Inhibition of action of lens mutarotase on glucose by cataractogenic sugars and corresponding polyols

AR(‘HIVES

OF

BICICHEMISTRY

Inhibition

AND

BIOPHYSICS

of Action

Cataractogenic

102, 306-312

of Lens Mutarotase Sugars

and

:ILl3E:RT Frovr

the Institute

(1963)

j’or Medical Received

on Glucose

Corresponding

by

Polyok’

8. KESTOX

Research

and Studies,

February

Sew

I-or/i,

.Tew

York

26, 1963

The enzyme mutarotase, which catalyzes the interconversion of anomeric forms of glucose and certain other sugars, is present in the tenses of various animals. The action of the enzyme on glucose is inhibited hy the cataract,ogenic sugars (o-xytose, D-gal&use, I,-arabinose) and, to a lesser extent, t)y the polyols resulting from the reduction of the sugars. The noncataractogenic sugars do not inhibit the enzyme. A possible relationstliu between inhibition of the enzyme mutarotase and the meclla&m of g:tl:tctose caltnrtlct is discussed.

accumulate Gal-l-l’. They postulated t,hat their findings may be involved in the genesis of the lesions associated with galactosemia. Schwarz and Golberg (8) found that Gal-l -I’ and galactose accumulate in lens capsules after galactose feeding, and suggested that Gal-l-l’ may interfere with metabolism of the lens in a similar manner to that in the erythrocytes. However, Korc (9) fouud only slight accumulation of Gal-l-P in lenses during the first, I1 days of galactose admillistration. The accumulation of Gal-l -1’ may thus not be of central importance ill cataractogenesis. I:urthermore, the involvement of phosphoglucomutase does not serve to rxplain the cat,aractogenic action of other sugars, such as xylose. .A more unitary hypothesis, involving the role of mutarotase iu the cataractogcnic action of galactose and other sugars, was suggested by .Mestou (10, 11). Darby and Day (la) reported that I)-galactose and I)-xylose, wheu fed to weanlitlg rats, produced cataract, while I)-arabillosc, [)-fructose, alld I)-maut1ose did Ilot’. I’atterson (13) and ICeston (14) have demoustratcd cataractogeuic action of I,-arabillosc, and pointed out t,hat the structures of various cat,aractogcllic aldoscs are similar to glucose on carbons I, 2, alld 3. Likcwisc, ill order for aldoscs to act a::

The enzyme mutarotase catalyzes the interconversion of the various anomeric forms of n-glucose and various other sugars (I-S), e.g., oc-I)-glucose s P-I)-glucose. A possible relationship between inhibition of mutarotase and the mechanism of galactose cataract will be discussed here. Galactosc cataract has been the subject of much study and speculation, particularly since the cataract of galactosemia may ininvolve similar factors. Kalckar, Anderson, and Isselbacher (1) have shown that galactosemia may be associated with a lack of P-gal-uridyl transferast. Schwarz et (11. (5) have shown that galactose 1-phosphate (Gal-l-P) accumulates in the tissues of galactosemic individuals. Sidbury (6) aud Ginsburg (7) reported that Gal-l-I’ strongly suggesting inhibits phosphoglucomutase, that this inhibition may bc a factor in the cataract of galactosemia. Schwarz et al. (5) found that oxygen uptake of galactosemic rrythrocytes ou glucose substrates was partially inhibited by the preseuce of galactose and that erythrocytes from galactosemics oil exposure to galact’ost I Ttris the U. AMo35m.

researrtl was supported 1)~ it grant, S. Public Ilrnltl~ Service, grant

from So. 3O6

M17TAROT.4S;P:

mutarotasc suhxtrates, their configurat’ion has t,o he similar t.o glucose on carbons I, 2,

