Inhibition ofPotatoTuberInvertase by … · catalytic activity of invertase. The optimal...

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Plant Physiol. (1980) 66, 451-456 0032-0889/80/66/045 1/05/$00.50/0 Inhibition of Potato Tuber Invertase by an Endogenous Inhibitor EFFECTS OF SALTS, pH, TEMPERATURE, AND SUGARS ON BINDING"2 Received for publication.August 17, 1979 and in revised form April 29, 1980 ROGER S. ANDERSON3, ELMER E. EWING4, AND ANNE HEDGES SENESAC Department of Vegetable Crops, Cornell University, Ithaca, New York 14853 ABSTRACT Binding between potato tuber invertase and its endogenous inhibitor followed second-order reaction kinetics. Binding rates were diminished by the presence of various inorganic salts, MgC12 being especially effective. This effect of MgCl2 was used in binding rate studies by adding the salt with sucrose to reduce binding during assay of previously unbound activity. The optimal pH for binding was about 4.8, similar to the optimal pH for catalytic activity of invertase. The optimal temperature for binding was about 45 C, approximately 5 C less than the optimum for catalytic activity. Sucrose at concentrations as low as 2 millimolar slowed binding, reducing sugars had little or no effect on binding or on catalytic activity. Pressey (10, 1 1) partially purified and characterized a naturally occurring inhibitor of invertase from potato tubers. The inhibitor is a protein with a mol wt estimated at 17,000 by gel filtration (I 1). It binds slowly and irreversibly to potato tuber invertase (3). The inhibitor does not appear to be an artifact of enzyme extrac- tion; there is evidence that it binds to invertase in situ. The invertase activity estimated by immersing thin slices of tuber directly into the sucrose assay medium corresponds to the activity of extracts from the same tissues which contain inhibited enzyme, not to that of extracts from which the inhibitor has been selectively removed (4). The inhibitor disappears from slices during "aging," and the disappearance is blocked by actinomycin D and cyclo- heximide (4). Tuber storage temperature also affects the inhibitor activity (13), suggesting that the inhibitor plays a physiological role in intact tubers. Although the binding between invertase and the inhibitor is irreversible under ordinary laboratory conditions, certain treat- ments will dissociate the complex and yield functional inhibitor or enzyme. Rapid stirring of extracts or passage of N2 bubbles through them produces a foaming that denatures the inhibitor and increases invertase activity (3, 10, 13). Conversely, low pH treat- ment of extracts selectively destroys invertase and increases inhib- itor activity (1). Passage of the complex over a concanavalin A- Sepharose column may also partiilly dissociate it, freeing inhibitor 'The major portion of this paper constitutes part of a Ph.D. thesis presented to Cornell University by R. S. A., the recipient of a Liberty Hyde Bailey Fellowship from the Department of Vegetable Crops. The work was also supported by Hatch Regional Marketing Funds granted to the Department of Vegetable Crops. 2This is Paper No. 755 of the Department of Vegetable Crops, Cornell University. 3Present address: Department of Nutrition and Food Science, Massa- chusetts Institute of Technology, Cambridge, Massachusetts 02139. 4To whom reprint requests should be addressed. and increasing invertase activity (1). Invertase inhibitors have been reported in five other species of higher plants (5, 7, 9, 12, 16). Although several of the other inhibitors bind slowly to the invertase, only in the case of the potato tuber inhibitor is there unequivocal evidence that the binding is essentially irreversible. We have examined further the binding reaction between potato tuber invertase and its inhibitor, and we describe here an improved method for assay of the binding rate. The method is used to show the influence of temperature, pH, sucrose, inorganic salts, and concentration of the reactants on binding rates. MATERIALS AND METHODS Enzyme and Inhibitor Purification. Invertase and inhibitor were partially purified from potato (Solanum tuberosum L.) tubers as previously described (1), except that (NH4)2SO4 fractionation was substituted for the cold ethanol fractionation in the enzyme prep- aration. The protein precipitating at pH 5.0, 2 C, from 50 to 90%o saturation with (NH4)2SO4 was saved. The specific activity of typical invertase and inhibitor preparations was, respectively, 150 and 78 units/mg protein. One unit of invertase activity was that amount which liberated 1 ,umol reducing sugar (or 0.5 ,umol glucose, where only glucose was assayed)/h under the conditions of the assay. One unit of inhibitor was the amount which, given infinitely long binding conditions, would inactivate 1 unit of invertase. The quantity of invertase used in most experiments corresponded to between 0.5 and 1.0 unit, and the inhibitor activity expressed in units was chosen to be equal to the units of invertase. Protein was determined according to Bradford (2), with BSA as the reference protein. Assay for Binding Rate. The rate of inhibitor binding to inver- tase was determined in 0.5-ml reaction mixtures containing en- zyme and inhibitor in 10 mm acetate (pH 4.8). Binding was started with the addition of the inhibitor and continued for the indicated time. Temperatures during binding were controlled in a Gilson Omnibath and, except in the experiment on the effect of temper- ature, were 37 or 2 C. The lower temperature was chosen for the experiments of Figures 4 and 5 as a matter of convenience so that longer incubation periods could be employed, thus minimizing the effects of errors in timing. Unbound enzyme activity was' assayed by adding 0.5 ml 0.5 M sucrose and 0.2 M MgCl2 in 10 or 100 mm acetate (pH 4.8), incubating 1 h, and measuring reducing sugars. The incubation temperature was 37 C unless otherwise stated. Assay for Reducing Sugars. Reducing sugars in most of the expenrments were analyzed by the Somogyi modification of the Ne4lson test, as previously described (3). In this procedure, the addition of Cu + inactivates the invertase at the end of the incubation in sucrose. Because Ca 2 salts interfere with the Nelson test, a glucose oxidase procedure was substituted for the experi- ment summarized in Table I. In this case, invertase was inactivated by heating the assay medium. CaCl2 and other salts also interfere 451 www.plantphysiol.org on June 14, 2018 - Published by Downloaded from Copyright © 1980 American Society of Plant Biologists. All rights reserved.

