JOURNAL OF BACTERIOLOGY, Aug., 1965 Vol. 90, No. 2Copyright @ 1965 American Society for Microbiology Printed in U.S.A.

Properties of a Purified Halophilic MalicDehydrogenase

P. K. HOLMES' AND H. ORIN HALVORSONDepartment of Microbiology, University of Illinois, Urbana, Illinois

Received for publication 12 March 1965

ABSTRACTHOLMES, P. K. (University of Illinois, Urbana), AND H. ORIN HALVORSON. Proper-

ties of a purified halophilic malic dehydrogenase. J. Bacteriol. 90:316-326. 1965.-Themalic dehydrogenase (MDH) from Halobacterium salinarium required high concentra-tions of monovalent ions for stability and activity. Studies of inactivation rates atdifferent salt concentrations suggested that approximately 25% NaCl (w/v) is re-quired to stabilize MDH. From 50 to 100% reactivation, depending on the salt con-centration present during inactivation, could occur in 2.5 to 5 M NaCl or KCI. Theoptimal salt concentration for activity of MDH was a function of the pH, and rangedfrom 1 to 3 M NaCl or KCl. The effect of salt concentration on the pH-activity curvesoccurred chiefly below pH 7.0. Inactivation of MDH with heat or thiol reagents showedthat the enzyme was more labile in the state induced by absence of salt. The activationof MDH by salts was attributed to a decreased rate of dissociation of MDH and re-duced nicotinamide adenine dinucleotide (NADH2). The inactivation of the enzymein the absence of salt could be largely prevented by the presence of NADH2. The S20,.of MDH decreased threefold at low salt concentrations. The enzyme was assumed tobe in its native compact configuration only in the presence of a high concentration ofsalt.

In the accompanying paper (Holmes and Hal-vorson, 1965), we described the purification ofnicotinamide adenine dinucleotide (NAD) malicdehydrogenase (MDH) from Halobacterium salin-arium. In the purified form, this enzyme retainsa requirement for salt. This paper presents theresults of experiments performed to explain thisrequirement.

MATERIALS AND METHODSThe enzyme preparations used were those

previously described (Holmes and Halvorson,1965). Preparations purified to different extentswere used in the various experiments as noted.The enzymatic activity was assayed by followingthe oxaloacetate-dependent oxidation of reducednicotinamide adenine dinucleotide (NADH2) witha Beckman (model DU) spectrophotometer.The inactivation studies were performed with

300-fold purified MDH. The partially purifiedenzyme was inactivated by extensive dilutionwith 0.03 M sodium phosphate buffer (pH 7.13)containing from 2 to 5% NaCl (w/v). The mix-ture was maintained in a water bath at 30 C. Thecourse of the inactivation was followed by trans-ferring samples of the diluted enzyme after various

1 Present address: Pioneering Research Divi-sion, U.S. Army Natick Laboratories, Natick,Mass.

time intervals into receiving tubes which held asaturated solution of NaCl containing undissolvedsalt. The receiving tubes and their contents weregently agitated until the undissolved salt crystalsdisappeared, which brought the NaCl concentra-tion to 25%. Introduction of the enzyme into thesalt solution apparently stopped the inactivationprocess instantly; after solution of all the undis-solved salt, the samples were assayed for MDHactivity.

Reactivation of the enzyme was accomplishedby dialysis in 0.5-inch (1.27 cm) diameter cellulosecasing, against the various salt solutions. Sampleswere removed at intervals, and activity was as-sayed in 25% NaCl. A plot against time of log[a/(a - b)], where a is the percentage of originalactivity recoverable and b is the percentage oforiginal activity found at each time interval, wasmade, and the slope of the initial portion of thiscurve was called the initial reactivation rate.For the pH studies, the activity of MDH was

assayed at several values (maleate buffer, pH5 to 6; potassium phosphate buffer, pH 6.3 to 8;glycine buffer, pH 9.5 to 10; all buffers 0.0625 M)and at various concentrations of both NaCl andKCl. The pH of the buffers was determined withthe glass electrode before the addition of thesalts. The enzyme used was purified 420-fold.The enzyme used for the kinetic study was

purified 250-fold. NADH2 assaying at 89% pure,assuming a molar extinction coefficient of 6.22

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X 106, was employed at concentrations rangingfrom 3 X 10-5 to 3 X 10-4 M. The concentration ofoxaloacetic acid (OAA) was varied over the samerange of concentrations. (Inhibition by bothsubstrates becomes apparent at concentrations ofabout 3 X 103 M). The reaction mixtures werebuffered with 0.05 M tris(hydroxymethyl)amino-methane (Tris)-acetate at pH 7.0, and were as-sayed spectrophotometrically at 25 C for NADH2oxidation.The heat sensitivity of the enzyme was assayed

