Partial Purification
and
Characterization
of
Lysine-Ketoglutarate
Reductase
in
Normal
and
Opaque-2 Maize Endosperms'
Marcia R. Brochetto-Braga2,Adilson Leite, and Paulo Arruda*
Departamento de Gen6tica eEvolusao, InstitutodeBiologia (M.R.B. -B., P.A.), andCentrodeBiologia Moleculare
Engenharia Genetica (A.L., P.A.), Universidade EstadualdeCampinas, 13081 Campinas, SP, Brazil
ABSTRACT
Lysine-ketoglutarate reductase catalyzes the first stepof ly-sinecatabolism inmaize (ZeamaysL.)endosperm.The enzyme condenses L-lysine and a-ketoglutarateintosaccharopine using NADPH ascofactor. Itisendosperm-specificandhasatemporal pattern ofactivity, increasingwiththe onsetofkernel develop-ment,reachingapeak 20to25 daysafter pollination, and there-after decreasingasthe kernel approachesmaturity.Theenzyme wasextracted from thedeveloping maizeendosperm and par-tiallypurified by ammonium-sulfate precipitation, anion-exchange chromatographyonDEAE-cellulose, andaffinity chromatography onBlue-Sepharose CL-6B. The preparation obtained from affinity chromatographywasenriched275-fold and hadaspecific activity of411 nanomoles perminute permilligram protein. The native anddenaturatedenzymeis a140kilodaltonproteinasdetermined by polyacrylamide gel electrophoresis. The enzyme showed specificity for its substrates and was not inhibited by either aminoethyl-cysteineorglutamate.Steady-state product-inhibition studies revealed thatsaccharopinewas anoncompetitive inhibi-tor with respect to a-ketoglutarate and a competitive inhibitor withrespect tolysine. This issuggestive ofarapid equilibrium-ordered binding mechanism with a binding order of lysine,
a-ketoglutarate, NADPH. Theenzymeactivity wasinvestigated in two maize inbred lineswith homozygous normal and opaque-2 endosperms.The patternoflysine-ketoglutaratereductase activ-ity is coordinated with the rate of zein accumulation during endosperm development. A coordinated regulation of enzyme activity and zein accumulation was observed in the opaque-2 endospermastheactivity and zein levelsweretwo tothreetimes lower than in the normal endosperm. Enzyme extracted from L1038 normal andopaque-2 20 days after pollinationwaspartially purified by DEAE-cellulose chromatography. Both genotypes showedasimilarelutionpattern withasingleactivity peakeluted atapproximately 0.2 molar KCL. The molecularweightand phys-icalproperties of the normal andopaque-2enzymeswere
essen-tially thesame. Wesuggestthat theOpaque-2gene, which isa
transactivatorof the22kilodalton zeingenes,may be involved in theregulation of thelysine-ketoglutaratereductasegene inmaize endosperm.Inaddition,the decreased reductaseactivity caused 'This workwassupportedin partbygrantstoP.A.from Financia-dora de EstudoseProjetosand Conselho Nacional de Desenvolvi-mento Cientifico e Tecnol6gico (CNPq). M.R.B.-B. received post-graduatefellowshipsfromFundagaodeAmparoaPesquisadoEstado deSaoPaulo andCNPq.P.A.andA.L.receivedresearchfellowships from CNPq. This research constituted partofthe Ph.D. thesis of M.R.B.-B.
2Present address: Depto Biologia, Instituto de Biociencias, UNESP/Rio Claro,Av. 24A,no 1515, 13500,RioClaro, SP,Brazil.
by the opaque-2 mutation may explain, at least in part, the ele-vated concentration oflysinefound in the opaque-2 endosperm.
There is little information on lysine catabolism in higher plants. Mostof the available datawereobtained in studieson
theincorporation and metabolism of radiolabeledprecursors
by plant tissues. Feeding experiments with
["4C]lysine
dem-onstrated the incorporation of radioactivity into a-amino adipic acid and glutamic acid in wheat (18)andinto sacchar-opineanddiaminopimelic acid in maize and barley (16, 26).Indeveloping endosperm of maize andbarley, radiolabeled lysine isincorporated primarily into glutamic acid and proline (4, 26). These findings indicate that lysine is catabolized in plantsviathesaccharopinepathway.
