Glutamine synthetase of pea leaves. I. Purification, stabilization, and pH optima

.\M’HIVES

OF

Glutamine

BIOCH~:MISTRY

AZNJ)BIOPHYSICS

Synthetase

169, 113-122 (1973)

of Pea Leaves. and

I. Purification,

Stabilization,

pH Optima

D. O’yEAL*

4x1)

I<. W. JOY

1. (;hltamine synthetase [I,-glutamate:ammonia ligase (AIIP), E.G. 6.3.1.21 was purified to apparent homogeneity from the shoots of light&grown pea seedlings. It, was found to be quite unstable, hut could be partially stabilized by the addition of divalent cation (Mg*+ or MnzC), and still furt,her by the addition of sucrose (0.5-15 M), fructose (l--2..! M), or ethylene&co1 @O-30”,). Stability varied considerably depending on pH and whet her Mg2+ or RZrr2+ was used. Under certain incubation conditions the inactivated enzyme could be partially reactivated. 2. The pH optimum for glutamine synthesis varied widely (.i.O-8.2) depending on the type and concentration of divalerrt cation (Mnz’, Co 2+, Mg*+) and the ATP concentration. grown in a greenhouse for 12-20 days, with daily watering with nutrient solution. Purine and pyrimidine nucleotides (Iisually best has been isolatctd from a number cjf sourc~~s, grade), enzymes, I,-glutamic acid monohydroxincluding shcqj brain (I, Z), E. coli (X-(i), amate, and all amino acids were obtained from rat liver (i, S), Racillus subfilus and othckr Sigma Chemical Co. Protamine Sulfate (Salmin), microorganism (9-1 I), pet swds (12-l 4), Norit B (Acid-washed), and sodium ribonncleate carrot (IS), :tnd rice: (1 A). This csnzymc: is (yeast) were from Nut,ritional Biochemical Co. An bc~licw~d to play a lacy role in the assimiltlalternative source of Norit A was Matheson, tion, stomgc, c‘rnd translocution of ammoni:~ (‘oleman, and Dell. Biuret reagent and 1, 2, 4 in higher plants (17-Z]), and glutaminc itwlf ;Imillollaphtholsulfollic acid was from Fisher is known to participatch in thch synthwis of Chemical Co. Calcium phosphate gel was prepared by the method of Keilin and Hartree (22). :I v:rricty of lwy mct~abolitos (5). Thr ~~nz~rnc $lutamincb qnth&sc: t,:tm:tt,c: ammonia ligascx (nl)l’) EC.

[L-glu-

6..1.1 .I-,]

Although glutaminc sgnth&w has bwn purified from plant sources and a number of its proportiw inwstigatcd, thaw still r(amains ;L grctt dral of chtlract,c,riz2ltit,11 to bc dorw wit,h the plant cnzyrnc’, espcciall~ with wgard t,o wgulatory propclrtics. This :~nd subsqucnt papers will dcwribc~, in considcrablc detail, the propwt~iw of :L highckr plant glutaminr: synthc+:w. Pea seeds (Piscc?,~sulis/rnl 1,. cv. 1Slr1rl3atltam) were obtained from Stokes Seed Co. (St. Cathcrines, Ont,ario) and after soaking overnight in tap watrr t.hey were planted in moist vrrmicldite and ________~ * Prrst>nt address: Biology l)epartment, I’L.P.T., Troy, KY 1218.

METHOIIS

il. PwiJication

113 C>o~,yright Ail rights

@ 19i3 by .Iradenric Press, of reproduction in any fr,rrn

Inc. reserved.

of the Enxple

All operations were conducted at 2--I%. Sfep 1. Clude ezlrnct. Freshly harvested pea seedling shoots were chilled in a cold room for an hour or two and then blended in a large Waring Blendor for 1 min at high speed, using 1.5 vol of cold 0.0,5 &XTris-TIC1 buffer, pH 7.9 (measured at, 23°C). The homogenate was filtered throllgh follr layers of cheesecloth. Step 2. Prolnnline slclfute. To the filtrate was added 7.5”; (v/v) of a 15; (w/v) solution of protamine sulfate (the opt,irnal amount may vary from 6.5 to 9’; with different lots of protamine sIllfate). After stirring 15 min the solution was ccntrifllged, the pellets discarded, and the pH of the allpernatant raised to pH 7.9 (2-4’C) by adding