as the decrease in activity resulting from a 5-minexposure to temperatures of 60 and 65 C. Afterheat treatment, but prior to assay, all sampleswere dialyzed against 25% NaCl for 20 hr at 5C. MDH, purified 65-fold, was used in threeforms: (i) the "native" form, in 25% NaCl, (ii)"salt-free" in deionized water, and (iii) the "salt-treated" form, which results when solid NaCl israpidly dissolved in the "salt-free" form, to makea 25% NaCl solution. The unheated controls wereheld at 25 C. The same enzyme preparation (65-fold purified) was used in the inhibitor studiesinvolving thiol reagents. The enzyme was incu-bated at pH 7.2 (0.01 M potassium phosphate) inthe presence of the thiol reagent for 24 hr at 4 C.Incubation mixtures with native enzyme and salt-treated enzyme contained 25% NaCl; the salt-free enzyme was incubated in the absence of salt.After the reaction period, all mixtures weredialyzed at 4 C against 25% NaCl-0.01 M phosphate(pH 7.2) for 24 hr, to remove the free reagents aswell as to reactivate the inactive protein.The sedimentation behavior was examined in

sucrose gradients, 5 to 15% sucrose in 0.1, 10, and27% NaCl, prepared by the method of Martin andAmes (1961). All gradients were buffered at pH7.2 with 0.005 M sodium phosphate. Immediatelyprior to centrifugation, the enzymes weresuspended in salt solution equivalent in strengthto the sodium chloride concentration in the cen-trifuge tube, and were layered on the surface ofthe gradients. The samples were mixtures of two orthree enzymes. Halophilic MDH, purified 750-fold (native enzyme), along with a similar prep-aration free from salt (inactive enzyme), wascombined with pig MDH (Calbiochem, LosAngeles, Calif.) and layered on the gradients con-taining 27 and 10% NaCl. Inactive halophilicMDH and pig MDH were used on the gradientcontaining only 0.1% NaCl. After centrifugationfor 24 hr, the tube contents were assayed for thethree malic dehydrogenases. The samples fromtubes containing 10 or 27% NaCl were first as-sayed in 10% NaCl for the native halophilic MDH.All samples were then dialyzed against 25% NaClat 25 C, and were assayed for the native plus thereactivated "inactive" halophilic MDH. Theactivity of the pig MDH, which was completelyinhibited by 10% NaCl, was then determined in asalt-free system. The samples from the tube con-taining 0.1% NaCl were first assayed for the pigenzyme, then reactivated with salt, and the ac-tivity of the halophilic enzyme was determined.

A quantitative estimate of the changes in S20o,produced by the salt was arrived at by the sedi-mentation velocity method (Schachman, 1957).The densities and specific viscosities of the grad-ient media were directly determined at the temper-ature of centrifugation with a pycnometer and aCannon-Fenske viscosimeter. The figures used incalculation were the average densities and vis-cosities of the specific media through which eachprotein peak had passed in the course of an ex-perimental run, as extrapolated from the data forthe media at the top and bottom of each tube.The density of the protein in solution was esti-mated by accepting the generally used averagefigure for the partial specific volume of non-halophilic proteins, 0.725 cc/g, derived chieflyfrom the work of Svedberg and Pedersen (1940).The distances travelled by the proteins weremeasured from the peaks of enzymatic activity.Only the lighter and major component of the pigenzyme was considered. The migration rates wereassumed to be constant.NADH2 and OAA were obtained from Sigma

Chemical Co., St. Louis, Mo.; iodoacetamidefrom Nutritional Biochemicals Corp., Cleveland,Ohio; N-ethyl maleimide from Mann ResearchLabs, New York, N.Y.; and p-chloromercuri-benzoate from Calbiochem.

RESULTS

Inactivation by removal of NaCI. At low con-centrations of salt, MDH was rapidly inactivated,and the inactivation could be halted by addingsalt. The rate of inactivation of 300-fold purifiedMDH was determined at several salt concentrs-tions in an attempt to ascertain the amount ofNaCl required to stabilize the enzyme. On= theassumption that enzymatic sites are equal andindependent, and are equally susceptible to in-activation, Van Eys, Ciotti, and Kaplan (1957)derived an equation for the relative rate (Vr) ofinactivation in the presence and absence of aprotecting substance:

Vr = 1/K(P)n + 1 (1)where K is the equilibrium constant for the in-activation, (P) is the concentration of protector,and n represents the number of moles of protectorrequired to protect 1 mole of enzyme. Equation 1may be written as:

log (1/Vr - 1) = n log (P) + log K (2)

From this equation it is evident that a plot of log(1/Vr - 1) vs. log (P) results in a straight lineof slope n.For the halophilic MDH, Vr, or the ratio of the

velocity constants in the presence (kp) and ab-sence (ka) of salt, cannot be determined, as in theabsence of salt the velocity constant ka presum-

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HOLMES AND HALVORSON

ably approaches infinity. For this reason,(ka/lkp - 1) will have a value very close to thatof ka/kp, and equation 2, as Baxter (1959) noted,becomes:

log (k./kp) = n log (P) + log K (3)

or

-log kp = n log (P) + log K - log ka (4)

From data for log kp and log (P) arranged accord-ing to equation 4, an estimate of n for MDH wasobtained.A plot of log (original activity/remaining activ-

ity) vs. time reveals a straight line (Fig. 1). Thus,the inactivation was assumed to follow first-orderkinetics. Table 1 compares the half-lives (t½2)and first-order velocity constants (k) found forthe denaturation at five different salt concentra-tions.

If, in accordance with equation 4, log k is plot-ted against. the. log of the NaCl concentration, a

_. 100 _

°080_

E .060 -

.040-

.>.0200

_'0.O't I

0 20 40 s0minutes

FIG. 1. Inactivation of MDH in 5% NaCl. Theenzyme was diluted into 5% NaCl and assayed aftervarious time intervals in 25% NaCl.

