that of the adult liver. To explore the above deranged flavin-nucleotide metabolism in foetuses consequent on immunoneutralization of the maternal

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1 Bichem. J. (1985) 23, Printed in Great Britain 363 Enzymic basis f deranged fetal flavin-nucletide metablism cnsequent n immunneutralizatin f maternal ribflavin carrier prtein in the pregnant rat Namita SUROLIA, Kavita KRISHNAMURTHY and P. Radhakantha ADIGA ICMR Center fr Advanced Study in Reprductive Bilgy, Department f Bichemistry, Indian Institute f Science, Bangalre-5612, India (Received 23 January 1985/12 April 1985; accepted 15 May 1985) A cmparisn f the kinetic and ther parameters f enzymes f flavin-nucletide metablism in the whle fetus vis-a-vis the maternal liver in the pregnant rat revealed relatively lwer activities f fetal flavkinase and FAD pyrphsphrylase. Passive immunneutralizatin f the maternal ribflavin carrier prtein suppresses fetal FAD pyrphsphrylase rather selectively. Additinally, althugh the activities f fetal nucletide pyrphsphatase and FMN phsphatase were unchanged wing t immunneutralizatin, higher activities f these enzymes in the whle fetus as cmpared with the maternal liver may be respnsible fr the drastic depletin f FAD levels that precipitates fetal degeneratin. Previusly we prvided bichemical (Muniyappa & Adiga, 198a) and immunlgical (Muniyappa & Adiga, 198b) evidence in the pregnant rat fr a specific ribflavin carrier prtein bligatrily invlved in transplacental flavin transprt t ensure uninterrupted vitamin supply t the develping fetuses. The functinal imprtance f this maternal vitamin carrier was shwn by passive immunneutralizatin, which led t drastic curtailment f vitamin influx int the fetplacental unit, culminating in fetal wastage and hence pregnancy terminatin (Murty & Adiga, 1981; Krishnamurthy et al., 198. Mre recently we demnstrated that ne f the majr cnsequences f such acute fetal flavin deficiency induced by immunlgical interference with the functining f this maternal prtein was a drastic disturbance in relative amunts f embrynic flavin nucletides, the mst cnspicuus being the severely depleted FAD cntent (Krishnamurthy et al., 198. Whereas the latter aspect f disturbed flavin metablism is reminiscent f that encuntered in a terminally differentiated tissue like the liver f pregnant rats fed either a ribflavindeficient diet alne r ne supplemented with galactflavin (Miller et al., 1962), r during hypthyrid cnditins (Rivlin & Langdn, 1965), the relatively less prnunced changes in fetal FMN and ribflavin cncentratins in the face f near-ttal depletin f FAD suggested that the develping and differentiating fetus may be endwed with a flavin metablic machinery qualitatively and/r quantitatively different frm Vl. 23 that f the adult liver. T explre the abve pssibility, the activities and kinetic prperties f the enzymes invlved in flavin-nucletide metablism f the maternal liver were cmpared with thse f the early fetuses. This als permitted the delineatin f the enzymic lesin respnsible fr deranged flavin-nucletide metablism in fetuses cnsequent n immunneutralizatin f the maternal vitamin carrier. Experimental Materials ATP, NADPH, phsphenlpyruvate, pyruvate kinase, L-lactate dehydrgenase and catalase were btained frm Sigma Chemical C., St Luis, MO, U.S.A. DL-Alanine was frm Kch-Light Labratries, Clnbrk, Slugh, Berks., U.K. The surces f ther chemicals and bichemicals were detailed previusly (Krishnamurthy et al., 198. [2-' 4C]Ribflavin (sp. radiactivity 31 mci/mml) was btained frm The Radichemical Centre, Amersham, Bucks., U.K. Methds Adult female albin rats (Wistar, 15g bdy wt.) were kept fr mating with fertile males and their pregnancies mnitred as described previusly (Murty & Adiga, 1981). Fr the assay f catablic enzymes, namely FMN phsphatase (acid phsphatase, EC ) and nucletide pyrphsphatase (EC ), as well as f bisynthetic enzymes, namely flavkinase (ribflavin kinase, EC ) and FAD pyrphsphrylase (FMN

364 adenylyltransferase, EC 2.7.7.2), the animals were killed n day 14 f pregnancy. In all cases, the rats were killed 24h after the administratin f either nn-immune rabbit serum (O.