The first enzymatic evidencefor the operation of the
sac-charopine pathway for lysine catabolism in plants was ob-tained with thedemonstrationofLKR3activityinimmature endosperm of maize (3). LKR(EC 1.5.1.8) condenses lysine and a-ketoglutarateintosaccharopine usingNADPH as
co-factor.
An understanding ofthepathways for lysine biosynthesis anddegradationinplants hasenormousimportancebecause of the limitingconcentration ofthis essential amino acid in major foodsourcessuchascereals. Valuableinformationcan
be obtained by the elucidation of the properties of enzymes involved in the biosynthesis andcatabolism oflysine and by
the useofmutantsinwhich the activities of theenzymesare
altered.
Since the discovery of the superior nutritive value of the high lysine maize mutant opaque-2 (15), there have been
many studies on the effects of this mutant gene on protein and amino acid metabolism in maize endosperm. The opaque-2gene is locatedonthe shortarm of chromosome7 and its major effect is the reduction of the maize storage
protein zein. This is a complex ofpolypeptides coded by a
multigenicfamilylocatedonchromosomes 4 and7(23).The
opaque-2 gene in the homozygous form reduces the zein
contentoftheendosperm byupto70% (6).Thereduction is 3Abbreviations: LKR,lysine-ketoglutaratereductase[L-lysine:
a-ketoglutarate: NADPHoxidoreductase(E-N-(glutary1-2)-lysine form-ing)]; DAP, days after pollination.
duemainlytoadecreasedsynthesisof the 22 kD a-zeins(12). The opaque-2 gene was recently cloned (17, 22). The gene encodes a 47 to 48 kD DNA-bindingprotein (1 1, 21) that binds to the 22 kD a-zein gene promoter and regulates its transcription(2 1).
Otherproteins and enzymesarealso affectedby the opaque-2 mutation. For example, ribonuclease activity is two- to
sixfold higherin opaque-2 endospermthan in normal endo-sperm(30).Theopaque-2 endospermlacksanalbumin of 32 kD(b-32) with unknownfunctionwhose accumulation pat-ternis coordinated with zein accumulation in normal endo-sperms(24).The geneencodingb-32is transactivatedbythe opaque-2 gene product (14). The opaque-2 mutation also affects lysine catabolism. Feeding experiments with
['4C]ly-sinehave demonstrated that theaminoacid isdegradedto a
lesserextentin the opaque-2 than in normal endosperm (26). Decreased lysine degradation has also been observed in the endospermofahigh lysinebarleymutant(4).
In thispaper, we reportthe partial purification and char-acterization ofLKR from immature maize endosperm and the activity of LKR in normal and opaque-2 endosperms duringkernel development. The results are discussed in the contextofthepropertiesandregulation ofenzymeactivity.
MATERIALS AND METHODS Chemical andChromatographic Materials
L-Lysine, a-ketoglutaric acid, NADPH, DEAE-cellulose, and other chemicals were purchased from Sigma Chemical
Company, Pharmacia Fine Chemicals,E. MerckDarmstadt, and Riedel-de Haen.
PlantMaterial
Maize(ZeamaysL.) inbred lineswerefrom thecollection of Universidade Estadual de Campinas. The inbred lines
L1038 andCat-100-1, bothcontaining homozygous normal andopaque-2 counterparts, were obtained by repeated
self-pollinationofheterozygous plantsafteratleast sixbackcrosses
withasourceof opaque-2mutantprovidedbyD. V.Glover (Purdue University). Plants were grown in the field, self-pollinated, and harvested from 10 to 50 DAP at 3- or 5-d
intervalsand stored frozenat-20°C.
