Biochem. J. (1980) 187, 623-636 623Printed in Great Britain
Purification and Physicochemical, Kinetic and Immunological Properties ofAllosteric Serine Hydroxymethyltransferase from Monkey Liver
Kashi S. RAMESH and N. APPAJI RAODepartment ofBiochemistry, Indian Institute ofScience, Bangalore-560012, India
(Received 17 September 1979)
1. The homogeneous serine hydroxymethyltransferase purified from monkey liver, bythe use of Blue Sepharose affinity chromatography, exhibited positive homotropicco-operative interactions (h = 2.5) with tetrahydrofolate and heterotropic interactionswith L-serine and nicotinamide nucleotides. 2. The enzyme had an unusually hightemperature optimum of 600C and was protected against thermal inactivation byL-serine. The allosteric effects were abolished when the monkey liver enzyme waspurified by using a heat-denaturation step in the presence of L-serine, a procedureadopted by earlier workers for the purification of this enzyme from mammalian andbacterial sources. 3. The enzyme activity was inhibited completely by N5-methyltetrahydrofolate, N5-formyltetrahydrofolate, dichloromethotrexate, aminopterinand D-cycloserine, whereas methotrexate and dihydrofolate were partial inhibitors.4. The insoluble monkey liver enzyme-antibody complex was catalytically active andfailed to show positive homotropic co-operative interactions with tetrahydrofolate(h = 1) and heterotropic interactions with NAD+. The enzyme showed a higher heat-stability in a complex with its antibody than as the free enzyme. 5. These resultshighlight the pitfalls in using a heat-denaturation step in the purification of allostericenzymes.
Serine hydroxymethyltransferase (5,10-methyl-enetetrahydrofolate-glycine hydroxymethyl-transferase, EC 2.1.2.1) has been purified fromseveral sources [rat liver (Nakano et al., 1968);rabbit liver (Fujioka, 1969; Schirch, 1971);Escherichia coli (Mansouri et al., 1972); ox liver(Jones & Priest, 1976); lamb liver (Ulevitch &Kallen, 1977)1. No evidence for co-operative inter-actions of substrates or effectors with the enzymewas obtained by these workers. It was shown,however, that the partially purified enzyme from pigkidney and monkey liver exhibited co-operativeinteractions with H4folate (Harish Kumar et al.,1976). In addition, the monkey liver enzymeexhibited heterotropic interactions with nicotin-amide nucleotides (Ramesh & Appaji Rao, 1978). Inview of the apparent contradictions between ourresults and those of earlier workers, it was necessaryto evolve a mild purification procedure for theisolation of an allosteric serine hydroxymethyl-transferase.
In the present paper we report a procedure for theisolation of the allosteric serine hydroxymethyl-transferase from monkey liver and its physico-chemical, kinetic and immunological properties.
Experimental
MaterialsThe following biochemicals were obtained from
Sigma Chemical Co., St. Louis, MO, U.S.A.:L-serine, DL-dithiothreitol, 2-mercaptoethanol,pyridoxal 5'-phosphate, EDTA (disodium salt),DL-fJ-phenylserine (threo form), 2,4-dinitrophenyl-hydrazine, D-cycloserine, glycine, Tris, ammoniumpersulphate, SDS, Blue Dextran, 3-(3-dimethyl-aminopropyl) 1-ethylcarbodi-imide, Folin-Ciocalteuphenol reagent, Coomassie Brilliant Blue G, folicacid, H2folic acid, DL-5-CH3-H4-folate (barium salt),ovalbumin, cytochrome c, (t-chymotrypsinogen,ferritin, haemoglobin, catalase (EC 1.1 1.1.6), humanIgG and conalbumin. Acrylamide and NN'-methyl-enebisacrylamide were purchased from Koch-LightLaboratories, Colnbrook, Bucks., U.K. CM-Sep-hadex C-50, Blue Sepharose CL-6B, Sephacryl S-200(superfine grade) and AH-Sepharose were fromPharmacia Fine Chemicals, Uppsala, Sweden.
Freund's complete adjuvant and agar were fromDifco Laboratories, Detroit, MI, U.S.A. DE-52DEAE-cellulose (microgranular) was purchasedfrom Whatman, Maidstone, Kent, U.K. CoomassieBrilliant Blue R-250 was a product of Bio-RadLaboratories, Richmond, CA, U.S.A. H4folate, pre-pared by the method of Hatefi et al. (1960), was agift from Dr. John H. Mangum, Brigham YoungUniversity, Provo, UT, U.S.A. 5-CHO-H4folate(folinic acid) was a product of ICN Phar-maceuticals, Plainview, NY, U.S.A. Methotrexate,dichloromethotrexate and aminopterin were kindlygiven by Dr. Robert Silber, New York UniversityMedical Center, New York, NY, U.S.A. UltrogelAcA-34 was a product of Industrie BiologiqueFranqaise and was a gift from Mr. Henrik Perlmut-ter, LKB-Produkter AB, Stockholm, Sweden. DL-[3-14CISerine (specific radioactivity 48.5 mCi/mmol)was purchased from New England Nuclear, Boston,MA, U.S.A.Animals
Adult bonnet monkeys (Macaca radiata) main-tained under standard experimental conditions oflight, temperature and diet and procured from theCentral Animal Facility, Indian Institute of Science,Bangalore, were used for the present study. Theanimals were killed by air-embolism.
sium phosphate buffer, pH 7.4, 1 mM-2-mercapto-ethanol, 10mM-EDTA, 0.2mM-pyridoxal 5'-phos-phate, 1.8 mM-H4folate, 3.6 mM-L-[3-'4C]serine,1.8 mM-dithiothreitol and an appropriate amountof the enzyme (0.5-2.O,g). The mixture was pre-incubated for 5min at 37°C and the reaction wasstopped by the addition of 0.1 ml of dimedone[0.4 M in 50% (v/v) ethanol]. The mixturewas heated for 5min at 1000C and [14CIfor-maldehyde-dimedone adduct was determined in aBeckman LS-IOOC liquid-scintillation spectrometer.One unit of enzyme activity was defined as theamount that catalysed the formation of lumol offormaldehyde/min at 37°C at pH 7.4. Specificactivity was expressed as units/mg of protein.Protein concentration was determined by the methodof Lowry et al. (1951), with bovine serum albuminas standard.Preparation of tissue homogenates and solublefractions
The following buffer was used for the preparationof the soluble fractions from various organs:50mM-potassium phosphate buffer (pH 7.4)/0.25M-sucrose / 20mm - EDTA / 1 mm - mercaptoethanol /0.05 mM-pyridoxal 5'-phosphate. Heart, lung andliver, removed immediately after the monkey hadbeen killed by air-embolism, were rapidly perfused
with ice-cold 0.25 M-sucrose to remove blood, blottedand immediately homogenized. All the other organswere washed three times with ice-cold 0.25 M-sucrose before homogenization. Except for skeletalmuscle, all the other organs were minced and thenhomogenized in 5 vol. of the buffer by using sixstrokes in a motor-driven Potter-Elvehjem hom-ogenizer with a tight-fitting Teflon pestle. Skeletalmuscle was ground with glass/sand and 5 vol. ofthe homogenizing buffer in an ice-cold mortar.Tissue homogenates were centrifuged at 10 5OOgfor 10min in a Sorvall RC-5-B Automatic Super-speed refrigerated centrifuge (DuPont Instruments)followed by centrifugation at 105 5OOg for 60minin a Beckman model L5-50 preparative ultra-centrifuge. The supernatant was dialysed for 24 hagainst buffer without sucrose.