and :< (2, 11). Because of this similarity in wirfigurational requirements of cataractogcnic sugars and mutarotase substrates, it may he worth investigating whether mutarotase is involved in galactose cataract. Whilr the role of mutarotase in t,he scheme of carbohydrate metabolism has not as yet bec~l clearly cstahlished, Keston has postulated a m~itary theory of glucose transport ilivolving mutarotase (3, 14). It’ is uot necessary to accept this unitary theory of glucose transport as the hasis for t,he role of mutarotase in cataractogenesis. At present it would seem reasonable to assume, at least, that mutarotase is involved in some aspect of carbohydrate metabolism, and to suggest that the carbohydrate metabolism of the lens may be affected by the competitive inhibitory effect of cataractogenic sugars (I)-xylose, u-galactose, L-arahinose) on mut,arotasr’s actiou. In this article me are focusing on the interference of cataractogenic sllgars with the action of mutarotasc on glucose. In the scheme of carbohydrate mct,aholism many substances, other than glucose, have the configurational requirement, of mutarotase substrates. The metabolism of these suhstallces must. also be cow sidercd t.o make the investigation of a possible role of mutarotase in cataractogenesis more complete. Recently van Heyningen (15) found very high concentrations of dulcitol (2.0 9) and xylit.4 (0.4 ‘X) in the lenses of rats fed galact,ose and xylosc, respectively. High lrvcls of these polyols were found in the early stages of the experiment, hcfore the cataract was visihle. While the polyols dulcit,ol (galactitol) and rylit’ol, which are the reduct,ion products of’ galactose and xylosr, respectively, are weaker inhihitors of mutarotase than the parent sugars, they also contribute significantly to the inhibition ol mutarotase when present in these high COIIcclltratiolls. Sorhitol levels ilr t,he lenses of alloxan diabetic animals reached 0.4 ‘:;, (1.5). It has hecn pointed out by Idwine, Goldstcill, cf. al. (16, 17) that, under the influetlce of insulin, sugars with the configuration similar to glucose on carbons 2 and :3 penetratc into cells more easily than sugars of

:307

ISHIRITION

other configuration. ;2ldoses which are mutarotase substrates also have this cow figuration. Galact,ose and xylose would thlts be among the sugars that would penetrat,c more readily. On this basis, their reduction products (dulcitol and xylitol) would tend to accumulate in the lens ill higher concew tration than the reduction products of sugars of other configurat,ion. In other words, the rate of transport of sugars might well be the determining factor affecting the extent of accumulation of the polyols which are their reduction products. Involvement of mutarotase in galactose cataract cannot be predicated unless it can he shown that (a) the enzyme is present in the lens; and (6) after feeding of cataractogenie sugars, in the early stages of cataract formation, the inhibitors of mutarotase are present in sufficient quantities to inhibit the enzyme markedly. The presence of the enzyme in the lens and the degree of its inhibition by cataractogenic sugars will be shown here.

a-u-Glwose was finely pulverized anhydrous Cerelose from Corn Products Co.; wwrt)itc~l (trydmt.e), rt-gxlmtose, wrylose, xmihinose, and wlevul~bse were from Pfanstiehl Lslwratories; dulcitol WE from Amend I)rug & Chemical Co., Inc., New York City; xylitol was from Nut,rit,ion:tl Hiochemicals Corp. The buffer was 0.025 .I1 phosphnte

buffer

:r;FFl 2’

I

= 2, prepared

from

t tw

sodiunl salts. Tlle steer lenses were excised from slaughterhouse :mirnals. The r&bit lenses xwre 0M:tined frozen from I’el Freeze Co., Rogers, A4rkans:rs. ,\[ETHOlW

TO dcrn0nstr:tt.c t Ire presence of mutarotase in, for example, steer lenses, 12 steer lenses wrr pooled, added to twice the weight of the bul’l’c~r used in the assay, hrnogenized in a 50-ml. vesw~ of the Send1 homogenizer for 2 min. at 80 v., anti wlltriflqyd in the 851i tread of the HKl Int.ernational centrifuge for 20 min. at 10,ooO r.p.m. The supernatant solut,iou was centrifuged in Spinco model I, ultracentrifuge for 90 min. using the IO-rotor at -W,OOO r.p.m. The centrifuged s,jlutic,ns of the lens hcmlogenate ~,ccasic~nall~ t)ec:irrie t,urhid. Freezing and t Il:lwing. f~~llo\YPri