Extraction and Quantification of Zein
Theendospermswere freeze-dried, groundtoaflour, and
defatted with acetone. After extraction ofsoluble proteins with 0.5MNaCl,zeinwasextractedatroomtemperaturewith 55% isopropanol containing 2% 2-mercaptoethanol accord-ingtothemethod of Paulisetal. (20). The nitrogencontent ofzein extracts wasdetermined by the method ofNkonge
andBallance(19).
LKRAssay
Theenzyme wasassayedat30°C ina 1mLreaction mixture
containing0.1 Mpotassium phosphatebuffer, pH 7.0, 20mM
L-lysine, 10 mm a-ketoglutarate neutralized with KOH, 0.1 mM NADPH, and 0.03 to 0.2 mg protein. Oxidation of
NADPH wasmonitored at 340 nm. One unit ofactivity was defined as the amount of enzyme required for the oxidation of 1 nmol NADPHmin-'at30C. The protein concentrations in the enzyme extracts were determined as described by Spector(27).
Preparation of Enzyme Extract
Immature endosperms of inbred line L1038 were hand-dissected from the seeds and homogenized in a Waring blenderwith bufferA(0.1 Mpotassium phosphate buffer, pH 7.0,1 mMEDTA, 1 mM DTT, 15%glycerol). The homogenate wasfiltered through four layers of cheesecloth andcentrifuged at22,1OOg for 30 min.
Solid ammonium sulfate wasslowly added to the superna-tantsolution at4°C to give 35% saturation. The solution was kept at 4°C for 1 h and the precipitate was removed by centrifugationat 22,1OOgfor 20 min. The supernatant fluid was brought to 60% saturationwith solid ammonium sulfate and, after centrifugation, the pellet was resuspended in a minimal volume ofbuffer A and desalted by filtration through aSephadex G-25 column. The desaltedfraction was centri-fugedat 105,000gfor1 h at4°C.
DEAE-CelluloseChromatography
Thesupernatant fraction from ultracentrifugationwas di-luted to a protein concentration of 3 mgmL-1 and applied to a column (18 x 2.5 cm i.d.) of DEAE-cellulose previously equilibrated with buffer A. The column was washed with buffer A until A280 < 0.05 and then eluted with a linear gradient of 0 to 0.5 M KCI in buffer A at a flow rate of 40 mL h-'. Fractions of5 mLcontainingLKRactivity were pooled, broughtto70% saturation withsolidammoniumsulfate, and centrifuged at 23,700gfor 20 min at 4°C. The pellet was resuspended in aminimal volume of buffer A and dialyzed overnight against2.7Lofthe samebuffer.
Affinity Chromatography
Thedialyzedsample wasapplied to a Blue-Sepharose CL-6Bcolumn(8 x 1.5 cmi.d.)equilibrated with bufferA.The column was washedwithbufferAand eluted with a 30mL, 0 to 15 mm NADPHgradientinbufferAat aflowrateof6.6
mL h-'. Successive 2-mL fractions were collected, dialyzed individually against bufferA, andassayed for LKRactivity. The fractions containing enzyme activity were dialyzed against2 x 2 L 10 mmpotassium phosphatebuffer, pH 7.0, containing 1 mMEDTAand 1 mmDTT.
PAGE
Discontinuous PAGE of nativeproteins was performed at pH 7.0 and 4C in 5 to 10% gradient slab gels. Enzyme activity in the gels was detected by a modification of the methodof Susor and Rutter (28). After electrophoresis, the gelwas washedwith0.1 M potassium phosphate buffer, pH 7.0, and incubated at 30C for 1.5 h in 0.1 M potassium phosphate buffer (pH 7.0) containing 20mML-lysine, 10mM a-ketoglutarate, and 0.5 mM NADPH. At the end ofthe incubationperiod, excess NADPH was removed by soaking
thegel for 10min in 0.1 Mphosphate buffer(pH 7.0). The
gel was immediately placedon the surface ofa
transillumi-nator with UVlightat 260 nm andphotographed. In these
experiments, the electrophoresed gel wasdivided into
dupli-cate sections, one of which was used to locate the enzyme
activity, as described above. The other section was stained
with Coomassie brilliant blue R-250tolocateproteinbands.