Isolation ofcell organellesfrom monkey liverNuclei were isolated by the method described by
Blobel & Potter (1966). Microsomal fraction,mitochondria and cytosol were prepared by theprocedure described by Conn & Stumpf (1972).
Coupling offolic acid to AH-SepharoseTo a mixture of AH-Sepharose (5 g) and folic acid
(330mg) in 40ml of deionized water, 5 ml of0.8 M- 3 - (3 - dimethylaminopropyl)- 1 - ethylcarbodi -imide was added. The pH was maintained at 5.5 bythe addition of 1 M-NaOH. After being stirredovernight at 40C in the dark, the gel was washedsuccessively with 1 litre each of 1 M-NaOH/1 M-NaCl, deionized water, 1 M-HCl/1 M-NaCl andfinally with 2 litres of deionized water. Folate-AH-Sepharose was stored in lM-potassium phos-phate buffer, pH 7.4, containing 1 M-NaCl andlOmM-2-mercaptoethanol in test tubes under N2 inthe dark at 40C.
Blue Sepharose CL-6BFreeze-dried powder of Blue Sepharose CL-6B
was washed free of dextran with O.1M-NaCl andthen with deionized water. The gel was stored in50 mM-potassium phosphate buffer, pH 7.4.
Purification of serine hydroxymethyltransferasefrom monkey liver
Potassium phosphate buffer, pH 7.4 (50mM),containing 1 mM-2-mercaptoethanol, 0.05 mm-pyridoxal 5'-phosphate and 20mM-EDTA was usedthroughout. All the purification steps were carriedout at 0-30C.
Step 1: preparation of crude extract. Liver(75-80g) removed immediately after the animal hadbeen killed by air-embolism was trimmed of excessfat, connective tissue and gall-bladder. It was dicedand homogenized with 220 ml of 50mM-potassiumphosphate buffer, pH 7.4, in a precooled industrial
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ALLOSTERIC SERINE HYDROXYMETHYLTRANSFERASE
Waring Blendor at high speed for 2 min. Thehomogenate was centrifuged at 27000g for 20min,and the supernatant designated as the crude enzymeextract.
Step 2: precipitation with ammonium sulphate.Solid (NH4)2SO4 (enzyme grade) was added to thecrude extract to give 25% saturation (13.4g/100ml).After equilibration for 15 min, the extract wascentrifuged at 27000g for 20min. To the super-natant solution solid (NH4)2SO4 was added to raisethe saturation to 50% (14.6g/l00ml). The pellet ob-tained after centrifugation (27 000g for 20min) wasdissolved in 100 ml of 50mM buffer and was dialysedfor 48h against eight changes of 2 litres of the samebuffer.
Step 3: CM-Sephadex C-50 ion-exchangechromatography. The clear supernatant obtainedafter centrifugation of the dialysed enzyme wasapplied to a CM-Sephadex C-50 column(2.5 cm x 36 cm) equilibrated with 50mM-potassiumphosphate buffer, pH7.4, at a flow rate of 12ml/h.The column was washed with 10 bed volumes of theequilibrating buffer, until the absorbance of theeluate at 280nm was less than 0.05. The enzymewas eluted with a linear 0.05-0.5 M-potassiumphosphate buffer gradient (250ml of each). Frac-tions (5ml) containing significant amounts of theenzyme (specific activity > 1.8 units/mg) werepooled, and concentrated by (NH4)2SO4 precipita-tion (50% saturation). The precipitate was dissolvedin 2ml of 50mM buffer and dialysed against 2 litresof the 50mM-potassium phosphate buffer for 24 h.
Step 4: molecular-exclusion chromatography onUltrogel AcA-34. The enzyme from the previous stepwas chromatographed on an Ultrogel AcA-34column (2.2cm x 115 cm), pre-equilibrated with50mM-potassium phosphate buffer, pH 7.4, con-taining 0.2M-KCI at a flow rate of 15 ml/h. Frac-tions (2.4 ml) with specific activity more than 2.9were pooled and dialysed against 5 litres of 50mMbuffer, pH 7.4.
Step 5: affinity chromatography on Blue Sepha-rose CL-6B (a) or folate-AH-Sepharose 4B (b)columns. Blue Sepharose CL-6B was packed in acolumn (1 cm x 20cm) and equilibrated with 50mMbuffer. The enzyme fraction from the previous stepwas loaded on to the affinity-gel column at a flowrate of 1 column volume/h. The column was thenwashed with the equilibrating buffer at a flow rate of2 column volumes/h, until the eluate had anabsorbance of 0.02 at 280nm. The enzyme wasdesorbed from the Blue Sepharose gel with thebuffer containing 1 M-KCI. Fractions (2 ml) con-taining serine hydroxymethyltransferase activitywere pooled and dialysed against 20vol. of 50mM-potassium phosphate buffer, pH 7.4.
Alternatively, folate-AH-Sepharose-4B could beused for the enzyme purification. Application of the
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sample, washing of the affinity matrix and de-sorption of the enzyme from the affinity gel weresimilar to the procedure described for Blue Sepha-rose CL-6B.
Regeneration of Blue Sepharose CL-6B andfolate-AH-Sepharose-4B matrices. The regenera-tion process includes washing the gel with 3 bedvolumes of 50mM-potassium phosphate buffer,pH 7.4, containing 3 M-KCI, followed by 40 bedvolumes of deionised water. The gel was stored inphosphate buffer containing 0.02% NaN3.
Purification of serine hydroxymethyltransferasefrom sheep and rabbit livers
The enzyme was purified by following theprocedure described above for isolating the enzymefrom monkey liver. The enzymes were homo-geneous as determined by polyacrylamide-gel discelectrophoresis.
