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Purification and properties of dl -lactate dehydrogenase from Leuconostoc mesenteroides
Purification
and
Properties Leuconostoc
of DL-Lactate
Dehydrogenase
from
rnesenteroides’
In the 30 years sinw Struub purifkd and charnctc~rizr~d t#hc>first DI’S+ 3-linl;c~d lact:tto d(~tr!.drogcrl:ls(, (1) from pig heart, scows of LDHs hnw been studicld. Al& of tJh(w h;~vc~been spwific for the L-isomer of Ixtats. Ho\\-cwr, DmI,DHs 1~:~~ bwn found in wrtain groups of bactck1, trspcci:Jly the l:lctobacilli, and, more rcwntly, in tlrcl ccllular slime molds (2), lowr fungi (3, 4), :wachnids, gnstropods, :md pol,vchnctw (5). The uL-lacbntc~-sprcific LDHs as :L class arc’ similar in their molecular wights, wliich arc :rround 140,000 daltons (6). ThcG ltint$ic prcqwrtiw wry bct\vwn those th:kt arc’ grcat’ly inhibited by high pyruwtc (WIIccmtr:~tions and thaw that, are not. Alt,hough few D-LDHs haw been wrll char:xtorized, thcxir proprtiw nppcar to br more wriod. Tlrc> Dl’S+-linlwd D-LDH from E. roli Ii:ts :I molecular \vc+ght * Contriblition K’o. 8% from the (:radlmte Department of Hiochemistry, Brandeis University. 2 Present address: l'hzyn~~ liesearch T,nbor:ttory, Massachusetts (:cneral Jlospital, Boston, hI:rssachusetts 02111. 3 Abbreviationi;: I)PN’ , diphosphol)yridille I),,dinucleotide) ; cleotide (llicotirl:Lmide-~dellirle LljH, lactate dehydrogennse; GTP, glmnosine triphosphnte; IAI, I~c~corws/oc mesenteroides.
Lezcconosloc ,,rcset,te,.oitles, strain 12291 (American Type Culture Collect ion), was grown ill .4C-1 media (Table I) at 30°C for 20 hr in 20.liter carboys with shaking. 1 )PN- alltl 1)PYH were obtained from P-I, Biochcmicals. The “Chronrntoprlre” grade was used ill thr kinetic st tidies. Sodillm pyruvnte and Tris (Trizma grade) were purchased from Sigma and lithilun u-lactate, from Miles. I)EhE-
30
cedure. L;\I 1, :I smaller preparation, 11:ld a spccifica nctivitj. of 5iOO units/mg :rntl LRI 2, 1100 rmits/mg.
with Ficoll to \yhich an equal amount of sllcrose had been added. When the volume had been reduced to 35 ml, the enzyme was dialyzed and chromatographed on :I 2-liter (5 X 100 cm) Scphadcx G-100 column v-it,h a one-to-ten dilntion of standard bluffer. After 100 ml had emerged from the colnmn, 15.ml frac.tions were collected. The enzyrnc was folmtl in tttbes 38-48. The active fractions were pooled, concentrated with Fir011 to a vohune of 7 ml, and rechromatogrn,phed on a 500ml (3 X 70 cm) (i-150 column. ;2fter ellltirrg 100 ml, 5.5-ml fractions were c*ollected. The activity and protclin peaks emerged at the same place and were spmmctrical, btlt the specific activity was slightly less at, the leading and t,railing edges. The frnctions of constant specific- activity in the center of the enzyme peak (tltbes 3053) were pooled (85 ml), concentrated with Ficoll and sI,crose to a volume of 5 ml, and dialyzed against a one-tenth dilution of standard brlffer for 3 hr, after which the voltlme was 10 ml. Cr~~stallizatio~~ (?i eTlal/?lle. Three milliliters of this solution, which rontained 5 mg/ml of protein, was brought gradually to 507, satllration with ammonium sulfate, at which point the enzyme began to crystallizr. ;\lthollgh the enzyme was kept at 4°C for spvcral months after this step, the crystals did not become large enough to photograph. The specific activity of the crystalline enzyme was identical to that of the uncrystallized preparation. The final specific activity was 3200 units/mg (this is for LplI 3, the third preparation). The plu+fication proredllrc is srmrmarixcd in Table II. Dihltions of the purified enzyme (Lhl 3) were used in the kinetic studies.
