Biosynthesis of bacterial glycogen IX: Regulatory properties of the adenosine diphosphate glucose pyrophosphorylases of the Enterobocteriaceae

ARCHIVES

OF

RIOCHEMISTRY

.lND

Biosynthesis Regulatory

142, 675-692

HIOPHYSICS

of

Properties

Bacterial

Department

of Biochemistry

and Biophysics, October

IX:

Diphosphate

Glucose

of the Enterobacteriaceae’

A. SABRAW,

Received

Glycogen

of the Adenosine

Pyrophosphorylases

G. RIBaREAU-GAYON,

(1971)

C. LAMMEL,

University

14, 1970;

The ADP-glucose pyrophosphorylases to be activated by fructose diphosphate, glycolytic int,ermediates. The enzyme monella typhimurium and Citrobacter

accepted

of California, December

from several enteric NADPH, pyridoxal-5’.P, was partially purified

AND

Davis,

JACK

PREISS

California

96616

3, 1970 organisms were found and by some other from ext,racts of Sal-

freundii and the effects of the activators on the kinetic properties of the enzymes were studied. The activators increased the apparent affinities of the enzyme for the various substrates and increased the maximal velocity of ADP-glucose synthesis several-fold for both partially purified enzymes. Both enzymes are inhibited by AMP, ADP, and Pi with AMP being the most potent inhibitor. The concentration of activator modulated the sensit.ivity of the S. typhimurium enzyme towards inhibition by AMP, ADP, or Pi . The enzyme is more sensitive to in-

hibition at lower and unsaturating concentration of the activators. However, in the absence of activator only 50-80~~ of t,he ADP-glucose synthesis rate could be inhibited even vator

at very greater

high concentrations than 95% inhibition

of 5’AMP (-2.5 of ADP-glucose

mM) while synthesis

in the presence of actioccurred at concentra-

tions of 0.5 mM AMP or less. The above findings are discussed with respect to the regulation of glycogen synthesis in the Enterobacteriaceae. Previous reports that the biosynthesis linkage of bacterial following reactions:

stimulate the rate of ADP-glucose synthesis while metabolites of energy metabolism, S’-adenylate, ADP, and Pi inhibit ADP-glucose synthesis (5-7). The nature of t’he activator varied in each bacterial system and seems to be related to nature of carbon metabolism carried out by the organism (7-9). The ADP-glucose pyrophosphorylase activity of Kscherichia coli B was found to be acbivated considerably by fructose diphosphate, SADPH, and pyridoxal $phosphate (6, 10, 11). Smaller magnitudes of activat’ion of the enzyme by 3-phosphoglyceraldehyde, phosphoenolpyruvate, and 2-phosphoglycerate were also observed. Since most members of t)he Enterobacteriaceae appear to metabolize glucose via the Embden-hIeyerhof pathway (12, 13), it was of interest t,o know if t,he activator spe&rum of t,he ADP-glucose pyrophosphorylase of other enteric bacteria

(l-4) have indicated of the a-l ,4-glucosidic glycogen occurs by the

ATP

+ or-glucose-l-P + ADP-glucose + PPi ADP-glucose + cu-1,4-glucan -+ a-1,4-glucosyl-glucan

(1) + ADP

(2)

The site of allosteric control of bacterial glycogen synthesis occurs at the enzymatic reaction where ADP-glucose is synthesized (s-7). Usually glycolytic intermediates 1 This research was supported

in part by United

States Public Health Service Grant AI 05520 from the National Institutes of Health. 2 Supported in part by a fellowship from the DQldgation G&&ale B la Recherche Scientifique et Technique. Present address: Department of Molecular Biology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, N. Y. 10461. 675

676

RIBEREAU-GAYON

was similar to that found for E. coli 13. This paper describes the properties of the ADP-glucose pyrophosphorylases of some enteric organisms. The ADP-glucose pyrophosphorylases of bot,h Salmonella typhimurium and Citrobacter freundii were partially purified and some of their propert,ies studied and described herein. The presence of ADP-glucose pyrophosphorylase in extracts of S. typhimurium Lt)-2 had been reported previously (14) but’ no mention of its regulatory properties was made. MATERIALS

AND

METHODS

Bacteria. The following microorganisms were obtained from Mrs. Gladys Cossins of t~he Department of Bacteriology, University of California, Davis: Aerobacter aerogenes, ATCC 13048; Aerobacter cloacae, ATCC 13047; Erwinia carotovora, ATCC 495; Proteus vulgaris, ATCC 13315; Serratia marcescens, ATCC 15365; Citrobacter freundii, ATCC 8090. Salmonella typhimurium Lt.2 was obtained from Dr. Mary-Jane Osborn of the Department of Microbiology, University of Connecticut School of Medicine and Escherichia aurescens, ATCC 12814, was obtained from the American Type Culture Collection. All organisms were maintained on nutrient agar slant,s and were grown aerobically in New Brunswick Fermentors either on 12 1. of enriched media containing 1% glucose, 1.1% K?HPO, , 0.85% KHzPO, , and 0.6% Difco yeast extract or 12 1. of minimal media containing 0.6% glucose, 0.12$& (NH,)&04 , 0.68% KH2P0, , 1.42oj, Na2HP04 , 0.0246’% MgS04.7H20, and 0.0011% CaClz The rate of aeration of the cultures was 16 l./min and the temperature was maintained at 37” except for S. marcescens which was grown at 30”and E. carotovora which was grown at 28”. After 16-24 hr of growth the bacteria were harvested wit.h a Sharples ult,racent.rifuge and stored as a paste at -12”. Chemicals. Glucose-‘V-1-P was obtained from Amersham/Searle, England, and 32PPi was obtained from New England Nuclear, Bost.on, Mass. DEAE-Cellulose was microgranular preswollen DE 52 (Whatman). All other chemicals were obtained at the highest purity available from commercial sources. Methods. Protein determinations were done by the method of Lowry et al. (15). The following systems were used for descending paper chromatography on Whatman No. 1 paper: Solvent A, 95% ethanol:1 M ammonium acetate, pH 3.8 (5:2); Solvent B, isobutyric acid:1 M NH3:O.l M EDTA, pH 7.4 (100:60:1.6); Solvent C, l-butanol:pyridine:HzO (6:4:3), Solvent D, 1-propanol:et,hyl acet,at,e:HzO (7: 1:2). The buffer system used for

EI’