Transcript of Inhibition ofPotatoTuberInvertase by … · catalytic activity of invertase. The optimal...

Plant Physiol. (1980) 66, 451-4560032-0889/80/66/045 1/05/$00.50/0

Inhibition of Potato Tuber Invertase by an Endogenous InhibitorEFFECTS OF SALTS, pH, TEMPERATURE, AND SUGARS ON BINDING"2

Received for publication.August 17, 1979 and in revised form April 29, 1980

ROGER S. ANDERSON3, ELMER E. EWING4, AND ANNE HEDGES SENESACDepartment of Vegetable Crops, Cornell University, Ithaca, New York 14853

ABSTRACT

Binding between potato tuber invertase and its endogenous inhibitorfollowed second-order reaction kinetics. Binding rates were diminished bythe presence of various inorganic salts, MgC12 being especially effective.This effect of MgCl2 was used in binding rate studies by adding the saltwith sucrose to reduce binding during assay of previously unbound activity.The optimal pH for binding was about 4.8, similar to the optimal pH forcatalytic activity of invertase. The optimal temperature for binding wasabout 45 C, approximately 5 C less than the optimum for catalytic activity.Sucrose at concentrations as low as 2 millimolar slowed binding, reducingsugars had little or no effect on binding or on catalytic activity.

Pressey (10, 1 1) partially purified and characterized a naturallyoccurring inhibitor of invertase from potato tubers. The inhibitoris a protein with a mol wt estimated at 17,000 by gel filtration(I 1). It binds slowly and irreversibly to potato tuber invertase (3).The inhibitor does not appear to be an artifact of enzyme extrac-tion; there is evidence that it binds to invertase in situ. Theinvertase activity estimated by immersing thin slices of tuberdirectly into the sucrose assay medium corresponds to the activityof extracts from the same tissues which contain inhibited enzyme,not to that ofextracts from which the inhibitor has been selectivelyremoved (4). The inhibitor disappears from slices during "aging,"and the disappearance is blocked by actinomycin D and cyclo-heximide (4). Tuber storage temperature also affects the inhibitoractivity (13), suggesting that the inhibitor plays a physiologicalrole in intact tubers.Although the binding between invertase and the inhibitor is

irreversible under ordinary laboratory conditions, certain treat-ments will dissociate the complex and yield functional inhibitoror enzyme. Rapid stirring of extracts or passage of N2 bubblesthrough them produces a foaming that denatures the inhibitor andincreases invertase activity (3, 10, 13). Conversely, low pH treat-ment of extracts selectively destroys invertase and increases inhib-itor activity (1). Passage of the complex over a concanavalin A-Sepharose column may also partiilly dissociate it, freeing inhibitor

'The major portion of this paper constitutes part of a Ph.D. thesispresented to Cornell University by R. S. A., the recipient of a LibertyHyde Bailey Fellowship from the Department of Vegetable Crops. Thework was also supported by Hatch Regional Marketing Funds granted tothe Department of Vegetable Crops.

2This is Paper No. 755 of the Department of Vegetable Crops, CornellUniversity.