SDS-PAGE of denatured proteinswasperformed in 5 to
20% gradient slabgels. Theapparentmonomeric molecular
mass of LKR was estimated by comparison with standard
molecular mass markers. Proteins were stained with
Coo-massie brilliant blue R-250 or acombined Coomassie
blue-silvernitrateprocedure(7).
Kinetic Studies
Samples ofLKRtaken from themostactivefractionsfrom
anindependent DEAE-cellulose chromatography experiment wereusedforkinetic studies. Steady-state product inhibition
studieswereperformedand the resultsinterpreted byaHanes
plotusingtheENZFITERprogram (13).
RESULTS Preparation and Purification of LKR
Forenzyme purification, a crudeextract wasprepared by
homogenizing 3000 20-DAP endosperms in buffer A. The
sequentialstepsofenzymepurificationareshowninTable I.
For the routine monitoring ofenzyme activity, except for crudeextract,thefractions obtained in each stepweredesalted
through Sephadex G-25 columns. Over 95% of the LKR
activity units was recovered in the 35 to 60% ammonium sulfate fraction. This preparation was enriched 3.5-fold in
relationto thecrudeextract. Theenzymeactivity recovered inthe 35 to60% ammonium sulfate fractionwasstable for
up to 2 weeks when stored at 0 to 4°C. This fraction was
clarified, desalted,andsmallamountsofprecipitated protein
weresedimented by centrifugation at 105,000g for 1 h. This
increased thespecific activity by45% in relationtothe35to 60% ammonium sulfate fraction.An increasedpercentageof
recoveryof totalactivitywasobserved after ammonium
sul-fatefractionationandultracentrifugation (Table I).Thiscould
be explained bythefactthat crudeextractwasassayedwithout
desalting given underestimated values. After ultracentrifuga-tion, the desalted preparation was loaded onto a
DEAE-cellulose column andelutedwithaKCI gradient inbufferA
(Fig. 1). A largeamountof protein lackingLKRactivity did not bind to the column. A single peak of activity eluted
between 0.2 and 0.3M KCI.Thespecific activity ofthemost
active fraction of this preparation was enhanced 97-fold in
relationtothe crudeextract(Table I). Thepurestpreparation
was obtained after binding the most active fractions from
DEAE-cellulosechromatographyontoaBlueSepharose
CL-6Bcolumn, whichwastheneluted witha0to15mmNADPH
gradient (Fig. 2).Theaddition of differentcombined
concen-trationsofL-lysine and a-ketoglutarate together withNADPH
did not improve the elution of enzyme from the affinity
column(datanotshown). The total recoveredenzymeactivity
after affinity chromatography was 13%, corresponding to a
specific activity of411nmolmin-'mg-'protein.Thepurified fractionwasenriched 275-fold (Table I).
Apparent Molecular Mass
The preparations obtained ateach step ofenzyme
purifi-cationweresubjectedtoSDS-PAGE. Aband of 140 kDwas
enrichedafter thepurificationstepsand became the
predom-inant band in the affinity chromatography fraction (Fig. 3, lane 5). Minor contaminant bands of lower molecularmass were present after the final stepofenzyme purification. To
verify that thepredominant protein in the affinity chroma-tographypreparation representedthe LKRenzyme,this frac-tionwassubjectedtonondenaturatingPAGE. Part of thegel
wasstained for protein and the remainderwasusedtolocate
enzymeactivity. The predominant band of 140 kD present
in the affinity chromatography preparation (Fig. 4, lane 1) correspondedto the LKR activitydetected in the duplicate gel (Fig. 4, lane 2).