Analytical polyacrylamide-gel disc electrophoresisPolyacrylamide-gel electrophoresis at 40C, with
0.5M-Tris/0.39M-glycine buffer, pH8.6, in 7% gelswas conducted at a current of 5mA/gel. Gels wereeither stained for protein [with 0.02%, CoomassieBrilliant Blue G in 3.5% (v/v) HCl04 at roomtemperature for 2 h] or for enzyme activity (byequilibration at 37°C for 10min in 0.1 M-DL-threo-f,-phenylserine in O.1M-potassium phosphate buffer,pH7.4, followed by incubation for 10min at 370Cin 0.1% 2,4-dinitrophenylhydrazine in 2.4M-HCl).Destaining of Coomassie Brilliant Blue G-stainedgels was not necessary (Reisner et al., 1975).Tube-gel isoelectrofocusing
The isoelectric point (pl) of the enzyme wasdetermined by isoelectrofocusing on polyacrylamidegels as described by Chua et al. (1978) with somemodifications. Electrophoretic runs were carried outwith LKB Ampholine carrier ampholytes, pH3.5-10.0. The polyacrylamide gel was prepared asfollows. A solution of acrylamide (1.42g) andNN'-methylenebisacrylamide (700 mg) in 18.5 ml ofdeionized water was filtered through Whatman no. 1filter paper. Sucrose (1 g), NNN'N'-tetramethylethyl-enediamine (13.4,ul), 0.004% riboflavin (1 ml), am-monium persulphate (6.67mg) and carrier Ampho-lyte, pH 3.5-10.0 (1 ml), were mixed with thefiltrate. The gel solution was deaerated and castin tubes (4mm x 120mm), and these were exposedto u.v. light for 30min. Anodic and cathodicchambers contained 10mM-H3P04 and 20mM-NaOH respectively. The gels were prerun at a con-stant voltage of 400V for 3h at 4°C. Samplescontaining 0.1% (v/v) Ampholyte were loaded onthe gels and electrophoresed. The power source wasadjusted to a constant voltage of 400V for a furtherperiod of 4h. After completion of the isoelectro-focusing, the gel was stained with the stain fixative
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K. S. RAMESH AND N. APPAJI RAO
prepared by mixing 100ml each of 2% (w/v)Coomassie Brilliant Blue R-250 and 2M-H2SO4. Theprecipitate formed was discarded and the greensupernatant was titrated with 10M-KOH, until thesolution turned pale blue. Trichloroacetic acid wasadded to the solution (12g/100ml) and used as stainfixative. An equivalent gel isoelectrophoresed underidentical conditions was sliced into 1 cm pieces andthe pH measured in water after 15 min equilibration.
Ultracentrifugal measurementsSedimentation-velocity studies were performed in
a single-sector cell with a Spinco model E analyticalultracentrifuge, equipped with schlieren optics. Theultracentrifuge was operated at 50740 rev./min forthe determination of sedimentation coefficient, whichwas corrected to S20,w (Schachman, 1957).
Molecular-weight determinationThe molecular weight of monkey liver serine
hydroxymethyltransferase was estimated by gelfiltration (Andrews, 1965) on Sephacryl S-200(superfine grade gel equilibrated with 50mM-potas-sium phosphate buffer, pH 7.4, containing 0.2 M-KCI.The following proteins of known molecular weightserved as markers: horse heart ferritin (480000),bovine liver catalase (232000), human IgG(156000) and horse heart cytochrome c (12384).The molecular weight was calculated from a plot oflog (molecular weight) against elution volume. Thepartition coefficient (KD) of standard proteins andthe enzyme were determined in separate runs. TheStokes radius of the enzyme was obtained byinterpolation on a calibration curve prepared byplotting Stokes radius of standard proteins versusKD+4The subunit molecular weight of serine hydroxy-
methyltransferase was determined by SDS/poly-acrylamide-gel disc electrophoresis as described byWeber & Osborn (1969).
Immunological techniquesAntibodies to the purified monkey liver serine
hydroxymethyltransferase were raised in male albinorabbits (about 2.5 kg). The enzyme from either step4 or step 5 in 50mM-potassium phosphate buffer,pH 7.4 (1 mg/ml), was emulsified with an equalvolume of Freund's complete adjuvant and injectedsubcutaneously at multiple points at weekly inter-vals. After 4 weeks a booster dose of 1 mg of enzymewas administered. After 7 days the rabbits were bledthrough the-marginal ear vein. Blood was allowed toclot initially for 2 h at room temperature and later for12h at 40C. Control sera (pre-immune sera) wereobtained from the rabbits before the first injection ofthe antigen. The IgG fraction was prepared byrepeated (NH4)2SO4 fractionation (0-52%saturation) and negative adsorption on DE-52
DEAE-cellulose. All sera and IgG fractions werestored at -200C. Ouchterlony double-immuno-diffusion analysis on agar was performed as de-scribed by Ouchterlony (1958). Immunoelectro-phoresis on agar was performed as described byClausen (1970).
Results
Distribution of serine hydroxymethyltransferase Inthe various organs ofadult male monkey
It is clear from Table 1 that the enzyme is presentin large amounts in the liver. In skeletal muscle,brain and pancreas the specific activity of serinehydroxymethyltransferase was about 10% and in thekidney 25% of that in the liver. For this reason liverwas chosen for further studies.Subcellular distribution of serine hydroxymethyl-transferase in monkey liverThe cytosol fraction contained 75% of the enzyme
activity, whereas the mitochondria had 5% and themicrosomal fraction 3% of the total activity. Thecytosolic fraction had the highest specific activity.The recovery was 83% (Table 2). The relatively lowrecovery was due to loss of the enzyme during thewashing procedure.
Table 1. Distribution of serine hydroxymethyltransferasein the organs ofthe monkey (Macaca radiata)
For details of the procedure used to obtain theenzyme from different organs and assay of theenzyme see the Experimental section.
Activity Specific activityOrgan (Units/g of tissue) (unit/mg of protein)
Table 2. Subcellular distribution of monkey liver serinehydroxymethyltransferase
For details see the Experimental section.
FractionHomogenateNucleiMitochondriaMicrosomal
fractionCytosol
Totalactivity(units)15.200.030.700.50
11.40
Specificactivity (unit/mg of protein
0.0200.0030.0090.007
0.029
Recovery(%)100
0.24.63.3
75.0
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ALLOSTERIC SERINE HYDROXYMETHYLTRANSFERASE
Enzyme purificationThe major protein peak eluted from the Blue
Sepharose CL-6B affinity column with 1 M-KCIcoincided with the enzyme activity (Fig. 1). L-Serine(10mM), NADH (10mM) or NADPH (50mM) failedto elute the enzyme from the Blue Sepharosecolumn. Polyacrylamide-gel disc electrophoresis offractions eluted from Blue Sepharose column re-vealed a single protein band [Fig. 1, inset (a)],
indicating the absence of contaminating proteins.The specific activity of the pooled fractions from thisaffinity column was 3.3 units/mg. The specificactivities of several preparations of the enzyme werein the range 3.2-3.5 units/mg.The enzyme from the Ultrogel AcA-34 step was
adsorbed on a folate-AH-Sepharose column.Elution with buffer containing 1 M-KCI gave a singlesymmetrical protein peak that coincided with the
ox4 1.0
0.10 _..- O
E ::
0.05 X
._:
0
u )
0 10 20 30 40 50 60Fraction no.