3Present address: Department of Nutrition and Food Science, Massa-chusetts Institute of Technology, Cambridge, Massachusetts 02139.4To whom reprint requests should be addressed.

and increasing invertase activity (1).Invertase inhibitors have been reported in five other species of

higher plants (5, 7, 9, 12, 16). Although several of the otherinhibitors bind slowly to the invertase, only in the case of thepotato tuber inhibitor is there unequivocal evidence that thebinding is essentially irreversible.We have examined further the binding reaction between potato

tuber invertase and its inhibitor, and we describe here an improvedmethod for assay of the binding rate. The method is used to showthe influence of temperature, pH, sucrose, inorganic salts, andconcentration of the reactants on binding rates.

MATERIALS AND METHODS

Enzyme and Inhibitor Purification. Invertase and inhibitor werepartially purified from potato (Solanum tuberosum L.) tubers aspreviously described (1), except that (NH4)2SO4 fractionation wassubstituted for the cold ethanol fractionation in the enzyme prep-aration. The protein precipitating at pH 5.0, 2 C, from 50 to 90%osaturation with (NH4)2SO4 was saved. The specific activity oftypical invertase and inhibitor preparations was, respectively, 150and 78 units/mg protein. One unit of invertase activity was thatamount which liberated 1 ,umol reducing sugar (or 0.5 ,umolglucose, where only glucose was assayed)/h under the conditionsof the assay. One unit of inhibitor was the amount which, giveninfinitely long binding conditions, would inactivate 1 unit ofinvertase. The quantity of invertase used in most experimentscorresponded to between 0.5 and 1.0 unit, and the inhibitoractivity expressed in units was chosen to be equal to the units ofinvertase. Protein was determined according to Bradford (2), withBSA as the reference protein.Assay for Binding Rate. The rate of inhibitor binding to inver-

tase was determined in 0.5-ml reaction mixtures containing en-zyme and inhibitor in 10 mm acetate (pH 4.8). Binding was startedwith the addition of the inhibitor and continued for the indicatedtime. Temperatures during binding were controlled in a GilsonOmnibath and, except in the experiment on the effect of temper-ature, were 37 or 2 C. The lower temperature was chosen for theexperiments of Figures 4 and 5 as a matter of convenience so thatlonger incubation periods could be employed, thus minimizingthe effects of errors in timing. Unbound enzyme activity was'assayed by adding 0.5 ml 0.5 M sucrose and 0.2 M MgCl2 in 10 or100 mm acetate (pH 4.8), incubating 1 h, and measuring reducingsugars. The incubation temperature was 37 C unless otherwisestated.

Assay for Reducing Sugars. Reducing sugars in most of theexpenrments were analyzed by the Somogyi modification of theNe4lson test, as previously described (3). In this procedure, theaddition of Cu + inactivates the invertase at the end of theincubation in sucrose. Because Ca2 salts interfere with the Nelsontest, a glucose oxidase procedure was substituted for the experi-ment summarized in Table I. In this case, invertase was inactivatedby heating the assay medium. CaCl2 and other salts also interfere

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ANDERSON, EWING, AND SENESAC

somewhat with the glucose oxidase and analysis by increasing thehydrolysis of sucrose during the heat inactivation of the enzyme.This problem was minimized by lowering the sucrose concentra-tion from 0.25 to 0.05 M in the invertase assay medium whenglucose oxidase was to be used. The optimum substrate concen-tration for potato tuber invertase is very broad, and enzymeactivity in 0.05 M sucrose is only slightly lower than at the highersucrose concentrations. At the end of the 1-h incubation periodfor invertase activity, the l-ml assay mixture was placed in aboiling water bath for 2 min and then cooled in an ice bath. Fourml freshly prepared reagent containing 0.16 mg peroxidase (SigmaP-8250, Type II, from horseradish, 167 units/mg), 12 units glucoseoxidase (Sigma G-6500, Type V, from Aspergillus niger, 1,400units/ml), and 0.26 mg o-dianisidine (Sigma D-3252) in 100 mmphosphate (pH 7.0) were added. After 20 min at room temperature,2 drops of 4 N HCI were added, and the A at 420 nm was measured5 min later. It was also necessary to use the glucose oxidaseprocedure for the experiment on effects of fructose on binding.

In the experiment on the effect of D-glucose on invertase activityand enzyme-inhibitor binding, the activity of the enzyme wasmeasured as the amount of [ H]fructose released from [3HJfruc-tose-labeled sucrose. Enzyme or enzyme and inhibitor were incu-bated in 10 mm acetate (pH 4.8), for 5 min at 25 C in 100-,ulreaction mixtures. Some of the reactions were made 100 or 250mM with D-glucose. The remaining unbound enzyme activity was

then assayed by making the reactions 0.25 M with sucrose and 100mM with MgCl2. The reactions were further incubated for I h at37 C. The reactions also contained 1.875 ,uCi [3HJfructose-labeledsucrose. They were stopped with the addition of 20 ,l 2% CuSO4and were diluted with 250 ll 10 mM acetate (pH 4.8). The labeledfructose resulting from invertase activity was separated from theremaining labeled sucrose by chromatographing a 10-,ul aliquot ofthe reaction mixture on a Silica Gel G thin-layer plate (15). Thechromatograms were developed three times in a butanol/acetone/water (4:1:5) solvent system. Sections of the chromatogram werescraped from the plate and the 3H was measured in a liquidscintillation counter. A counting fluid consisting of 5 g PPO and0.1 g POPOP/1 of toluene was used.