SubstrateSpecificity
Thesubstratespecificity ofLKR hasbeentestedpreviously by adding several amino acids and ketoacids such as
L-ornithine, L-glutamine, L-asparagine, D-lysine, diaminopi-melic acid, pyruvic acid, and oxalocetate to the incubation
TableI. Purification ofLysine-KetoglutarateReductase fromImmature MaizeEndospermsHarvested
at20 DAP
PurificationStep Volume Total Total Specific Recovery
Protein Activity Activity
mL mg/mL units' units/mg % Crudeextract 1158 3.7 6514 1.5 100 35-60%(NH4)2SO4 76 20.2 8023 5.2 123 precipitate Ultracentrifugationat 76 15.4 8919 7.6 137 105,000g DEAE-cellulosechroma- 3 5.8 2534 146.0 39 tography Blue-Sepharosechroma- 8 0.3 822 411.0 13 tography
0 K0 5 0 10 20 30 0 5 6
FRACT ION
Figure 1. Elution pattern of LKR activity and protein from theDEAE-cellulosecolumn.(0)Enzyme activity; (0)proteinmeasured by absorbance at 280 nm;(-) KCIconcentration gradient.
#
l
~~~~wI
>0q
t.
<5
o ,8 so -0.4~~~~~~o0.
o E~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~c 40 -~~~~~~~~~~~~~~~' 0.2 4 10 20 ' O 10 20FRACTION
Figure2. Elution pattern of LKR activity and protein fromBlue-Sepharose CL-6B column. (0)Enzyme activity; (0)protein measured by the Coomassie bluemethod; (-)NADPHconcentrationgradient.
(A)
1
2
3
4
5
220
-
36-
18-Figure3. SDS-PAGE ofenzymepurificationsteps.Thedenaturated
proteins were subjected to electrophoresis through a 5 to 20%
gradient slab gel. The numbers at the left indicate the following
standardproteins: ferritin,220kD; albumin, 67 kD; lactate
dehydro-genase, 36kO;ferritinsubunit, 18.5kD. Lane1,crudeextract; lane 2,35to60% ammonium sulfateprecipitate;lane3,ultracentrifugation
at 10OSOQ0g; lane 4, DEAE-cellulose columnchromatography; lane 5,Blue-Sepharose CL-6B3 chromatography. Laneswere loaded with ito10ug ofprotein.
1
I', (B) [LYSINE] mM 0 1 2 3 [a-KETOGLUTARATE]mMFigure5. Inhibition oflysine-ketoglutaratereductaseactivity by sac-charopine. A, Lysine concentration was varied from 0 to 30 mm. Saccharopine concentrationswere0(@),0.1 (0),0.2 (A), and 0.4 (A) mM. B,a-Ketoglutarateconcentrationswerevariedfrom0to 4 mM. Saccharopine concentrationswere0(0),0.1 (0),0.2 (A), 0.4 (A)mm. InAand B, NADPH was at0.1 mM.
2
440-232%_..
140-
Us-
l
67-Figure 4. PAGE undernondenaturating conditions of the enzyme
preparation obtained afterchromatography on Blue-Sepharose CL-6B. The numbers at the left indicate thefollowingstandardproteins: ferritin, 440 kD; catalase,232 kD;lactate dehydrogenase, 140kD; albumin, 67kD. Lane1,protein stained with Coomassieblue-silver;
lane2,duplicate gel assayed forLKRactivity.Thearrowindicate the band of LKRactivity.
mixture.Noneofthesecompoundswasabletosubstitute for L-lysineora-ketoglutarate(2, 3). Theenzyme wasactive with NADPH, butnot with NADH(3). Inaddition, the capacity ofS-2-aminoethyl-L-cysteine and L-glutamate to substitute for substratesand/or inhibitLKRactivitywastested.Neither compound couldsubstituteforL-lysine ora-ketoglutarateor
inhibitLKRactivity (datanotshown).
KineticAnalysis
Steady-state product-inhibition analyses revealed that
sac-charopinewas acompetitive inhibitor with respectto lysine (Fig. 5A), with a Ki value of0.14 mm as estimated by the replotofthe datafromFigure 5A(saccharopineconcentration
versusKm). Saccharopinewasanoncompetitive inhibitor with
respect to a-ketoglutarate (Fig. 5B), with a Ki value of 0.21
mm as estimated by a replot of the data from Figure 5B
(saccharopineconcentration versus 1/kkat). Itwasnot possi-ble todetermine the kinetic properties ofLKR with respect toNADPH undertheexperimental conditions used.