114

O’SISAI,

cold 1 Y Tris. This was followed by the addition of 1.5’; (v/v) of a 1 M ILlgSOa solution, and mercnptoethanol to 7 rnll. Sfep 3. .Yorif A treatment. Norit) h carbon was added to the protamine treated supernatant, the optimum amount (310-420 mg/lO ml) being determined in each case in a pilot experiment. After stirring for .i min, and a 12-14 mill centrifugation (2O,OOOg), the supernatant was filtered throrrgh two layers of Miracloth. The Norit A step illcreases purit,y only slightly, hut does remove a considerable amount of nonprotrin uv nbsorbitlg material (probably phenolic compounds and n11cleic acids). Tt was folmd that this step collld hr omitted without milch decrease of final pluity. Step 4. Calcium phosphate gel r. A pilot test was performed to determine the optimal amollnt, of gel to add. The pH of a sample of the Norit S-trratrd enzyme was lowered to 6.8 by slow addition of 1 s acetic acid while stirring. Calcium phosphate gel (14--20 mg solids/ml) was added ill var?;itlg amormts to small aliquots of the enzyme, alIt after 20 min stirring each aliquot was centrifnged 4 min (lO,OOOg), and the supernatant, assayed. The optimal proteinjgel ratio (ammmt of gel needed to bind about 907;, of the enzyme) was 3-.i (rng prnt./mg gel) in most cases, hilt varied signilicantly with age of the gel preparation. hluch more gel was required if the gel was less than 4 w-k old 01 grearer than 12 wk old, the optimal age being 8-10 Wk. After adjusting pH, treatiirg the hulk of the enzyme with the correct amolmt of gel and CCIItrifuging, the enzyme was elutcd from the gel pe!icts with MgSO,. .4lthough phosphate buffer works well, the st,abilizing effect of J’Ig*- is lost due to i?Ig:~(PO,)~ precipitat,ion. Solutions of 0.25 M, 0.30 M, 0.40 M, and 0.50 M MgS04 dissolved itI 0.O.i M Tris-HCl buffer (pH 7.7 at 23°C) containing lA.‘ir, et,hylenc glycol were WISPYfor ellltion. Thrl gel pellets were first eluted 1hrce or four 1imcs with 0.2,j M MgHOa by stirring the sllspension for 20 mill followed by a 4-min centrifugation. The first (0.25 M) ellltion llsed about, 5 vol of Mg buffer per volume of sediment,, while subseql~ent elutions Ilsed 1 or 1.5 vol. The gel pellets were subsequently eluted three times with 0.3 M MgAOa , three to four timrs with 0.4 M i&SO, , and follr to six times wit)h O..i M MgSOa , the last elution being overnight. The 0.4 M and O.,; M >fgSOr eltiates contained most of thr nctivitp and were pooled. Step 6. Calciwn phosphafe gel II. The poolrd MgSOl ehlates were diluted with 0.9 vol of water. A second pilot test was performed [Ising small amounts of the diluted elunte to determine the amount, of gel to adsorb 90c; of the enzyme. This was usually a protein/gel ratio of 1.3-2.0 (mg/mg). The calcium phosphate gel was diluted to contain

AS11 .JOY i-10 mg solids/ml. The bulk of thr crlzymr was treated with the optimal amount c,f grl, st irrcd 20 mill, alrd centrifuged .i min, tlisc:ti,ditlg t hc s,lpctrIlatan1. The gel lxllcts wcrc clr~lctl thrclc times (I%30 min each time) with 0.28 M hIgSO1 solu(io~~s (as above brit containing ot11>- 10’ ( cthylf:nc glyicol), then thloe to four times with 0.4 \f 1IgWd solLltic)n, and fii~ally four or five timrs with n O..i 21 hIgW0, solution, the last elntion t)ritlg ovrrnight The 0.4 and 0.5 M eluatcs were pooled. Sfcp