Influence of Salts on Binding. To compare the effects of six saltson binding during the assay, 0.8 ml of a 62.5 mm sucrose, 125 mmsalt solution was mixed with 0.1 ml 100 mm acetate. Where thesalts to be tested were acetates, the pH of the sucrose-salt solutionwas adjusted to 4.8 with acetic acid before making the solution up

to volume. The pH of the 100 mm acetate was adjusted for eachtreatment so that the pH of the final assay medium would be 4.8.A 75-,ul aliquot of 10 mm acetate (pH 4.8) containing 0.56 unitinhibitor was added, followed by a 25-,ul aliquot of the same buffercontaining 0.48 unit invertase. Thus, all six treatments contained11 mM acetate buffer and had a final pH of 4.8. Ignoring theNaC2H302 present in the buffer of all treatments, the six treat-ments were 100 mm for NaCl, MgCl2, CaCl2, NaC2H302,Mg(C2H302)2, and Ca(C2H302)2, respectively. The 1-h assay forinvertase activity started with the addition of the enzyme. Per centinhibition during the assay was calculated by comparing theresulting activity to the activity of similar treatments that received75 [1I 10 mm acetate buffer rather than the inhibitor.The experiment on effect of salt concentration was identical,

except that the salt concentrations in the final assay medium were10 mM NaCl, 30 mM NaCl, 90 mm NaCl, 10 mM MgCl2, 30 mMMgCI2, and 90 mM MgCl2, respectively.The experiments shown in Figures I and 2 were performed with

the Nelson test for reducing sugars.

RESULTS

Influence of Salts on Binding. Preliminary experiments indi-cated that various inorganic salts interfered with the binding ofthe inhibitor to potato tuber invertase and that the degree of

interference increased with salt concentration. Table I shows theeffects of three chlorides and three acetates on binding. Inhibitorwas added to invertase at the time of the sucrose addition. Theenzyme activity in the presence of inhibitor was compared to theactivity in its absence to obtain the per cent inhibition during theinvertase assay period. There was significantly less inhibition withchlorides than with acetates, indicating that the chlorides gavemore interference with binding. There was also a highly significanteffect of cations; MgCl2 interfered more than any of the other saltstested. In a similar experiment, three concentrations of NaCl andMgCl2 were compared. Values of 29, 25, and 15% inhibition wereobtained for 10, 30, and 90 mm NaCl concentrations, respectively.Values for the same three concentrations of MgC12 were 15, 13,and 6%, inhibition, respectively, during the invertase assay. MgC12at 100 mm was selected for further examination of the salt effect.

Figure I shows the effects of adding 100 mM MgCl2 at thebeginning of binding between enzyme and inhibitor compared toaddition at the end of the binding period. The double-reciprocalplot of the data (Fig. 1B) indicates that, at extended incubationtimes, the same amount of binding could be expected in thepresence or absence of MgCl2. That is, the presence of MgC12reduced the rate but not the final extent of binding.MgCl2 did not cause the dissociation of the complex once

formed (data not shown), and it had only slight effect on theactivity of the enzyme in the absence of inhibitor. In four experi-ments, each replicated six times, 100 mM MgCl2 was added eitherat the beginning or at the end of the incubation period withsucrose. The quantity of reducing sugars formed after the earlyaddition of MgCl2 averaged only 3% less than the amount formedwhen MgCl2 was added at the end. There was a 14% reduction ifthe MgCl2 was added 1 h in advance of the assay, compared tothe control which received buffer I h in adveince and MgCl2 at theend of the assay. MgCl2 lowers the sensitivity of the Nelson testfor reducing sugars, but there is a linear response ofA to reducingsugar concentration at the MgCl2 concentration used.Assay for Binding Rate. The observations that 100 mM MgC12

slowed the binding of enzyme to inhibitor but that it neithercaused dissociation of the complex nor affected seriously thecatalytic activity of invertase suggested that MgCl2 should beuseful in measuring binding rate. If binding rate is measured asthe loss of invertase activity with time, there is a problem ofcontinued inhibitor binding during the assay of any remainingunbound enzyme. The addition of MgCl2 to the sucrose substratesolution used in measuring unbound enzyme should significantlyreduce this problem by retarding binding during the invertaseassay.