Fig. 1. Elution profile ofmonkey liver serine hydroxymethyltransferasefrom a Blue Sepharose CL-6B affinity columnThe enzyme (step 4, Table 3; 111 units; specific activity 3.04 units/mg) was loaded on a Blue Sepharose column(1 cm x 20cm). The column was washed free of unadsorbed protein with 250ml of 50mM-potassium phosphatebuffer, pH 7.4, containing 0.05 mM-pyridoxal 5'-phosphate, 1 mM-2-mercaptoethanol and 20mM-EDTA. The enzymewas eluted with the buffer containing 1 M-KCI, and fractions (2.0 ml) were assayed for protein (A280) (-) and enzymeactivity (0) as described in the Experimental section. The arrow (W) indicates the point at which 1 M-KCl wasincluded in the eluting buffer. The inset (a) shows polyacrylamide-gel disc electrophoresis of fractions 37, 38 and 39.Inset (b) shows the electrophoretic pattern of the pooled fractions. Gels were stained for protein (bl) and for activity(b2).
Table 3. Summary ofpurification ofserine hydroxymethyltransferasefrom monkey liverFor details see the Experimental section.
Step1. Crude extract (from 80 g of liver)2. (NH)2SO4 precipitate
graphy (pooled fractions)4. Ultrogel AcA-34 gel chromatography5. (a) Blue Sepharose CL-6B affinity
chromatography(b) Folate-AH-Sepharose 4B
Total protein(mg)83263320
68
36.523
21
Total activity(units)150140
131
11176
71
Specific activity(units/mg of protein)
0.0180.042
1.93
3.043.3
3.4
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Recovery(%)10093
87
7451
47
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K. S. RAMESH AND N. APPAJI RAO
enzyme activity. Electrophoresis of the enzymeeluted from the folate-AH-Sepharose affinitycolumn also gave a single protein band and wasidentical in all respects with the enzyme prepared byusing Blue Sepharose (results not given).
All the studies reported in the present paper werecarried out with the enzyme purified by using theBlue Sepharose affinity column rather than the folateaffinity matrix. The five-step purification proceduredescribed in the present paper resulted in an overallpurification of 180-fold, with 50% recovery. Table 3summarizes the results of the purification. Enzyme(2mg/ml) could be stored at 0°C without loss ofactivity for at least 2 months, and it was stable at-200C for 6-8 months. Pyridoxal 5'-phosphate,2-mercaptoethanol or dithiothreitol and EDTA werenecessary for the stability of the enzyme.
Criteria ofpurityOn polyacrylamide-gel disc electrophoresis a
single protein band was obtained that coincided withthe enzyme activity band [Fig. 1, inset (b)]. Tube-gelisoelectrofocusing on polyacrylamide gels gave asingle protein band, located by staining with Coo-massie Brilliant Blue R-250 stain fixative. Enzymeactivity could not be detected in a similar gel run
below pH 5.0 owing to inactivation of the enzyme(results not given). The pI of the protein wasdetermined to be 4.2 + 0.1. Immunoelectrophoresisof the purified enzyme gave a single precipitin line(Fig. 2a) on either side of the antigen track.Ouchterlony double-immunodiffusion assay withrabbit IgG rasied against the partially purifiedenzyme gave a single precipitin line with the purifiedenzyme (well 4) and two precipitin bands with theenzyme at the Ultrogel AcA-34 step (well 3 in Fig.2b). Normal rabbit IgG (well 2), however, did notgive any precipitin line with the anti-enzyme IgG. Onultracentrifugation of the purified enzyme a singlesymmetrical sedimenting peak was obtained with ans20W. value of 8.9 + 0.1 S (Fig. 3). The enzyme, onpassage through Sephacryl S-200 (superfine grade,gel column, emerged as a single symmetrical peakwith constant specific activity (results not given).
Catalytic properties ofthe enzymeCatalytic-centre activity of the enzyme was
calculated to be 1.7 x 10-3s-, on the basis of amolecular weight of 2.08 x 105 (4mol of pyridoxal5'-phosphate/mol of the enzyme) and the Vmaxcalculated from the double-reciprocal plot. The
(a) Agar (1.5%, w/v) in 60mM-sodium veronal buffer, pH 8.6, was layered on a glass plate (3.5cm x 9.0cm) andallowed to form an uniform layer of gel. The enzyme (40,g) was placed in the well punched at the centre of the plate,and electrophoresed at 4°C for lOh at 250V. After electrophoresis, troughs running through the entire length of thegel were cut on either side of the antigen well. Rabbit anti-enzyme IgG (antibody raised against the enzyme at step 4,5 mg/0.5 ml) poured in each trough and allowed to diffuse overnight in a humidity chamber at 37°C and for 1 weekat room temperature (Clausen, 1970). The gel was deproteinized with 0.15M-NaCl, and stained with Amido BlackIOB and destained with 7% (v/v) acetic acid. For details see the text. (b) Agar (1.25%, w/v) in 25mM-potassiumphosphate buffer, pH 7.4, was poured into Petri dishes and allowed to gel. Four wells were punched in the plate. Well1 contained 500,ug of rabbit anti-enzyme IgG; well 2, 500,ug of control IgG (pre-immune sera); well 3, 600,ug ofenzyme from step 4 (Table 3); well 4, 300,ug of the purified enzyme (step 5). The plates were kept in humiditychambers for 24 h at 370C and for 1 week at room temperature. Deproteinization of the gel, staining and destainingwere performed as described for (a). For details see the text.
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loo..:3
ALLOSTERIC SERINE HYDROXYMETHYLTRANSFERASE
Fig. 3. Sedimentation pattern of purified monkey liverserine hydroxymethyltransferase
Ultracentrifugation analysis of the monkey liverenzyme (5 mg/ml) in 50mM-potassium phosphatebuffer, pH 7.4, was performed at 180C in a Spincomodel E analytical ultracentrifuge with schlierenoptics. The pattern reproduced here was taken 7 minafter a speed of 50740 rev./min had been reached,with the phase-plate angle at 400. The enzyme
moved as a single symmetrical peak during centri-fugation for 75min. Sedimentation is from left toright.
purified enzyme showed (i) a linear relationshipbetween initial reaction rate and enzyme concen-tration up to 28,ug/ml of reaction mixture, (ii) a
linear rate of formaldehyde formation for 30minwhen serine (3.6mM), H4folate (1.8 mM) and protein(up to 28,ug) were used, and (iii) optimum enzymeactivity at pH7.4 and 60°C at 3.6mM-serine and2mM-H4folate concentrations.