308

KESTON

1)~ recentrifugation, helped reduce the turbidity so that it did not interfere with the polarimetric assay. The assay medium was the bufl’er described in the Materials section. For the assay of the steer lenses, 0.8 ml. of the lens homogenate was added to 7.2 ml. of the bufl’er. This was brought to 24°C. 1)Jr being placed in a water bath. Tllen 25 mg. of finely powdered solid a-glucose was added and quickly dissolved, and the solut,ion was transferred to the 7.5.~~~. polarimeter tube. When sugars other than glucose were being measured, the appropriate solid sugar was used in place of a-glucose. When the 5-11~1. polarimeter tube h-as used, tire total volutiie of I)ull’er and enzyme solution was 5 ml.* The mutarotation of the sugar was followed in the pllotoelectric polarimetric attxhment previously described (18, In), hut with c::lcite polariaing prisms instead of polarizing filters in t,he optical field. h water-jacketed 5- or 7.5.cm. Koehler Instrument Company polarimeter tube was maintained at 24.0%. The polarimetric attachment, was in tile ligllt path of either a Zeiss PQM or a Beckman 1)U spec:tropllotometer. Since the Zeiss instrument, aut~~nistically sets the reference beam at lOOr; transmission (a requirement of the polarimetric method), it, was possible to nlakc as many as 6 readings/min. wit11 this unit,. To insure that equilibrium Ilad been reaclled, wit,lrin the limit,s of the esperimental error, nieasurements were continued for about ten times t,he reaction half-time. Since the kinetics of the mutarot,ation of glucose follow a first-order reaction rate, t.he plot of log iat - a=) as the ordinate against, time (1) as the abscissa is linear (01 is the optical rotation). Gallop (20) leas shown a linear relationship of optical rotation wit11 “optical density” readings (D) when t,his type of polarimet,er is employed. Tllercfore, we have plott,ed on seinilogaritlllrlic paper t,lie values of (D, - DIj) instead of (a, - ax) against time (1). Straight lines were drawn through the points, and the halftime of the reaction was determined from the plot. Kinetics of the reaction are independent, of the wavelength at whicll tile polarimet,ric readings * Preparations of lens extracts. Preparation 81 has been described in the text. The following were prepared similarly with the exception that : Rl: the weight of the rabbit lenses was 14.5 g and they were IIomogenized with 2I4.0 ml. of assay buffer; R2: the weight of tile rabbit lenses was 29.5 g. and they were homogenized with 59.0 ml. of assay buffer; RTl : tlie lenses from 50 rats, whose average weigllt was 20 g., neiglled l.ii g. and were Iromogenized with 8.9 ml. of assay bufter; I)1 : the weigllt of the dog lensc~ was 3.3 g. and were homogenized wit11 10 ml. of assay buffer. These lrnscs had been stored in the freezer for 1 months.

are obtained. Gsually a fixed wavelength between 500 and 600 rnp was selected. In all cases the spontaneous uncutslyzed rate was determined in the absence of the enzyme, and equal volumes of buffer were substitut,ed in place of the enzyme solution. Variations from this general procedure will be mentioned with the appropriate t,ables. The portion of the rate of t,he mutarotation reaction due to catalysis hy the enzyme is V = (k, - h-.,)S. h-, and h-, are the mut:trotat,ion cocfficient,s of the reactions carried out in the presence and absence of the enzyme, respcct,ively. The nil]tarotation coefficient is the sulll of the rate constants of the fornard and backward components of the rcvcrsible mutarotation reaction and is equal to 0.693/tl,s. S is subst,rafe concentration. In order to test t,lre rffect. of inhibitors, the inhibiting substance was added to the appropriat,e amount of buf?er and enzyme. Where the inhibitor was a sugar, tile solution was allowed to stand long enough to come to rrilltarot:ttioliitl eyuilihbrium (at, least ten times the estimated reaction half-time for the pnrt,icular inhibitor). Solid cu-glucose was then added, and the course of t,lre reaction was followed as described previously. The fractional residual activity is calcul:ited 3s follows :

r1 1 = fractional

residual

activity

=

17 t1*2s l1/2r0, r ---1 l-l

l-l

where tllBlll is the measured h:tlf~t,ime in prescncc of enzynie; fliQs is the half-time of spontaneous reaction (without enzyme); and IlrZn,, is the halftime in presence of enzyme and inlrit)itor. “;, inllibition

= 100 X (1 -

1’)

To determine t,lre nIi~tluelis-blrrlt.rn constants (21) of the enzyme, Lineweaver-13urk (22) plots were constructed using data obtained sequentially on the same day with the same conrentrxtioll of enzyme but with different, concentrations of substrate. Figure 1 shows determin:tt,ion of Ilalft,imes for one of these sets. Figure 2 shows Lineweaver-13urk plots for two setSa of detcrll~ill:tt,il,rls of the K, of glucose for rabbit lens mutarotase. The nveragc K, value det,erniinrd f’ronr these plots is 0.011 M. Inhitjitor constants (K, 1 wlliclr may he usrtl in calculating tlte degree of inhibition wit Ii various colrcent,ratic,rls of intribit,ors and substrates, c:ul I)e obtained from IQ. (1) which describes eonpetitive inhibition in accordance wit11 Mir~!;:trlisMenten theory (23) :

iWJTAROTAGE

309

INHIBITIOS

40 30 4

\

\

I 70 Minutes I~‘Ic;. 1. Effect 112 from Table

of concentrntiorr I). Total glucose:

of glucose top curve

X6

on action of r:lW)it, 100 mg., middle curve

lens

mntarotnse mg., bottom

67.2

(data curve

of Prepn. 35 mg.

too I/V

80

t

80 FIG.

2. J,inewenver

of P~-P~Ix~. ru and

Wu,

60 Burk plots Table I).

40 of data

I

20 for

ralhitj

lens

I

I

I

20

40

60

mutarotase

with

glucose

as R snhstmte

(data.