Figure 2 shows the benefit of the MgCl2 addition. Binding wasallowed to proceed for the indicated periods prior to the assay of

Table 1. Influence of 100 mM Salts on Invertase-Inhibitor BindingIn addition to the 100 mM concentrations of the various salts which

were added with the sucrose, all solutions received sufficient sodiumacetate-acetic acid buffer (I I mm for acetate in the final assay medium) tomaintain the pH at 4.8. Half of the samples received inhibitor at the sametime. Percentages represent the reduction in invertase activity in thepresence of inhibitor plus salt compared to the enzyme activity in thepresence of the salt only. Values are means of six replications. Main effectsof both cations and anions were statistically significant at the 0.01 level.The interaction was not significant.

% Inhibition during Invertase Assay

Cl- C2H302 Mean

Na+ 30 38 34Ca2+ 27 29 28Mg2+ 14 27 21Mean 24 31 28

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BINDING OF POTATO INVERTASE TO INHIBITOR

p

t, min

0

P 002 -

no MgCI2

0.01

C

0 0.10 0.20 0.30

1, min1I

FIG. 1. Influence of 100 mm MgCl2 on binding of invertase to inhibitor.Enzyme and inhibitor were incubated at 37 C in 100 mm NaC2H302 (pH4.8) with or without 100 mM MgCI2. MgCl2 (100 mM) was added to thesecond treatment at the time of the sucrose addition. Binding is directlyproportional to per cent inhibition (p), which is estimated as the per centreduction in enzyme activity from the activity at binding time = 0. Eachpoint represents the mean of six determinations. A: Data plotted as pversus binding time in min (t); B: same data in form of equation 5; C: samedata in double reciprocal plot, after equation 6. Lines in B and C were

calculated by least squares analysis.

remaining invertase activity. When MgCl2 was added with sucroseat the beginning of the invertase assay, the observed binding ratewas very different from that observed when only sucrose wasadded. The difference was especially pronounced with short pe-riods of binding. Addition of MgCl2 gave a binding curve whichmuch more nearly approached that expected of a second-orderreaction.

In the remaining experiments, MgCl2 was added with the su-crose at the beginning of the assay period to block further bindingand thus permit an examination of the effects of various factorson binding rate.

Second-order Reaction Kinetics. A test for second-order kineticswas derived from the kinetic equation for a second-order reactionwhen the two reactants are of equal concentration:

--=kt(I)c co

100[ I

80

0 60m

Z 40ae

20

0 10 20 30 40 50

BINDING TIME, MIN60

FIG. 2. Predicted binding of invertase to inhibitor compared withobserved, with and without addition of 100 mm MgCl2 to assay medium.The apparent rate of binding in 10 mm NaC2H302 (pH 4.8) was measuredby making the assay medium 0.25 M with sucrose only or by making it0.25 M with respect to sucrose and 0.1 M with respect to MgCl2. Data pointsobtained by adding MgCb2 with the sucrose conformed more closely to thecurve calculated from second-order reaction kinetics. Each point representsthe mean of 10 determinations.

where c0 = the original concentration of each reactant, c = theconcentration of each reactant remaining at time t, and k = thereaction rate constant. By definition, the percentage binding be-tween enzyme and inhibitor, p, is:

p=~ ~~c

or

100 -p100

Substituting for c in equation 1 gives:1 100 1

IO 10-p = kt.

Rearranging gives:

P c0kt.100 p

Inversion and rearrangement gives:

1 1 110.01+

p Il00c,0k t

(2)

(3)

(4)

(5)

(6)

Equation 5 yields a straight line through the origin ifp/(100 -

p) is plotted against t. The slope of the line multiplied by 100 givesthe initial binding rate expressed in terms ofp since, when t = 0,

dp/dt = 100kco. The reciprocal of the slope gives the half-time ofthe reaction. Neither invertase nor the inhibitor have been com-pletely purified, and it is not possible to express their concentra-tions in molarities. However, if constant volumes of partiallypurified invertase solution are incubated with varying volumes ofpartially purified inhibitor solution for 3 days at 2 C, then plottingp versus the volume of added inhibitor yields a straight line up toat least 60%o inhibition (3). Extrapolation of the line to 100%oinhibition indicates the volume of inhibitor solution required toreact with the given amount of enzyme. The quantity of inhibitor,which according to such extrapolation would react with I unit of

o. - - 6o

00 , '

0

0o0o

* no MgCIlo with MgC12--- 2nd order curve

lI

70

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ANDERSON, EWING, AND SENESAC

invertase, is defined as 1 unit of inhibitor. In all remainingexperiments, the units of inhibitor present were equal to the unitsof invertase with which the inhibitor was incubated. Bindingbetween inhibitor and enzyme was allowed to proceed for varyingperiods of time. Plotting the data in the form ofequation 5 resultedin a straight line that passed through or near the origin (Fig. 1B).