6. Conwn

Irnlion

mtl

O-200

S~pirtiti~x

~lflo-

n~a/ograph~~. To I hc pooled fxlrlatrs W:LS :~dtlctl >igSOa , at t hp rate of 0.37 g/ml, mft st irritlg w:~s cant itrlled lllrtil the RIgSO, had tlissolvcd. The p1J was lowPrfxl to 4.C4.1 by slow dflitiorl ot’ cold 1 N acetic acid. I’rotcin precipil:ttioil ~~s~~ally trf,g;tth at pH 4.3 4.7. After 10 min lhc protein was rcntrifllged down (10 mill at 20,000g). ‘l’hcl pellf,ts wf’rc flissolvcd ii1 it mitlimal voliimc of 0.O.i >I Tria IlCl 1.i m\~ hrgso,. hiifTf~r cprI7.7 at 23°C:) containing At times, the pll 4.0-4.1 sllprrn:~tni~t Ix,c:trnr slightly clo~ldy after stallding anofhcr30 mill or so, ill which case it was rccclltrifllgcd and t IIF rcsul( ing protein pellet was tlissolvefl alrd ;ddrd lo 1hc enzymr prcpnrnt iota. Abont 45 90 illill was :rllowf~l for the protrin I)rccipit:ttc: 10 dissolvf~, :illd the small nmorult of rf~maillillg s;lisprnsioll \~iis rcmoved 1)y a IO-rnirj cciltrifllgnt ioll (20,OOO~g).

step was oi11y c:rnplo~.efl whetl

I hr itlitiid

spf,cific

PEA

LEAF

GLUT.QIINE

nitrogen gas. Ethylene glycol was added to a final concentration of 25”,, and the enzyme was stored at 0-3°C. (For optimal stability the enzyme was dialysed overnight in the cold against 200 vol of 0.05 M Tris-TIC1 (pH 7.9 at 23°C) containing 25$ v/v ethylene glycol.) Some protein precipitated from the solution during the first’ several weeks and was removed by centrifugation. Co~~??~c:nison the isolafion procedure. To avoid excessive loss of activity due to enzgmc instability, it is best to perform the steps (except the last two) on consecutive days. The protamine sulfate or Norit A enzyme cottld be stored about 2 days with ,?-lSe;, activity loss, but storage for longer periods or during later stages was best avoided, as was dialysis of these prepnrations l>uring the CT-200 Sephadex step, usually lo-255 of the activity was lost, and often with 2 or 3 days of storage the remaining activity decreased anothrr 10-20’5, accompanied by some protein precipitation. Thereafter, the rate of inactivst,ion decreased considerably, and it was possible to store the enzyme 3 mo and retain 30-.jOc; of the initial activit,y of the G-200 step enzyme. The critical stages in purificatiou were the two gel steps. Creater purification was achieved using calcitun phosphate gel aged 8-10 wk. Several purification techniques which were used with great effect in the pea seed enzgmc preparations (12, 14) were not sttccessfttl, including RNA precipitation aud charcoal adsorption and elution. Use of acetcme or ethanol inactivated the enzyme by over 7~~;) even in the presence of Rig*-, but the more effective stabilizers h2n2” plus sttcfose were not t&cd in this respect.

I?. Defemination Ettzyme methods.

activity

1. Bios!prlhelir

of Enzyme dcfiuitq cotdd

assay

be assayed

based on glulanqll

by three

0.20 M TCA’. Centrifugation to remove protein was needed only where steps l-3 enzyme was used. The amount of r-glut,amgl hydroxamate formed was determined calorimetrically (540 nm) by comparison with a standard curve made using attthentic r-glut amy hydroxamute in the presence of all assay components except ATP.

2.

Hioslynthetic

1 Abbreviations. IITP.4 = diethq-lelletri:~mille pentaacetic acid; ME = mercaptoethanol; TCA trichloroacetic acid; PEP = phosphoelrolp?rllvate.

=

a%X~J

based

on release

oj” inor-

ganic phosphate. Performed as above, except that S’H,Cl (4 mM) replaced n’H2011. The Pi liberated was measured by the Fiske-SubbaRow (23) or Bayer CL al. (24) trchniqttes, except that) in the former method the volume was scaled down to 2 ml (to increase sensitivity), and to 1.82 ml in the latter method. Where still further sensitivity was desired, the absorption was read at 333 nm (FiskeSubbaRow technique) or 366 nm (Bayer rt al. technique). 3. Biosqnthefic assay phofomrfric assay. This