2 364 adenylyltransferase, EC ), the animals were killed n day 14 f pregnancy. In all cases, the rats were killed 24h after the administratin f either nn-immune rabbit serum (O.5 ml/rat) fr the cntrl grup r the antiserum (.2ml/rat) t purified ribflavin carrier prtein raised in rabbits. This dse f antiserum had previusly been shwn t be effective fr 1 fetal rejectin between 24 and 48h f antiserum administratin. A similar sequence f events was bserved when the antiserum was administered n any day between day 6 and day 16 f pregnancy (Muniyappa & Adiga, 198b). The antiserum t chicken ribflavin carrier prtein culd neutralize 48 Mg f prtein/ml f serum at equivalence pint. Sera were administered intraperitnially t day-1 3 pregnant rats. At 24h after antiserum administratin, drastic fetal wastage, as indicated by 5-6% decrease in fresh weight, was clearly evident. Purificatin f chicken's-egg ribflavin carrier prtein and elicitatin f antibdies t the purified prtein in rabbits was described previusly (Murty & Adiga, 1981). After the animals had been killed by decapitatin, whle fetuses were remved and dissected free f the maternal uterine cvering, placenta and the crd. The fetuses were then washed several times with buffer until mst f the bld was remved. Befre hmgenizatin maternal livers were perfused in situ fr IOmin with the buffer used fr the particular enzyme assay until all the bld was remved. Hmgenizatin was perfrmed at -4C with a Ptter-Elvehjem hmgenizer with seven t ten strkes f the Tefln pestle. Fr all enzyme assays, the fetuses and the maternal liver were separately prcessed at 4 C, unless therwise stated, and 1-2% (w/v) hmgenates were prepared, depending n the enzyme t be assayed. Prtein was determined by the methd f Lwry et al. (1951), with crystalline bvine serum albumin as standard. Sheep kidney ap-(d-amin-acid xidase) was partially purified by the methd f Burtn (1955) and was stred as a freeze-dried pwder at -5 C until used. One unit f flavkinase r FAD pyrphsphrylase activity is that catalysing the frmatin f 1 nml f prduct/h under the experimental cnditins emplyed. Similarly, ne unit f nucletide pyrphsphatase r FMN phsphatase activity is expressed as 1 nml f substrate hydrlysed/min. Fr all enzymes, the specific and ttal activities are expressed as units/mg f prtein and units/g f tissue respectively. Km and Vma,. values were determined by the Lineweaver-Burk plt and the h value was calculated frm the slpe f Hill plt by pltting lg[(v/vmax.)-v] against lgs n a linear scale (Segal, 1975). N. Surlia, K. Krishnamurthy and P. R. Adiga Enzyme assays FMN phsphatase. The enzyme extract was prepared fr assay as described by McCrmick & Russel (1962). The assay mixture (1.ml) cntained 7.5 mm-ptassium acetate buffer, ph 5., 2mM-FMN and.5mg f the enzyme prtein (McCrmick, 1962). After incubatin fr 1min at 37 C in subdued light, the reactin was terminated by sptting 1uI f the reactin mixture n t a Whatman n. 3 paper. Flavins were reslved by descending chrmatgraphy using butanl/acetic acid/water (12:3:5, by vl.), and ribflavin quantified flurimetrically with a Perkin-Elmer 23 spectrflurimeter. Nucletide pyrphsphatase. Fr reprducible quantificatin, it was fund necessary t purify this enzyme activity partially befre assay by the methd f Krishnan & Ra (1972). The assay mixture (1.ml) cntained 2mM-sdium carbnate/sdium bicarbnate buffer, ph9.7,.2mm- FAD and the enzyme prtein (.28mg). The reactin mixture was incubated at 3 C fr 1.5 min and the reactin terminated by additin f 2 ml f ethanl. The amunt f FAD hydrlysed was determined by measuring increase in flavin flurescence (Bessey et al., 1949). FAD pyrphsphrylase. FAD pyrphsphrylase was partially purified frm the maternal liver as well as frm the fetuses as described by DeLuca & Kaplan (1958). The reactin mixture (McCrmick, 196 cntained 25 mm-ptassium phsphate buffer, ph 7.5, 1 mm-atp, 1 mm-mgcl2,.5mm- FMN and the enzyme preparatin (1.mg) in a ttal vlume f 1.ml. Incubatin was carried ut in the dark fr 6min at 37 C and the reactin terminated by heating at 8 C fr 5min. After remving the precipitated prtein by centrifugatin, the supernatant was assayed fr FAD by the spectrphtmetric methd f McCrmick (196 as mdified by Rivlin (1969a), which emplyed ap-(d-amin-acid xidase). Flavkinase. Flavkinase was partially purified by the methd f McCrmick (1962) and the activity determined as described by Merrill & McCrmick (198) with slight mdificatins. Assay mixtures (.5 ml) cntained.1 M-Tris/HCl, ph 8.2 at 37 C, 1 mm-atp, 1 mm-zncl,,.1 mm- [2-'4C]ribflavin and the prtein (hepatic, 1 mg; fetal, 12 mg). After incubatin fr 6min in the dark at 37 C, prtins f the reactin mixture were applied t Whatman n. 3 paper and chrmatgraphed with butanl/acetic acid/water (12:3:5, by vl.). Radiactivities assciated with FMN and ribflavin regins were quantified by liquidscintillatin spectrmetry (Merrill & McCrmick, 198). Fetal flavkinase was assayed essentially as 1985