Regulatory properties ofthe enzymeThe purified enzyme exhibited a sigmoid
saturation pattem when H4folate was the variedsubstrate (Fig. 4). The S0.5 (H4folate) and h value of0.90mM and 2.5 were calculated by using the Hillequation (Segel, 1975). The L-serine saturation curvewas hyperbolic with aKm (L-serine) value of 0.70mm(inset to Fig. 4). NADH (10mM) and NADPH(50mM) abolished the sigmoidicity of the H4folatesaturation curve, and an h value of 1.1 was obtained.On the other hand, NAD+ (50mM) increased thesigmoidicity and gave a h value of 3.3. These resultsare in good agreement with our previous findings
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with the partially purified monkey liver enzyme(Ramesh & Appaji Rao, 1978).
Reconstitution and reactivation ofthe enzymeEnzyme purified in the absence of pyridoxal
5'-phosphate, EDTA and a thiol compound (dithio-threitol or 2-mercaptoethanol) had a specific activityof 0.12 unit/mg, as compared with 3.3 units/mg,when the enzyme was purified in the presence ofthese compounds. Addition of any one of these threecompounds separately resulted in a 10-fold enhance-ment of the specific activity. However, dithiothreitol(1 mM), pyridoxal 5'-phosphate (1 mM) and EDTA(20mM) added together led to a 29-fold increase inthe specific activity. The reactivation of the apo-enzyme by EDTA could be due to the removal ofcontaminating metal ions from the apoenzyme or theprotection of H4folate against air oxidation, or itcould be acting as electron donor, similar to itsfunction in the photoreduction of flavin (Strickland& Massey, 1973). These results are summarized inTable 4.
Visible-absorption spectrum of monkey liver serinehydroxymethyltransferaseThe spectrum of the purified enzyme had an
absorption maximum centred at 420nm (Fig. 5),characteristic of pyridoxal 5'-phosphate-dependentenzymes, due to the internal protonated Schiff basebetween pyridoxal 5'-phosphate and the e-aminogroup of a lysine residue of the enzyme (Fasella,1967).
Molecular weight of monkey liver serine hydroxy-methyltransferase
The molecular weight of the native enzyme wasestimated to be 208 000 + 5000, by using a cali-brated Sepachryl S-200 (superfine grade) gel column.The Stokes radius of the enzyme (KD = 0. 18),determined from the regression line of standardproteins, was 4.0nm.
SDS/polyacrylamide-gel disc electrophoresis ofthe purified enzyme revealed a single protein band(inset to Fig. 6). The molecular weight of thispolypeptide chain was estimated to be52 000 + 1400. The validation experiment for theSDS/polyacrylamide-gel disc electrophoresis deter-mination of molecular weight indicated that theenzyme does not have anomalous properties, sincethe position in a plot of relative electrophoreticmobility versus log (molecular weight) falls on astraight line generated by four standard markerproteins (Fig. 6).
Thermal stability ofthe enzymeThe enzyme was stable at 500C for 20min. At
65°C there was a gradual loss of enzyme activity(80% loss in 20min). However, at 70°C the enzyme
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K. S. RAMESH AND N. APPAJI RAO
0 0.6 1.2 1.8 2.4
[H4 folatel (mM)Fig. 4. Co-operative interactions of H,folate and the effects of nicotinamide nucleotides on these interactions with
monkey liver serine hydroxymethyltransferaseThe enzyme (1.O,ug/lOO,ul of reaction mixture) was preincubated with the concentrations of H4folate (0)indicated and the reaction was started by the addition of 3.6mM-L-[3-14C]serine. After incubation for 15minat 370C, the amount of [14C]formaldehyde formed was determined (Taylor & Weissbach, 1965). The enzymeafter preincubation with H4folate was incubated for 5min with lOmM-NADH (0) or 50mM-NAD+ (A) andassayed for enzyme activity. The inset shows the saturation pattern of the enzyme with L-serine, at saturatingconcentration of H4folate (1.8 mM).
Table 4. Reactivation of monkey liver serine hydroxy-methyltransferase
Enzyme preparation A was purified in the absence ofpyridoxal 5'-phosphate (pyridoxal-P), 2-mercapto-ethanol and EDTA. Each of these compounds wereadded to the enzyme and incubated for 30min at370C, and samples were assayed for enzyme activity.Enzyme preparation B was purified in the presenceof EDTA (20mM), 2-mercaptoethanol (1 mM) andpyridoxal 5'-phosphate (0.05mM). For details seethe Experimental section.
Enzyme preparationEnzyme AEnzyme A + 20mM-EDTAEnzyme A + 1 mM-pyridoxal-PEnzyme A + 1 mM-dithiothreitolEnzyme A + 1 mM-pyridoxal-P+ 20mM-EDTA
Enzyme A + 1 mM-pyridoxal-P+ 1 mM-dithiothreitol
Enzyme A + 1 mM-pyridoxal-P+ 20mM-EDTA + 1 mm-dithiothreitol
Enzyme BEnzyme B + 1 mM-pyridoxal-P+ 20mM-EDTA + 1mM-dithiothreitol
The spectrum of the enzyme (1.94 mg/ml) in50mM-potassium phosphate buffer, pH 7.4, wasrecorded in a Cary model 14 spectrophotometer.
was rapidly inactivated, with complete loss ofactivity occurring in 6min. Considerable protection(80%) of the enzyme against thermal inactivationwas observed when L-serine (18mM) was added.Protection of the enzyme by H4folate against ther-
1980
630
ALLOSTERIC SERINE HYDROXYMETHYLTRANSFERASE
1.0
0
Ei 0.8._
o 0.60.
0
"." 0.4u
.?; 0.2
3..8 4.0 4.2 4.4 4.6 4.8 5.0
log (Molecular weight)
Fig. 6. Subunit composition and determination of themolecular weight of monkey liver serine hydroxymethyl-
transferaseThe enzyme (50,ug) was denatured in the presence of2-mercaptoethanol and SDS, each at 1% (v/v)concentration in 0.43 M-glycine/Tris buffer (7: 1),pH 8.4, by heating at 900C for 5 min. The denaturedsimple was mixed with lO,l each of 0.05%Bromophenol Blue and 8 M-sucrose. The markerproteins 1 (cytochrome c, mol.wt. 12 384), 2(a-chymotrypsinogen, mol.wt. 25 600), 3 (oval-bumin, mol.wt. 43000) and 4 (conalbumin, mol.wt.68000) were similarly denatured and subjected toelectrophoresis along with the denatured monkeyliver enzyme (5), at 8 mA/tube at 300C, until themarker dye reached the end of the tube (about 6h).The proteins were stained with Coomassie BrilliantBlue R and the mobility of the proteins relative tothe migration of the dye were plotted againstlog (molecular weight). The subunit molecularweight (52000) of the enzyme was determined byextrapolation. The inset shows the electrophoreticpattern of monkey liver serine hydroxymethyl-transferase on SDS/polyacrylamide gel, demon-strating the presence of identical subunits.
mal inactivation could not be attempted because ofthe instability of H4folate. Allosteric effectors(NADH, NAD+ and NADPH) failed to protect theenzyme against thermal inactivation.
