Rapid purification of human placental aldose reductase

Zp\:\t

‘rTI(

,,,

114, 53-58

13lOC ttt.MISTKY

Ralpid

(1981)

Purification

PETEK F. Lahoraror~,

of Human

KADOR,

04‘ Vision

DEBORAH Hrsenrc~h,

CARPER,

.Vational

Ej,e

Bethesda.

Aldosc

with

reductasc

(alditol:NADP

which haa been implicated from the human placenta. apparent

placed

on

homogeneity

reductabc apparent

H. Kt~owt-rn

JIN Nariorml

1mtitu~es

in three

21,

EC

1980

I.i.l.tl)

an cn~ymc

steps.

An

initial

30-70.~ ‘7 ammonium

AH-Sepharosc an enzyme

48 fraction

I”

ihl:

pt)ly~l

raised either

a

sulfate

fraction

wa\

column where upon clulion separated from the major

buffer. pH 6.2, of this enzyme fraction on an Amicon with phosphate buffer containing 0.1 mM

represents

c
in the pathogenesis of diabcric complications ha\ been Using affinity chromatography the en7ymc can bc obtained

of high purity. Antibodies angle line of rdenttty with

complicaltions

Reductase

20205

oxidorcduclax

in rabbits against the crude or purified aldose

Aldose redcuctase (alditol:NADP oxidoreductase EC I. I. 1.2 I ) is an enzyme in the polyol pathway which reduces glucose to sorbitol. The accumulation of intracellular sorbitol in turn can cause a hyperosmotic effect which results in cellular swelling. In diabetic animals aldose reductase has been shown to initiate cataract formation ( 1). Evidence implicating aldose reductase as possibly playing a role in the pathogenesis of other diabetic complications including neuropathy (7). retinopathy (3), nephropathy (4), platelet aggregation (5). and cornea1 reepithelialization (6) is also accumulating. The potentiltl involvement of aldose reductase in diabetic complications has spurred increased interest in both the biological role of this enzyme and in the development of specific inhibitors. Since currently no agents for the general control of diabetic complications are known, the use of aldose reduct;ise inhibilors for the delay or prevention of diahclic

Insrirurc~.

November

a 4-carboxybenzaldehyde-coupled

with 0.1 M Nu,K-phosphate protein peak. Chromatography ligand column and clution

Aldose

AND

Marrjland

Received

pathway purified

Placental

Matrex NADPH purilicd reductasc.

Orange yielded cnqme

A dye aldose gave

an

ductase inhibitors (6,7). Recent investigations, however. indicate that there are specific differences in the susceptibility of aldose reductases from various tissues to be inhibited (8. 10). To more closely study the biochemical role of aldose reductase and the relationships between aldose reductascs from different tissues and species, methods for the rapid purification of the enryme and specific antibodies against Ihe puriticd enqme arc necessary. Here, we report 3 method for the rapid purification of human placental aldose reductase and the preparation of specific antibodies against this en/ymc. MATERIALS

AND METHODS

Mafrrials. Unless otherwise stated. ~111 chemicals were of reagent grade quality. Ammonium sulfate was of enzyme grade obtained from Bethesda Research l.;lborarories, Inc. NADPH (Type I ), NAD (Grade V), D(+)-xylose, DL-Glyceraldrhyde and mercaptoethanol were obtained from Sigma Chemical Cornpliny. L-Gulonate was prepared from L-gulonolactone ( ICN) by heating with tin equivalent amount of NaOH for

phar-

m:lcologically attractive approach lo the control of these complications. In diabetic rats the onset of cataract formation and decreased cornea1 reepithelialization can be prevented by administration of aldose re-

10

min

at

60°C

(I

1 ). Certified

digitonin

53

0003-X97/X

I /090053-06$0’.00/0

was

54

KADOR.

CARPER.