310

KESTON

I s-l=

(1)

A-X:

where h, = Michaelis-Menten constant of sub skate, C, = concentration of substrate, Ci = concentration of inhibitor, and Ki = inhibitor constant. In t,hese experiments t.tlc sugar act,ing as conpetitive substrate was present at Inut:rrotational equilibrium, and only the substrate (glucose) contributed to t,lre measured changes of optical rotation. IJnder these conditions KC equals K,,, of

the inhibitor

sugar (21).

Table I shows that mutarotase is present in the lens of several speciesof animals. It can be seen that lens mutarotase has cousiderable activity in catalyzing the mutarotation of sugars. The enzymic nature of the catalysis is illustrated by its stereospecificity. The system catalyzes mutarotation of n-xylose and L-arabinosc, but does not

Prepn. X-0.

lens

Sl

$1 Sl Sl

Rabbit lens

lhg

lens

25

$1 Rl Rl

67.2

R2 R2 112 R2n R2a R2:t Rat lens

25 25 25 25 25 50

81 Sl

similar animals were maintained on a normal diet, tile lens concentrations were essentially

0 for galactose and 0.5 mAI for glucose.

Sugar ml.

mg.

Steer

catalyze the reaction of L-xylose and u-arabinose. r>-Galactose is also a substrate. Table II illustrates that the cataractogenic sugars, aud the polyols dulcitol and xylitol, do inhibit the action of mutarotase on glucose,and that’ the noncataractogenic sugars do not. The calculations from these data illdicatc marked inhibition of mutarotase’s action at concentrations of inhibitors of the order of magnitude that may occur in tissue when the animals received a cataractogenic sugar in their dirt. Iiinoshita (25) reported the levels of galactose and glucosein lenses of rats on various diets. In lensesof animals receiving (Lataractogenic amounts of galactose, the concentrations were G md/ for galactose and 0.3 mM for glucose on the fifth day of their diet, just prior to thr onset of cataract. When

42 67.2 35 100

50 25 80

D-GhKoSe L-Arabinose D-Sylose D-Galactose L-Xylose

0.8 0.8 0 .8 0.8

o-Arabinose n-Levulosea

0.8 0.8 1 .o

u-Glucose wGlucose o-Glucose r)-Glucose n-Glucose o-Glucose u-Glucose D-Glucose

2 0 2.0 2.0 2.0 2.0 2.0 2.0

(7.5)” 17.5) (7.5) (7.5) (7.5) 17.5) (7.5) (7.5)

1.0

RTl RTl

35

D-Glucose n-Glucose

5.0

18

I)1

84

D-Glucose

1.5

(7.5) (7.5)

1 .o

a Temperature was 19.5”C. Cacodylat,e buffer, 0.05 X B/A h The values in parentheses are the average of several value for the spontaneous t,;> is independent of concentration

8.5

(7.5)

= 2.33, wits used. determinations made in same in the range measured.

3 2

buffer.

TIE

:31 1

GhKCW

Inhibitor

Inhibitor

Prep.

Inhibition

Buffer

---___~~

','fi.

1l11.

25 25 25 25 25 25 25 25

0 0 20

26 26 26 20 26

(ii.2 (ii.2 (ii 2 67.2 (ii.2 (ii. 2 (ii.2 (ii.2 (ii 2 Iii.2 (ii.2 (ii.2 (i7.2 67.2 (ii. 2

L’or rabbit lens mutarotsse, our data yield tmhr value of 0.014 A/ for tjhe K,, of glucose alid 0.004 .I/ for the /Ci of gala&our. :Zssumilg Kilwshita’s values for gala&se alld ~lucosr ill the rat 1~11saud using FIq. (l), it caii br calculatrd that in Go the extent of’ ilrtiibitioii of ICIIS mutarotase’s action on glrlcosr bv galact,osr would be about GO”;. ‘I’hr I\‘; :;lld K,, values for rabbit lens mutarotasc wrrc usrd in thrsr calculatioiw, siiw it was llot rspedirnt t,o make rxtrilsivc kiilrtic st,lidirs of rat lens niutarotasr ii) viw of t,hr tow wright of each rat lrlis aild its rctativrtv tow tcvel of mutarotase coiit,rntj. Ttirsr ciifEcultirs would have greatly tw ~IKT~ thr accurxy of’ t,lw \xlues obt,aitwd with nirt~tiods avaitat~te. tiitwshita (20) also descritwd ill vitro studirs iti which cataracts were produced in young rabbit, trns t)y gala&se wi-ithin 3 days and t)y sylosr iti I day. His medium cotltaitied .i nimotrs glucosr I. ai~t rithrr 130 mdl

0 0 10 10 10 00 150 150 0 160 210 0 250 0 1.50

C’<-

7111.