TABLE 1. Inactivation of MDH at various saltconcentrations*

NaCl concn t§ k

% min

5 189 0.00374 14.3 0.04753 3.8 0.1842.5 3.1 0.2202.0 0.8 0.815

* The course of inactivation of MDH was fol-lowed in different concentrations of NaCl. Thesymbol k represents the first-order velocity con-

stant for inactivation; t3/ is the half-life in min-utes, derived from k.

straight line with a slope, n, of 4.5 is consonantwith the data (Fig. 2). A 4.5 M solution of NaClapproximates the tonicity of the growth mediumfor this organism.

Reactivation with salt. Salt-free preparations ofMDH regained 50 to 60% of the original activityif dialyzed against solutions containing NaCl.The activity could be completely restored to prep-arations in which a small amount (0.5%) of so-dium chloride had been allowed to remain.

Salt requirements for reactivation. No reactiva-tion of salt-free preparations by dialysis occursunless the salt solution contains at least 15%NaCl, and maximal reactivation occurs only inthe presence of 20% NaCl and above (Table 2).KCl reactivated the enzyme to the same extent

as did NaCl, although the variability of results

0

-

0

00

-%

en

2-

01

0

J0

0

-1.o0-

0

-2.0-_

0

-.5 -.4 -.3 -.2 -.1 0LOG (NaCI)

FIG. 2. Influence of NaCl concentration on therate of inactivation of MDH. The slope of the lineis 4.5, and corresponds to the number of moles ofNaCl required to prevent inactivation of the enzyme.

TABLE 2. Reactivation of MDH from salt-free stateby various concentrations of NaCl*

NaCl concn Reactivation

0 05 010 015 2020 6025 5730 54

* Reactivation was by dialysis against the saltconcentrations shown, and was continued untilno further increase in activity was detectable, ap-proximately 36 hr.

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HALOPHILIC MALIC DEHYDROGENASE

was in general greater. The reactivating ability ofother salts is seen in Table 3. The sodium andpotassium halides other than the chlorides wereineffective; lithium chloride likewise did not re-activate. It is noteworthy that the sodium saltsof the lower aliphatic acids had reactivatingpower which decreased with increase in molecularweight.The enzyme could also be assayed in other

salts. If crude MDH was dialyzed against 4.3 MNaCl, KCI, sodium formate, or potassium for-mate, and then assayed in the corresponding saltsolution at 0.86 M, activity was found in all cases(Table 4); 4.3 and 0.86 M correspond, respec-tively, to 25 and 5% NaCl (w/v). MDH appar-ently does not have a specific requirement forsodium chloride.

Reactivation kinetics. The reactivation of salt-free MDH preparations by dialysis against salt

TABLE 3. Reactivation of salt-free MDH withvarious salts*

Salt Reactivation

NaCl............................. 60Sodium formate ..................... 25Sodium acetate..................... 8Sodium propionate .......... ........ 6KCI.............................. 60Potassium acetate .......... ........ 0.8NH4Cl ............................. 1

* Salts which gave no reactivation were NaF,Nal, NaBr, NaNO3, Na2HPO4, KF, KI, KBr,LiCl, MgCl2, CaC12. All salts except NaF weretested for reactivation at a concentration of 4.3 Mand at 30 C, and enzymatic activity was subse-quently assayed in 5% NaCl. The pH of the saltsolutions containing sodium or potassium wasadjusted to about pH 7 with the correspondingalkali hydroxide. The NaF used was a saturatedsolution, approximately 1 M.

TABLE 4. Effect of the species of salt onMDH activity*

Salt (4.3 m) Maximal

NaCl.............................. 84KCI.............................. 100Sodium formate ..................... 25Potassium formate................... 45

* Samples of crude extract were dialyzedagainst the salts shown, then assayed in 0.86 Msolutions of the corresponding salt (isotonic with5% NaCl). The assay system also contained 0.01M potassium phosphate (pH 7.2).

solutions was not a simple reaction. A semilogplot (Fig. 3) shows that the process did not followfirst-order kinetics, and that it may be the sum oftwo first-order reactivations. An initially rapidreturn of approximately one-half of the activitywas followed by a much slower return of the re-mainder. That this biphasic reaction was dueto the decreasing gradient of NaCl concentrationacross the dialysis membrane was unlikely, asequilibrium was established across the membranewithin 45 miin and the initial rapid reactivationlasts considerably longer than this. The reactiva-tion kinetics are essentially the same for MDHboth in crude extracts and in the partially purifiedform.

Reactivation and pH. The initial rate of reacti-vation showed a broad optimal range above pH7.0. Table 5 compares the initial rates of reactiva-tion of 300-fold purified MDH in buffers of differ-ent pH. Crude extracts, which are internally buf-fered by protein at about pH 7.2, are reactivatedat a maximal rate in the absence of any buffer.