J~oleculur wi~jlrt. Tl~c molcculx \\-oiglits of tlic, tlirw prcy1’:ltions rvcrc rompared b?- the intwfwcww scdimc~ntation cyulibrium mc~thotl of I-pll:rntis (12). TIIP cyuntion used was MW = [2R7’/1( --J+p)w2] X [cl(ln c);‘cI(~.?)].At pH 8.6 in 0.05 RI (SH& COe, thcl molfculnr xiglits were dcterminc~tl to bc %,500, 63,000, and C,2,700 for the first, sccontl, :uld third rnzyme preparations, wapwtwely. Plots of t’he log of t’he fringe displacements wrsus the square of the distance from the center of rotntjion are straight lines of constant. slope, indicating homogeneity. The data for LRI 2, the lrast active preparation. is slion-n in Fig. 2. The> sxO,2,,for LM 3 was determined by t:str:~polating the line draxvn through the sedimentation const:tnts at two different protcin concentrations (3.2 and 10 m&l) to the> intcwcyt at, zero ~oncc~nt,rnt’ion, and \\-a~ 3..5%S. Tllcl I&,u value was dctcrmined to be 5.0 X lo-’ cm”i’rnl at one protein concentmt~ion. Wlim tlic sedimentation and diffusion constants wro srtbstitutcd in tlrc
KESULTS Criteria of puu’ty. The purified enzyme appeared homogeneous in the ultmcentrifuge (Fig. 1) mcl on starch gel. Ho’~i’ev~r, there was :I discrqxmc?- bctwcen the specific activity of this preparation and two previous ones purifird by csscntially the same proTABLE PGRIFICATION Purification
II
OF Leucor~ostoc J2esenteroide.s L.xT.~TI~~ ~I~II~DI~~~~~~x.~sI~
step
Protein bd
Activity units (~moles/min)
137,000 28,800
654,000 490,000
Specific activity (unitsimg)
\Yeltl cc;d
~__~ Crllde extract (NH&S04 (40-755;) DE-11 chromatography DEAE-Scphadex chromatography Sephadex G-100 chromat,ography Sephadcx G-150 chromatography
5,620
580 185 78
256,000 245,000
204,000 118,000
4.7 17
100 i5
46 425 1050 1510
39 37 31 18 -__
40
GARLAND
.50
FIG. 2. Sedimentation equilibrium experiment with preparation No. 3 of I,. mesmleroides LDH for determination of molecular weight. The logarithm of the fringe displacement is plotted as a function of the square of t,he distance from t,he center of rot,ation.
r 60 e
-
i:o 40 0
-
o 01 M K-Pod,
pH75
0 01 M Tris-HCI,
pH 8.5
20 -
T
was held 10 min at the designated temperature. con of rcaaction velocity versus DPSH rclntrution at diffwcxnt fixed lrvc~ls of pyruvntcl intcrscctcld on th x-axis (Fig. 4). This indicatrs that the> K, :md dissociafor DI’NH W: idt>ntical tion constants (16). At pH 7.5 in 0.1 ;\I phosphnto buffr>r :lt %“C, t,his V&P is 6.7 X lo-” N. A similnr plot of rcciproc*:tl pyruvUte concentrations gives a K, for pyruvatc of 1.0 X 10e3 ;1I (Fig. 5). Thr turnover numb~~r for LA1 ?I calculatcld from thr 1’ is 9.S X lo4 molts DPNH per molt> of cnzymcl per minute under the abovc conditions. For the mor(l uctivc LM 1 the valuct is 1.7 X IO”, which is comparable to that of thcl most active IV-type animal cnzymc’s (15) or thca E. coli D-LDH on a molar basis (7).