Effects of antifolate drugs on monkey liver serinehydroxymethyltransferase
Inhibition of the enzyme activity by metho-trexate, aminopterin and dichloromethotrexate fol-lowed different patterns (Fig. 7). Inhibition bymethotrexate was partial (67% at infinite con-
centration determined by extrapolation; inset to Fig.7), whereas that by aminopterin was sigmoidal.Dichloromethotrexate inhibited the enzyme activityin a hyperbolic manner. The concentrations of the
Vol. 187
100 5.0
3.0
1.0
0 0.2 0.4 0.6CU ~~~~~~~~~1/AMe-thotrex-atel (mm")
05
or
0 10 20[Inhibitorl (mM)
Fig. 7. Inhibition of monkey liver serine hydroxymethyl-transferase by antifolate drugs
The enzyme was preincubated successively for 5 minat 37°C with H4folate (1.8mM) and methotrexate(0) or aminopterin (0) or dichloromethotrexate(A). The enzyme activity was assayed as describedin the Experimental section. The enzyme activity inthe absence of inhibitor was normalized to 100. Theinset shows the plot of reciprocal of fractionalinhibition (i) versus the reciprocal of methotrexateconcentration. Aminopterin also gave similar results,whereas dichloromethotrexate completely inhibitedthe enzyme activity.
drugs required for 50% inhibition were 1.2mM(dichloromethotrexate), 8.1 mm (methotrexate) and14.7mm (aminopterin).
Effects offolate analogues on the enzyme activityFolate, H2folate, 5-CH3-H4folate and 5-CHO-H4-
folate inhibited the monkey liver serine hydroxy-methyltransferase activity to different extents, andthe inhibition was concentration-dependent (Fig. 8).Partial-inhibition analysis (Webb, 1963) showedthat H2folate was a partial inhibitor (inset to Fig. 8),whereas 5-CH3-H4folate and 5-CHO-H4folate com-pletely inhibited the enzyme activity. An interceptvalue greater than 1 (inset to Fig. 8) when 1/i was
plotted against 1/[II suggested that H2folate was apartial inhibitor (Webb, 1963), whereas 5-CH3-H4folate and 5-CHO-H4folate completely inhibitedthe enzyme activity, indicated by the intercept valueof I (not shown in the inset). The theoretical basisfor the partial inhibition analysis is as follows.When III is a partial competitive inhibitor:
1 (tKi(Km+IS])1 1 aKm+ SI1_ = _+ I-
Km((t'1) -ilKIK (a-i)]
(1)
2 3XX~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~5 +
4I
631
K. S. RAMESH AND N. APPAJI RAO
100 4
0 0.2 0.4
1/tHfiblatel (mm-')
50
0~~~~~~~~~0
0 10 20[Inhibitor] (mm)
Fig. 8. Effects of folate derivatives on monkey liverserine hydroxymethyltransferase
Enzyme (1 jugll/00ul of reaction mixture) wasincubated with folate (A), H2folate (A), 5-CH3-H4folate (0) or 5-CHO-H4folate (0) after prein-cubation with 1.8mM-H4folate for 5min at 37°C.The enzyme activity in the absence of any inhibitorwas normalized to 100. The inset shows thereciprocal of fractional inhibition (i) versus recipro-cal of H2folate concentrations. A similar analysiswith folate, 5-CH3-H4folate and 5-CHO-H4-folate,however, gave an intercept value of 1.0, indicatingthat these inhibited the activity completely at infiniteconcentration.
where i = 1- V1/V, Vi being the velocity of thereaction at a fixed substrate concentration in thepresence of the inhibitor and V being the velocity ofthe reaction at the same substrate concentration inthe absence of the inhibitor. The intercept on theordinate of a I/i versus 1/lI] plot is given by:
aKKm + [S]Km(a-1) (2)
Table 5. Inhibition data for the effects of glycine andD-cycloserine on monkey liver serine hydroxymethyl-
transferaseFor details see the text.
Varied Inhibition Slope K1substrate Inhibitor pattern replot (mM)L-Serine Glycine Competitive Linear 2.0H4folate Glycine Non-competitive Linear 10.5L-Serine D-Cyclo- Competitive Linear 0.27
serine
100
U;:Yc)
E 50N
i)
10
._
[IgGI (mg)
Fig. 9. Interaction of anti-(monkey liver serine hydroxy-methyltransferase) IgG with the purified serine hydroxy-methyltransferases from monkey, sheep and rabbit liversThe antibodies raised in the rabbits to the homo-geneous monkey liver enzyme (step 5) were in-cubated for 30min at 370C and then for 12h at 40Cwith 20,ug of monkey liver enzyme (0) or with 22,ugof sheep liver enzyme (0) or with 20,g of rabbitliver enzyme (A) or 20,ug of control IgG (pre-imunesera) with 20,g of monkey liver enzyme (A). Theprecipitated enzyme-antibody complex was centri-fuged at 3000g for 10 min and the enzyme activityin the supernatant was measured. The immuno-precipitate obtained with 350,ug of IgG antibodiesand 20,ug of monkey liver enzyme was dispersed inthe buffer and enzyme activity was assayed. (B).The activity in the absence of added antibodieswas normalized to 100.
whose value is always greater than 1 because a isgreater than 1. When ( = oo, the equation forcompletely competitive inhibitor is given by:
I= [Ki 1( -+I)]I 1(3)
The ordinate intercept value is always equal to 1. a
in eqn. (1) and expression (2) represents the factorby which K, and Ks are altered when El and ESreact with the substrate or inhibitor.
Effects of glycine and D-cycloserine on the enzymeactivity
The product of the reaction, glycine (2-30mM),competitively inhibited the enzyme when L-serinewas the varied substrate at saturating concentrationof H4folate (1.8 mM) and non-competitively whenH4folate was the varied substrate at 3.6 mM-L-serine.
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632
ALLOSTERIC SERINE HYDROXYMETHYLTRANSFERASE
-0.8
1.0
0os C e h2.5
-1.6
-2.4 _l
-1.6 -0.8 0
log llH4folatel (mM)O
Fig. 10. Hill plots showing the H,folate saturation ofmonkey liver serine hydroxymethyltransferase-anti-
body complexThe enzyme (40pg) was incubated for 30min at370C and then for 12h at 4°C with 700,ug ofanti-(monkey liver enzyme) IgG. The immuneprecipitate obtained on centrifugation at 3000g for10min was dispersed in 50mM-potassium phos-phate buffer, pH 7.4. This dispersion (corres-ponding to 1.1,ug of the free enzyme/lOO,d ofreaction mixture) was used for the H4folatesaturation (0). A mixture of 40,ug of enzyme and700pg of control IgG (pre-immune sera), treatedsimilarly, served as the control for H4folatesaturation (0).