Alternatively, plotting the data in the form of l/p versus /Italso gave a straight line, as predicted by equation 6 for second-order reactions where the reactants are equal in concentration(Fig. IC). Interception of the ordinate at I/p = 0.01 indicates thatthe reactants were equal in concentration. The reciprocal of theslope provides an estimate of the initial binding rate, expressed aspercentage inhibition/min when t = 0.

If binding follows second-order kinetics, then a simultaneousdoubling of the concentrations of enzyme and inhibitor shoulddouble the initial binding rate and reduce the half-time by 50%o.To test this, 15 ,il invertase, 50 ,ul inhibitor, and 435 ,ul buffer wereincubated at 2 C for eight periods of time ranging from 50 to 300min. During the same eight times, 30 ,ul invertase, 100 ,lI inhibitor,and 370 ,ul buffer were incubated together. The initial bindingrates calculated from plotting as in Figure lB were 0. 15%/min forthe lower and 0.30%o/min for the higher concentrations. Thecalculated half-times for the two reactions were 667 and 333 min,respectively.Inasmuch as the experiments with concentration of invertase

and inhibitor were consistent with second-order kinetics, equation5 was used as the basis for studying the effects of temperature,pH, and sucrose on binding. Initial binding rates were obtainedby plotting the data as in Figure lB and calculating the slopes ofthe "best-fit" lines.

Temperature. The catalytic activity of the enzyme reached amaximum at about 50 C (Fig. 3). The relationship of binding rateto temperature followed a similar pattern, although the optimaltemperature was 5 to 10 C lower. Binding measurements above40 C become increasingly difficult to interpret because "decay" ofthe enzyme accelerates rapidly as temperature increases (unpub-lished data).pH. The optimal pH for initial binding rate was within the same

0.5

|> 0.4

_ x

'0

en z

I-50.2

X0.

0 10 20 30 40 50

TEMPERATURE, °C60

range as that reported for the catalytic activity of invertase. Pressey(10) found that potato tuber invertase activity was greatest at pH4.75. Of six pH values between 3.6 and 5.6, binding was mostrapid at pH 4.8 (Fig. 4). Pressey (10, 11) reported that invertaseassay in the presence of inhibitor displayed a double pH optimumfor catalytic activity. This would not be expected in the case ofirreversible complex formation. We found no evidence for adouble optimum whether inhibitor was present or absent (Fig. 4).Pressey (10, 11) made no mention of a preincubation period forbinding of invertase and the inhibitor, which may explain thediscrepancy. If insufficient time were allowed for completion ofthe binding reaction between enzyme and inhibitor, then the morerapid binding at pH 4.8 might well give the appearance of adouble optimum in catalytic activity.

Sugars. To study the effect of sucrose on binding, it wasnecessary to correct for the sucrose hydrolysis that occurred duringthe binding period. We did this by stopping half of the reactionsat the beginning of the assay period, when the additional sucroseand MgCl2 were added. The quantity of reducing sugars formedduring the binding period then was subtracted from the totalquantity of reducing sugars formed by the end of the invertaseassay. The difference was a measure of the invertase activityremaining after binding.

Sucrose concentrations as low as 2 mm in the binding mediumhad a pronounced inhibitory effect on the initial binding rate (Fig.5). Relatively small decreases in binding resulted from furtherincreasing the sucrose concentration. In contrast with sucrose,neither D-fructose nor D-glucose appeared to have a major effecton inhibitor binding or on catalytic activity of invertase. The effectof fructose was examined by adding 0.56 unit inhibitor andvarying concentrations of fructose along with sucrose at the begin-ning of the 1-h invertase assay. (In this experiment, MgCl2 wasomitted from the assay medium so that inhibition during the assay

25 Pa

(CO20 WOZ0

c0o

15O Z Z

m-oI0 X

4X

_-z

5 Z w

FIG. 3. Influence of temperature upon the catalytic activity of invertaseand upon binding of invertase to inhibitor. Invertase activity was assayedat the indicated temperatures. Values are means of two replications. In a

separate experiment, 0.44 unit invertase were incubated with 0.44 unitinhibitor for varying periods of time at the indicated temperatures, andinitial binding rates were calculated. Assay of unbound invertase activitywas at 37 C when binding temperatures were less than 30 C; otherwise,the assay was carried out at the binding temperature. Each rate was basedupon five replications and four periods of time and was calculated as inFigure IB.