/nhiDifo~s. Sodium oxam:~tc, a cwmpc?tiw inhibitor of pyruvafo reduction b> many lnctatcl tlcli?drogcn:wcs (l’i), inhibitcld thch Leucowstoc D-LDH 50 ‘2 at 3 X 1OP 11 under th(l usual assay csonditions. This can b(> compnwd to a K; of 1.S X 10e2 AI oxumntr~ for t’lw n-J,DH from I:‘. wli. Howver, the E. coli cnzymck rquirw high concentrations of subntratw to rcarh masimum wlocity (th(> K, for pyruwto is ‘7.2 x 1O-3 M). The D-LDH from L. pla~rta~w~~ is not inhibited by S X 1OV AI oxnmatc>, but the L~LDH from tlw s:lmc’ organkm is inhibited 50% by 5 X 10e4 11 osamat
(1 (S) .
0
1
2
1/[DPNl+ld4M-
3
n
5
0
2
4
I/ [ PYRUVATE]
6
10
8 X?C
-3
M
-1
Frc;. 5. l,illrffr:tvCl.~ljllrk plot for reverse rciL,a1ion with pgr\lv:itc :w thfs varinhle sllbst r:rtcg. 11PNH conc‘entrat ions were %.O X lO-> 11 (circles), 3.0 X lo-” hl (triarrglrs), 5.0 X lo-” u (sqllarcs;), 7.5 X lo-” 11 (crosses), :ind 1.0 X 1OW .M (inverlcd t rianglesj The o( hpr condit iorls u-er(l the same il,q for Fig. -1.
42
CARLAED
0
1
2 I/[DPN+IxIO
-3
3 b?’
FIG. 6. Lineweaver-Burk plot for the forward reartion with L. t,?csenle~,oic(c.s LDH with L)l’?;+ as variable substrate. Lactate concentrations, from the top to bottom c,,rves in order, were 6.7 X 10-3 -52, 1.0 X 1O-2 M, 1.7 X 10d2 M, and 3.3 X 1OW Y. The brlffer m-as 0.1 Y Tris--IICl, pI1 8.7, and the temprrature was 25°C. The reaction was started with 0.01 ml of :i 2.5 pg,‘ml solrltion of enzyme (L&I 3). II = AA &min.
substrate nctivntion reported by Raloustian and I&plan (19) for the L-LDH from lobster musclc~. They drmonslrated that the lobster enzyme is nctiwted by the product, DPNH, so tllat the rxtc :wc+ratcs during thr course of tlitk reaction. T:LrmJ, :uld Kaplan (7) also observed sipmoid kinetics \vith the D-IJIH from E. di ~~llcn pyruwtc is the substratt. This effect is :rssocinted wit11 a lag in tlw initial rcwtion rate at low pyruvate conccsntratiwis, howver, and is abolished by pr(!incubation of tlrc cwymc with p\.ruvate. l-‘hysiologic~al
-1
I/ [LACTATE]
b1
FIG. 7. Lineweaver-Burk plot of the forward reaction with lactate as variable substrate. DPZjconcentrations, from the top to bottom cluves in order, were 3.0 X lo+ M, 5.0 X lo+ M, 7.1 X 10m4M, and 1.0 X 10-a 11. The buffer was 0.1 M Tris-HCl, pII 8.i, and the temperature was 25°C. The reaction was started with 0.04 ml of a 2.5 pg,/ml solution of enzyme (L&l 3). 2) = AA340 nm/min.
role of the enxyme. Leuconostoc
is :I mcmbcr of the lactic acid group of bacteria, \vllicAh 1~s :t vq. rwtrictetl respir:rtoq capwit\* (20). Uriliko the coliform hattcria \\-llich fwmcnt sugars only in t I)(’ :tbwncc of oxygen, these fxult:~tiw xwerobes arc nbligntcly fcrmcntativc. Lactic witI and &I~\-1 :k~~hol are the products of Leucomstoc’s lic~terolacti~ fermentation. Leuconostoc mesol~ewifles :Iccumul:rtcs lactate even llnd~i ;Icrobic conditions \vlwrc DPKH oxid:~tion b\- DPNH osidnw is thc~rn~od\~n:~n~ic::lII~ fkrvowtl (21). Tlicl lapid diffusion of l:wt:ltcs into
tllcx rncdiuin
\V:IS suggrstotl
:I,< ;I11 (‘s-
3. 5. l-i. 7. 8. !I. 10. 11. 12. I:<. 14 15.