Reactivation and temperature. The initial reac-tivation rate was maximal at 32 C; at highertemperatures, the rate fell off rapidly and reachedzero somewhere between 40 and 50 C (Fig. 4).This decrease in reactivation rate probably re-flected a permanent denaturation of the enzyme.If once subjected to a temperature of 40 C, theprotein did not regain a maximal rate of reactiva-tion if returned to 32 C, nor was the maximalrate or total activity restored if the salt concen-tration of the (40 C) sample was lowered to 0.5%and a new attempt at reactivation made at 32 C.The data for rates of reactivation between 10

and 32 C can be plotted against reciprocal abso-

.0

0I0

-i

0 6 12 18 24 30TIME, hours

FIG. 3. Reactivation of MDH with NaCl at 5 C.The curve may represent the sum of two first-orderreactions. The "a" represents the percentage oforiginal activity recoverable; "b" is the percentageof original activity found at each interval.

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TABLE 5. Effect of pH on the reactivation of MDHby 25% NaCi at 30 C

Initial rateof reactiva-Buffer tion, % ofmaximum

Citrate, pH 5.0...................... 0Citrate, pH 5.8....................... 0Acetate, pH 5.8...................... 0Phosphate, pH 6.3 ................... 0Phosphate, pH 6.6 ................... 2Phosphate, pH 7.0 ................... 84Phosphate, pH 7.3 .. 88Phosphate, pH 7.6 .. 99Phosphate, pH 8.0 .................. 96Tris, pH 8.0 .............. 99Tris, pH 8.4......................... 99Tris,pH 8.7 ............... 100Tris, pH 9.0 ......................... 95

0

.OE

E

x0

EIle

2.5t-

c

,; 2.0LI

I. 1.5

0. 1.0J

.00320

0

.00340I4-]l

.00360/ Tabs

FIG. 5. Arrhenius plot for reactivation of MDH,10 to 32 C. The slope corresponds to 24,600 cal.

TABLE 6. Effect of salt concentration and pH onMDH activity*

Salt

0 20 40TEMP, °C.

FIG. 4. Effect of temperature onof reactivation of MDH at pH 7.5.

NaCl

KCI60

the initial rate

lute temperature to yield a line (Fig. 5), fromwhich the Arrhenius function, A = 24,600 calmay be derived.

Reactivation by direct addition of solid NaCl. Thereactivation of MDH from the salt-free state bythe direct addition of solid NaCl to the proteinsolution ("salt-treated") has shown highly vari-able results which are without simple explanation.In general, this method resulted in a much lowerrate of reactivation, and little of the enzyme be-came reactivated. Yet such salt-treated samplescould often be satisfactorily reactivated by dial-ysis against 25% NaCl.

Interdependence of pH optima and optimal saltconcentrations. The concentration of salt at whichAIDH exhibits maximal activity varied with thepH as well as with the species of salt. The optimalpH was likewise shifted with a change in salt con-centration. Maximal activity was found (Table6) with a pH of 8.0 to 8.5 and a NaCl or KCIconcentration of about 0.9 M (5% NaCl).

Increasing the salt concentration from 0.04 to4.3 M lowered the pH optimum about 1.5 units,

M

0.040.861.72.63.44.3

0.040.861.72.63.44.3

pH

5.8 6.3 7.0 7.5 8.0 8.4 9.0 9.5

06070686056

05878888076

86272727052

26186888272

128482736151

137490908682

229085756652

248693938780

489288756250

439895918273

549072605036

5010076665652

526752433226

507256463932

424842352522

425653422620

* In these assays, NADH2 was added to MDHbefore the salt concentration was lowered to theconcentrations shown. The protection againstinactivation afforded by NADH2 allowed thedetermination of activity at low salt concentra-tions. The results are expressed as percentagesof maximal activity.

from 8.6 to 7.1. Sodium chloride had approxi-mately the same effect as does potassium chloride,and it may be said that within limits the lowerthe salt concentration, the higher the pH requiredfor optimal activity. Conversely, the optimal saltconcentration declined with increase in pH. Asthe pH decreased to about 7.0 or 7.5, there was arather abrupt increase in the amount of salt re-quired to maintain optimal activity (Table 6).

Effect of salt on the kinetics of the enzyme reac-tion. At pH 7, the activity of MDH was greaterin 12% KCl than in 6% KCI. To decide whetherthe salt increased the number of active molecules,or whether salt increased the turnover number,we determined the relationships between initial

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velocity and the concentrations of the substratesat different salt concentrations.

Initial velocities were determined at variousinitial concentrations of one substrate at a con-stant initial concentration of the second sub-strate. From these data, Lineweaver-Burk (1934)plots of 1/v vs. 1/s were constructed. The appar-ent substrate constants, KN1ADH, and KOAA ("ap-parent," not real, as they are a function of theconcentration of the second substrate) were eval-uated as the abscissa intercepts (Table 7). Thesedata allow one to assume that more than one en-zyme-substrate complex is formed in the courseof the reaction, for if the mechanism was re-stricted to the formation of only one binary com-plex, one of the apparent substrate constantsshould be a linear function of the concentration ofthe second substrate (Alberty, 1956). As this wasnot the case, a mechanism involving at least twocomplexes, each composed of a minimum of twoconstituents, must be considered.The general mechanism for two-substrate en-

zymes proposed by Theorell and Chance (1951)involves two binary complexes, and may be writ-ten:

E + DPN.H E DPN-Hk_,

E.DPN-H + OAAk

E-DPN + malatek-s

E.DPN Ik' E + DPN

where k represents the first-order velocity con-stant. The initial velocity (v) in the steady state,where no changes occur in the concentrations ofthe enzyme-substrate complexes, and where theconcentration of products is assumed to be neg-ligible, is related to the total enzyme concentra-

TABLE 7. Apparent substrate constants as afunctionof the concentration of the second substrate*

Concn of the second Apparent substrate constantssubstrate (NADH2

or OAA) K'NADH K'OAA

M M Mt

3.0 X 10-5 4.2 X 10-6 1.0 X 10-43.8 X 10-5 5.3 X 10-6 1.2 X 10-45.5 X 10-5 7.0 X 10-6 1.5 X 10-49.1 X 10-5 9.5 X 10-6 1.7 X 10-43.0 X 10-4 1.4 X 10-5 1.8 X 10-4

* The apparent substrate constants were evalu-ated as abscissa intercepts of Lineweaver-Burkplots made by varying the concentration of onesubstrate while maintaining constant the concen-tration of the second substrate.

tion and the initial substrate concentrations(NADH2) and (OAA) by the formula:

v = ek3 + ekh(NADH2) + ek2(OAA)

+ (NADH2)(OAA)k-I

e 1 1 1v k3 k,(NADH2) k2(OAA)

+ k-,

k1 kc2(NADH2)(OAA)

(I)

(II)

Dalziel (1957) introduces the general form of thislatter equation as:

e XI X2 X12v = [S11_ S [v [SdJ [S21 [S1][S21

(III)

in which the kinetic coefficient Xo represents thereciprocal of the maximal velocity at zero-orderconditions, X1 and X2 are reciprocal rate con-stants of the second order, and X12 is a reciprocalthird-order rate constant. Equation III is equiv-alent to:

e X2 1 X12- = Xo + [S] + [Si] (S) (IV)

and as Dalziel (1957) has shown, graphs of theslopes and intercepts of Lineweaver-Burk plots(made at several constant initial concentrationsof Si and S2) against 1/S, or 1/SA will yield thefour kinetic coefficients directly as either slopesor intercepts.Data for the halophilic enzyme plotted in this

manner are shown in Fig. 6 and 7. Figure 6 is agraph of the ordinate intercepts (1 IV' max) fromLineweaver-Burk plots plotted against the recip-rocal of substrate concentration at two differentconcentrations of KCl. Straight lines connect themajority of the points in each series, indicatingthat the functions plotted are reciprocal second-order rate constants, i. e., X1 and X2. With anincrease in KCl concentration, there was a de-crease in Xo, i. e., an increase in maximal velocityextrapolated to infinite substrate concentrations.Increasing with the increase in KCl were X1 andX2, the reciprocal second-order kinetic coeffi-cients.

Figure 7 relates the slopes (1/v)/(1/S) of theLineweaver-Burk plots to the reciprocal of sub-strate concentration; the resulting slopes repre-sent the value for X12. At 12% KCl, it is apparentthat the third-order rate constant X12 was veryclose to zero. At the lower salt concentration, thiskinetic constant increased appreciably. The in-crease with salt concentration of X1 and X2 is bestexplained by postulating a substantial decrease

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E

8

6

-I-Z_ I cn

\ 4

-I >

2

0 5 10

40X103 XM-'

I / (S]

OAA { maintained at fixed initial conc., 3 X104 M

NADH2{ 8

FIG. 6. Effect of KCl concentration on the sub-strate con.stants of MDH. Reciprocal substrateconcentration is plotted against the ordinate inter-cepts obtained from Lineweaver-Burk plots.

in k-1; i. e., the dissociation of a complex of theenzyme and the first substrate (presumablyNADH2) is reduced by increasing the KCI con-

centration.Substrate binding, enzyme stability, and salt con-

centration. The rate of inactivation of MDH was

inversely proportional to the salt concentration,and, at concentrations below 2%, it was too highto measure accurately. This rate of inactivationcould be greatly reduced by the presence ofNADH2, and the activity of the enzyme couldthus be measured at very low concentrations ofNaCl.Enzyme purified 250-fold was incubated for

various periods of time in 0.05 M potassium phos-phate or Tris buffer at pH 7.0, containing a finalconcentration of 0.4% NaCl, at 25 C. At the endof the incubation period, oxaloacetate was addedand the activity was determined. At this salt con-

centration, the half-life of the enzyme in the ab-sence of substrate is considerably less than 1 min;yet, in the presence of 0.0009 M NADH2, the half-life was approximately 48 hr (Table 8).The protection is apparently specific to the

substrate; reduced nicotinamide adenine dinu-cleotide phosphate (NADPH2) conferred no pro-tection, nor did nicotinamide. NAD had a small

6% KCI

-a--12% KCI

6%KCI

II I15 X 03 XM-

/ LS]

° } OAA maintained at fixed initial concentration

* } NADH2 " " " " It

FIG. 7. Effect of KCl concentration on the sub-strate constants of MDH. Reciprocal substrate con-

centration is plotted against the slopes obtained fromLineweaver-Burk plots.