AL

high voltage paper elect,rophoresis was 0.05 M citrate buffer, pH 3.9. Assay of ADP-glucose pyrophosphorylase. Assay A (activated pyrophosphorolysis). Pyrophosphorolysis of ADP-glucose cat.alyzed by S. typhimurium or C. freundii extracts was followed by the formation of ATP-32P from ADP-glucose and “PPi (16). The reaction mixture, which contained 20 pmoles of Tris-Cl buffer (pH 8.5), 2 pmoles of MgClz , 1OOpg of bovine plasma albumin, 0.5 pmole of 32PPi (5-50 X lo5 cpm/pmole), 0.2 fimole of ADP-glucose, activator at the indicated concentration, and enzyme in a final volume of 0.25 ml, was incubated at. 37” for 10 min. The react.ion was t,erminat.ed by the addition of 3 ml of cold 5% trichloroacetic acid and assayed as described previously (16). Assay R (unactivated pyrophosphorolysis). In this assay pyrophosphorolysis of ADP-glucose was measured in the absence of activator. When S. typhimurium enzyme was assayed the amounts of ADP-glucose and MgCls were increased to 0.5 and 5pmoles, respect.ively. The amount,s of other components of the reaction mixture and the conditions of the assay were the same as described in Assay A. In reaction mixtures containing C. freundii enzyme, the amounts of all components remained the same except for ADP-glucose which was increased to 0.3 rmole. Assay C (activated synthesis). The synthesis of ADP-glucose was measured by following the formation of ADP-glucose-‘4C from glucose-‘%1-P (16,17). The reaction mixture for measuring ADPglucose synthesis catalyzed by S. typhimurium enzyme which contained 0.1 pmole of glucose-14C1-P (5.0-7.5 x 106 cpm/pmole), 0.3 Imole ATP, buffer, 1.0 pmole of MgC& , 20 pmoles of Tris-Cl pH 8.4, 100 pg of bovine plasma albumin, 0.24 rg of yeast inorganic pyrophosphatase (Worthington Freehold, N. J.; 850 unit.s/mg), activator and enzyme in a total volume of 0.2 ml was incubated at 37” for 10 min. The reaction was terminated by heating the mixture for 1 min in a boiling water bath and then assayed as described previously (16, 17). The conditions and reaction mixture for measuring ADP-glucose synthesis catalyzed by the C. fret&ii enzyme were essentially the same as above except the buffer was Bicine-KOH,3 pH 8.0, 3 Abbreviations used: DTE = dithioerythritol; Bicine = N, N’-bis (2-hydroxyethyl).glycine; PLP = pyridoxal-5’-phosphate; FDP = fructose diphosphate; Gald-3-P = glyceraldehyde-3-P; 2PGA = 2-phosphoglycerate; 3PGA = 3.phosphoglycerate; PEP = phosphoenolpyruvate; KDPG = 2.keto,3-deoxy-phosphogluconate; HEPES = N-2.hydroxy-ethylpiperazene-N’-2ethanesulfonic acid.

llEGULBTIO?J

OF

AlIP-GLUCOSIS

and the amounts of ATP and MgClz were 0.2 and 0.5 rmole, respectively. Assay D (unaclivaled synfhesiw). The reaction mixture for measuring ADP-glucose synthesis catalyzed by the 8. typhimurirrm enzyme in the absence of activator was the same as in Assay C except that glucose-l-P, ATP, and MgCl2 were increased to 0.2, 1.5, and 5.0 rmoles, respectively. When measuring unactivated AI)P-glucose synthesis by C.fretrndii extracm the reaction mixtures were the same as described in Assay C except, that the amount of MgC12 was 1.0 pmole and the amount, of ATP was 0.5 rmole. The concentrations of metal ion and substrat,es used in the act,ivated and unactivated assays described above are those concentrations giving optimal activity in the presence and absence of t,he activator, respect,ively. Since the activat,or increases the apparent affinity of the substrates for the enzyme, the optimal concentrations of substrates are invariably lower in the reaction mixtures containing activator. All kinetic studies were done in the range where velocity of ATP or ADP-glucose formation is proportjional to protein concentration and t,ime. Purification of ADP-Glucose Pyrophosphorylases 1. Disruption of cells: S. typhimurium enzyme. Frozen cells, 50 g, obtained from growth on synthet,ic media were mixed with 40 ml of cold 0.05 M glycylglycined mM dithiot,hreitol buffer, pH 7.0, and 150 g of Superbriteglass beads. This mixture was homogenized at medium speed in a glass Waring Blendor for a total t,ime of 15 min with frequent interruption of the grinding for cooling in an ice-salt bat,h in order to keep the temperature below 15”. At the end of the grinding period 150 ml TABLE PURIFICATION

OF

ADP-Gr,ucoss

SYNTHESIS

677

of the above buffer was added and the mixture stirred at low speed for an additional 10 min. The beads were allowed to settle and the supernatant solution was decanted. The beads were rinsed with an additional 100 ml of buffer with stirring at, low speed for 5 more min. The supernatant solutions were combined and centrifttged at 18,OOOg for 15 min. 2. Heat trealment. The supernatant fluid was made 0.03 M with respect, to phosphate by the addition of 1 M phosphate buffer, pH 7.0. This solut,ion was transferred to a 2800.ml Fernbach flask and heated to 60” in a 75” water bat,h (12 1.). The supernatant, fraction was maintained at this temperature for 5 min. The solut,ion was quickly cooled to 5” and centrifuged at 18,0COg for 15 min. 3. Ammonium suljutejractionution. To the supernatant fluid of t,he heat-treated fraction was added an equal volume of sat,urated ammonium sulfate solution. After st.irring for 10 min the resulting suspension was centrifuged for 15 min at 30,0009. The precipitat,e was dissolved in 0.05 M Tris-Cl buffer, pH 7.2, containing 0.005 M DTE and di alyzed against 1 1. of this buffer overnight. 4. DEAE-Cellulose chronzalogruphy. A column (2 X 17 cm) containing DEAE-cellulose was equilibrat,ed with 0.015 hz phosphate-5 mM DTE buffer, pH 7.5, and the ammonium sulfate fraction was adsorbed ont,o the column. The column was washed with one resin bed volttme of the phosphate buffer (54 ml) and the gradient, which contained 800 ml of t,he 0.015 Y phosphate-5 my DTE buffer, pH 7.5 in the mixing chamber and 800 ml of 0.1 &I phosphate buffer, pII 7.0, containing 0.3 M KC1 and 5 mM I>TE in the reservoir chamber, was start,ed. Fractions of 40 ml were collected and the fract,ions which contrained enzymatic act,ivity were located with ilssay A, pooled (225 ml), and I

PYROPHOSPHORYLASFS

FROM

S.

typhimurium

C. fret&ii

.\NV

Fraction

A. Salmonella l~/phimurimt

B. Citrobacter jremdii

with

0 One unit 1.0 rnM

1. 2. 3. 4.

Crude extract. Heat step Ammonium sulfate DEAE-cellulose chromatography

240 213 7.5 5.3

432 1044 832 458

19.8 6.5 72.2 9.0

0.091 0.75 1.5 9.6

1 8.2 16.3 105

1. 2. 3. 4.