1Ci. 17.
18. I!). 20.
21.
and
Properties Leuconostoc
of DL-Lactate
Dehydrogenase
from
rnesenteroides’
In the 30 years sinw Struub purifkd and charnctc~rizr~d t#hc>first DI’S+ 3-linl;c~d lact:tto d(~tr!.drogcrl:ls(, (1) from pig heart, scows of LDHs hnw been studicld. Al& of tJh(w h;~vc~been spwific for the L-isomer of Ixtats. Ho\\-cwr, DmI,DHs 1~:~~ bwn found in wrtain groups of bactck1, trspcci:Jly the l:lctobacilli, and, more rcwntly, in tlrcl ccllular slime molds (2), lowr fungi (3, 4), :wachnids, gnstropods, :md pol,vchnctw (5). The uL-lacbntc~-sprcific LDHs as :L class arc’ similar in their molecular wights, wliich arc :rround 140,000 daltons (6). ThcG ltint$ic prcqwrtiw wry bct\vwn those th:kt arc’ grcat’ly inhibited by high pyruwtc (WIIccmtr:~tions and thaw that, are not. Alt,hough few D-LDHs haw been wrll char:xtorized, thcxir proprtiw nppcar to br more wriod. Tlrc> Dl’S+-linlwd D-LDH from E. roli Ii:ts :I molecular \vc+ght * Contriblition K’o. 8% from the (:radlmte Department of Hiochemistry, Brandeis University. 2 Present address: l'hzyn~~ liesearch T,nbor:ttory, Massachusetts (:cneral Jlospital, Boston, hI:rssachusetts 02111. 3 Abbreviationi;: I)PN’ , diphosphol)yridille I),,dinucleotide) ; cleotide (llicotirl:Lmide-~dellirle LljH, lactate dehydrogennse; GTP, glmnosine triphosphnte; IAI, I~c~corws/oc mesenteroides.
Lezcconosloc ,,rcset,te,.oitles, strain 12291 (American Type Culture Collect ion), was grown ill .4C-1 media (Table I) at 30°C for 20 hr in 20.liter carboys with shaking. 1 )PN- alltl 1)PYH were obtained from P-I, Biochcmicals. The “Chronrntoprlre” grade was used ill thr kinetic st tidies. Sodillm pyruvnte and Tris (Trizma grade) were purchased from Sigma and lithilun u-lactate, from Miles. I)EhE-
30
cedure. L;\I 1, :I smaller preparation, 11:ld a spccifica nctivitj. of 5iOO units/mg :rntl LRI 2, 1100 rmits/mg.
with Ficoll to \yhich an equal amount of sllcrose had been added. When the volume had been reduced to 35 ml, the enzyme was dialyzed and chromatographed on :I 2-liter (5 X 100 cm) Scphadcx G-100 column v-it,h a one-to-ten dilntion of standard bluffer. After 100 ml had emerged from the colnmn, 15.ml frac.tions were collected. The enzyrnc was folmtl in tttbes 38-48. The active fractions were pooled, concentrated with Fir011 to a vohune of 7 ml, and rechromatogrn,phed on a 500ml (3 X 70 cm) (i-150 column. ;2fter ellltirrg 100 ml, 5.5-ml fractions were c*ollected. The activity and protclin peaks emerged at the same place and were spmmctrical, btlt the specific activity was slightly less at, the leading and t,railing edges. The frnctions of constant specific- activity in the center of the enzyme peak (tltbes 3053) were pooled (85 ml), concentrated with Ficoll and sI,crose to a volume of 5 ml, and dialyzed against a one-tenth dilution of standard brlffer for 3 hr, after which the voltlme was 10 ml. Cr~~stallizatio~~ (?i eTlal/?lle. Three milliliters of this solution, which rontained 5 mg/ml of protein, was brought gradually to 507, satllration with ammonium sulfate, at which point the enzyme began to crystallizr. ;\lthollgh the enzyme was kept at 4°C for spvcral months after this step, the crystals did not become large enough to photograph. The specific activity of the crystalline enzyme was identical to that of the uncrystallized preparation. The final specific activity was 3200 units/mg (this is for LplI 3, the third preparation). The plu+fication proredllrc is srmrmarixcd in Table II. Dihltions of the purified enzyme (Lhl 3) were used in the kinetic studies.