3 Enzymic basis f disturbed fetal flavin-nucletide metablism in the rat 365 described abve, except that the reactin mixture additinally cntained an ATP-regenerating system (2pg f pyruvate kinase and 1 mm-phsphenlpyruvate). Under the cnditins f the assay emplyed, all the reactin rates were linear with time and prprtinal t the amunt f enzyme prtein used. The substrate cncentratins emplyed gave ptimal activities. I.. ~. S-. ce,. ce,,* Cl. +1 cl -l Results FMNphsphatase activities in the maternal liver and the fetus The kinetic prperties f the enzyme frm the tw tissues are summarized in Table 1. Hepatic as well as fetal FMN phsphatase shwed cmparable h values. Further, bth the specific and ttal activities were unaltered by passive immunneutralizatin f maternal ribflavin carrier prtein (Table 2). The Km values f the enzyme fr FMN differed nly marginally, being.3mm and.7 mm fr liver and fetus respectively. -.. C-Cl-.) co cl Li. -6 C- ne _ m C- al CliC 1-~ C Nucletide pyrphsphatase activities in the tw tissues As shwn in Table 1, the Km fr FAD is almst the same fr the fetus enzyme as fr that frm maternal liver. Hwever, the fetal pyrphsphatase activity is abut 4-fld higher than its hepatic cunterpart in terms f bth specific as well as ttal activity. Interestingly, n significant alteratin in the activities f the enzyme was nticeable n immunneutralizatin f ribflavin carrier prtein in the mther (Table 2). Flavkinase activities in the fetal and the hepatic tissues The Km f the hepatic flavkinase fr ribflavin was 14mM as cmpared with 7pM fr the fetal enzyme (Table 1). It may be mentined that the fetal flavkinase culd be assayed nly after the inclusin f an ATP-regenerating system in the reactin mixture, which is suggestive f intrinsically higher ATPase activity, thus necessitating a cntinuus supply f ATP fr FMN prductin in the fetal tissue. It is als ntewrthy that, in cntrl animals, the hepatic flavkinase activity was severalfld higher than its fetal cunterpart; hwever, under cnditins f immunneutralizatin there was a 5-6-fld increase in fetal flavkinase activity ver the cntrls, whereas hepatic activity remained unaltered under these cnditins (Table 2). *J43 -- ^O ^ 11 t +1.D C:, Cd... z L. CO *- wc. C). Z2S. N.. ce 41) i.4 4.) cn Cl r^ - _ s Clt ^ O~ -_ ~ m4 FAD pyrphsphrylase in the tw tissues The Km values fr FAD f the fetal and the hepatic enzymes were fund t be.3 and.1 mm- E-- *, Vl. 23