based

on coupled

specfro-

assay coupled the AIIP produced to pyruvate kinase and lactic dehydrogenase. In addition to the normal components, the reaction mixture contained 60 m&f KCl. l>PNH (0.O.i ml of a 10 mg/ml stock solution), 1 mu PEP’, and lo-20 units each of pyruvate kinase and lactic dehydrogcnase. The final volume was 1.,50 ml and the rate of decrease in absorbance at 340 111nwas recorded. This assay was performed at room temperature (22-24°C) using a Zeiss recording spectrophotometer. The reaction was started with XfT,Cl after preincubating the other components (plus enzyme) for 5-10 mitt, or trntil absorbance was constant. Tn all three of these assay methods, the activity was approximately equal in terms of pmnles product (AlIP, P, , or glut amyl hydroxamate) released per mg protein per min at 35°C. One tuiit of activity was defined as the amount of enzyme catalyzing the release of 1 pmole of prodttct per hr.

h!jdvos-

n,,tnte s!ynfh.esis. The standard reaction misttuc cottsisted of 0.10 M tricine-KOH buffer containing 20 m&r &LgSCl ) 1 mM lITPA’, 80 ml% r,-glutamate, 6 nmbr SHzOI-T, and 8 rnlr ATT’, with the final pH adjusted to 7.8 (23°C). In some experiments, Ml?t was added to 8 IDM final concentration. The rexction was started after an initial 2 min preincttbation at 3,5”C, by the addition of the enzyme or the NH~OH (pH previottsl>- adjusted to 7.0). The total reaction volume was 1.0 ml. .4fter incubat.ion 6 min at 3.?“C, t.he reaction was stopped with 1.0 ml of a soltttion containing 0.37 M FeCL , 0.67 x HCI. and

115

STKTHETASE

C. Protein Deferknation Protein was determitled by the method of Lowry rt nl. (25) using bovine serum albumin as a standard. For all except the step li (G-200 Sephadex) errzyme the protein was first precipitated by 8% TCA. After carefully washing the precipitated protein pellet (obtained by centrifugation) with cold water, the pellet was dissolved in 1 x NaCrR. 111this proccdttre, the Xa2CO3 solut,ion used in the I.owr~- r/ al. procedure had no added NaOH, but the fi~lal NaOff concctttration was adjttstrd to the llsll:rl

lcvrl.

D. Disc Gel Elecfrophoresis This was performed on ,j or C,C; acrylamide gefs usirlp Tris-glycine httffer (pH 8.3) with or without,

116

O’NEAL

stacking gel, (26). The protein Coomassie blue.

was stained

with

RESULTS

Purity A summary of the purification of one lot of seedlings is given in Table I. The enzyme (step 6) appeared to be nearly homogeneous with respect to disc gel electrophoresis (Fig. 1). It is interesting to note that the final specific activity in t,his preparation (i.e., approx 2000) is similar to that reported with the pea seed enzyme (sp act = 1845) which behaved as a homogeneous protein in the analytical ultracentrifuge (14). The highest specific activity in five different preparations was 2036, and the lowest 1050 (step 6). However, owing to the pronounced instabilit’y of the enzyme, t’he act’ual specific activity was probably underestimated at times owing to progressive inact’ivation. Molecular Weight An approximation of the molecular weight was obtained using a calibrated G-200 Sephadex column (1.6 X 90 cm). Using this technique the molecular weight appeared to TABLE PTRIFICATIOK

AND

JOY

be in the range 330370,000. The peak of glutamine synthetase activity- appeared in the same 1.2 ml fraction as the ptxak of bovine glutamic dehydrogcnasc (applied at, 1.5 mg/ml) and one fraction later than a commercial preparation of ovine glutamine synthetase. The former rnzymc (at, the prot,ein concentration used) is believed to have a molecular weight of 330-360,000 (27) while t,he latter enzyme has a molecular weight of 392,000 (7).

The ratio of absorbance at 280/260 nm was between 0.81-0.86 in step 1 and step 2 enzyme, 0.88-0.95 in st’ep 3, 1.&l .7 in step 4, and 1.77-1.89 in steps 6 or 7 preparations. Stability

The enzyme isolated in this study was found to be quite unstable during all stages of purificat,ion, and thus it was necessary to improve its stability in order to purify and characterize it, as well as to provide possible information as to the nature of the enzyme.