.-

0.0 0.5 0.5 0.5 0.5 0.5 0.5 0 .5

5 1.5 1.5 1.5 4 5 4 .5 4.5 -4 .5

T.5 2.4 3 .7 1.5 4..i 2.5 2. 4 2.4

0 2.0 2.0 2.0 2.0 2.0

8 6.0 6.0 ti.0 (i .o

7.5 2. :?I:< 3 00 3 56 3 !I7 3.1; 3.01 3 2.5 2 .:10 2 !)8 2.78 2.40 2. !I:% 2.19 3.00

2 0

(i .O fi .O

2 0 2 0 2.0 2 0 2.0 2.0 2.0

(i .o (i .o li .o ti .O li .o 6.0

2 0

6.0

(i. 0

51 (i!) G 1 0 0

33 50 GO 48 33 11

0.016 0.008 0.001 0.013 0 ,057 0.035

33 ?1

0.051 0 OO!J

:3i

O.O!E

38

0. O-Hi

galactose, or 30 m31 sytose.” 4\s can he srrn from F:q. (l), 30 mill concentration of galactosr (26) would inhibit rabbit lens mut,arotase about 85 T; , while the sylosr would it1 hibit it’ about, OO’G. (The K, for sylosr calculated from our data is 0.015 :I/.) The averagr I<, vatiie for rabbit, letIs miitarotasr for xylitol calculatrd from out’ data is 0.05 111,and t,ht I\‘, for dulcit,ot is 0.04 .I/. I-sing these valurs, tiiltostiit~a’s vatur of 0.3 mJ/ glucose, and vat1 Hryninge~r’s valurs (15) for conceiitratiolls of the potyols ~01’. rected for the 1e11s solids (0.1 fi 111 dulcitot and 0.038 A/ xytitot), the calculatioiw itldicate that the degrer of’ itlhibitioll of’ rabbit tells mutaiutasr t)y tlirsr polyots would t)r ahut 80 “; for dl&:it~ot atld 412“; for sylit,ot in viw Sorbitot trvrls iii ttw triises of’ altosalk diabetic rats, corrected for trtls solids, aw shit

0.03

.I/

(1.S).

t:rom

0111’ data

ttir

K,

calculated for sorbitol is about 0.10 M. Thus, the degree of inhibition of rabbit lens mutarotase by sorbitol would be about 2-l ‘5,. On the basis of the hypothesis that mutarotase is involved in cataractogenesis, a question might be raised whether the high glucose levels in diabetes might conceivably also contribute to cataract’ogenesis, if they produced very high levels of sorbitol in t,he lens. Against this argumed is the possibility that the higher glucose levels would promote higher carbohydrate utilization so as to tend to compensat’e for the inhibition. It should be pointed out that the COP centration of the inhibitors in the lens epithelium, which is the region of highest carbohydrate metabolism, would be higher than the analytical values obtained for the total lens. Accordingly, their effects on rabbit lens mutarotase might he expected to be higher than the calculations involving these substances indicate. .ACKNowLEI )( :hrE:n-‘l The antllor wislles to express his npprwiation to Mrs. Bert h:i Bout is for her technid assist,:ulce ill this work. liP:FE:r1P:~cI~S 1. KEILIN, 60, 2.

I)., 341

LEVY,

(:.

67, 50 3.

KESTON,

I\NL)

B.,

II.

BACIJER,

15. F.,

Hioc~hem.

./.

.4~,)

(holi,

14;. s.,

hxhem.

.I.

(lwl).

SGmz

rZ. H.,

-1. KALCKAR,

H.~I~‘Iww,

(1952).

31..

120,355

:~M)ERSON,

J., Pm,.

K.

(1054). E.

I’.,

Ayatl.

:icad.

L.,

KOM~OWER,

AND

ISSEL-

&?.,

t!. 8.

42, 49 (1051i). 5.

SCHWARZ, ANI)

V., HOI.ZER,

(;oI~ER(:, A.,

Biwhm.

.I.

G.

&I.,

62, 34 (1956).