TABLE 8. Substrate protection against enzymedenaturation at 0.4% NaCl*

Substrate Concn Approximate half-life ofenzymatic activity

M

Malate ......... 0.0035 <0.25 minNAD ........... 0.0001 1.0 min

0.0010 4.0 minOAA............ 0.0030 <0.25 minNicotinamide... 0.0030 <0.25 minNADPH2..... 0.0002 <0.25 min

0.0030 <0.25 minNADH2 .0......O.0001 6.0 min

0.0002 25.0 min0.0003 11.0 hr0.0009 48.0 hr

* MDH in 25% NaCl was diluted with bufferedsubstrate to a salt concentration of 0.4%. At in-tervals, samples were removed, oxaloacetate wasadded, and the MDH activity was recorded.

protective effect; protection by OAA was so slightthat it had to be measured at higher salt concen-trations (2% NaCl). Malate offered no apparentprotection.

Heat inactivation and salt concentration. MDHwas more sensitive to heat denaturation in thepresence of salt than in its absence, but the mostheat-sensitive state of the enzyme was the salt-free form to which salt had been rapidly added("salt-treated"). Whereas the native enzyme lost

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TABLE 9. Heat sensitivity of native, salt-free, andsalt-treated MDH*

Relative specific activity (%)

Heat treatmentNative Salt-freet trSeated

Control (25 C) ..... 100 51 4860 C, 5 min ........ 85 48 365C,5min ........ 44 53 1

* All samples were heated at the temperatureindicated, then dialyzed against 25% NaCl for24 hr before assaying MDH activity.

t As these samples were dialyzed against water,not dilute NaCl, to render them "salt-free,"approximately one-half of the original activityis all that reactivation will return to the sampleunder optimal conditions; i.e., there is no effectof heat on the activity of the salt-free sample.

TABLE 10. Effect of thiol reagents on the recoverableactivity of MDH after treatment in the

presence and absence of salt*

Relative specific activityrecovered (%)

Reagent

Native Salt- Salt-treatedfree

Control ................ 100 51 50IAA, 0.009 m........... 100 39 38NEM, 0.017M.90 0 0PCMB, 0.001 M ........ 100 64 63

* MDH with and without NaCl was incubatedwith the reagents shown (IAA; iodoacetamide;NEM: N-ethylmaleimide; PCMB: p-chloromer-curibenzoate), then reactivated by dialysisagainst 25% NaCl prior to assaying.

more than one-half its former activity at 65 C, thesalt-free sample was stable at this temperature(Table 9). The presumably intermediate form,the salt-treated enzyme, was by far the most heat-labile of the three.

Chemical inactivation and salt concentration.The effects of alkylating and mercaptide-formingagents on the activity of MDH depended uponthe salt concentration as well as upon the state ofthe enzyme. Three reagents capable of reactingwith the thiol groups of protein, iodoacetamide(IAA), N-ethylmaleimide (NEM), and p-chloro-mercuribenzoate (PCMB), were tested for theireffectiveness in decreasing the activity of theMDH in the native, salt-free, and salt-treatedforms. The results of the assay for MDH activityremaining after treatment are seen in Table 10.The native enzyme was relatively unaffected bytreatment with IAA and NEM, but a substantial

decrease in activity resulted in the case of thesalt-free and salt-treated forms. PCMB, in con-trast, enhanced the return of activity to the twolatter forms of the enzyme. It is possible thatcombination of PCMB with protein was reversedat high salt concentrations, owing to the mass-action effect of chloride ion on the reaction.

Effect of salt concentration on the sedimentationcoefficient of malic dehydrogenase. If the removalof salt results in a change in the state of the hal-ophilic MDH molecule, this change should bereflected as a change in the sedimentation coeffi-cient of the enzyme. To determine whether theS20o. does change, the sedimentation of the pro-tein was examined by gradient centrifugation inthe presence of 27, 10, and 0.1% NaCl. For com-parison, a nonhalophilic MDH from pig heartwas included with each sample.

If NaCl was rapidly added to salt-free MDH,at 5 C, the reactivation occurred very slowly, andit was hoped that an effective separation of the

E10iE 27%NaCI

E

100

25 20 15SAMPLE NUMBER

2II'I

25 20 15

25 20 15

o native halophilic enzymea 5 reactivated "o pig enzyme

FIG. 8. Effect of salt concentration on the sedi-mentation of MDH. Bottom of the tubes is to theright. As the salt concentration changes, the halo-philic MDH and the nonhalophilic MDH exchangetheir relative positions in the tube.

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HOLMES AND HALVORSON

TABLE 11. Effect of salt concentration on the S20,.of halophilic and nonhalophilic malic

dehydrogenaseS20,.

Salt concnPig MDH Halophile MDH

27 1.5 9.510 1.9 9.50.1 5.6 2.4

active native form from the inactive form mightbe accomplished by centrifugation in 25% NaClin the cold. Experiments showed, however, thatthe distribution of the native and the inactiveforms precisely overlapped after 12 hr in the cen-

trifuge at 5 C. It was, therefore, assumed that,even though the enzymatic activity had not re-

turned during this 12-hr period of exposure toNaCl, the bulk of the change in molecular shapeoccurred soon after the introduction of the en-

zyme into the salt medium. The sedimentationbehavior was therefore studied at different saltconcentrations, and compared with the behaviorof the nonhalophilic MIDH.The relative positions of the three enzymes in

the centrifuge tubes from one experiment may beseen in Fig. 8. In the presence of 27 and 10%7NaCl, the native and the inactive halophilic en-

zymes traveled together and ahead of the pigenzyme. In 0.1% NaCl, however, the halophilicenzyme trailed the enzyme from the pig.The S20,, values for the halophilic and pig malic

dehydrogenases, calculated on the assumptionslisted in 1Iaterials and Methods, are listed inTable 11. The sedimentation coefficient for thehalophilic enzyme decreased with the decrease insalt concentration, whereas that for the pig en-

zyme increased. There was little change in thesedimentation coefficients as the salt concentra-tion was increased from 10 to 27o%.

