Methionine adenosyltransferase and S-adenosylmethionine in the developing rat lens

Eq,.

Eye RPS. (1988)

Methionine

47, 197-204

Adenosyltransferase in the Developing

and S-Adenosylmethionine Rat Lens

ARTHUR M. GELLER*. MALAK E’. s. KoTutf, HOWARD M. JERNIGAN, JR*§ AXD NI(:HOLAS M. KREDICH$, Departments Memphis.

of *Biochemistry

and

§Ophthalrr,oloyy,

CTniversity

TN 38163, and the SHoward Hughes Medical Institute, Medical Center. Durham, NC’ 27710. U.S.A. (Received

12 May

19X7 altd accepted in revised form

of Tennessee, Duke University

1.5 October 1987)

Methionine adenosyltransferase (MAT) activity, and the concentration of its reaction product, Sadenosylmethionine (AdoMet), were measured in the lenses of rats of different ages ; ranging from 1 day of age to over 1 yr. The highest specific activity of MAT was found in the lenses of the one day old rats (sp. ac. 0327 units/mg-’ protein). After 1 week the specific activity had dropped to 6067, and by 6 weeks had declined to adult levels (602 units/mg-’ protein). AdoMet concentrations were measured by HPLC in perchloric acid extracts, The highest concentration of AdoMet was found in the lenses of day-old rats (48.2 PM), and gradually declined with increasing age, reaching 55 ,UM in the oldest rats. In addition, the specific activity of MAT was found to be higher in the lens epithelium than in the cortex plus nucleus. The specific activity of MAT is almost an order of magnitude higher in the lens epithelial fraction (0.099 units mg-’ in the combined cortex plus nucleus fraction (0.011 units mg’ protein). Key words: S-adenosylmethionine ; methionine adenosyltransferase ; lens ; aging; ornithine decarboxylase ; putresrine ; polyamines.

protein)

than

development

1. Introduction S-Adenosylmethionine (AdoMet) has many important metabolic roles, which include serving as a methyl group donor in transmethylation reactions, participation in the biosynthesis of cysteine, glutathione, and taurine through the tram-sulfuration pathway, and functioning as a source of propylamine groups for the biosynthesis of the polyamines, spermine and spermidine (Borchardt. Creveling and Ueland, 1986). In the lens, AdoMet may be involved in the methylation of aged or damaged proteins by protein carboxyl methyltransferase (McFadden and Clarke, 1986), and in the differentiation of chicken lens fiber cells (Zelenka, Beebe and Feagans, 1982). In addition, sulfur from 35S-labeled methionine is incorporated into cysteine and taurine (Gupta and Mathur, 1983), and glutathione can be synthesized in the lens (Reddy. 1971). AdoMet is synthesized from methionine and ATP by the enzyme methionine adenosyltransferase (MAT). This enzyme is widely distributed in mammalian tissues due to the essential role of AdoMet in transmethylation reactions. In previous studies we reported that partially purified MAT from rat lenses is very similar to the highly purified MAT isozyme from human lymphocytes (Geller and Jernigan, 1985; Kotb and Kredich, 1985), and that both enzymes seem to have similar properties and regulatory mechanisms, including inhibition by dimethylsulfoxide (Geller, Kotb. Jernigan and Kredich, 1986). We have extended these studies t,o investigate the role of AdoMet in lens development. Since AdoMet appears to be important for reactions which occur in both young and aged animals, it was thought that both the activity t Present address: Department Please

address

all correspondence

00144835/88/08OI97+08

of Medicine, University to Dr A. M. Geller.

$03.00/O

of Tennessee,

Memphis,

TN 38163,

0 1988 Academic

L1.S.A.

Press Limited

.\

19x

of’ MBT

\r.(:Er,I,I~:K

b:1

\I

and/or

thv ~onc,erltl.;lt,ioli of BdoMet might lx, important fi)r l)oth thv and aging of lenses. To inrrstigatc the possible role of X&Met in Iens development. both the activity and properties of MAT. as well as the cwncwttration of AdoMet,, were measured in lenses of rats of different ages: from 1 da). t)c, over 1 ~1’ of ape. In addit,ion. the location of MAT in lenses \vas tl&erminrd. differentiation