Crude extract Heat step Ammonium sulfate DEAE-cellulose chromatography

260 215 35 8

1320 1155 650 290

20.7 6.8 17.7 8.1

0.25 0.79 1.05 4.5

1 3.2 4.2 18

is eqtml to 1 @mole of ATP FDP as the activator.

formed

in 10 min

under

the

conditions

specified

for

Assay

A

675

RIBEREAU-GAYON

brought to 80% saturation with solid ammonium sulfate. The precipitate obtained after centrifugation for 15 min at 16,OOOg was dissolved in 0.05 M Tris-Cl buffer, pH 7.2, containing 5 mM DTE, and dialyzed against 1 1. of this buffer overnight. Table IA is a summary of the purification procedure used for preparation of the S. typhimurium ADP-glucose pyrophosphorylase. A unit of enzyme activity is based on the conditions of Assay A in the presence of 1 mM FDP and is defined as that amount required for formation of 1 pmole of ATP in 10 min at 37”. C. freundii enzyme. ADP-glucose pyrophosphorylase was partially purified from 6. freundii in essentially the same way as described for the enzyme from 8. typhimurium. The major difference was in the heat-treatment step where the enzyme fraction from C. freundii wss heated to 65” instead of 60”. Table IB summarizes the purification procedure for the C. freundii enzyme. A unit of enzyme activity is defined in the same way as for the S. typhimurium enzyme. The DEAE-cellulose fractions for both enzymes had negligible degrading activities towards ADPglucose, glucose-l-P, ATP, or pyrophosphate. RESULTS

Levels of the glycogen biosynthetic enzymes in bacterial extracts. Table II shows the levels and of ADP-glucose pyrophosphorylase ADP-glucose : cY-glucan transferase in various cell-free extracts of enteric bacteria. Significant activity of bot’h enzymes was observed except in extracts prepared from P. vulgaris and E. caratovora. The ADPglucose pyrophosphorylase activity observed in the extracts was activated by fructose diphosphate except in the case of the 8. marcescen8 extract where no activation was observed. In general the properties of the transferase in the crude extracts of C. freundii, S. typhimurium, S. marcescens, E. aurescens, A. cloacae, and A. aerogenes were the same. In the absence of a glycogen primer the glucoseJ4C transfer from ADP-glucose was reduced to 10% or less of the rate observed in the presence of glycogen primer. Glucose14C transfer was inhibited completely (> 99 %) when 20 pg of B. subtilis cr-amylase was present in the reaction mixture. Substitution of either UDP-glucose, TDPglucose, CDP-glucose, GDP-glucose, or glucose-l-P for ADP-glucose in reaction

ET

AL. TABLE

LEVELS

OF

II

ADP-GLUCOSE

PYROPHOSPHORYLASE

AND ADP-GLIJCOSE:LY-1,4-GLUCAN~GLUCOSYL TR~NSFERASE

Organism

IN

ENTERIC

BACTERIA-

ADP-glucose pyrophosphorylase (nmoles 10 min-1 +$;pM

-FDP

%z(nmoles 10 min-1 w-9

56 20 205 51 100 85 3 1

90 2,000 660 3,300 2,300 910 1.7 31

mgl)

-

C. S. S. E. A. A. P. E. -

freundii typhimurium marcescens aurescens cloacae aerogenes vulgaris caratovora

100 89 210 780 740 517 4 1

a The assay of ADP-glucose pyrophosphorylase was the same as described for S. typhimurium in Assay A. Transferase activity was measured as previously described (1). The reaction mixture contained in a volume of 0.2 ml, 10 pmoles of TrisCl buffer, pH 8.5, 2 pmoles of GSH, 5 pmoles of KCl, 0.10 mg of bovine plasma albumin, 1.0 pmole of MgClz ,0.5 mg of rabbit liver glycogen, enzyme extract, and 0.15 pmole of ADP-glucose-W (4-8 X lo6 cpm/pmole). The enzyme extract was prepared by subjecting a suspension of 0.5 g of bacteria (wet wt) in 10 ml of 0.05 M glycylglycine buffer, pH 7.0, containing 5 mM DTE to sonic oscillations for l-3 min with a Biosonik III probe sonic oscillator. In the case of S. marcescens 4 g (wet wt) of bacteria was used. The assay of the transferase was done with the uncentrifuged sonic extracts while the assay of the pyrophosphorylase was performed on a supernatant solution obtained after centrifugation of the sonic extract at 30,OOOg for 15 min.

mixtures resulted in less than 2% of the activity observed for ADP-glucose. The rate of glucose transfer from deoxy-ADPglucoseJ4C to glycogen was about the same observed for ADP-glucose. Transferase activity in the various extracts was not stimulated by the presence of either 0.5 mM pyridoxal-5’-P or 1.0 mM glucose-6-P, fructose-g-p, fructose-l, 6-dip, 3-phosphoglycerate, pyruvate, or 1.0 rn%I NADPH. In each system the radioactive alcoholinsoluble product was hydrolyzed with pamylase at pH 5.0 and in each case a radioact,ive material was obtained which cochromatographed with maltose in solvent

REGULATION

OF

ADP-GLUCOSE

systems C and D. This indicated that new cu-1,4-glucosidic linkages were being formed by transfer of glucoseJ4C from ADPglucose to the primer glycogen. Activation of the enteric bacterial ADPglucose pyrophosph.orylases. Table III shows the activator specificity for the ADPglucose pyrophosphorylase in t,he various organisms. The cell-free extract of E. aurescens, A. cloacae, and A. aerogenes and the DEAE-cellulose fractions of S. typhimurium and C. freundii were used for the study. PLP, FDP, NADPH, and the 3-carbon glycolytic intermediates, glyceraldehyde-3-P, 2-phosphoglycerate, 3-phosphoglycerate, and phosphoenolpyruvate, gave tfhe highest stimulations of ADP-glucose synthesis catalyzed by t,he various enzymes. Usually lesser stimulation was seen for the compounds glucose-l ,6-dip, 6-phosphogluconate, NADP, 2-keto-3-deoxy-6-phosphogluconate, ribose-5-P, and glycerol-l, 3-dip. Negligible activation of ADP-glucose synthesis was seen for the following compounds which were tested at concentrations of l-2 rn>f; glucose-6-P, fructose-6-P, dihydroxyacetone phosphate, pyruvate, deoxyriboseTABLE ACTIVATION

OF

ADP-GLUCOSE

None PLP, 0.05 rnM FDP NADPH, 1.0 mM NADP, 1.0 mM Gald-3-P 2-PGA 3-PGA PEP Glucose-1,6-diP 6-P-gluconate KDPG Ribose-5-P Glycerol-1,3-diP Pyridoxal, 0.5 mM D-Arabinitol-1,5-dip,

5-P, acetyl CoA, oxaloacet,ate, citrate, isocitrate, cr-ketoglutarate, fumarate, succinate, malate, glutamate, alanine, pyridoxal, pyridoxamine&P, NADH, NAD, FAD, E’MN, and aspartate. The product synthesized from ATP and glucose-14C-l-P in the presence and absence of the activators, fructose-dip, PLP, NADPH, glyceraldehyde-3-P, and glycerol1,3-dip, by the various crude extracts and partially purified enzymes, cochromatographed wiOh ADP-glucose in solvent systems A and B and migrated with ADPglucose in electrophoresis at, pH 3.9. Figure 1 shows the effect, of fructose-l ,6dip, NADPH, pyridoxal-5’-P, glyceraldehyde-3-P, and glycerol-l ,3-diphosphate concentrations on the rate of ADP-glucose synthesis catalyzed by the S. typhimurium enzyme. The increase in rate under the conditions of Fig. 1 ranged from about, 35-fold wit’h saturating concentrations of PLP to about 16-fold for glycerol-l, 3-dip. The concent,rations of PLP, fructose-dip, NADPH, glyceraldehyde-3-P, and glycerol1,3-diP giving 50 % of maximal activation were 9.4, 98, 105, 690, and 305 ~$1, respecIII