I

OF PEA SHOOT GLUTIMINE SYNTHETASE~

step

Total protein (md

Total units*

I Crude extract. II Protamim SO4 III Norit A IV Cap04 Gel I V CaPO4 Gel II VI G-200 Sephader

19,375

344,190

17.7

100

iG,516

361,050

21.8

104

302,160 243,700

32.2 673

88 78

9390 3G2

Specific activity

Yield (G/o)

.I-

105.5

145 ) 100

1371

42

53.4

108,700

2036

32

a 1.63 kg of &day-old seedlings were used in this preparat,ion. b One unit of activity is that amount of enzyme catalyzing synthesis of 1.0 rmole glut,amyl hydroxamate/hr at 3.i”C (Mg2+ in assay).

FIG. 1. Acrylamide gel electrophoresis of 60 rg of step 6 (G-200 Sephadex) enzyme, 670 gel. Details given in Methods and blaterials.

PEA LEAF GLUTAMINE

TABLE III

TABLE II EFFECT OF METI\LS, SWROSE, ~KD MER~~PTOETHdNOL ON STABILITY OF PEA SHOOT GL~TAMI~E S~STHET.\SE~

Treatment -~ Control (-Mg*+) MgCl:, (19 rnM) MgCl2 (38 mM) MgCl, (70 mu) MgSO~ (38 mM) MgCl, (37 mm) bIgC1, (36 mM) MgCL (37 rnM) M&l, (33 mu) (7 rnM)

+ ME (18 rnsl) + EIITA (4 mM) + sucrose (0.75 M) + I,-glutamate

117

8YNTHETASE

EFFECT OF~IET.LLS, SWROSE, AND FRUCTOSE STABILITY OF PEA SHOOT GLuTAMIriE S~NTHET.LSE"

Treatment

Final activity (5; of initial) 0 G3 GO .53 GO 100 G3 i7 61

0 Step 4 enzyme was used, which was freed of Mg2+ by G-2.5 sephadex chromatography. The protein concentration was 1.5 mg/ml and the enzyme was in 0.17 M Tris-HCl buffer, pH 7.7 (measnrcd at 23°C). The enzyme samples were heated at 33°C for 90 min and then placed in ice water.

Slagnesium and manganese increased stability markedly at all stages, and still further improvement’ occurred upon the addition of sucrow (051.5 JI), fructose (1.3-2.4 AI), or ethylene glycol (10-4070) (Tables II and III). It was subsequently discovered that the stage 6 enzyme (at O-3%) was more stable without ;\I$+ if 1.0 JI sucrose or 20-30% ethylene glycol were present. The pH optimum for &ability seemed t,o vary with the nature of the divalent cation (Mgz+ vs Mn2+), stage of and bct,wcen preparations. In purification, all cases, Mn?+ was much superior to 1\Ig”+ at pHs below 7.6-7.2, while at pH 7.7was almost as good as RTn?+ 5.3 n:fg2+ (Table IV). XE (wit’h Mg2+) swmed to enhance stability only at high temperatures (Tables II and V), except’ in t,he prcscnw of high concentrations of MgSO, (the 0.30.5 31 MgSO, eluates of calcium phosphate gel in st’eps 4 and C;), where it decreased stability. At low temperatures (0-3°C) ME (4 mlr) or dithiothreitol (2 rn.\r) cause a significant decrease in st.abilit,y of stage 6 enzyme. Where Mnz+ was used in place of hIg*+, ME decreased stability at all km-

.-~ A. Cont,rol (-Mgz+) MgS04 (15 mM) + sucrose (1 11) MgS04 (15 m&f) + M&O4 (0.23 mM) YlgSO, (I.5 mkl) + MnSOI (1.0 mM) MIlSO~

(0.25 rnM) l\-lnS0, (1.0 mM) MnSOa (1.0 rnhf) + sucrose (1 >f) JIgSO, (15 mhx) + blnS04 (1 mM) + sucrose (1 3~) hIgS0, (1.5 mM) + ?vInSOI (1 mM) + fructose (1.8 a~)

B. Control (-?vIg2+) MgSOl (20 rnhl) Sucrose (1 M) S\lcrose (1 M) + Mg2+ (20 mhl) Ethylene glycol (lo?;;.) Ethylene glycol (20:/i,) Ethylene glycol (305 ) Ethylene glycol (209 ) + RIg2+ (20 mM)