DISCUSSIONThe experiments showed that MDH purified

from H. salinarium is profoundly affected bychanges in the surrounding salt concentration.This protein may be described as extremely hal-ophilic; the salt concentration required for main-tenance of the enzymatic activity is vastly greaterthan concentrations normally associated with en-

zyme cofactors. The halophilic enzyme requireslarge amounts of monovalent ions, and is totallyinactive in their absence. Moreover, the enzymein the absence of salt appears to have some of theattributes generally associated with denaturedproteins.

In the salt-free or denatured state, MDH ex-hibits a marked difference in susceptibility tosalting-out. Native MDH is not precipitated from25% NaCl solutions containing 22% (NH4)2SO4(w/v); in the absence of NaCl, a 10 to 12% (w/v)solution of (NH4)2SO4 precipitates the enzyme.Crude extracts as a whole act thus, indicatingthat the change in solubility after salt removal isnot limited to MDH.The inactivation is not a partial one, but is

total at all salt concentrations tested (0 to 5%/oNaCl). The first-order relation of activity to time(over at least two logio units) implies that mostof the MDH molecules are equally sensitive tosalt deprivation.The slow return of activity after addition of

salt suggests that the reactivation is not a simpleprocess, and that it may reflect a conformationalchange occurring in the molecule. A study of therate at which activity returns shows either thatreactivation did not follow first-order kinetics, orthat more than one species of MDH, each withits characteristic reactivation rate, was present.

Presumably, some critical amino acid residueor residues must be ionized for reactivation totake place; this ionization must occur at a pHvery close to 7. Very little reactivation could beachieved below this pH, yet almost full activitycould be restored anywhere between pH 7 and 9.It is possible that a certain suppression of theionization of basic residues is a prerequisite forreactivation.The temperature dependency of reactivation,

and the relatively low Arrhenius constant derivedfrom these data, suggest that reactivation is moreclosely akin to a bimolecular reaction (involving,for instance, salt and protein) than it is to a mono-molecular renaturation involving only protein.However, if the number of internal bond rear-rangements necessary for reactivation is small,the process might be essentially monomolecular.The reactivation is not absolutely dependent

upon the presence of NaCl as the renaturingagent. The salt-free enzyme could, for instance, bepartially reactivated and assayed in solutions ofsodium formate or potassium chloride. The spe-cific requirement for the cation seems to be moreexacting than that for the anion. Yet the decreasein reactivating ability which accompanies the in-crease in molecular weight of the organic acidsreflects the necessity for a suitable anion if reac-tivation is to occur.The inactivation is markedly inhibited by the

presence of the reduced coenzyme, NADH2,whereas the addition of NADH2 to the denaturedenzyme resulted in no renaturation. It seems rea-sonable to suppose that the coenzyme is boundrather firmly to the native protein.

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HALOPHILIC MALIC DEHYDROGENASE

The magnitude of the effect of thiol reagentsin the presence and absence of salt upon the en-zvmatic activity of the halophilic MDH was as-sumed to be a measure of the availability of -SHgroups in the protein. These reagents appear toaffect the enzyme activity only if added in theabsence of salt or if added just after the rapid re-turn of salt. Under these conditions, the enzymeis assumed to be denatured, and it is possible thatthere is a coincident greater availability of thiolgroups for reaction.The fact that the sedimentation rate (S20,,) Of

the halophilic MDH decreases upon inactivationcan implicate molecular shape change, disaggre-gation, or an increase in partial specific volume.It is unlikely that the enzyme disaggregates atlow ionic strengths since, under optimal condi-tions, virtually all of the enzyme may be reactiv-ated, even in crude extracts. It is conceivable thatthe molecule reversibly binds a large number ofions in strong salt solutions. The effect of pH onthe optimal salt concentration for MDH could beconstrued as an effect of ion binding. The moststriking differences in the pH-activity relation-ships determined at different concentrations ofsalts are found on the acid sides of the curves. AtpH 6, the activity is much more dependent on thesalt concentration than it is at pH 9; i. e., at pH9.0, MDH is more active in 0.3% KCI than in19% KCl. In general, increasing concentrationsof NaCl or KCI shift the pH-activity curves tothe acid side, without much effect on the curvesabove pH 8.5.As Massey (1953) postulated, the binding of an

ion to a group adjacent to the active site and theconcomitant suppression of a charge can effectthe pK of the group in the active site responsiblefor the pH-activity curve. The observed shift ofthe pH optimum for the halophilic MDH could beexplained if the enzyme binds cations (Na or Kions), and if the degree of binding is concentra-tion-dependent up to 3 or 4 M. Some of the ob-served shift is due, of course, to the effect of salton the pH of the buffer. The prediction, on gen-eral principles, that salt will lower the pH of weakacid-strong base buffers is upheld by the readingsobtained with the glass electrode-calomel elec-trode pH meter (Table 12). It is apparent that