PYROPHOSPHORYLASES

BY

(nmoles

Activator

1 mM

679

SYNTHESIS

V.~RIOUS

Organism ADP-glucose formed

METABOLITES~ -10

min-1)

A. cloacae

A. aerogmes

E. auresce?zs

C. freundii

1.2 9.6 8.5 7.5 5.3 7.5 8.9 6.2 8.6 3.75 3.5 5.6 3.2 6.1 0.96 -

0.70 21.1 19.3 17.1 15.1 17.8 20.0 12.7 21.9 11.8 7.8 17.9 5.7 15.6 0.74 -

0.40 20.9 15.8 13.5 2.1 12.0 5.8 1.45 1.60 4.55 0.99 4.32 2.72 7.5 0.40 -

6.7 lQ.8 18.5 16.0 7.5 15.0 12.5 6.7 17.3 6.0 8.8 6.9 6.7 -

S. lyfihimurium

0.46 22.5 15.6 13.9 4.4 16.6 8.2 1.42 6.2 2.2 1.3 6.8 0.67 10.6 0.50 19.1

a The assay used was that described for S. lyphimurium enzyme in Assay C. Unless indicated otherwise, the concentration of activator was 1.5 mM. The DEAE-cellulose fractions of the C. freundii and S. typhimurium enzymes and sonic extracts of the other bacteria prepared as described in Table II were the source of ADP-glucose pyrophosphorylases used in this experiment.

680

RIBRREAU-GAYON

0.05

PLP,

mM

ACTIVATOR

ET AL.

0.1

CONC.,

mM

FIG. 1. Activation of S. typhimurium ADP-glucose pyrophosphorylase. The conditions of the assay are those of Assay C. Ao.5 is concentration of activator giving 5001, of the maximal stimulation. The inset in the upper part of the graph is a plot of the data according to the Hill equation (18). Vm,, was estimated from reciprocal plots of rate vs. activator concentration. Au is the increase in velocity due to addition of activator, i.e., the velocity obtained upon an addition of activator to the reaction mixture minus the velocity of the reaction mixtures containing no activator. fi is the interaction coefficient of the Hill equation.

tively. The activation curves of Fig. 1 were sigmoidal in shape. Hill plots (18) of the data gave values for the apparent order of reactions, ti, for the above activators of 2.2 or greater. In experiments with the C. freundii ADPglucose pyrophosphorylase similar activation curves for PLP, fructose diphosphate, NADPH, and glyceraldehyde-3-P were seen. The concent’rations of activators required to give 50% of maximal activation

were somewhat lower for the C. freundii enzyme than for the S. typhimurium enzyme. The values for PLP, NADPH, fructose-dip, and glyceraldehyde-3-P were 7.4, 78, 32, and 117 PM, respectively. Sigmoidal curves for these activators were also seen with the 6. freundii enzyme. However, the apparent order of reactions, fi, obtained from the Hill plots were in the range of 1.5-1.6 and were significantly lower than those observed for S. typhimurium system.

REGULATION

OF

ADP-GLUCOSE

Effect of Activators on the Properties of the ADP-Glucose Pyrophosphorylases of C. freundii and S. typhimurium

SYNTHESIS

681

Since the stimulation by activator was greatest in magnitude in Bicine buffer, pH 8.0, kinetic studies were done at this pH. The pH optimum for pyrophosphorolysis of ADP-glucose by C. freundii enzyme in TrisCl buffer was 5.5. @Feet of activators on nucleoside triphosphate and nucleotide diphosphate sugar specifkity. Replacement of ADP-glucose in Assay A by either GDP-glucose, IDPglucose, TDP-glucose, CDP-glucose, or GDP-mannose in reaction mixtures containing either the partially purified enzyme from C. freundii or S. typhiwhurium resulted in less than 1% the activit,y observed with ADP-glucose. Pyrophosphorolysis of deoxyADP-glucose was catalyzed by both the C. freundii and S. typhimuriuwl enzymes at about 7-10 % the rate seen for ADP-glucose. The presence or absence of activators fructose-dip or PLP did not effect the above results (Assay A or B). The partially purified enzyme from C.

pH Optiwla. The pH optimum for ADPglucose synthesis or pyrophosphorolysis catalyzed by the S. typhimurium enzyme in the presence or absence of fructose-dip in Tris-Cl buffer was broad and in the range of pH 7.5-5.5. Figure 2 shows the pH optimum of ADP-glucose synthesis catalyzed by the Citrobacter freundii enzyme in various buffers and in the presence and absence of the activator fructose-dip. In the presence of fructose-dip the pH opt’imum in Bicine and HEPES buffers was S.0 while in the presence of Tris-Cl buffer the optimum pH was 5.5. In the absence of activator the pH optimum increased to about 9-9.5. Thus the activator fructose diphosphate shifts the pH optimum for the C. freundii enzyme to a more neutral range. Essentially similar results were obtained with pyridoxal-5’-phosphate as the activator.

PH FIG. 2. pH optimum of ADP-glucose synthesis catalyzed by C. freundii ADP-glucose pyrophosphorylase. The conditions of the experiments were Assay C (activated synthesis in the presence of 1 m FDP; A-A, O--O, O---O, and X--X) and Assay D (unactivated synthesis; A-----A, O-----O, O-----O, and X-----X).

682

RIBfiREAU-GAYON

freundii synt’hesized sugar nucleotide with TTP or UTP as the nucleoside triphosphate at 5% the rate observed when ATP was present in assay reaction mixtures. These rates were not increased in the presence of activator and probably are activities catalyzed by contaminating enzymes. The ADPglucose pyrophosphorylase partially purified from Salmonella, however, was inactive with either UTP, GTP, TTP, or ITP. About 9-10% of the activity seen for ATP was observed when either dATP or CTP was present in reaction mixtures (Assay C). Sugar nucleotide synthesis with dATP was stimulated by fructose diphosphate or PLP but sugar nucleotide synthesis with CTP was not. Most likely sugar nucleotide synthesis from CTP is due to a contaminating pyrophosphorylase activity. Crude extracts of Citrobacter freundii catalyzed synthesis of TDP-glucose and UDP-glucose from their respective nucleoside triphosphates at 10 times the rate of synthesis of ADP-glucose under the conditions of Assay D (unactivated synthesis). Fructose-diP did not stimulate the synthesis of either TDP-glucose or UDP-glucose (Assay C). Crude extracts of S. typhimurium also catalyzed the synthesis of TDPglucose, UDP-glucose, and CDP-glucose from their respective nucleoside triphosphates at lo-14 times the rate observed for ADP-glucose under the conditions of Assay D. PLP or fructose-dip did not activate the synthesis of TDP-glucose, UDPglucose, or CDP-glucose in the S. typhimurium sonic extracts under the conditions of Assay C. E#ect of activators on the kinetic parameters of the ADP-glucose pyrophosphorylases from C. freundii and S. typhimurium. Figure 3 shows the ATP saturation curve for the S. typhimurium enzyme in the presence and absence of the activators, PLP, NADPH, and FDP. In the absence of activator the shape of the ATP saturation curve is slightly sigmoidal and the concentration of ATP required for half-maximal velocity (SOJ is 2.4 mM. The inclusion of saturating concentration of activators reduced significantly the S0.5 value for ATP. In the presence of PLP, NADPH, or FDP the So., value for ATP was reduced to 0.22,

ET AL.