ON

Final activity (% of initial) __.-I 33 4 i

3 2G 80 G4 GO

27 44 90 .iO (il 88 89 03

n Step 3 enzyme (Norit -4) was employed here. Iu part A, ME (8 mM) was preseut in all cases and the enzyme was heated at 40°C for 80 min. The pH was 7..i (23”(Z), the buffer 0.0.5 11 Tris-HCl. In part B, no ME was present. The euzyme (Ci-200 step) was stored at 2%4°C for 2 u-k diluted to 0.1 mg/ml protein concentration. The pH was 8.0 (2°C)) 0.0.; M Tris-HCl.

prraturcs, exwpt in the prcwnce of ATP (Table V). EDTA or DTPA (l-4 mAI) wcrc without significant effect’ provided enough uncomplexcd Mg2+ or Mn2+ remained in solution. ATP (6 mAI> decreased st’abilit’y if present with lower concentrations of SIg”+ (or ;\In2+), probably by complrxing all of the divalcnt cations, but whcrc I\Ig?+ or >In’+ were present in 4 rnlr excess over =\TP, ATP increased stability slightly. L-glutamate (7 mlr) in the prcwncc or abscncc of 1\Igz+ had lit.tle or no effect., nor did KHJ+in the presence or absence of i\lgz+ or 1In’f. With respect to st’ability at 35-6O”C, an cquimolnr concentration of l\In:iZTP (op-

118

O’NEAL

TABLE IV EFFECT OF pH ON STABILITY OF PES SHOOT GLUTAMINE SYNTHETASEQ Treatment (pH) divalent ion present

Final activity (% of initial)

pH 5.86 Mg2+ Mn2+ pH 6.39 Mg2+ Mn2’ pH 6.78 Mg2+ Mn2+ pH 7.43 Mg2f Mn2+ pH 8.10 Mg2+ Mn2+

3 7 4 35 14 82 78 102 94 85

V

EFFECT OF ELEVATED TEMPERATURE ON STABILITY OF PEA SHOOT GLUTaMINE SYNTHETASE~ Treatment

Find activity soy, 20 mm

1. MnS04 = 4 mM, MgSOa = 2.8 mM, ATP = 4 mM 2. MnSOa = 8 mM, MgSO, = 2.8 mM, ATP = 8 mM 3. As #2, plus 4 mM ME

(% of initial)

SST, 15 min

JOY

and 8.4 occurred at pH 7.3 in t,he presence of 0.25 m>I MnSOq , such that after 2 mo storage 98y0 of the initial activity remained. In nearly all cases, manganese (plus sucrose or et’hyleneglycol) was the most effective divalent cation for retaining activity in purified preparat’ions. The effect of protein concentrations on stability was not critically examined, although a trend toward increased stability at higher protein concentrations was apparent. Reactivation.

(1The enzyme used was from step 3 (Norit A) prepared without Mg2+. The pH values (at 2-3°C) were adjusted with 0.1 N acetic acid or 0.2 M Tris base. The MgS04 concentration was 20 mM, and the Mn2+ concentration 2 mM. ME = 8 mM. After 6 days of storage at 24°C the final activity was measured. (Mg2+ dependent activity.) TABLE

AND

60% amin

95%

66%

29yc

94

60

25

134

112

101

D Diluted step 6 enzyme (G-200 Sephadex) was used. Protein concentration was 0.32 mg/ml. The pH (measured at 22°C) was 8.4 (0.125 M Tris-HCl buffer). The “control”act,ivityforeach treatment (before heating) was set as 100%. All enzyme solutions contained 6.7% ethylene glycol.

timal level of 2 rn>f or less) was superior to Mn2+ alone, while MnATP (1: 1) plus 4-5 mM ME was best of all (Table V). This is in contrast to decreased stability in the presence of ME where Mn2+ exists without ATP. Using step 5 enzyme (in 0.4 RI JIgSO,), stored at 2-4’C for several months, t’he optimal stability measured between pH 7.3