CoordinateRegulationofZein Synthesisand LKR
Activityby theopaque-2Mutation
Zein begins toaccumulate in both normal and opaque-2 endospermsaround 10 DAP.Afterashortlagphaseupto 12
to 15 DAP, zein increases exponentially, reachingits maxi-mum rateof accumulation at 25 DAP(Fig.6). Itisimportant to notethatzeinaccumulationreaches its maximum rateat thetime of maximumLKRactivity (see below).Asshownin Figure6, the opaque-2 mutationreduceszein accumulation in both inbred lines, but the reduction is higher in L1038 (Fig. 6A)than in Cat-100-1 (Fig. 6B). Thereductioninzein synthesis affected
primarily
theaccumulation of the22 kDa-zein in both inbred lines (data not shown). These results demonstrate that the opaque-2 mutation inourinbred lines exhibitsthe same characteristic alterations in zein synthesis that havebeenreportedby otherinvestigators (12).
LKR assays were carried out with endosperms obtained fromthe sameears as those usedforthezeinstudies. Crude extracts, prepared in parallel from 20 normaland opaque-2 endospermssampled at eachdevelopmental stage, were frac-tionated withammoniumsulfate,desaltedthrough Sephadex G-25 columns, and assayedfor LKR activity. The enzyme activitywascharacterized byasimilardevelopmental pattern in all materials analyzed, increasing in immature kernels, reachinga peak at anintermediatestageofdevelopment and decreasingasthekernels approached
maturity
(Fig. 6). How-ever,the totalactivitywastwofoldhigher inL1038(Fig. 6C) than in Cat-100-1 (Fig. 6D). In general, this difference be-tweeninbred lineswas also observed for zein accumulation andit may reflect differencesin the genetic background of theinbred lines. GreatdifferencesinendospermRNase activ-ity have been observed in different maize inbred lines and hybrids(29). The LKRactivity ofopaque-2endosperms was two tothreetimes lower than that ofnormalendosperms.As was observed for zein synthesis, the effect ofthe opaque-2mutation on LKR activity was more marked in L1038 (Fig. 6C) than in theCat-100-l inbred line (Fig. 6D).
Because the enzyme activity of normal endosperm was higher in the L1038 inbred line and the difference between normal and opaque-2 endosperms was more accentuated, L1038 was usedfor partial purification of LKR. Sixty normal and 60 opaque-220-DAPendosperms werehomogenizedin parallelwith60 mLofextractionbuffer andfractionatedwith ammonium sulfate. The fractions obtained between 35 and 60% saturation were desalted through Sephadex G-25 col-umns, and22 mg ofprotein, representingthe same amount ofendosperm fromeach genotype, was loaded onto DEAE-cellulose columns. The unbound proteins wereremoved by washing and the enzyme eluted with a 0 to 0.5 M linear gradient ofKC1. The enzymeelution profiles are shown in Figure 7. The amount of unbound proteinwas similar for both genotypes and did not contain any enzyme activity. Single peaks of activity from both normal and opaque-2 endospermswereelutedatthesamepointin theKClgradient. However, the LKR peak was two to three times lower in opaque-2than in normalendosperm.Thesepartiallypurified fractions were used for a comparison of enzyme properties of normal and opaque-2 endosperms. Both enzymes possessed similar physical and electrophoretic properties (data not shown).
Extractsfrom differentseed andseedling tissues from nor-mal and opaque-2 genotypes were prepared using the same methodology that wasutilizedin theendospermstudies. No LKR activity was detected in extracts from immature em-bryos, shoots,orroots(data notshown).