0.37, and 0.48 mM, respectively. However, these activators had different effects on the shape of the ATP curve. The presence of PLP caused it to be hyperbolic (a = 1); NADPH had no effect on the shape of the curve and fructose diphosphate caused the ATP saturation curve to become even more sigmoidal increasing fi from 1.5 to 2.2. Essentially similar results were obtained for the ATP saturation curve for the C. freundii enzyme (Fig. 4). Fructose diphosphate reduced the S0.svalue for ATP from 0.72 to 0.26 m while in the presence of 0.05 M PLP the ATP S0.6 value was 0.15 rnn[ (Table IV). In the caseof the C. freundii enzyme both FDP and PLP decreased the sigmoidicity of the ATP saturation curve. rt, the interaction coefficient, decreased from a value of 2.5 to 1.5 in the presence of 1.0 mM fructose-dip. PLP at a concentration of 0.05 mM converted the ATP saturation curve from a sigmoidal form to a hyperbolic form. Figure 4 also shows t’hat the So,s and fi values were the same at pH 8.0, the optimal pH for synthesis in the presence of activator, and pH 9.0, the optimal pH for synthesis of ADP-glucose in the absence of activator (Assay D). Table IV summarizes some of the kinetic parameters obtained for the various substrates and divalent cations for the partially purified ADP-glucose pyrophosphorylases from S. typhimurium and C. freundii. The activators NADPH, PLP, and FDP at saturating concentration lower the apparent affinity (S0.5) of the S. typhimurium enzyme for glucose-l-P about 4- to 5-fold and for ADP-glucose about 6- to la-fold. These same activators reduced the So.6 value of glucose-l-P for the C. freundii enzyme about 1.5- to 2.6-fold. Pyrophosphorolysis of ADP-glucose catalyzed by the C. freundii enzyme was studied with only one activator, FDP. The apparent affinities of both pyrophosphate and ADPglucose (&.h) for enzyme were reduced about 2- to S-fold. For both enzymes t’he glucose-l-P and pyrophosphate saturation curves were hyperbolic in the presence or absence of activator. The ADP-glucose saturation curves were also sigmoidal in shape in the absence of activator for both enzymes. The presence

REGULATION

7 +PLP 1.0-e +FDP +NADPH

OF ADP-GLUCOSE

SYNTHESIS

n

SO., 0.22mM 0.48 0.37

1.0

0.04 0.070.1 0.2 ATP

ATP.

0.4 0.7 1.0 2.0 CONC.,

4.0 7.0

mM

mM

FIG. 3. The effect of ATP on ADP-glucose synthesis catalyzed by the S. typhimurium ADP-glucose pyrophosphorylase. The conditions of the experiment were those of Assay C of (a---0, A---A, and O--O) and Assay D (A-7 A). 80.~ is the concentration substrate required for 5v0 of maximal velocity.

of fructose-l ,6-diP and NADPH in reaction mixtures containing X. typhimurium enzyme although reducing the XO.s value of ADP-glucose did not alter the shape of the ADP-glucose saturation curve. However, the presence of PLP did change the shape of the ADP-glucose curve to a more hyperbolic form. In this instance fi, the interaction coefficient, changed from 1.8 to 1.1. Studies with the C. freundii enzyme revealed that the presence of fructose-l ,6diP convert’ed the sigmoidal shape of the ADP-glucose curve to a more hyperbolic form; R being 2.0 in the absenceof activator

and 1.3 in the presence of 0.75 mM fructosedip. Optimal pyrophosphorolysis rates with the S. typhimurium enzyme occurred at 8 mM MgCls in the presence of activators and 12.520 rnlu in the absenceof activator under the conditions of Assays A and B while with the C. jreundii enzyme optimal pyrophosphorolysis occurred between 6 to 8 m&r MgCl,. Optimal synthesis of ADPglucose with the S. typhimurium enzyme occurred at 5 rnM MgClz in the presence of the activators FDP, NADPH, and PLP under the conditions of Assay C. In the

RIBEREAU-GAYON

684

ET AL. + FDP.

ATP QO

Log

CONC.,

pH 80 +FDP

pH 8.0

mM

-5 1.5

so.3 0.26mM

” “m-v O.O-

-“‘tc/

[

0.02

0.04 0.07 0.1

0.2

I

;RrnM

0.4

FIG. 4. The effect of ATP on ADP-glucose synthesis catalyzed by the C. freundii ADPglucose pyrophosphorylase. The conditions of the experiment were those of Assay C (1.0 rnM C, X-----X) and of Assay D (O-----O, A---A). The buffer used in these FDP; Cexperiments was Bicine. The amount of enzyme used for the pH 8.0 and 9.0 studies in Assay C was the same. However, three times as much enzyme was used for the Assay D, pH 9.0 experiment as in Assay C and six times as much enzyme was used for the Assay D, pH 9.0 experiment as in Assay C.

absence of activator under the conditions of Assay D, 25 mM MgCl, was required for optimal rates of ADP-glucose synthesis. In reaction mixtures containing C. freundii enzyme 2.5 mM MgCl, was required for optimal rates of synthesis in the presence of activators (Assay C) while 5 mM MgClz was needed for optimal rates in the absence of activator (Assay D). For both enzymes and under the assay conditions tested, the MgClz saturation curves were highly sig-

moidal giving Hill constants fi greater than 3. The activators also increased the maximal velocity of both ADP-glucose synthesis and pyrophosphorolysis catalyzed by the S. typhimurium enzyme (Table IV). Maximal velocity of ADP-glucose synthesis was stimulated 4-fold by FDP, 5fold by PLP, and 3.5-fold by NADPH. Max of pyrophosphorolysis was stimulated 2.4fold by FDP, 2.8-fold by PLP, and 2.2-fold by

REGULATION

OF ADP-GLUCOSE TABLE

KINETIC

P.~RAMETERS

OF THE

-

SYNTHESIS

IV

ADP-GLUCOSE PYROPHOSPHORYLlSES AND C. freundi+

Substrate

635

li

Activator

FROM

8. typhimurium

(flmoles

VIII,,

mg-1 10 min-1)