Secondary slope and intercept replots were linearand K, values were determined from the slopereplots. D-Cycloserine, a rigid cyclic analogue ofD-alanine, inhibited the activity of the enzymecompetitively when L-serine was the varied sub-strate at 1.8 mM-H4folate. The secondary slopereplot was linear. These results are summarized inTable 5.
Interaction of rabbit anti-enzyme IgG with monkeyliver serine hydroxymethyltransferase
A progressive loss of enzyme activity in thesupernatant solution occurred when increasingamounts of anti-enzyme antibodies were added (Fig.
The enzyme-antibody complex obtained asdescribed in the legend for Fig. 10 was disperseduniformly in 50mM-potassium phosphate buffer,pH 7.4. This dispersion was heated at 70°C, andsamples were withdrawn at regular time intervals,and rapidly cooled in ice and assayed for enzymeactivity (A). A mixture of the enzyme and normalIgG (pre-immune sera) (0) or the enzyme alone(0), treated identically, served as controls. Theactivity of the unheated enzyme was normalized to100.
9), and the inhibition pattern was not altered in thepresence of NADH or NAD+. It was noted thatantibodies to the monkey liver enzyme cross-reactedand inhibited purified sheep liver serine hydroxy-methyltransferase, although at fairly high concen-trations of IgG. In contrast, the purified rabbit liverenzyme neither cross-reacted nor was inhibited bythe antibodies to the monkey liver enzyme. Com-parable quantities of control antibodies (pre-immunesera) had no effect on the enzymes from sheep,monkey and rabbit livers.
The insoluble enzyme-antibody complex, whendispersed in the buffer, had 90% of the originalactivity. The complex gave a hyperbolic saturationpattern with both H4folate and L-serine. In contrastwith the free enzyme, the enzyme-antibody complexgave an h value of 1.0 and s05 (H4folate) 0.7mM(Fig. 10). NADH and NAD+ had no effect on theH4folate saturation pattern obtained with the en-zyme-antibody complex. The enzyme-antibodycomplex was heat-stable. In contrast with the freeenzyme, the complex retained 70% of the activitywhen heated at 700C for 20min (Fig. 11).
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K. S. RAMESH AND N. APPAJI RAO
DiscussionSerine hydroxymethyltransferase has been iso-
lated from several mammalian and microbial sources
(Nakano et al., 1968; Fujioka, 1969; Schirch, 1971;Jones & Priest, 1976; Ulevitch & Kallen, 1977;Mansouri et al., 1972). None of these preparationsshowed any co-operative interactions with sub-strates or other metabolites, whereas the partiallypurified enzyme from pig kidney and monkey liverexhibited both homotropic and heterotropic inter-actions (Harish Kumar et al., 1976; Ramesh &Appaji Rao, 1978). This apparent discrepancyseems to be resolved by the isolation of an
homogeneous enzyme exhibiting allosteric properties(Table 3 and Figs. 2-4).The special features of the present method for
purification of the monkey liver enzyme were the use
of a Blue Sepharose affinity matrix and the omissionof thermal denaturation in the presence of L-serine,which is a major step used by earlier workers for thepurification of this enzyme (Nakano et al., 1968;Fujioka, 1969; Akhtar & El-Obeid, 1972; Cheng &Haslam, 1972; Rowe & Lewis, 1973; Palekar et al.,1973; Jones & Priest, 1976; Ulevitch & Kallen,1977). The allosteric effects of nicotinamide nucleo-tides on the monkey liver enzyme (Ramesh &Appaji Rao, 1978) suggested that the Blue Sepha-rose affinity matrix could be effectively used in thepurification of the serine hydroxymethyltransferase.It is well known that Blue Sepharose binds proteinsthat contain a dinucleotide fold in their secondarystructure (Thompson et al., 1975). The enzymecould also be purified by using a column offolate-AH-Sepharose. However, the Blue Sepharoseaffinity procedure was preferred, as this matrix was
stable.The monkey liver enzyme was homogeneous as
determined by polyacrylamide-gel disc electro-phoresis [Fig. 1, inset (a)], isoelectrofocusing,immunoelectrophoresis (Fig. 2a), immunodiffusion(Fig. 2b), analytical ultracentrifugation (Fig. 3) andgel filtration on Sephacryl S-200 (superfine grade)columns. The enzyme had a molecular weight of208 000 + 5000 and was composed of four identicalsubunits of molecular weight 52000 + 1400 (Fig. 6).The monkey liver enzyme was similar in itsmolecular weight and s20,w value to the enzyme
isolated from rabbit liver (Fujioka, 1969; Martinez-Carrion et al., 1972), lamb liver (Ulevitch & Kallen,1977) and sheep liver (R. Manohar, personalcommunication). The monkey liver enzyme, like theenzyme from other sources, contained boundpyridoxal 5'-phosphate (Fig. 5). The pH optimum ofthe monkey liver enzyme determined at 3.6 mm-serine and 1.8mM-H4folate was 7.4, and this valuewas similar to that reported for this enzyme fromother sources. The pl of the monkey liver enzymewas 4.2+0.1, although the enzyme was adsorbed
strongly on CM-Sephadex, suggesting that enzymehad a net positive charge. The reason for thisdiscrepancy can be ascertained only after thedetermination of the amino acid composition of theenzyme. The catalytic-centre activity of the monkeyliver enzyme calculated by using the extrapolatedvalue of Vmax with L-serine as the substrate was1.7 x 10-3s-; values for this enzyme from othersources with L-serine as the substrate are notavailable. However, when L-threonine was used asthe substrate (this reaction does not require H4-folate), the values were 0.76 x 10- s-' trabbit liver(Schirch & Diller, 1971)] and 0.09s-5 [lamb liver(Ulevitch & Kallen, 1977)].The next important property of the purified
monkey liver serine hydromethyltransferase thatdistinguishes it from those of rabbit, lamb, rat andbovine livers is the sigmoid saturation with H4folate(Fig. 4). The presence of positive homotropicco-operative interactions with H4folate was indi-cated by the curvilinear double-reciprocal plot and hvalue of 2.5. As already mentioned, the presence ofL-serine [a positive heterotropic effector (Ramesh &Appaji Rao, 1978)] and the use of a heat-de-naturation step during purification of this enzymefrom other sources may have resulted in the loss ofan allosteric binding site (Harish Kumar et al.,1976). Evidence in support of this argument isprovided by the oservations that (i) the monkey liverenzyme purified by using a heat-denaturation step(70°C for 5min) in the presence of L-serine (15 mM)failed to exhibit positive homotropic co-operativeinteractions with H4folate or heterotropic allostericeffects with nicotinamide nucleotides. When thepurified enzyme was heated in the presence ofL-serine, the allosteric properties were abolished, (ii)purification of this enzyme from rabbit, sheep andrat livers by the procedure adopted for the monkeyliver enzyme gave a homogeneous enzyme thatretained positive homotropic co-operative inter-actions with H4folate (K. S. Ramesh & N. AppajiRao, unpublished work), (iii) all these enzymepreparations showed positive heterotropic inter-actions with nicotinamide nucleotides and (iv) theSo.s (H4folate) values determined by us were 0.90mM(monkey liver), 0.50mM [mouse liver (HarishKumar et al., 1976)], 0.74mM (sheep liver) and1.21 mm (rabbit liver) compared with the Km values(H4folate) of 0.07mm [ox liver (Rowe & Lewis,1973)], 0.0072mM [rat liver (Nakano et al., 1968)]and 0.077mM [rabbit liver (Schirch, 1971)]. Thedecrease in Km or SO. values as a consequence ofdesensitization of allosteric enzymes has been exten-sively documented (Gerhart & Pardee, 1962; Man-sour & Martensen, 1978). Thus it is clear from ourobservations that the predominant form of theenzyme and its kinetics depend on the method ofisolation and assay of the enzyme.