(SN-

-3 z

ZEa X

no-J Z<

pH

FIG. 4. Influence of pH upon invertase activity in the presence or

absence of inhibitor and upon binding of invertase to inhibitor. Excessinvertase was incubated for h at 37 C in 100 mm NaC2H302 (pH 4.8) inthe presence (O-O) or absence (x X) of inhibitor. The remaininginvertase activity was measured as the amount of reducing sugars formedafter making the reaction mixtures 0.25 M with sucrose and incubating Ih at 37 C. The broken line (@ - -0) summarizes a separate experimenton how pH affects initial binding rate between equal amounts of enzymeand inhibitor at 2 C. Data points are means of six determinations in thefirst experiment; binding rates were based upon eight replications and fiveperiods of binding, calculated as in Figure IB.

binding rate

0

/ II'\ * *nzyme; activityI 0

0xxI

I t I .zI

T I I I A I

I I i I i0% 0

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BINDING OF POTATO INVERTASE TO INHIBITOR

nQl

c4Jc-

w cO.E*17

< E0.4

4Z-.

ae0.2pi

0 10 20 30 40 50 250SUCROSE CONCN. IN BINDING MEDIUM, mM

FIG. 5. Effect of sucrose concentration on binding between invertaseand inhibitor. Enzyme and inhibitor were incubated at 2 C for varyingperiods of time in the presence of the indicated sucrose concentrations,and in 100 mm acetate buffer (pH 4.8). Initial binding rates were calculatedas in Figure IB from four incubation periods, each replicated five times.

would be promoted.) There were 12 replications. The enzyme

activity measured in the presence of 0, 10, 50, and 250 mm fructosewas 0.48, 0.47, 0.48, and 0.41 unit, respectively, in the absence ofinhibitor. The lower invertase activity in 250 mm fructose was

significant at the 0.01 level. The per cent inhibition caused byinhibitor added at the beginning of the assay was 34, 32, 30, and35%, respectively, for the control and three fructose concentra-tions; differences were not significant at the 0.05 level. In aseparate experiment, fructose concentrations of 0, 25, 250, and 500mm had no significant effect on initial binding rates betweeninvertase and inhibitor prior to the addition of sucrose.We used sucrose containing D-[3H]fructose to determine how

D-glucose affected the catalytic activity of invertase. The presenceof D-glucose in the assay medium at 0, 100, and 250 mm concen-

trations resulted in 4,148, 4,548, and 4,015 cpm, respectively, inthe D-fructose released by the enzyme. Incubation of inhibitorwith invertase for 5 min reduced by 56% the amount of D-

[3H]fructose released when no D-glucose was added during binding(1,749 cpm in the presence of the inhibitor versus 3991 cpm in itsabsence). The addition of 250 mM D-glucose during bindingresulted in 51% inhibition (2,115 cpm versus 4,319 cpm).More experiments would be necessary to determine precisely

whether the reducing sugars have any effect on binding; but itappears from the present data that, if there is such an effect, it issmall in comparison to the pronounced effect of sucrose.

DISCUSSION

A difficulty in measuring the rate of binding between potatotuber invertase and its proteinaceous inhibitor is that the bindingcontinues during the assay for remaining enzyme activity. Thisgives an erroneously high estimate of the binding rate. Theproblem is not unique to the Nelson or glucose oxidase tests usedin these experiments; it is inherent in any method of measuringremaining invertase activity that depends upon monitoring therate at which sucrose is converted to reducing sugars, unless some

method is found to stop binding at the time of the sucrose addition.In this respect, it is fortunate that sucrose itself slows the rate ofbinding, thus reducing the error (Fig. 5). Still further decreases inbinding rate are possible through the addition of MgCl2 alongwith the sucrose. Partial correction may be made for the smallamount of binding that occurs even in the presence of sucrose andMgCl2 (Fig. 2) by including a "zero-time" treatment and comput-

ing the per cent inhibition in terms of this control, as was done forthe data of Figure 1.From the binding studies carried out, it is clear that binding

between potato tuber invertase and its inhibitor follows second-

order kinetics. The graph of per cent inhibition plotted againstbinding time (Fig. 2) closely approximates that predicted for a

second order reaction. Simultaneous doubling of the two proteinsgave a doubling of the initial binding rate. When the activities ofenzyme and inhibitor, expressed in units, were equal, a straightline was obtained by plotting the reciprocal of the per centinhibition versus the reciprocal of binding time (Fig. IC andequation 6) or by plotting the data in the form of equation 5 (Fig.1B). These observations were consistent with second-order kinet-ics.