-S. typhimuriw

1

ATP

Glucose-l-P

ADP-glucose

ATP

C. freundii

Glucose-l-P

2.4 0.48 0.22 0.37 0.21 0.034 0.042 0.039 0.57 0.10 0.046 0.10

1.5 2.2 1.0 1.5 0.9 1.06 1.05 1.06 1.8 1.9 1.1 1.7

None FDP, 1.0 mM PLP, 0.05 rnM

0.72 0.26 0.15 0.13 0.05 0.07 0.09 3.4 1.0

2.5 1.5 1.0 0.9 0.9 1.0 0.9 3.5 3.5 0.92 1 0.84 2.0 1.3

None

MgC& (synthesis ) Pyrophosphate ADP-glucose -

None FDP, 1.0 mM PLP, 0.05 mM NADPH, 0.5 mM None FDP, 1.0 mM PLP, 0.05 rnM NADPH, 0.5 mM None FDP, 0.8 mM PLP, 0.04 rnM NADPH, 0.8 mM

-

FDP, 1.0 mM PLP, 0.05 mM NADPH, 1.0 mM None FDP, 1.0 mM None FDP, 0.75 mM None FDP, 0.75 mM

z3 0.38 0.12

No activator + FDP, 1.0 rnM + PLP, 0.05 rnM + NADPH, 1.0 mM

1.2 4.8 6.4 4.3

No activator + FDP, 0.8 mM + PLP, 0.04 rnM + NADPH, 0.8 mM

4.0 9.6 11.2 8.6

No activator + FDP, 1.0 mM + PLP, 0.05 rnM + NADPH, 1.0 mM

1.10 7.2 9.3 5.0

No activator + FDP, 0.75 mM

4.0 4.5

1

a The assays for synthesis and pyrophosphorolysis of ADP-glucose are described in the text. fi is the Hill constant (18) and So.6 represents the concentration of substrate or metal ion required for half of maximal activity and was obtained from Hill plots after maximal velocity was determined from the intercepts of reciprocal plots of velocity vs. substrate concentration.

NADPH. V,,, of ADP-glucose synthesis catalyzed by t’he C. freundii enzyme in Bitine buffer, pH 8.0, was increased about 6.5-fold by FDP, 8.6-fold by PLP, and 4.5 fold by NADPH. In Tris-Cl buffer, pH 8.5, FDP gave little or no stimulation of the maximal velocity of ADP-glucose pyrophosphorolysis catalyzed by t’he C. freundii enzyme. Inhibition of ADP-glucose synthesis by 5’-AMP, ADP, and phosphate. Both the C. freundii and S. typhimurium ADPglucose pyrophosphorylases were inhibited by 5’-adenylate, ADP, and Pi. Figures 5 and 6 show t’he inhibition of the S. typhimurium enzyme by the tZhree inhibitors in the presence of the activators FDP and

PLP. The most effective inhibitor was 5’AMP and the sensitivity of the enzyme towards inhibition was modulated by the concentration of activator present. In the presence of 1.0 mM fructose-dip or 0.049 rnnf PLP (concentrations of activator giving maximal stimulation) about 0.11-0.13 rnM 5’-AMP was required for 50 % inhibition (IoJ. If the fructose diphosphate level was decreased to 0.25 mM, a concentration giving 80 % of the maximal velocity of ADP-glucose synthesis, the concentration of 5’-AMP causing 50 % inhibition was decreased about 4-fold to 0.028 m&I. If the PLP concentration was decreased to 0.01 mM, a value that gave rates of ADPglucose synthesis 60-65% of the maximal

686

RIBEREAU-GAYON

INHIBITOR

0

ET AL.

CONC., mM

0.5

INHIBITOR

1.0

I .5’ 2.0

5.0

CONC., mM

FIG. 5. Inhibition of SaZmoneZZa typhimurium ADP-glucose pyrophosphorylase. The conditions of the experiment are those of Assay C (activated ADP-glucose synthesis) with the indicated concentrations of FDP. One hundred per cent activity in A is 15.4 nmoles of ADP-glucose formed in absence of inhibitor in 10 min and in B is 12 nmoles of ADPglucose formed in 10 min.

rate, the concentration of 5’-AMP causing 50% inhibition was lowered to 0.019 mM. The sensitivity of the S. lyphimurium enzyme to ADP and Pi inhibition was also increased in the presence of lower concentrations of activator (Figs. 5 and 6). Figure 7 shows the Hill plots of the data of Figs. 5 and 6. All curves are sigmoidal giving Hill coefficients fi of 1.6 or greater. It appears that at lower concentrations of activator the inhibition curves are less sigmoidal giving lower values for 6. Table V summarizes the inhibition data for the S.

typhimurium enzyme when KADPH is the activator. Essentially similar results are obtained. The enzyme is most sensitive to inhibition by 5’-adenylate and a decrease in NADPH concentration caused the enzyme to be more sensitive to inhibition by AMP, ADP, and Pi. Table V also shows inhibition data for the C. jreundii ADP-glucose pyrophosphorylase. This enzyme was also most sensitive to inhibition by j/-AMP. Inhibition by ADP and Pi was also observed. The concentrations of activator used in these experiments were those giving 90-

REGULATION

OF ADP-GLUCOSE

SYNTHESIS

687

INHIBITOR CONC.. mM

R

P1P.f

INHIBITOR

CONC.,mM

FIG. 6. Inhibition of ADP-glucose synthesis catalyzed by 8. typhimurium ADP-glucose pyrophosphorylase. The conditions of the experiment are those of Assay C with the indicated concentrations of PLP. One hunderd per cent activity in A is 12.6 nmoles of ADPglucose formed in absence of inhibitor in 10 min and in B is 13.0 nmoles of ADP-glucose formed in 10 min.

100 % of the maximal stimulation of ADPglucose synthesis. In the presence of PLP the enzyme was least sensitive to inhibition. In fact no inhibition by 10 mM phosphate was observed in the presence of 0.045 rnM PLP. Figure 8 shows the inhibition of the S. typhimurium and C. freundii enzyme under unactivated conditions (Assay D). Although in most cases some inhibition occurred, the enzymes were not as sensitive to inhibition as when activator was present. In addition part of the enzyme activity is refractory to inhibition. About 50% of the

S. typhimurium ADP-glucose pyrophosphorylase is inhibited by 0.18 mM 5’-AMP under conditions of Assay D. However, elevation of the 5’-AMP concentration up to 2.5 mM causes no further inhibition. In the absence of activator 10 mM Pi does not inhibit the ADP-glucose synthesis catalyzed by the S. typhimurium enzyme. Essentially similar results are observed with the C. freunclii enzyme in the absence of activator. Inhibition of the activity never exceeded 80% in the absence of activator whereas in the presence of activator inhibition was 95 % or greater.

ET AL.

RIBEREAU-GAYON

688 A. FDP

4. *

..- IO

7

3l-l

an

M

-TNHKToK

B. PLP

Inn

mnfl

ENC+M

‘---

1.0.. PLPI,, n p”M mM \

o--o 0.0.. e--. )(-w+. 1.0.. n

AMP

49 4.6 4.3 IO 1.35 2.3 49 1.5 3.7 IO 0.22 1.6 49 0.13 2.3 IO

0.0192.0 \ i.3

I

4

7 IO

20

40 70 loo INHIBITOR CCW./.I M

1000

10000

FIG. 7. Hill plots of the data of Figs. 5 and 6.10.5 is the concentration of inhibitor giving 50% inhibition under the conditions of the experiment. Vo is the velocity obtained in the absence of inhibitor. TABLE INHIBITION

CONSTANTS

FOR

THE

ADP-GLUCOSE AND c.