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ALLOSTERIC SERINE HYDROXYMETHYLTRANSFERASE
In view of the high temperature optimum (60°C)of the monkey liver serine hydroxymethyltransferaseand the extensive use of heat-denaturation steps inthe purification of this enzyme from other sources, itwas decided to study the thermal stability of themonkey liver enzyme. It is evident that L-serineprotects the enzyme against thermal inactivation,whereas allosteric effectors such as NAD+ andNADH failed to afford any protection. It is wellknown that substrates or effectors often influence thestability of native enzyme conformations duringthermal denaturation. The thermal denaturation of aprotein occurs co-operatively, starting from an opensite. The ligand-induced conformational changesmight either hinder or facilitate the accessibility ofthe open site for initiation of the denaturationprocess. In the presence of L-serine this open sitemay be blocked and hence denaturation prevented.This protection by the substrate is unusual, sincedenaturation of allosteric proteins is usuallyfacilitated by substrate binding, owing to weakeningof subunit interaction (Bernhard, 1968).
Antifolates have greater affinity for dihydrofolatereductase (5,6,7,8-tetrahydrofolate-NADP+ oxido-reductase, EC 1.5.1.3) than for any other enzyme offolate metabolism (Bertino, 1963; Werkheiser,1963). The beneficial effects of heavy-dose chemo-therapy with methotrexate (Frei et al., 1975)suggested the prevalence of secondary site(s) ofinteraction with the drug. The inhibition of monkeyliver serine hydroxymethyltransferase by classicalantifolate compounds (Fig. 7) suggests that thisenzyme might be an alternative target, especiallywhen the circulating concentration of this drug isvery high. A noteworthy feature of the inhibition bymethotrexate is the partial inhibition observed evenat infinite concentration of the drug (determined byextrapolation), suggesting that this vital enzyme inthe folate-metabolic pathway is not completelyblocked in the methotrexate therapy. As a conse-quence the reactions requiring folate coenzyme,except thymidylate synthetase (EC 2.1.1.45) maycontinue to function even at high concentrations ofmethotrexate.
It is to be expected that folate coenzymes regulatetheir metabolism by affecting the first enzyme in thefolate pathway, namely serine hydroxymethyltrans-ferase. 5-CH3-H4folate and 5-CHO-H4folate, the endproducts of the pathway, inhibited the enzyme, theconcentrations required to cause 50% inhibitionbeing 3.0mM and 1 mm respectively. The predomi-nant folate derivative in the liver is 5-CH3-H4folate(Noronha & Silverman, 1962; Shin et al., 1972;Brown et al., 1974). H2folate was a partial inhibitorof the enzyme. Folate activated the enzyme at lowconcentrations (< 1 mM) and inhibited in a sigmoidalfashion with increasing concentrations, suggestingthat folate interacted at multiple site(s) on the
enzyme. The product of the reaction, glycine, and asubstrate analogue, D-cycloserine, competitivelyinhibited the enzyme when L-serine was the variedsubstrate (Table 5).
Recovery of full catalytic activity in the insolubleenzyme-antibody complex (Fig. 9) suggests thateither the substrate can diffuse through the complexto reach the active site or the antigenic determinantsite may be located distal to the substrate-bindingsite. Antibody binding to the enzyme has resulted inthe loss of the nicotinamide nucleotide-binding siteand also the site involved in the positive co-operativeinteractions with H4folate (Fig. 10). The loss ofpositive homotropic co-operative interaction offructose 6-phosphate with the phosphofructokinase(EC 2.7.1.1 1-antibody complex has been observed;however, the allosteric interaction with fructose1,6-bisphosphate was not lost on binding of theantibody to the enzyme (Bartholome-D6nnicke &Hofer, 1975). The loss of allosteric properties ofserine hydroxymethyltransferase on its binding toantibody is reminiscent of the observation that thisenzyme from L-1210 solid tumours in mice failed toexhibit homotropic interactions with H4folate. Theenzyme isolated from liver and kidney of tumour-bearing mice also failed to exhibit homotropicinteractions (Harish Kumar et al., 1976). Theenzyme from tumour tissue regained its allostericproperties on (NH4)2S04 fractionation of the homo-genate, suggesting that a dissociable factor was lostduring purification (P. M. Harish Kumar, J. H.Mangum & N. Appaji Rao, unpublished work).These results were interpreted to suggest the pos-sibility of the production of a chemical messenger bythe tumour that is transported to, and producesbiochemical changes in, other tissues of the animal(Harish Kumar et al., 1976). The loss of allostericproperties of the monkey liver enzyme on com-plexing with its antibody suggests that the pro-duction of a proteinaceous factor by the tumourcould be one of the mechanisms by which theenzyme activity is regulated in neoplastic tissues.The protection of the enzyme against thermalinactivation by enzyme antibodies (Fig. 11)suggests that the binding of antibody prevents theinitiation of denaturation. These results demonstratethat the specific antibodies to the enzyme induce aconformational change that is manifested by alteredcatalytic, regulatory and physicochemical proper-ties.
The results described in the present paper clearlyindicate that serine hydroxymethyltransferase is aregulatory protein, and emphasize the importance ofusing gentle methods of purification of allostericenzymes.
We thank Professor C. S. Vaidyanathan and ProfessorJohn H. Mangum for their valuable discussion and
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636 K. S. RAMESH AND N. APPAJI RAO
suggestions. The technical assistance of Miss V. Sow-mithri is acknowledged. This investigation was supportedby a research grant from the Department of Science andTechnology, Government of India, New Delhi, India.K. S. R. is the recipient of a Senior Research Fellowshipof the Department of Atomic Energy, Government ofIndia, India.
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