2. Materials

and Methods

Matfvials TES (N-Tris[hydroxymethyl]methyl-S-aminoethanesulfonic acid), oL-dithiothreitol (DTT). and S-adenosylmet,hionine were purchased from Sigma Chemical C’o. ATP was from PL-Biochemicals, and [‘“C’]methyl-Lmethionine (40-N m(‘i mmol-‘) was obtained from h’ew England Nuclear. Partisil- 10 WX HI’L(’ columns and P-81 ~)hos~)ho~ellulosr ionexchange paper were obta,ined from Whatman. Inc. Rat lenses Lenses were obtained from SpragueeDawley rats of different ages. Eyes were either used immediately or frozen and stored at -70°C. Lenses. removed by posterior excision, wen> homogenized in ground-glass homogenizers. Following homogenization in 5-10 volumes of 10 mM TES, pH 7.3, 100 mM KU, 1.0 mM DTT, 05 InM EDTA, and 50 ,UM phenylmethylsulfonylfluoride, the homogenate was centrifuged in an Eppendorf Microcentrifuge to remove insoluble material. In experiments involving the capsule/epithelial layer, t,he capsule/epithelial layer was first separated from the cortex/nucleus by dissection under a dissecting microscope. and then homogenized as described above. Lens MAT activity was determined by measuring the formation of labeled AdoMet from [‘%]methyl-L-methionine and ATP as described by Geller. Kotb, Jernigan and Kredich (1986). Protein was measured either b! the dye-binding method of Bradford (1976), using a Bio-Rad reagent kit and serum albumm as the standard. or by measuring the absorbance at 280 nm (and assuming that an absorbance of 1.0 is equivalent to a protein concentration of 1.0 mg ml-‘). One unit of MAT activit: is defined as nmol AdoMet, formed hr.‘, and specific, activity is defined as units MAT act’lvlty rng-’ protein. Determination of AdoMet AdoMet was determined by a modification of the method of Kredich and Hershfield (1979). Rat lenses were excised, weighed, and homogenized in perchloric acid (PCA). The homogenate was centrifuged in an microcentrifuge for 2 min to remove insoluble materials. The aqueous with a combination of KOH and layer was quantitat’ively removed. and neutralized KHCO,. AdoMet was determined in the neutralized PCA extracts by HPLC analysis using a Whatman Partisil-10 SCX column (@46 x 25 cm). The AdoMet was eluted isocratically at’ 1.0 ml min-’ with 019 M ammonium phosphate containing 2% acetonitrile (v/v). and adjusted to pH 2.6 with H,PO,. AdoMet was quantified by simultaneously monitoring absorbance at X54- and 280 nm. AdoMet standards were prepared as described by Kotb and Kredich (1985), and standardized spectrophotometrically assuming an atso of 14.6 x lo3 Me1 cm-‘. The amount of AdoMet per injection volume was calculated from a standard curve of peak height vs. AdoMet concentration. decarboxylaase assay Ornithine decarboxylase (ODC) activity was determined by measuring [‘“C]CO, from l-[Wlornithine as described by Lau and Slotkin (1980). Ornithine

the release of

3. Results Changes

in lenticular

MAT

activity

with

age

MAT activity was measured in the lenses of rats of ages varying from 1 day to over 1 yr of age. Lenses from newborn animals averages 5.7 + 1.17 mg per lens; lens weight

METHIONINE

ADENOSYLTRANSPERASE

AC’TIVITY

199

rapidly increased during the weeks of life, followed by a marked decline in the growth rate (Table I). When MAT activity was measured in the lenses of animals of different ages, it’ was found that the specific activity of lenticular MAT was highest in bhe youngest animals, and then decreased with increased age (Fig. 1). The specific activity of 033 units mg-’ protein, found in l-day old rat pups. is over 16 times higher than TABLE

Units

of MAT

a&z&y

I

per 1en.s as a function. Lens wt (mg)

Age (days) 1 9 10-22 32 Adults Old adults

of age and lens weight

[‘nits

of MAT

per lens

570+ 1.16 1504*091 2190+361 2656+2,78

W501& 0097 @372+0.116 0.259 + @078 0.239 + 0.052

4543*3TGo 61.92fCX3

w348*0.037 0581$-PO19

The ages of the l-, 9., and B-day-old rats were known with certainty. while the remaining animals were grouped within known age limits (for example, lG22 days). Adult lenses were obtained from a mixed population whose weights varied from 200- to 400 g. Lenses from ‘old’ rats were obtained from rats known to be over 1 yr of age and weighing over 500 g. The average lens weights were calculated in the following manner : for each age, several sets of lenses, each set consisting of from one to 111 lenses, were weighed and the average weight per lens was calculated for each set. The weights reported in the table are the weighted-average for all sets of lenses at each age. MAT activity was determined as described in Matrhals and MGhods. 0.4%

1

I

9

IO-13

15-22 Age

FIG. 1. Changes in the specific activity were determined as described in Materials

32

42

Adults

Old adults

(days)

of rat lens MAT and Methods.

with

age.