J~oleculur wi~jlrt. Tl~c molcculx \\-oiglits of tlic, tlirw prcy1’:ltions rvcrc rompared b?- the intwfwcww scdimc~ntation cyulibrium mc~thotl of I-pll:rntis (12). TIIP cyuntion used was MW = [2R7’/1( --J+p)w2] X [cl(ln c);‘cI(~.?)].At pH 8.6 in 0.05 RI (SH& COe, thcl molfculnr xiglits were dcterminc~tl to bc %,500, 63,000, and C,2,700 for the first, sccontl, :uld third rnzyme preparations, wapwtwely. Plots of t’he log of t’he fringe displacements wrsus the square of the distance from the center of rotntjion are straight lines of constant. slope, indicating homogeneity. The data for LRI 2, the lrast active preparation. is slion-n in Fig. 2. The> sxO,2,,for LM 3 was determined by t:str:~polating the line draxvn through the sedimentation const:tnts at two different protcin concentrations (3.2 and 10 m&l) to the> intcwcyt at, zero ~oncc~nt,rnt’ion, and \\-a~ 3..5%S. Tllcl I&,u value was dctcrmined to be 5.0 X lo-’ cm”i’rnl at one protein concentmt~ion. Wlim tlic sedimentation and diffusion constants wro srtbstitutcd in tlrc
KESULTS Criteria of puu’ty. The purified enzyme appeared homogeneous in the ultmcentrifuge (Fig. 1) mcl on starch gel. Ho’~i’ev~r, there was :I discrqxmc?- bctwcen the specific activity of this preparation and two previous ones purifird by csscntially the same proTABLE PGRIFICATION Purification
II
OF Leucor~ostoc J2esenteroide.s L.xT.~TI~~ ~I~II~DI~~~~~~x.~sI~
step
Protein bd
Activity units (~moles/min)
137,000 28,800
654,000 490,000
Specific activity (unitsimg)
\Yeltl cc;d
~__~ Crllde extract (NH&S04 (40-755;) DE-11 chromatography DEAE-Scphadex chromatography Sephadex G-100 chromat,ography Sephadcx G-150 chromatography
5,620
580 185 78
256,000 245,000
204,000 118,000
4.7 17
100 i5
46 425 1050 1510
39 37 31 18 -__
40
GARLAND
.50
FIG. 2. Sedimentation equilibrium experiment with preparation No. 3 of I,. mesmleroides LDH for determination of molecular weight. The logarithm of the fringe displacement is plotted as a function of the square of t,he distance from t,he center of rot,ation.
r 60 e
-
i:o 40 0
-
o 01 M K-Pod,
pH75
0 01 M Tris-HCI,
pH 8.5
20 -
T
was held 10 min at the designated temperature. con of rcaaction velocity versus DPSH rclntrution at diffwcxnt fixed lrvc~ls of pyruvntcl intcrscctcld on th x-axis (Fig. 4). This indicatrs that the> K, :md dissociafor DI’NH W: idt>ntical tion constants (16). At pH 7.5 in 0.1 ;\I phosphnto buffr>r :lt %“C, t,his V&P is 6.7 X lo-” N. A similnr plot of rcciproc*:tl pyruvUte concentrations gives a K, for pyruvatc of 1.0 X 10e3 ;1I (Fig. 5). Thr turnover numb~~r for LA1 ?I calculatcld from thr 1’ is 9.S X lo4 molts DPNH per molt> of cnzymcl per minute under the abovc conditions. For the mor(l uctivc LM 1 the valuct is 1.7 X IO”, which is comparable to that of thcl most active IV-type animal cnzymc’s (15) or thca E. coli D-LDH on a molar basis (7).