4 366 N. Surlia, K. Krishnamurthy and P. R. Adiga C's c 'A ;I (A cs: CZ ;N L. P. Q... CLI. ct x C) 6 +l+ l +l respectively (Table 1). The higher Km f the fetal pyrphsphrylase fr FMN is suggestive f a lwer affinity f the enzyme fr its substrate. The hepatic pyrphsphrylase level was 2-fld higher than its fetal cunterpart and was unchanged n immunneutralizatin f ribflavin carrier prtein in the pregnant rat. Cntrastingly, a 5% decrease in the fetal enzyme activity was manifest under the abve cnditins (Table 2). Since bth specific and ttal activities exhibited parallel trends, it wuld appear that the decreased enzyme activity in the affected fetuses reflects the situatin in viv and is nt a cnsequence f accelerated depletin f its prtein cntent by, presumably, catablism. x,c ;; ct Cl t (X-. R C,, COv C) '4 ) 4 -~-~CZ t= 4l ).) 3~- C C,, az - c C) _ - CO.. Cq+1. CdC) - 8 O ri = -'.kh CO H n\ 4 ct V. I c. Ca. ZCZ. - ~.) C)-~ N a.. a. et v C's. v CO cs 6 I - - 'IC r'i C r- t - - 'I tt t -- O C cmn t - Cn ri " Rf i 6 * =C CO C'. -i-; < U Discussin One f the remarkable features f the acute flavin deficiency precipitated in the develping fetus as a cnsequence f immunlgical interference with the maternal vitamin carrier is that, instead f entering a quiescent stage due t develpmental arrest, the fetus underges rapid degeneratin, culminating in death. This cntrasts with the situatin in certain tumurs where vitamin deficiency inhibits grwth rather than bringing abut degeneratin; the tumur tissue is apparently mre resistant than nrmal rgans t deficiency (Rivlin, 1973). Similarly, in adult animals, dietary vitamin deficiency nly suppresses grwth rate withut causing immediate death. These bservatins seem t suggest subtle differences in detail between the enzymes cncerned with flavin metablism and regulatin theref in the adult liver and fetus. The present investigatins largely supprt the abve premise. Thus, with regard t flavin-nucletide-bisynthetic enzymes, the significantly higher Km f the fetal FAD pyrphsphrylase fr FMN vis-a-vis its hepatic cunterpart (Table 1) is suggestive f a need fr a relatively higher intracellular FMN cncentratin t drive the reactin in favur f FAD synthesis. Frm the catalytic indices (represented by the Vmax/.Km rati), it is clear that bth the bisynthetic enzymes f flavin-nucletide metablism frm the maternal liver are relatively mre efficient than their fetal cunterparts (Table 1). Thus it wuld appear, perhaps surprisingly, that, at the early stages f fetal grwth, these anablic enzymes are relatively sluggish in activity and less adapted t their functin. A plausible M.,,,explanatin fr this finding may be cncerned with the fact that these bisynthetic enzymes in the adult tissue are mdulated by the thyrid hrmnes (Greengard, 1971); n the ther hand, in develp- C _ ing rdent fetuses the thyrid becmes gradually functinal nly during days 16-2 f gestatin. It was als shwn that these enzymes increase slwly 1985

Enzymic basis f disturbed fetal flavin-nucletide metablism in the rat 367 during fetal develpment t reach adult levels just after birth (Rivlin, 1969b).