MAT

activity

and protein

analysis

the specific activity of MAT found in adult rats. MAT specific activity then declined by almost 80 % during the first 8-9 days of life, and then gradually declined for about 4-5 weeks until adult levels (sp. ac. 002) were reached. In addition to the large change in the specific activity of MAT with age, the total units of enzyme per lens also varied with age (Table I) ; the number of units declined following birth, and then increased with increasing age. Therefore, although the specific activity of MAT decreases by a factor of 16 with increasing age, the amount of MAT per lens varies less than threefold with age.

Init,ial attempts to measure t,he concentrat,ion of AdoMet in neutralized perchloric~ acid lens homogenates using the conclit,ions of Kredich a,nd Hershfleld (1979) were equivocal due t.o the large amount of mat.erial elut.ing at t.ha sa,me t,imc as AdoMet.. However, when the ammonium phosphate concentration was reduced from 0.15 to 0.16 M. the retention time for AdoMet increased. and it was possible tjo separate AdoMet from the bulk of the eluting material. To confirm that the identity of the eluted material was AdoMet. t,wo experiments were run. First, a known amount of a highly purified AdoMet standard was added to a lens homogenate. The AdoMet, standard was found to co-elute wit,h the putative AdolIet peak, and the area of the AdoMet peak was increased by an amount equal to t,he amount of AdoMet added. Second, since AdoMet, is unstable in alkaline medium, the put.ative AdoMet completely disappeared, while the remainder of the sample appeared to be unchanged, after a sample of a lens homogenate was incubated for 60 min with KOH (Fig. 2).

FIG. 2. Determination of AdoMet in lens homogenates. Aliquots of neutralized PCA extracts lenses were analyzed for AdoMet as described in Materials and Methods. HPLC tracings of: AdoMet standard purified as described in the text, (13) an aliquot of sample of a neutralized PCA of rat lens, (C) a neutralized PCA extract containing a known quantity of an AdoMet standard, a neutralized PCA extract following incubation with KOH. AdoMet standards were purified. concentration determined on a Whatman Partisil- 10 SCX column as described in the text.

Concentration

of AdoMet

in

of rat (A) an extract and (D) and the

rat lenses

To determine if the concentration of lenticular AdoMet was related to the activity of lens MAT, the concentration of AdoMet was measured in lenses of rats from different age groups. Like the specific activity of MAT, the AdoMet concentration was found to decline with increasing age; however, the decline in the MAT specific activity and AdoMet concentration followed different time curves. Lens AdoMet concentration was about 48 ,u~ on the day of birth, decreased to 18.0 ,UM by the third week, and continued to decline throughout life, reaching a concentration of 5.5 ,UM in the oldest lenses (Fig. 3). During the first week of life, the decrease in AdoMet concentration was not as steep as the decline in the specific activity of MAT, which declined by 80% from day 1 to day 8, compared with a 23 % drop in the AdoMet concentration. In the

METHIONINE

ADENOSYLTRANSFERASE

AC’TIVITY

201

50 40 9 ;

30

z i

20

IO

0

FIG.

described

I

9

IO-13

3. Changes in rat lens AdoMet in the text.

oldest animals, the MAT 6- and 11 “A;,, respectively. Localization

15-16 22 Age (days) concentration with

32

Adults

age. Ad&let

specific activity and the AdoMet of the initial values.

Old adults

was measured

concentration

in rat

lens as

dropped

t’o

MAT

of lenticular

To determine the location of MAT in the lens, the capsule/epithelium layer was carefully removed, and MAT specific activity in that, layer was compared with MAT specific activit,y in the cortex/nucleus. It was found that the MAT specific activity in the caapsule/epithelium fraction was about nine bimes higher than the activity in the cortex/nucleus fract,ion in both young and adult rats. When compared with a wholelens homogenate. the activity in the capsule-epithelium was enriched five-fold, while the activity in the cortex/nucleus fraction was significantly reduced (Table II).

Specific

activity

of MAT

Le,rw

epithelium was removed analysis were described ornithine

decarboxylase

different

portions

3’.day-old rats 0.1.7.7 SL 0,014 0.036

Capsule/epithelium Cortex/nucleus Whole lens Lens protein

in

under a dissecting microscope. in Materials and Methods.

of the lens

Adult rats 0.0999 O-01 11 0.0194 Determination

of MAT

activity

and

activity

To determine if AdoMet is utilized in rat lens by reactions other than the methyltransferase, we measured the activity of ornithine decarboxylase (ODC), which synthesizes putrescine from ornithine, and whose activity can be used as a indicator of polyamine synthesis, since it is the initial reaction in the formation of the polyamines, spermine and spermidine, from putrescine and decarboxylated AdoMet. A homogenate prepared from 28 lenses from day-old rat pups was assayed for ODC activity, and the specific activity was found to be 1.09 nmol CO, evolved hr-l g-l of lens. This indicates that lenses have the capability of utilizing AdoMet via the synthesis of the polyamines.