/nhiDifo~s. Sodium oxam:~tc, a cwmpc?tiw inhibitor of pyruvafo reduction b> many lnctatcl tlcli?drogcn:wcs (l’i), inhibitcld thch Leucowstoc D-LDH 50 ‘2 at 3 X 1OP 11 under th(l usual assay csonditions. This can b(> compnwd to a K; of 1.S X 10e2 AI oxumntr~ for t’lw n-J,DH from I:‘. wli. Howver, the E. coli cnzymck rquirw high concentrations of subntratw to rcarh masimum wlocity (th(> K, for pyruwto is ‘7.2 x 1O-3 M). The D-LDH from L. pla~rta~w~~ is not inhibited by S X 1OV AI oxnmatc>, but the L~LDH from tlw s:lmc’ organkm is inhibited 50% by 5 X 10e4 11 osamat
(1 (S) .
0
1
2
1/[DPNl+ld4M-
3
n
5
0
2
4
I/ [ PYRUVATE]
6
10
8 X?C
-3
M
-1
Frc;. 5. l,illrffr:tvCl.~ljllrk plot for reverse rciL,a1ion with pgr\lv:itc :w thfs varinhle sllbst r:rtcg. 11PNH conc‘entrat ions were %.O X lO-> 11 (circles), 3.0 X lo-” hl (triarrglrs), 5.0 X lo-” u (sqllarcs;), 7.5 X lo-” 11 (crosses), :ind 1.0 X 1OW .M (inverlcd t rianglesj The o( hpr condit iorls u-er(l the same il,q for Fig. -1.
42
CARLAED
0
1
2 I/[DPN+IxIO
-3
3 b?’
FIG. 6. Lineweaver-Burk plot for the forward reartion with L. t,?csenle~,oic(c.s LDH with L)l’?;+ as variable substrate. Lactate concentrations, from the top to bottom c,,rves in order, were 6.7 X 10-3 -52, 1.0 X 1O-2 M, 1.7 X 10d2 M, and 3.3 X 1OW Y. The brlffer m-as 0.1 Y Tris--IICl, pI1 8.7, and the temprrature was 25°C. The reaction was started with 0.01 ml of :i 2.5 pg,‘ml solrltion of enzyme (L&I 3). II = AA &min.
substrate nctivntion reported by Raloustian and I&plan (19) for the L-LDH from lobster musclc~. They drmonslrated that the lobster enzyme is nctiwted by the product, DPNH, so tllat the rxtc :wc+ratcs during thr course of tlitk reaction. T:LrmJ, :uld Kaplan (7) also observed sipmoid kinetics \vith the D-IJIH from E. di ~~llcn pyruwtc is the substratt. This effect is :rssocinted wit11 a lag in tlw initial rcwtion rate at low pyruvate conccsntratiwis, howver, and is abolished by pr(!incubation of tlrc cwymc with p\.ruvate. l-‘hysiologic~al
-1
I/ [LACTATE]
b1
FIG. 7. Lineweaver-Burk plot of the forward reaction with lactate as variable substrate. DPZjconcentrations, from the top to bottom cluves in order, were 3.0 X lo+ M, 5.0 X lo+ M, 7.1 X 10m4M, and 1.0 X 10-a 11. The buffer was 0.1 M Tris-HCl, pII 8.i, and the temperature was 25°C. The reaction was started with 0.04 ml of a 2.5 pg,/ml solution of enzyme (L&l 3). 2) = AA340 nm/min.
role of the enxyme. Leuconostoc
is :I mcmbcr of the lactic acid group of bacteria, \vllicAh 1~s :t vq. rwtrictetl respir:rtoq capwit\* (20). Uriliko the coliform hattcria \\-llich fwmcnt sugars only in t I)(’ :tbwncc of oxygen, these fxult:~tiw xwerobes arc nbligntcly fcrmcntativc. Lactic witI and &I~\-1 :k~~hol are the products of Leucomstoc’s lic~terolacti~ fermentation. Leuconostoc mesol~ewifles :Iccumul:rtcs lactate even llnd~i ;Icrobic conditions \vlwrc DPKH oxid:~tion b\- DPNH osidnw is thc~rn~od\~n:~n~ic::lII~ fkrvowtl (21). Tlicl lapid diffusion of l:wt:ltcs into
tllcx rncdiuin
\V:IS suggrstotl
:I,< ;I11 (‘s-
3. 5. l-i. 7. 8. !I. 10. 11. 12. I:<. 14 15.
1Ci. 17.
18. I!). 20.
21.