TABLE 12. Effect of salts on the pH of buffers, asmeasured with the glass electrode

Buffer (0.05 m) Salt added pH change(4.3 m) observed

NaOH-potassium phosphate NaCl -1.2NaOH-potassium phosphate. KCI -0.6NaOH-glycine ............... NaCl -0.6

the observed pH change is dependent upon thespecies of salt added as well as upon the bufferused.What relation the observed pH bears to the

true pH is uncertain. Some observations on thebiological effect of pH lead one to believe thatthe observed pH in salt solutions is not the truepH of the system. For instance, a series of salt-free phosphate, Tris, and glycine buffers may beprepared covering the pH range 6.0 to 9.0. Ifthese are then made up to 25% NaCl and usedfor the determination of the pH optimum ofMDH, one finds a smooth pH-activity curvethrough the three buffer systems. If the observedpH (after the addition of salt) reflected the truepH, one would not expect the pH-activity curveto be unimodal, owing to the different effectsNaCl has on the apparent pH changes in the dif-ferent buffers. Also, the pH optima found forMDH in various molar concentrations of NaCland KCl were close to identical. On the basis ofthe observed pH in high salt concentrations, adifference in pH optima of 0.6 would be expected.Baxter (1959) argued that the inactivation ofhalophilic protein may be due to internal elec-trostatic repulsions of charged residues, and thatsalts reduce electrostatic effects due to the ionicatmosphere formed around such charges. Thus, ifthe native configuration and activity of MDH de-mand the propinquity of positively charged resi-dues, the "neutralizing" effect of high concentra-tions of ions could allow such a propinquity as wellas explain the acid shift of thepH curve. The neces-sity for "neutralizing" positively charged groupsmight also be reflected in the fact that reactiva-tion of MDH occurs only if the pH is above neu-trality.The fact that the initial slopes of velocity-time

curves for the enzyme reaction made at differentKCl concentrations were linear indicates thatthis salt serves to activate MDH as well as toprotect it from denaturation. It is clear that theactivation is not an all-or-none effect; the appar-ent substrate constants change with the KClconcentration.The model involving two binary complexes and

an ordered binding sequence was chosen chieflyfor its simplicity. The conclusion drawn, however,would not be invalidated if we accept the modelof Raval and Wolfe (1962), one which involvesordered binding and at least one ternary complexwhich breaks down much faster than do the bi-nary complexes. A 100-fold decrease in the en-zyme-coenzyme dissociation constant could ex-plain the observed increases in the substrateconstants and the maximal velocity which occurupon increasing the ionic strength. There is noreason to propose, at the moment, a rigid model

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326 HOLMES ANI

for salt stabilization and activation of halophilicenzymes. It is conceivable that when a model iscompounded, it will be based upon the concept ofhalophilic proteins which become less compactin the absence of salt.

ACKNOWLEDGMENTSThis investigation was supported by contract

1834(30) from the U.S. Office of Naval Research,and by training grant 2G-510 from the U.S. PublicHealth Service.

LITERATURE CITEDALBERTY, R. A. 1956. Enzyme kinetics. Advan.Enzymol. 17:1-64.

BAXTER, R. M. 1959. An interpretation of theeffects of salts on the lactic dehydrogenase ofHalobacterium salinarium. Can. J. Microbiol.5:47-57.

DALZIEL, K. 1957. Initial steady state velocities inthe evaluation of enzyme-coenzyme-substratereaction mechanisms. Acta Chem. Scand. 11:1706-1723.

HOLMES, P. K., AND H. 0. HALVORSON. 1965.Purification of a salt-requiring enzyme from anobligately halophilic bacterium. J. Bacteriol.90:312-315.

) HALVORSON J. BACTERIOL.

LINEWEAVER, H., AND D. BURK. 1934. The deter-mination of enzyme dissociation constants. J.Am. Chem. Soc. 66:65-666.

MARTIN, R. G., AND B. N. AMES. 1961. A methodfor determining the sedimentation behaviourof enzymes; application to protein mixtures. J.Biol. Chem. 236:1372-1379.

MASSEY, Y. 1953. Studies on fumarase. 2. Theeffects of inorganic anions on fumarase activity.Biochem. J. 53:67-71.

RAVAL, D. N., AND R. G. WOLFE. 1962. Malicdehydrogenase. III. Kinetic studies of the re-action mechanism by product inhibition. Bio-chemistry 1:1112-1117.

SCHACHMAN, H. K. 1957. Ultracentrifugation, dif-fusion, and viscometry, p. 32-103. In S. P. Colo-wick and N. 0. Kaplan [ed.], Methods in enzy-mology, vol. 4. Academic Press, Inc., New York.

SVEDBERG, T., AND K. 0. PEDERSEN. 1940. Theultracentrifuge. Clarendon Press, Oxford, Eng-land.

THEORELL, H., AND B. CHANCE. 1951. Studies onliver alcohol dehydrogenase. II. Acta Chem.Scand. 5:1127-1144.

VAN Eys, J., M. M. CIOTTI, AND N. 0. KAPLAN.1957. Yeast alcohol dehydrogenase. Biochim.Biophys. Acta 23:581-587.

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