V PYROPHOSPHORYLASES

OF S. typhimurium

fWUndiia

li

1o.s b.0

Activator

AMP

ADP

Pi

AMP

ADP

Pi

S. typhimurium

NADPH, NADPH,

0.5 nw 0.20 mM

0.079 0.016

1.0 0.25

2.7 0.96

2.4 1.5

2.5 1.5

3.0 2.0

C. freundii

FDP, 0.10 mu NADPH, 0.13 mM PLP, 0.645 mM

0.022 0.013 0.60

0.33 0.18 4.8

1.8 0.9 -

1.6 1.3 2.3

1.6 1.3 2.5

2.3 1.3 -

a 10.6is the concentration ment (Assay C).

of inhibitor

giving 50% inhibition

DISCUSSION

The above studies suggest that glycogen synthesis in the Enterobacteriaceae is regulated at the enzymatic reaction where ADPglucose synthesis occurs. Some glycolytic intermediates and cofactors, notably fructose diphosphate, NADPH, and pyridoxal-P are activators of the enzyme, ADP-glucose

under

the conditions

of the experi-

pyrophosphorylase, while AMP, ADP, and Pi are inhibitors. AMP is the most potent inhibitor. The properties of the ADPglucose pyrophosphorylases from Salmonella typhimurium and Citrobacter freundii were studied in more detail and the properties of these enzymes were found to be very similar to the properties noted previously for the E.

REGULATION

OF ADP-GLUCOSE

SYNTHESIS

689

Pi

8

A. S.typhimurium

20 ,;;.

0

I.0 INHIBITOR

2.0 CONC. mM

8. C. freund

t

;

:

:

0 FIG. 8. Inhibition

;

:

:

I .o of ADP-glucose

synthesis

coli ADP-glucose pyrophosphorylase (6, S, 10, 11). The activators not only increase the maximal velocity of ADP-glucose synthesis 4- to S-fold but also increase the apparent affinity for the substrates ATP, glucose-l-P, ADP-glucose, pyrophosphate and the required divalent cation, Mg2+, anywhere from 2- to 11-fold (Table IV). The activat,ors seem to modulate the sensitivity of the S. typhimurium enzyme towards inhibition by AMP, Pi, or ADP. Lower and unsaturating concentrations of activators increased the sensitivity of the enzyme to inhibition as lesser concentrations of inhibitors were needed for 50 % inhibition (Figs. 5-7, Table V). However, in the absence of activator the inhibition is strongly reduced with both the S. typhi?nuriurn and C. freundii enzymes. In this

4

: 6

, 8 IO

ii

:

i

:

!

[I

2.0 under unactivated

:

4 6 8 conditions

(

IO (Assay D).

case a part of the activity, 20-40% with AMP and ADP, 30-100% with Pi, is insensitive to inhibition (Fig. S). A striking example of this is where phosphate gives maximal inhibition of the S. typhimurium enzyme (>90 %) in the presence of the various activators and no inhibition in the absenceof activators. Thus the activators appear to have four effects on the enzyme; (1) they increase Vmax of ADP-glucose synthesis, (2) they increase the appearent affinities of the various substrates and Mg2+, (3) they modulate the sensitivity of the enzyme to inhibition, and (4) their presence allows the enzyme to become fully inhibited by 5’AMP, ADP, or Pi. All of these effects by the activators had been seen with the E. coli ADP-glucose pyrophosphorylase (6, 8,

690

RIBGREAU-GAYON

10, 11). The inhibition of the E. coli ADPglucose pyrophosphorylase by AMP is either mixed or noncompetitive with the substrates ATP and glucose-l-P (10). However, this aspect was not studied with either the S. typhimurium or C. freundii enzymes. Some small differences in the properties of the E. coli ADP-glucose pyrophosphorylases and the two enzymes currently studied are noted. Whereas the presence of activators FDP and NADPH do not significantly change the sigmoidal shape of the ATP saturation curve for the E. coli enzyme (6, S), there is a marked decrease of the sigmoidicity (Hill r~.varies from 2.5 to 1.5) for the same curve with the C. freundii enzyme in the presence of FDP. On the contrary, ti for the ATP saturation curve of the S. typhimurium enzyme remains unchanged in the presence of NADPH and increases in the presence of FDP. For all three enzymes PLP converts the sigmoidal ATP curve of the unactivated reaction to a hyperbolic curve (fi = 1). The concentration of PLP necessary for 50% of maximal activation (&.rJ seemed to be the same for the three enteric bact,erial ADP-glucose pyrophosphorylases. However, the Ao.s values for FDP and NADPH seen for the C. freundii were slightly but significantly lower than the AO.S values observed for the E. coli (11) and S. typhimurium enzymes. The above results suggest ADP-glucose synthesis and therefore glycogen synthesis is regulated in part by the energy charge (19-22) of the cell and by the availability of an excess carbon source that would cause accumulation of glycolytic intermediates. Because of its sigmoidal saturation curve, ATP may be considered as an activator as well as a substrate for ADPglucose synthesis, Since AMP and ADP are inhibitors of ADP-glucose pyrophosphorylase, ADP-glucose synthesis may be considered regulated by the energy charge of the cell as defined by Atkinson (19, 20). Under conditions when there is “high energy charge” ADP-glucose and glycogen synthesis would occur whereas under conditions of “low energy charge” glycogen synthesis would be inhibited. Glycogen

ET AL.

accumulation occurs as a result of limiting growth conditions in the presence of excess carbon source (23-25). Under these conditions it is believed that the rate of ATP production is in excess of its utilization since energy for the biosynthesis of constituents necessary for cell growth and division are not required. The necessity for excess carbon being present for glycogen synthesis to occur is also consistent with the observations that glycolytic intermediates, which may accumulate under the conditions of limiting growth and excess carbon, are activators of ADP-glucose pyrophosphorylase. Furthermore, under conditions of limited growth NADPH may also accumulate since the reduced pyridine nucleotide would be no longer needed for the numerous biosynthetic reactions. The studies of Model and Rittenberg (26) have suggested that the hexose monophosphate shunt in E. coli is regulated by the availability of oxidized NADP. As bacterial growth ceases in the presence of excess glucose the available NADP supply would be converted to NADPH which would accumulate because of its decreased utilization. The accumulat’ion of NADPH in the stationary phase would cause activation of enteric bacterial ADP-glucose synthesis which in turn would promote glycogen synthesis. Due to the inhibition of the hexose monophosphate shunt pathway both glucose-6-P and fructose-6-P may also accumulate from glucose due to glucokinase and phosphohexoseisomerase action. The increased accumulation of fructose-6-P would overcome the inhibition of phosphofructokinase by ATP (27, 28) at the high energy charge levels present in these conditions thereby causing formation of fructose-dip. Accumulation of fructose diphosphate would thereby also cause activation of ADP-glucose pyrophosphorylase. Fructose-dip and other glycolytic intermediates that stimulate ADP-glucose synthesis (2-phosphoglycerate, phosphoenolpyruvate, glyceraldehyde-3-P) may also be pertinent in the activation of glycogen synthesis when the organism is grown on carbon sources such as tricarboxylic acid intermediates, amino acid, or glycerol. At present it is difficult to explain the