5 Enzymic basis f disturbed fetal flavin-nucletide metablism in the rat 367 during fetal develpment t reach adult levels just after birth (Rivlin, 1969b). In cntrast with the anablic enzymes, the Km values f the fetal flavin-degrading enzymes, namely nucletide pyrphsphatase and FMN phsphatase, are mre-r-less cmparable with their hepatic cunterparts (Table 1). Furthermre, whereas FMN phsphatases f the tw tissues are similar in terms f their catalytic indices, the fetal nucletide pyrphsphatase has a 7-8-fld higher catalytic efficiency than the crrespnding maternal liver enzyme. It wuld appear therefre that, superimpsed n an inherently inefficient bisynthetic machinery cncerned with the elabratin f flavin cenzymes, acute flavin deficiency precipitated by immunneutralizatin f the carrier prtein brings abut a 5% decline in fetal FAD pyrphsphrylase in the treated mthers (Table 2). It is pssible that a crrespnding increase in fetal flavkinase (Table 2) may represent an adaptive, but unsuccessful, fetal stratagem t ffset the depressed FAD levels. It is ntewrthy that, unlike the situatin in the flavindeficient adult liver, the fetal FAD is nt cnserved at the expense f the mre dispensable FMN by an increase in FAD pyrphsphrylase (Rivlin, 1969a). It wuld als appear that the FAD pyrphsphrylase f the fetus is the pace-setting enzyme in flavin-nucletide bisynthesis and is mre stringently regulated by the available intracellular ribflavin. It is intriguing that the degradatin f FMN and FAD in fetuses seems t be independent f ribflavin status, since the degrading enzymes remain unaltered bth in flavindeficient fetuses and in the vitamin-deficientadult hepatic tissue (Lee & McCrmick, 1983; Fass & Rivlin, 1969). Frm the abvementined bservatins, it is clear that there are certain basic differences in enzyme prfiles cncerned with flavin-nucletide metablism between the early fetus vis-a'-vis the adult liver; in particular, the fetal bisynthetic enzymes seem t be relatively sluggish, whereas the reverse seems t be the case with the catablic enzymes. Althugh it is cnceivable that sme f the differences in enzyme characteristics bserved are related t the different nature f the tissues used fr cmparisn, i.e. liver in the mther and whle-bdy rgans in the fetus. Interference with the carrier-prtein-mediated transplacental vitamin-delivery mechanism, with the resultant acute flavin deficiency (Murty & Adiga, 1981; Krishnamurthy et al., 198, seems t inhibit FAD pyrphsphrylase selectively, leading t curtailed FAD prductin. This situatin, cupled with relatively higher activities f FAD-catablizing enzymes in the fetus, apparently depletes the vital cenzymes t such critically lw levels that the affected fetus is cmpelled t degenerate rapidly in the absence f ther regulatry (hrmnal?) mechanisms t ffset this derangement. Further experiments n the dynamics f flavin flw int different flavin cenzymes in the affected fetuses shuld substantiate the abve cnclusin. Our grateful thanks are due t Prfessr N. Appaji Ra fr many helpful discussins and t the Indian Cuncil f Medical Research and the Family Planning Fundatin, New Delhi, fr financial supprt. References Bessey,. A., Lwry,. H. & Lve, R. H. (1949) J. Bil. Chem. 18, Burtn, K. (1955) Methds Enzyml. 2, DeLuca, C. & Kaplan, N.. (1958) Bichim. Biphys. Acta 3, 6-11 Fass, S. & Rivlin, R. S. (1969) Am. J. Physil. 217, Greengard,. (1971) in Bichemical Actins f Hrmnes (Litwack, G., ed.), vl. 1, pp , Academic Press, Lndn and New Yrk Krishnamurthy, K., Surlia, N. & Adiga, P. R. (198 FEBS Lett. 178, Krishnan, N. & Ra, N. A. (1972) Arch. Bichem. Biphys. 149, Lee, S. & McCrmick, D. B. (1983) J. Nutr. 113, Lwry,. H., Rsebrugh, N. J., Farr, A. L. & Randall, R. J. (1951) J. Bil. Chem. 193, McCrmick, D. B. (1962) J. Bil. Chem. 237, McCrmick, D. B. (196 Nature (Lndn) 21, McCrmick, D. B. & Russel, M. (1962) Cmp. Bichem. Physil. 5, Merrill, A. M. & McCrmick, D. B. (198) J. Bil. Chem. 255, Miller, Z., Pncet, I. & Takacs, E. (1962) J. Bil. Chem. 237, Muniyappa, K. & Adiga, P. R. (198a) Bichem. J. 187, Muniyappa, K. & Adiga, P. R. (198b) FEBS Lett. 11, Murty, C. V. R. & Adiga, P. R. (1981) FEBS Lett. 135, Rivlin, R. S. (1969a) Adv. Enzyme Regul. 8, Rivlin, R. S. (1969b) Am. J. Physil. 216, Rivlin, R. S. (1973) Can. Res. 33, Rivlin, R. S. & Langdn, R. G. (1965) Adv. Enzyme Regul. 4, Segal, I. H. (1975) Enzyme Kinetics: Behavir and Analysis f Rapid Equilibrium and Rapid State Enzyme Systems, pp. 365 and 374, Jhn Wiley and Sns, New Yrk and Lndn Vl. 23

The demonstration of lysosomes by the controlled temperature freezing-sectioning method By LUCILLE BITENSKY

The demonstration of lysosomes by the controlled temperature freezing-sectioning method By LUCILLE BITENSKY 205 The demnstratin f lyssmes by the cntrlled temperature freezing-sectining methd By LUCILLE BITESKY (Frm the Department f Pathlgy, Ryal Cllege f Surgens f England, Lincln's Inn Fields, Lndn, W.C. 2)