obtained from the Fisher Scientitic Company. AH-Sepharose 48 was obtained from Pharmacia, Inc.. and Matrex Gel Orange A was obtained from Amicon Corporation. All electrophoretic reagents were obtained from BioRad Laboratories. En~~~nzr analysis. Enzyme activity was photometrically followed by the decrease in the concentration of NADPH at 340 nm as previously described (8). Unless otherwise stated, DL-glyceraldehyde was used as the substrate. Kinetic analyses were conducted using the PROPHET computer system by titting the means of n = 4-8 determinations to the enzyme kinetic equation t‘ = V,,:,, [S]/ [Sl + K,, where 1; represents the initial velocity of the enzyme reaction, V,,:,, represents the maximum velocity, IS] represents the substrate concentration and K,,, represents the Michaelis-Menten constant. The fit was iteratively carried out using a Taylor expansion of the nonlinear parameter, K,,. Protein concentrations were spectrophotometrically determined according to the method of Bradford (I 2) or Kalckar (I 3). Human lens and rat lens aldose reductase were prepared as previously described (9). Purification of human placental aldose reductase. All steps were conducted at 4°C. Amnmniur~z sulfatefractionation. Human placentas were frozen immediately after delivery and stored at -20°C. In batches of 30 or more the thawed placentas were homogenized in a Waring blender with 2 vol (w/v) of Na,K-phosphate buffer, pH 6.8, and a 30-704;’ ammonium sulfate precipitate was prepared by the NIH National Institute of Arthritis, Metabolism and Digestive Diseases Pilot Plant. The precipitate dissolved in a minimum amount of distilled water was dialyzed against 2 X SO vol of 0.1 M Na. Kphosphate buffer, pH 6.2, containing 1 1nM mercaptoethanol and stored at -78°C in 250-ml aliquots. .4H-Sepharose 4B chronzatograph~~. One hundred-milliliter aliquots of the dialyzate were placed on a K 50/30 Pharmacia column containing 360 ml of AH-Sepharose 4B

AND

KINOSHITA

coupled to 4-carboxybenzaldehyde as previously described (8). The protein solution was then eluted with 0. I M Na,K-phosphate buffer containing 5 rnv mercaptoethanol and .02% sodium azide at the rate of 28 drops/min and collected in 200 drop fractions. Fractions containing aldose reductase activity were combined and concentrated on an Amicon CEC-I column eluant concentrator equipped with a PM-10 membrane filter to a final volume of ca. 30 ml. An appropriate amount of glycerol to make a 5% (v/v) solution was then added to the concentrate. Mutres Orange .4 c,hronlatograpfl~.. The 30 ml concentrate containing 5% glycerol was added to a BioRad 2 X 30-cm Econocolumn containing 75 ml of Matrex Gel Orange A. After I h equilibration. the column was eluted with 0. I M Na,K-phosphate buffer containing 5% (v/v) glycerol. 5mn mercaptoethanol and 0.02% sodium a/ide at a rate of 28 drops/min and collected in 200 drop aliquots. After 20 200 drop fractions had been collected 50 ml of buffer containing 0.6 mM NAD was added to the column followed again by elution with buffer (20 fractions). This was followed by the addition of 50 ml of buffer containing 0.5 mM digitonin and elution with buffer (20 fractions). Finally, IO0 ml of buffer containing 0.1 KlM NADPH was added followed by elution with buffer. Fractions containing aldose reductase activity from the NADPH wash were combined and concentrated on an Amicon CEC-1 column eluant concentrator equipped with a PM-IO membrane lilter. Pol?lacrylanzide gel electrophowsis. Purity of the preparations was estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis’ (SDS-electrophoresis) using either disc gels ( 14) or slab gels employing a 15% acrylamide separating gel (15). Inln2unoreactivit~. qf’ aldosr rrduc~tusr. Virgin female New Zealand white rabbits ’ Abbreviation

used: SDS.

sodium

dudccyl

\ulfatc.

RAPID

PURIFICATION

OF

HUMAN

of approximately 4 kg body wt were subcutaneously injected with 30 pg of purified enzyme mixed I: I with complete Freunds adjuvant. This was followed with boosts of 50 and 100 pg enzyme in 1: 1 complete Freunds adjuvant at 2- and 4-week intervals. The antibody was then partially purified from serum by a 33% ammonium sulfate precipitation and dialysis with phosphate-buffered saline. Reactivity of the antibody was determined on Ouchterlony plates and with Laurel1 rocket immunoelectrophoresis ( 16). RESULTS Human placental aldose reductase was initially purified from crude placental homogenate by a 30--70% ammonium sulfate precipitation. Purification of the dialyzed ammonium sulfate fraction, however, was not calculated because aldose reductase activity could not be accurately determined in the initial crude preparation. The first chromatographic step utilized a 4-carboxybenzaldehyde-coupled AH-Sepharose 4B column which has previously been reported to bind aldose reductase until elution with a salt gradient (8). The specific binding of aldose reductase to the column, however, is rapidly decreased upon repeated use of the column by nonspecific binding of contaminants which can not be removed by either washing with high-ionic-strength (2 M) buffers or mixtures of chloroform-methanol, dioxane-water, or tetrahydrofuranwater. As the binding of aldose reductase to the column decreased, it was noted that the elution of unbound enzyme was always retarded past the initial void volume which contained most ‘of the protein peak. Moreover, this retention of the enzyme did not significantly decrease upon repeated column use when each elution was followed by a final wash with buffer containing 2 M sodium chloride. Therefore, overloading the 4carboxybenzaldehyde-coupled AH-Sepharose 4B column with the dialyzate and then

