh d CNBr Cledvd e PEAR or DMEDWEAR *al drrrqlved ,n 97% form< d<,d and then d,luted to

elamred water solud 048, was added W,OO mal 1 mol PEAR) and the ~ol”tmn wat~ncubated for 24or48 h at22”C M guamdme-HCI acldlfaed ,wth TFA

Afte,lyophihzat~on. the rer,dueward,rrolved I” 6 To prevent the a<,d~,nduced cleavage at Asp~x bonds

observed 8” the above PEAR,CNBrd,gelt, thecarboxyl grouprof~rp and GIu were am,dated w,th DMEDatcordmg taTarr(19.36)

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Sequence Analysis of Aldose Reductase

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Sequence Analysis of Aldose Reductase 3635

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C DoughtyS Z Schade, S L Early, T R Williams, F J Kézdy, R L Heinrikson, C E Grimshaw and C

Sequence analysis of bovine lens aldose reductase.

1990, 265:3628-3635.J. Biol. Chem. 

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