REGULATION

OF

ADP-GLUCOSE

participation of pyridoxal phosphate in the regulation of ADP-glucose and glycogen synthesis. It is possible that PLP synthesis in stationary phase becomes derepressed enabling its concentration to increase under the conditions that glycogen synthesis occurs. Alternatively, under conditions of nikogen limitation it is also possible that the tobal pyridoxine pool in the cell is mainly in the form of pyridoxal phosphate, since the pyridoxamine-P levels may be lowered when a nitrogen source is not available. The PLP concentration might increase under nitrogen-limiting conditions at the expense of the pyridoxamine-P pool and a greater concentration of pyridoxal-P may then be available for the activation of the ADP-glucose pyrophosphorylase when protein synthesis and amino acid metabolism are limited. Recent results indicate that mutants of E. coli that accumulate more glycogen than their wild-type strains contain an ADP-glucose pyrophosphorylase that have a higher affinity for FDP and a lower affinity for 5’-AMP than the parent-strain enzyme (8, 29-32). The activation by NADPH and PLP has been studied for one of these “glycogen excess” mutant ADP-glucose pyrophosphorylases and it was found to have a higher apparent affinity for these activators than the wild-type enzyme (31). It appears therefore, that the activation and inhibition phenomena observed in vitro are physiologically significant. It should be pointed out that studies of the regulation of ADP-glucose pyrophosphorylases from other bacteria and from plants reveal that in these instances other glycolytic intermediates are much more effective activators than FDP and that NADPH and PLP are totally ineffective activators (6-9, 17, 33, 34). The nature of the activat,ors of the ADP-glucose pyrophosphorylase of the organism or tissue appears to be related to its mode of carbon metabolism and t’his has been discussed previously (7, 8). The only ADP-glucose pyrophosphorylase so far studied having no activation phenomenon associated with it is that found in S. marcescens extracts. This enzyme is inhibited, however, by 5’ adenyl-

SYNTHESIS

ate (unpublished experiments) the subject of a future report.

691 and will be

REFERENCES 1. GREENBERG, E., AND PREISS, J., J. Biol. Chem. 239, 4314 (1964). 2. SHEN, L., GHOSH, H. P., GREENBERG, E., AND PREISS, J., Biochim. Biophys. Acta 89, 370 (1964). 3. SHEN, L., .~ND PREISS, J., J. Biol. Chem. 240, 2334 (1965). 4. GREENBERG, E., .\IVD PREISS, J., J. Biol. Chem. 240, 2341 (1965). 5. SHEN, L., AND PREISS, J., Arch. Biochem. Biophys. 116, 374 (1966). 6. PREISS, J., SHEN, L., GREENBERG, E., AND GENTNER, N., Biochemistry 6, 1833 (1965). 7. FURLONG, C. E., .~ND PI~EISS, J., J. Biol. Chem. 244, 2539 (1969). 8. PREISS, J., in “Current Topics in Cellular Regulation” (B. L. Horecker and E. R. Stadtman), Vol. 1, pp. 125-160. Academic Press, New York (1969). 9. EIDELS, L., EDELMANN, P., BND PREISS, J., Arch. Biochem. Biophys. 140, 60 (1970). 10. GENTNER, N., AND PREISS, J., J. Bid. Chem. 243, 5882 (1968). 11. GENTNEI~, N., GREENBERG, E., AND PREISS, J., Biochem. Biophys. Res. Commun. 36, 373 (1969). 12. CHELDELIN, H. V., WANG, C. H., AND KING, T. E., in “Comparative Biochemistry” (M. Florkin and H. 8. Mason), Vol. III. Academic Press, New York (1962). 13. FRAENKEL, D. G., AND HORECKER, B. L., J. Biol. Chem. 239, 2765 (1964). 14. CHOJNICKI, T., S.I~ICK.~, T., AND KORZYBSKI, T., Acta Biochim. Pal. 16, 293 (1968). 15. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., AND Rrl~~.\~~, R. J., J. Biol. Chem. 193, 265 (1951). 16. SHEN, L., AXD PREISS, J., Biochem. Biophys. Res. Commun. 17, 424 (1964). 17. GHOSH, H. P., AND PREISS, J., J. Biol. Chem. 241, 4491 (1966). 18. CHANGEUX, J. P., Cold Spring Harbor Symp. Quant. Biol. 26, 497 (1963). 19. ATKINSON, D. E., AND WALTON, G. M., J. Biol. Chem. 242, 3239 (1967). 20. ATKINSON, D. E., Biochemistry ‘7, 4030 (1968). 21. KLUNGSYOR, L., HAGEMAN, J. H., FALL, L., AND ATKINSON, D. E., Biochemistry 7, 4035 (1968). 22. SHEN, L., AND ATKINSON, D. E., J. Biol. Chem., in press, 1970. 23. D~WES, E. A., AND RIBBONS, D. W., Bacterial. Rev. 28, 126 (1964).

692

RIBGREAU-GAYON

24. HOLME, T., AND PALMSTIERNA, H., Acta Chem. &and. 10, 578 (1956). 25. SIGAL, N., CATTANEO, J., AND SEGEL, I. H., Arch. Biochem. Biophys. 108, 440 (1964). 26. MODEL, P., AND RITTENBERG, D., Biochemistry 6, 69 (1967). 27. ATKINSON, D. E., AND WALTON, G. M., J. Biol. Chem. 240, 757 (1965). 28. BLBNGY, D., But, H., AND MONOD, J., J. Mol. Biol. 31, 13 (1968). 29. GOVONS, S., VINOP~L, R., INGRAHAM, J., AND PREISS, J., J. Bacterial. 97, 970 (1969). 30. DAMOTTE, M., CATTANEO, J., SIGAL, N., AND PUIG, J., Biochem. Biophys. Res. Commun. 32, 916 (1968).

ET

AL.

31. PREISS, J., GOVONS, S., EIDELS, L., LAMMEL, C., GREENBERG, E., EDELMANN, P., AND SABRAW, A., in “Proceedings of the Miami Winter Symposia” (W. J. Whelan). North-Holland, Amsterdam, in press, 1971. 32. CATTANEO, J., DAMOTTE, M., SIGAL, N., SANCHEZ-MEDINA, P., AND PUIG, J., Biochem, Biophys. Res. Commun. 34, 694 (1970). 33. SANWAL, G. G., AND PREISS, J., Arch. Biothem. Biophys. 119, 45 (1967). 34. SANWAL, G. G., GREENBERG, E., HARDIE, J., CAMERON, E. c., .~ND PREISS, J., Plant Physiol. 43, 417 (1968).