The inhibitor binds irreversibly to invertase (3) and, therefore,gives noncompetitive inhibition (3, 11). Presumably, binding ofthe inhibitor to the enzyme causes a blocking of or a change inthe catalytic site. There were interesting parallels between theeffects of various factors on binding and on catalytic activity.These include the effects of temperature, pH, and sugars. Theinterpretation of effects of temperatures higher than 40 C iscomplicated by the instability of the enzyme during binding andassay at these temperatures but, up to 40 C, the curves were

similar. Binding and catalytic activity both showed broad rateoptima between pH 4.4 and 5.2, although the effects of pH weremore pronounced on binding that on catalytic activity. Sucrose,the substrate for the enzyme, inhibited binding at concentrationswell below the 4 mM Km value (4). Neither fructose nor glucosehad detectable effects on binding. Fructose at 250 mm depressedcatalytic activity slightly. The catalytic activities of invertases fromseveral other species are reportedly inhibited by glucose andfructose. Neurospora invertase was inhibited by both sugars (14).Sweet potato alkaline invertase was inhibited by glucose and byglucose-6-P, although the acid invertase from sweet potato wasnot inhibited by glucose or fructose (8). Kuhn and Munch (6)found that inhibition of yeast invertase varied with the yeast strainstudied and that inhibition by glucose and fructose was competi-tive.The sensitivity of binding to salts and pH would be consistent

with an electrostatic reaction. It is also possible that conforma-tional changes must occur in one or both proteins before bindingcan take place. Apparently, the reaction is not a covalent onewhich drastically modifies either protein. Although normally thereaction is irreversible, functional enzyme or inhibitor can berecovered from the complex through rather extreme treatments(1, 3, 10). It is hoped that the data and methods described herewill facilitate further examination of the binding reaction.

Acknowledgments-We thank A. T. Jagendorf and R. R. Alexander for helpfulsuggestions during the course of these experiments. We are also grateful to G. G.Hammes for reviewing the derivation of equation 6, for suggesting equation 5 as an

alternative basis for plotting, and for a critical reading of the manuscript.

LITERATURE CITED

1. ANDERSON RS, EE EWING 1978 Partial purification of tuber invertase and itsproteinaceous inhibitor. Phytochemistry 17: 1077-1081

2. BRADFORD MM 1976 A rapid and sensitive method for the quantitation ofmicrogram quantities of protein utilizing the principle of protein-dye binding.Analytical Biochem 72: 248-254

3. EWING EE, MH McADOO 1971 An examination of methods used to assay potatotuber invertase and its naturally occurring inhibitor. Plant Physiol 48: 366-370

4. EWING EE, M DEVLIN, DA McNEILL, MH McADOO, AM HEDGES 1977 Changesin potato tuber invertase and its endogenous inhibitor after slicing, includinga study of assay methods. Plant Physiol 49: 925-929

5. JAYNES TA, OE NELSON 1971 An invertase inactivator in maize endosperm andfactors affecting inactivation. Plant Physiol 47: 629-634

6. KUHN R, H MUNCH 1925 Uber Gluco- und Fructosaccharase. Z Physiol Chem150: 220-242

7. MALIK CP, R SOOD 1976 Ecophysiological regulation of invertase activity in pea

pollen. Indian J Ecol 3: 44-488. MATSUSHITA K, K URITANI 1974 Change in invertase activity of sweet potato in

response to wounding and purification and properties of its invertase. PlantPhysiol 54: 60-66

9. MATSUSHITA K, K URITANI 1976 Isolation and characterization of acid invertaseinhibitor from sweet potato. J Biochem 79: 633-639

10. PRESSEY R 1966 Separation and properties of potato invertase and invertase

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inhibitor. Arch Biochem Biophys 113: 667-67411. PRESSEY R 1967 Invertase inhibitor from potatoes: purification, characterization,

and reactivity with plant invertases. Plant Physiol 42: 1780-178612. PRESSEY R 1968 Invertase inhibitors from red beet, sugar beet, and sweet potato

roots. Plant Physiol 43: 1430-143413. PRESSEY R, R SHAW 1966 Effect of temperature on invertase, invertase inhibitor,

and sugars in potato tubers. Plant Physiol 41: 1657-166114. TREVITHICK JR, RL METZENBERG 1964 Kinetics of the inhibition of Neurospora

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invertase by products and aniline. Arch Biochem Biophys 106: 260-27015. WIEST SC, PL STEPONKUS 1977 Accumulation of sugars and plasmalemma

alterations: factors related to the lack of cold acclimation in young roots. J AmSoc Hortic Sci 102: 119-123

16. WINKENBACH F, P MATILE 1970 Evidence for de novo synthesis of an invertaseinhibitor protein in senescing petals of Ipomoea. Z Pflanzenphysiol 63: 292-295

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