Figure 6. Zein accumulation and lysine-ketoglu-tarate reductase activity in normal (@) and opaque-2(0)endosperms. Zein wasextracted from freeze-dried immatureLi038 (A) and Cat-100-1 (B) inbred line endosperms with 55% isopropanol containing 2% 2-mercaptoethanol. Immature Li038 (C) and Cat-100-1 (D) endo-sperms sampled during kernel development were homogenized with extraction buffer and fractionated with ammonium sulfate. The frac-tionsobtained between35 and 60% saturation weredesaltedthroughSephadex G-25 columns andassayed for enzyme activity. Equalamounts ofendosperm wereused for each genotypeat each stage of kemel development. Each point represents duplicateassays.
c E o E aa a X 0I z a c0 F U Du0 -A *normal
45\
oopaque-2 450- / 1500 // lpO0 0 -0 10 20 30 40 50 64 0.%E 0a C;
c ouu -B * normal oopaque-2 450-300-O-
¢_°/
O-No\
=
150/
N\~
0/
1 0 0 10 20 30 40 50 6 35 0D normal oopaque-2 25--20-. 15-.10
-/
0 ) 0 10 20 .30C 40 50O 6( ,0Figure7. Elutionpattemoflysine-ketoglutarate reductase activity from DEAE-cellulose columns withnormal and opaque-2 endosperm extracts from Li038 maize inbred line sampled at 20 DAP.(0)LKR activity;(0)protein measured by absorbance at280 nm; (-) KCI concentration gradient.
5 ri
x
DISCUSSION
Ithas beendemonstratedinthis andinanearlier
publica-tion (2)that LKR isvery active indeveloping maize
endo-sperm. Themaize enzymeexhibitsspecificityforlysineand
a-ketoglutarate, NADPH is requiredas acofactor, and
sac-charopineistheproductofthe reaction(2, 3).
Theapparentmolecularmassof maizeLKR,asdetermined
by denaturating and nondenaturating PAGE, is approxi-mately 140 kD(Figs. 3, 4). Thesimilarityofmolecularmass
estimations obtainedby gel electrophoresisunder
denaturat-ing and nondenaturatdenaturat-ing conditions suggests that the
holo-enzymeisamonomer.
The enzyme binding mechanism and order of substrate
addition, examined by product inhibition kinetics, showed
competitiveinhibitionby saccharopinewithrespect tolysine andnoncompetitiveinhibitiontowarda-ketoglutarate.
Com-paringwiththe results ofproductinhibitionkinetics obtained for LKR purified from human placenta (9), the kinetics observed for maize LKRsuggestarapidequilibrium-ordered
binding mechanism in which lysinebinds first, followedby a-ketoglutarateandNADPH,leadingtothe release ofNADP+
andsaccharopine. For the humanenzyme, asimilar ordered mechanismwas suggested, exceptthat a-ketoglutaratebinds
first followed bylysine (9).
This is thefirstreportof LKRbeing highly purifiedfroma
plantsource, sotheonlyinformationavailable for
compara-tivepurposes aredatapertainingtothemammalianenzyme
(9). The solubility properties differasthe human enzymeis
recovered between 26.5 and 32.5% ammonium sulfate (9),
whereas the maize enzyme is precipitated between 35 and
60% saturation(Table I).The two enzymesdiffer markedly withrespecttomolecularmass, asthe humanenzyme,
puri-fiedfromplacenta (9),ismultimeric withanative molecular mass of480 kD, whereas the maize enzyme is apparently composed of a single polypeptide chain with an apparent
molecular massof 140 kD (Figs. 3, 4). The maize enzyme
also differsfrom the humanenzymewithrespect toitskinetic properties; maizeLKRisnotinhibitedby S-2-aminoethyl-L-cysteine,nordoes thislysine analogsubstitute forlysineas a
substrateasit does with thehumanenzyme(9).The
compet-itivepattern obtainedforlysine and the noncompetitive pat-ternobtained fora-ketoglutaratecontrastwith thoseobserved with LKRpurified from humanplacenta, inwhich
sacchar-opinewas a competitiveinhibitor withrespect to
a-ketoglu-tarateand a noncompetitive inhibitorwithrespect tolysine
(9).