4. Discussion total units of &IT act,ivity, X(loMrt In this study MAT spec*ific. ac$ivity. concentrat,ion and lens weight were measured in rats as a function of’ age. Measurements of lens weight,s coonfirm previous studies which show that the growth curve of the rat lens is asymptot,ic (Norrby, 1958), with the most rapid period of growth occurring within the first, few weeks of life. Both the specific: activity of MA’1 and the concentration of AdoMet were found to be age dependent, being highest in the youngest animals, and decreasing with increasing age. For example. t,he specific activit’y of MAT in the lenses of i-day-old rat pups is over 16 times higher than the specific activity of MAT in the lenses from adult animals. The decrease in specifics activity during the first’ weeks of life is largely due to the rapid growth. and accumulation of protein, that occurs during this period; however, it is also due to a decrease in total activity of MAT in individual lenses. The number of units of MAl activity per lens decreased during the first 5-6 weeks of life and then began t’o increase again. Since the specific activity of MAT remains constant’ during adult life. and the size of the lens increases with age, then it is not, unexpected that the t’otal MA’1 activity also increases wit’h age. When the specific activity of MAT in the lens was determined in different, portions of the lens, it was found that the specific activity in the epithelium was almost 10 times higher than in the combined cortex plus nucleus fraction. The properties of lenses from young rats also appeared to be similar to t’hosr inhibition by AdoMet, phosphate. and pyrophosphat,e of older rats, showing (unpublished results). The AdoMet concentration also declined with increasing age ; however, the decline in the AdoMet concentration was not as rapid as the changes in the specific activit’) of MAT. During the first weeks the decline in AdoMet concent,ration appeared to parallel the drop in the total units of &MAT. Changes in AdoMet concentration with age could be a reflection of changes in synthesis or utilizat’ion. Bge-related changes in AdoMet concentration (Baldessaiini and Kopin. 1966), and enzymes (Paik and Kim. 1974) ; McFadden and Clarke, 1986) that utilize AdoMet have been reported in other tissues, and the act’ivity of a number of lens enzymes are known to change with age. A detailed analysis of the metabolism and role of Ado,Met in lenses has not been carried out. Labeling studies have shown that methionine sulfur can be incorporated into taurine in the lens (Gupta and Mathur, 1983). The polyamines are also found in lenses, but little is known about, their synthesis and metabolism. Kremzner, Roy and Spector (1983) found polyamines crosslinked to lens proteins in cataractous lenses. In cataracts, the concentration of glutathione and taurine are known to decrease: however. it is not known whether there is any relationship between AdoMet’ concentration and the synt’hesis of polyamines. glutathione. or t,aurine in cataract,ous lenses. In addition, the relative significance of synthesis of these c~ompounds vs. transport into the lens is unknown. Lenses also have an active arginase whose function is unknown (Jernigan. 1983). but a possible role is the synthesis of ornithine, which is subsequently converted to putrescine by ODC. We have det,ected ODC activity in day-old lenses, but studies on ODC or polyamine synthesis have not been carried out in lenses. Diamine oxidase. which can oxidize polyamines, is present in bovine aqueous humor, but not in lens (Crabbe, 1985). Shiono. Kador and Kinoshita (1985) have demonstrated that ornithine can be transported int,o lenses. and a deficiency of ornithine transaminase