PLACENTAL

ALDOSE

REDUCTASE

55

eluting with 0.1 M Na,K-phosphate buffer, pH 6.2, resulted in the elution pattern of Fig. I. Combining and concentrating the enzymatically active fractions resulted in an average 16-fold purification with 70% yield. The activity of the purified enzyme rspecially in dilute solution was quite unstable. Enzyme activity, however, was found to be stabilized upon the addition of 5% (v/v) glycerol to the buffer. Unfortunately, viscosity problems made the addition of glycerol during the first chromatographic step unfeasible. The enzyme concentrate with 5% (v/v) glycerol was placed onto a Matrex Orange A column equilibrated with 0.1 M Na.Kphosphate buffer, pH 6.2. containing 5% (v/ v) glycerol. After 1 h equilibration of the enzyme solution with the gel the column was washed with the phosphate buffer containing glycerol (Fig. 2). This was then followed with glycerol -phosphate buffer washings containing 0.6 mM NAD and 0.5 mM digitonin. respectively. These washings removed a contaminating protein with an apparent molecular weight of 67K on SDS electrophoresis which otherwise would elute in the following NADPH wash. This protein formed a line of identity with human serum albumin antibody. The washes also eluted a protein comigrating with sldose reductase

Fraction

Number

FIG. 1. Elution pattern of the initial dialyzed 30-70% ammonium sulfate fraction of 4-carbunybenraldehydecoupled AH-Sepharose 4B column eluted with 0.1 \I Na,ti-phosphate buffer. pH 6.2. containing 5 mM mercaptoethanol and 0.02’% sodium resents milligrams per milllllter line represents oldose reductase dehyde as substrate expressed in units/4 min/ml. (See Material

aTide. Solid line repof protein while dashed activity wtth glyceralterms of change in OD and Methods.)

S6

KADOR.

CARPER,

FIG. 2. Elution pattern obtained on Matrex Gel Orange A dye-ligand column eluted with 0.1 v Na, I
which had no aldose reductase activity but formed a line of identity with the aldose reductase antibody, along with occasional 30 and 42K proteins present in trace amounts. The order of the NAD and digitonin washings was not important with most of the contaminating proteins being removed in the first wash with either. Finally, upon elution with 0.1 mM NADPH dissolved in the glycerol -phosphate buffer and concentration on a PM-IO membrane filter aldose reductase was obtained with an average 556-fold purification and 20% yield. The purified enzyme preparation gave a single band corresponding to an apparent molecular weight of 37K on 15% SDS-electrophoresis. Occasionally upon overloading electrophoresis the 67K contaminant (
AND

KINOSHITA

strate indicating that the purified enqme is an aldose reductase, rather than an l-hexonate dehydrogenase ( EC I. I. I. 19). Antibodies raised in rabbits against this enLyme formed one line of identity on Ouchterlony plates with either the purified enzyme or the crude ammonium sulfate dialysate and no line with human serum (Fig. 3). Similar results were also obtained with the more sensitive Laurel1 rocket immunoelectrophoresis in which only one peak was observed with either crude or purified enzyme preparations. This indicates that the antibodies were directed against human placental aldose reductase. These antibodies also formed a line of identity with human lens aldose reductase but no line with rat lens aldose reductase on either Ouchterloney plates or rocket immunoelectrophoresis. Upon repeated bleedings of the rabbits antibodies directed against human serum albumin also developed. These, however, could be removed upon absorption of the antiserum with human serum albumin. DISCUSSION Human placental aldose reductase was tirst purified by Clements and Winegrad in 1972 (17). Using an involved six-step pro-

FIG. 3. Immunoreactivity on Ouchterlony plate of antibodies prepared in rabbits against purified human placental aldose reductase.