It isnoteworthythat thepatternof LKRactivityisrelated totherateof zein accumulation andtotalnitrogen inputin maize endosperm(Fig. 6) (2). This couldbe ofphysiological importance for the followingreasons.First,zein isdevoid of
lysine (6) and, because zein is the most abundant protein
accumulated in theendosperm,thedemand forlysine during
T E 0 N m00 C ._ 0 OD X
'-7z
_a 4t0 C_ E _- ?'t- .4-aX c 0,5 0.4 0.3 0,2 0,1 0,5 04 3 02 0,1 Fractionendosperm development wouldappear to be very low. Sec-ond, although lysine is synthesized in the developing
endo-sperm(25),substantialamountsof this amino acid (approxi-mately5%), believedtobeproduced by hydrolysis of soluble proteins accumulated in the leaves before anthesis,are trans-located to the endosperm during development (1). In such circumstancesonewouldexpect an excessoflysineto accu-mulate in the developing endosperm. This is not the case,
however, as total lysine is approximately 1.5% (2) and the free lysine concentration in the endosperm is maintained at
low levels throughoutdevelopment(2). The freelysineshould bemaintainedat alowconcentrationbecausethis amino acid isafeedback inhibitorofenzymesinvolved in earlystepsof the aspartate pathway (8, 10). The inhibition ofaspartate
kinase (8), the firstenzyme in thepathway, results in alack ofintermediary substrates for the synthesis of methionine. Because onlysmall amountsofmethionineare translocated
tothekernel(1), this could limitprotein synthesisandhave adverse effects on the development ofthe endosperm. The high activity ofLKR during endosperm development may preventtheaccumulation offreelysine,and if this is thecase
it would be expected that both zein synthesis and lysine catabolism operate under the same regulatory system. We
suggest that the genes coding for LKR and zeins could be under the controlofthesameregulators.
Thereis also evidenceto suggestthatLKRcould be under the controlofopaque-2,agenethattransactivatesthe
expres-sionofthe22 kDa-zeingenesinmaizeendosperm. Indica-tions that lysine catabolism is higher in normal than in opaque-2 endospermwereobtainedby feeding ['4C]lysineto
developing ears segregating for normal and opaque-2
endo-sperm(26). Further evidence wasobtained byanalyzing the lysine concentration in the vascularsap ofa segregatingear
containing normal andsugary-opaque-2 endosperm. The ly-sine concentration in vascular sapwas doublethat observed in normal, but similar to that found in sugary-opaque-2, endosperm (5). In thecurrentstudy, two maize inbred lines containing homozygous normal andopaque-2 versionswere
usedtoinvestigatewhethertheopaque-2 mutation affectsthe activity of LKR. The opaque-2 inbred lines exhibited the characteristic reduction in zein accumulation
(Fig.
6), due mainlyto arepressionin thesynthesisof the22 kDzein class. The activity ofLKR was greatly reduced in the opaque-2 endosperm at all stages ofdevelopment, butthis effect wasaccentuated in the L1038 inbredline, which also showed a more marked reduction in zein accumulation (Fig. 6). The results demonstrate that the opaque-2mutation affectszein synthesis andLKR activity in a similar developmental
pat-tern. The partial purification on DEAE-cellulose (Fig. 7) of
enzyme extracted from normal and opaque-2 endosperms suggested that the opaque-2 mutationmayaffect thesynthesis ofLKR rather than its structure. Therecentcloning of the Opaque-2 gene has demonstrated that Opaque-2 codes fora DNAbinding proteinthattransactivates the22 kDzeinand
b-32genes(14, 17,21, 22). Itispossiblethat thesame
trans-acting factoris involved in theexpression of the LKRgene.
Furthermore, low LKR activity in the opaque-2 endosperm
maybe responsible forthe reducedlysine catabolism in the mutant endosperm and thuscontribute, at least in part, to
the elevated concentration oflysine in opaque-2 compared withnormal endosperm.
ACKNOWLEDGMENT
Wewouldlike to thankDrAlanCrozier (GlasgowUniversity, UK) for the criticalreading ofthemanuscript.
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