METHIONINE

ADENOSYLTRANSFERASE

BC’TIVITY

203

has been shown to cause gyrate atrophy of the choroid and retina (Kaiser-Kupter, \‘alle and Del Valle, 1978), which is associated with increased aqueous levels of ornithine, and cataracts (Kaiser-Kupfer, Kuwabara, Uga and Valle, 1983; Simmel and Takki, 1973). It is clear from this and other studies that both MST and AdoMet are present in lenses, and that AdoMet is an essential intermediat’e in lent,icular sulfur metabolism. Additional studies are needed to quantitatively assess the role of each of the pathways which utilize AdoMet in the lens. and the relat)ive significance of each of these pathways as a function of age and metabolic state. AC’KXU’OWLEDGMEXTG This work was supported by USPHS research grants EY 06540 (AMG), AM 12828 (NMK). and EY 02665 (HMJ). The authors wish to thank Dr Theodore A. Slotkin and Dr Frederic J. Seidler (Duke Cniversity Medical Center. Department of Pharmacology), Dr K. V. Rajagopalan (Duke University Medical Center, Department, of Biochemistry), Dr Elizabeth Fields (Duke University, Depart,ment of Anatomy), and the Duke rniversity Vivarium for generously contributing rat eyes. We also wish to thank Dr Frederic J. Seidler for the ornithine drcarboxylase assay. RFFERFNCE6 3 J Baldessarini. R. J. and Kopin, I. J. (1966). S-Adenosylmethionine in brain and other tissues. .J. NEUROCHRM. 13, 76S77. Borchardt, R. T.. Creveling, C. R. and Ueland. P. M. (1986). Biological Methyl&ion and Drug Design. Pp. 1457. Humana Press: Clifton, NJ. Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72. 248-54. Crabbe, M. J. C. (1985). Ocular diamine oxidase activity. Exp. Eye Res. 41, 777-8. Geller. A. M. and Jernigan, H. M.. Jr (1985). S-Adenosylmethionine synthesis in rat. lens. Fed.

Proc.

Fed.

An&. Sot.

Exp.

Biol.

44,

1225.

Gellrr. A. M., Kotb, M. Y. S., Jernigan, H. M., Jr and Kredich, N. M. (1986). Purification and properties of rat lens met’hionine adenosyltransferase. Exp. Eye Res. 43, 997-1008. Gupta. K. and Mathur, R. L. (1983). Studies on the synthesis and uptake of taurine in rat lens in vitro. Exp. Eye Res. 37, 385-91. Jernigan. H. M., .Jr (1983). Urea formation in rat. bovine. and human lens. Exp. Eye Res. 37. 551-8. Kaiser-Kupfer, M. I., Kuwabara, T., Uga, S. and Valle, D. (1983). Cataract in gyrate atrophy : clinical and morphologic studies. Iwest. OphthuEmoE. Vis. Sci. 24. 432-6. Kaiser-Kupfer, M. I., Valle. D. and Del Valle. I,. A. (1978). A specific enzyme defect in gyrat.r atrophy. Am. J. Ophthalmol. 85, 2OCL-4. Kotb. M. and Kredich, N. (1985). S-Adenosylmethionine synthetase from human lymphocytes: purification and properties. J. Biol. Chem. 260, 39X-30. Kredich. N. M. and Hershfield. M. S. (1979). S-Adenosylhomocpsteine toxicity in normal and adrnosine kinase-deficient lymphoblasts of human origin. Proc. ,Vat. Acad. Sci. tTS.A. 76. 24,564. Kremzner, L. T.. Roy, D. and Spector. A. (1983). Polyamines in normal and catarartous lenses evidence for post-translational modification. E:cp. Eye Res. 37, 649-59. Lau. C. and Slotkin, T. A. (1980). Regulation of ornithine decarboxylase activity in the developing heart of euthroid or hyperthyroid rats. Mol. Pharmacol. 18. 247-52. McFadden. P. Ir’. and Clarke, S. (1986). Protein carboxyl methyltransferase and methyl accepter proteins in aging and cataractous tissue of the human eye lens. Mech. ilgeing Dev.

34,

91-105.

McFadden, P. N., Horowitz, J. and Clarke, S. (1983). Protein carboxyl from cow epe lens. Biochem. Riophys. Res. Commun. 113. 418-24.

methyltransferase

2 )4

.\

;\iorrby. rat. Paik.

Aike.

(1958).

=Ictn

Ophthnlmol.

On thta growth Suppl.

n1. ~:EI,I,Eli

of the 49.

cqxtallinr

ET

AI,.

lrns.

the eyeball

and

thr

vornc’a

in tht.

l-12.

W. K. and Kim. S. (1974). ~:psilon-alk~llysine. Kew assay method. purification. and biological significanrr. .1 rch. Biochem. R~ophy~. 165. 369--78. Keddv. \‘. i%. (1971). Metabolism of glut,athione in the lens. h’xp. fiye Hes. 11. 310-Z. Shiono. T.. Kador, I’. F. and Kinoshit,a. J. H. (198.i). (Irnithine awumulation and metabolism in rat lens. E.xp. Eyp tlp.s. 40, 421-Y. Smell. 0. and Takki, Ii. (1973). Raised plasma-ornithine and gyrate atrophy of’ the choroid and retina. Lancet 1. 1031-3. Zrlrnka. P. S.. Beebr. 1). C’. and Feagans, D. E. (1982). Transmethylation of phosphatidylethanolaminr : and initial event in differentiation of chicken lens fiber cell. Sciww 217. 12&F7.