It was observed on several occasions wit’h step 6 enzyme, st’ored for several days or weeks, that Mn2+, Mn:ATP (molar ratio l:l), or Mn:ATP (molar ratio 1: 1) plus hIg2+ actually activated or react,ivated the enzyme when incubated for 40-120 min at, 35-45°C (with ME), or for a period of several days to several weeks at 2-4°C (without ME) (Table V). The amount of activation was variable, with increases of act’ivity of up to 50% being observed, more usually 15-357,; occasionally reactivation did not occur. The optimal conditions for such reactivation remain to be det’ermined. Linearity

with Time

With purified enzyme (steps 6 or 7) the reaction was linear at 35°C for 8-10 min at pH 5.2 (Mn2+ divalent cation) using the biosynthetic Pi assay, and also at pH 7.8 (hIg2+). With preparations from steps l-4, the reaction at’ pH 7.8 (Mg*+) n-as usually linear only for 4-5 min, falling off rapidly thereafter, even in the presence of an ATPregenerating system (PEP and ppruvat’e kinase). pH Optimu8m The pH optimum varied considerably depending on the divalent cation used and its concentration. With Mgz+, the pH optimum varied from 8.2-8.6 to 7.3-7.4 depending on the Mg2+ concentration (Fig. 2). The pH optimum using one level of Co*+ is shown in Fig. 3. With Mn*+ even greater variation in pH optimum occurred. In this case, it was possible to shift the pH optimum about 2 pH units, depending on thr Mn/ATP ratio and

PEA

I

6.0

6.4

LEAF

GLUTAMINE

6.8

119

SPNTHETASE

7.2

8.0

7.6

8.4

8.8

PH

FIG. 2. The effect of MgSOa concentration on the pH optimum. ATP = 8 mM, NH,Cl = 5 mM, L-glutamate = 60 mM, DTPA = 1 mx, and 0.10 M tricine-KOH buffer (pH 7.2-8.4) or MES-KOH buffer (pH 6.369). (X-X-X) MgSO, ,8mM; ATP, 8mnf; (O-O-O) MgSO, , 20 mM; ATP, 8 mM; (@--O--O) MgSO, ,60 mu; ATP, 8 mM.

OL 6-O

6.9

7.2

7-6

8.0

PH FIG. 3. The pH optimum with CoC12. ATP = 6 mM, CoC12 = 10 m&f, r>-glutamate mM, UTPA = 1 mM, PIPES-NaOH buffer, 0.10 Y.

the Mn2+ concentration, as shown in lcigs. 4 and 5. These results part’ly explain the observation that there is an optimal 1\In2+ concentration for each level of ATP at any given PH.

Inactivation

During

Concentration

Upon concent’rating the enzyme prrparations (G-ZOO-enzyme or DEAE-enzyme)

= 40

under nitrogen pressure using an Amicon stirred cell (43 mm size) and XM-1OOA membrane, it was found that significant activity was present in the ultrafiltrate and that when the activity in both fract’ions (retentate and ultrafiltrate) were summed, it was oft’en less than one-third of the initial activity. This was surprising in view of the high molecular weight of t’his enzyme and the

120

O’NEAL

48

5.6

5.2

.4Nn JOY

6.0

6.4

6.8

7.2

PH

FIG. 4. The effect of Mn*+ and ATP concentration on pH optimum. L-glutamate = 60 mM, NH,Cl = 5 rnivr, MES-KOH buffer, 0.10 M. (X-X-X) MnSOd , 12 mM; ATP, 7 InM; (O--O--O) MnSO, ,4 mM; ATP, 7 mM; (O-0-0) MnPOl , 1 mix; ATP, 7 my.

IOO,:

,

‘\, ’\

: /

‘b

,

80.

‘\ / P

20.

I’

1 O4

4-C

I’

‘. ‘0

/’

4.8

5.6

52

6.0

6.4

6.8

7.2

PH

FIG. 5. The effect of Mn2+ and ATP concentration on pH optimum. As above, but 0.10 IM RIES-KOH buffer used from pH 5.4-6.2 and succinate-KOH buffer between pH 4.0-5.4 (0.13 M). (X-X-X) MnSOI , 0.5 mu; ATP, 0.5 m&r; (O--O--O) 3’lnSO~ , 10 mM; BTP, 10 mlr; (@-O--O) MnS04 , 12 m.w; ATP, 7 mM. 100,000 lllW

cut,-off of the X34-1OOA

mem-

brane. However, where the XWSOA mcmbrane was used under the same conditions, most (over 9.Yjj0) of the cnzymc was retained, although here too the recovery was often only 50-75°10 (ewn at a slow stirring rate, or when Amicon Centriflo membrane cones were

used in a centrifuge).