RAPID

PURIFICATION

OF

HUMAN

PLACENTAL.

cedure which culminated with two isoelectric focusing columns, enzyme with 17X0-fold purification (specific activity 1675 pmol/ min/g) was (obtained from the original homogenate supernatant. The purified enzyme yielded a single band on electrophoresis. Using affinity chromatography, the purification of this enzyme from the readily available placenta has been reduced to three steps. As summarized in Table I, chromatography of the dialyzed 30-70s ammonium sulfate fraction on 4-carboxybenzaldehyde-coupI,-d AH-Sepharose 4B and dyeligand Matrex Orange A yielded aldose reductase with very high specific activity and 8900-fold purification. The purified enLyme peak gave a single band on SDS-electrophoresis with an apparent molecular weight and K,,, in close agreement to previously published results ( 17). Several dye ligand affinity columns are able to bind aldose reductase. Cibachrom blue F3GA affinity ligand which binds to the dinucleotide fold of enzymes has been used in the purification of porcine brain aldose reductase ( 1X). Human placental uldose reductase also binds to this dye ligand as well as to Imperial Chemical Industries Procion Kcd HE-3B, an NADPH enzyme selective dye- and to Amicon Orange A dye ligand. The apparent .jpecific removal of aldose reductase from the dye ligand with NADPH strongly suggests that the enzyme is also specifically bound to the dye through its nucleotide fold. The orange A dye ligand differs from the other two ligands in that it only

units

(pmol/min)

hlatrcx ” Mean\

Gel

Oranfc of three

(NH,),YO,

3666.4 ‘775.5

9312

440 /I 10 five determinations

0.158 baxd

55.1 on

57

I

Total

X-70’; JB

REDUCTASF

binds 8 20%~ of the total proteins bound by either the blue or red dyes ( 19). With human placental aldose reductase the Orange A dye ligand is, therefore, superior to the other two dyes in that it binds significantly less contaminating proteins. The only aigniticant contaminating protein observed in this procedure is a h7K protein, immunologically identified as serum albumin. which can essentially be removed by washing with either NAD or digitonin. Moreover. the serum albumin may be only indirectly bound to the column through a protein protein interaction with aldose reductase since serum albumin has been reported to stabilire aldose reductase activity. During the wash a small amount of inactive aldose reductase is also removed with the serum albumin suggesting that either inactive aldose reductase is less tightly bound to the dye ligand so that it can more easily be removed by ;I nonspecific nucleotide or that there may be more protein protein interaction between inactive aldose reductase and serum albumin. Specific antibodies produced in rabbits against the purified human placental aldose reductase gave an apparent single line of identity with either human placental or lens aldose reductase on both Ouchterlony plates (Fig. 3) and the more sensitive rocket immunoelectrophoresis. No line of identity was formed with rat lens aldose reductase indicating immunological species-specific differences between rat lens and human lens aldose reductase. This adda evidence to the suggestion that differences in the inhibitor

TABLE

Dialywte from At I-Sepharosc

ALDOSE

100 ml uf dialysatc

Specilic activity Purifcation

(fimol/min/g)

394 6304 3.5 ;r IO” roughly

corresponding

(1) 16 XX96 to one-half

Recovery (‘5)

(100) 76 I5 placenta.

58

KADOR.

CARPER,

AND

structure activity relationships observed between rut lens aldose reductase and human lens and placental aldose reductase or the apparent brain lens aldose reductase are due to tissue-specific differences in the susceptibilities of the enzyme to be inhibited.

6.

fate fraction, nical assistance David

Drs. Regina Skelly and lgal on the rabbit immunizations.

Greenhouse

for

technical

Gery and

for techto Mr.

7.

J. H.. L. 0.

Fukurhi, (1980)

Fukushi.

(1979)

S., Kador.

Mc~taholisnr

P.. and

28,

S.. Merola. L. 0.. Invesf. Ophrhalnwl.

and

462

Mer-

469.

Kinoshita. Vis. Sci

J. H. 3 13-

19,

316. 8.

Kador, P. F.. Merola. L. 0.. and Kinoshita. J. H. (1979) Docum Ophthal. Proz. Ser. 18, 117-l 24.

9.

Kador. P. F., Kinoshita. J. H.. Tung. Chylack. L. T.. Jr. (I 9X0) Inwsr. Vis.

IO.

Sri.

I I.

W. H., Ophrhalnwl.

and

19. 9X0-982.

Hoffman,

P. L..

J. P. ( 1980) Yoshitake.

M.

B., and

Swuki.

N.

Bradford,

Wermuth. J. Nrurochmt.

M..

mazono, 12.

help.

Kinoshita. ola.

ACKNOWLEDGMENTS We wish to thank Dr. Joseph Shiloach of the National lnstitutc of Arthritis. Metabolism and Digestive Diseases Pilot Plant for preparation of the ammonium sul-

KINOSHITA

K.. Yamada.

( I96 I ) J. Biochem. M.

van

(1976)

Warburg.

35, 354-366.

.4nal.

K., and 49.

Shi-

6 I X-634.

Biochenr.

72.

24X

254.

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