During

concentration using X,11-1OOA membranes the solution rapidly became turbid, owing t’o precipitated protein, even though the protcin concentnttion was less than 2-3 mg/ml. I)IScUSSION

Green pea shoots were used for the enzyme source for sew& reasons: a. the initial

PP:A LI
GLUTARIIIW

specific activit’y of these preparations n-as over sevenfold greater than that obt’ained from dry pea seeds, the source used by earlier investigators (14) ; b. The seeds (var. Blue Bantam) we obtained had only 40-45ye of t.he act,ivity found by Varnrr and Webster (13), and c. it, was considered a possibility that, the seedling might contain a different glutaminc synthctase t’han the seed, for the seed enzyme is found chiefly in the cotyledons, which arc specialized storage organs, while the growing pw shoots reprcsent a different developmental and metabolic situation. The glutamine synthetase of pea leaves appears to have a molecular weight similar to that, reported for t’he pea seed enzyme and the ovine brain and rat liver enzymes (about 360,000) (7). All of these enzymes are smaller than the E. coli and R. subtilis enzymes, which have molecular ncight of about 600,000 (7). On the other hand, the pea leaf GS has a higher molecular weight) than the carrot GS, lvhich is estimated (by t’he scrylamidr gel method) to consist of two fractions of molecular weights 150,000 and .56-79,000 (15). The pea leaf GS is apparently much less stable than the pea seed GS, which has been report,ed as being stable at O-4’% (l Y-14), as is enzyme from bact’erial and animal sources (l-lo). Sta.bility data are not’ given for the rice enzyme, but the carrot enzyme is quite unstable; after 2 hr at 35°C (in the presence of 20 mnr Rig?+ and 5 rnlr dithiothrcitol) less than one-third of t.he initial activity rcmained. Interest~ingly enough, the carrot enzyme was stabilized by similar compounds as the pea leaf enzyme; namely, glycerol (or sucrose) and JIgSO . The cause of the inst’ability of the pea leaf or carrot GS is unknown. Given the high purity of these preparat,ions, the instability is probably innate and due to spontaneous denaturation rat,hcr than to the influence of proteases or inhibit’ory compounds. This hypothesis is st,rengthened by the observation t,hat even aftrr several momhs storage, still only one major band was seen upon elcctrophoresis, although over two-thirds of the initial activit’y had been lost. The nature of the stabilizing action of >I,@+ and I\In2+ is

121

SYNTHET.4HE

not. known, although the E. coli enzyme is kno\vn to contain bound Nn2+ which presumably hold subunits together (29). It is interesting to note that the ovine enzyme is protected from thermal drnaturation at 60°C only by XTP plus JIg2+ (ratio I : 2 to I : 5), but not by either compound alone (I) in contrast to our observations that the pea leaf enzyme is stabilized by h4g2+ in the absence I of ATP. The explanat,ion for the inactivation (and partial precipitation} of the pea leaf enzyme upon concentration in an Amicon concrntrator is obscure, as is the fact t’hat the X11-100 A membrane does not retain much of the enzyme. A similar inactivat’ion during pressure concentration or pressure dialysis has been found to occur wit,h spinach fruct,ose diphosphatase (28), due t’o the format,ion of inactive subunits. The effect of divalent cations and their concentrat’ions on the pH optimum of the pea leaf GS is striking. L4 similar phcnomenon occurs with pea seed enzyme (30) n-here the pH optimum for Mg2+ can be shift#ed from 8.5 to 7.0 or to intermediate values. Equimolar Nn:ATP gave a pH optimum of 5.2, while with ATP in excess the optimum was 6.2. With bovine and ovinr brain GS the pH opt*imum varied between 6.5-7.6 (Mg”+), and 5.2-6.1 (CW+) (2). The E. coli enzyme behaved somewhat similarly (3). The pH optimum with the carrot enzyme n‘as 7.2 with >Ig2+, a very sharp opt’imum of Z..ij with 11In?+, and 6.5-7.2 wit,h Co2+ (1.5). An explanation of these shifts in pH opt.imum with divaIcnt catsion source is not known, but it is likely t’hat specific conformat)ional states of the protein result from addition of 1Ig, ?\In, and Co. Other evidence supporting this hypothesis will be presented in subsequent papers, including data on K,s and the considerable influence of divalent cation species on substrate and inhibitor specificity. ACKNOWLEDGMENTS The work was srqq>ortetl National Research Council

by a (irant of Canada.

from

the

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122

O’NEAL

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