metabolism reserve polysaccharide streptococcus …jb.asm.org/content/127/1/281.full.pdfparticulate...

JOURNAL OF BACTERIOLOGY, July 1976, p. 281-290Copyright C 1976 American Society for Microbiology

Vol. 127, No. 1Printed in U.S.A.

Metabolism of the Reserve Polysaccharide of Streptococcusmitior (mitis): Is There a Second a-1,4-Glucan Phosphorylase?

ALEXANDRA PULKOWNIK AND GWEN J. WALKER*Institute ofDental Research, Surry Hills, Sydney, New South Wales 2010, Australia

Received for publication 29 December 1975

The a-1,4-glucan phosphorylase (a-1,4-glucan: orthophosphate glucosyltrans-ferase; EC 2.4.1.1) associated with the particulate cell fraction of Streptococcusmitior strain S3 was compared with the soluble maltodextrin phosphorylase thathad been previously isolated from the same organism (Walker et al., 1969). Theparticulate enzyme was more sensitive to the glycogen content of the cell thanthe soluble enzyme; its activity was highest when the cells were grown underconditions favoring high glycogen storage. Substrate specificities of the twophosphorylases were also different. The particulate phosphorylase exhibited ahigh activity towards endogenous glycogen, whereas low-molecular-weight mal-todextrins were the preferred substrates for the soluble phosphorylase. Thepurification of the particulate phosphorylase included incubation of the particu-late fraction in 160 mM sodium phosphate-10 mM sodium citrate-O.1% (wt/vol)Triton X-100 buffer (pH 6.7) and ion-exchange chromatography on diethylamino-ethyl-Sephadex A-50. The purified enzyme was fully soluble. The value for thepurification factor was variable and depended on (i) the substrate used and (ii)whether the synthetic or the degradative reaction was being measured. Thesolubilization resulted in considerable changes in the properties of the phospho-rylase: the pH optimum for activity was raised from 6.0 to 7.0-7.5 and thesubstrate specificity was altered. Consequently, the purified enzyme boregreater similarity to the soluble maltodextrin phosphorylase. The reportedresults are best explained in terms of a single phosphorylase, the specificity ofwhich is determined by its binding state in the cell. The enzyme acts as aglycogen phosphorylase in the particulate state and as a maltodextrin phospho-rylase when soluble. The equilibrium between the two forms is related to theglycogen content of the cells.

The function of glycogen and starches as en-ergy reserve materials in animals and plants iswell established; a similar role has been attrib-uted to microbial glycogen (8). Microbial cellscontaining intracytoplasmic glycogen granuleshave been detected in large numbers in dentalplaque, particularly in the regions overlyingcarious lesions (11). The link between the cari-ogenicity of these organisms and glycogen stor-age has been explained in terms of greatly pro-longed production of lactic acid, which initiatesthe demineralization of enamel. Detailed stud-ies of glycogen storage in two oral streptococci,Streptococcus mitior (12) and S. salivarius (16),substantiate this hypothesis.Glycogen phosphorylase has been detected in

all cells that store glycogen. Although the en-zyme can catalyze both the synthesis and thedegradation of a-1,4-glucans in vitro, its physi-ological function is considered to be degradative(25). Glycogenolysis in animal cells is regulatedby the presence of two forms of the enzyme; the

interconversion of these forms is finely con-trolled by hormonal and functional require-ments (25). There is no evidence for intercon-vertible forms of phosphorylase in plants, andthe glycogen phosphorylases isolated from Neu-rospora crassa (31) and Saccharomyces cerevi-siae (10) are the only microbial phosphorylasesthat resemble the animal enzymes in beingpresent in two interconvertible forms. BothEscherichia coli and S. salivarius cells elabo-rate two phosphorylases of different specifici-ties: one is a glycogen phosphorylase, whereasthe other phosphorylase acts preferentially onlow-molecular-weight, linear a--1,4-glucans,maltodextrins (19, 26).

Several enzymes involved in glycogen metab-olism have been isolated from the soluble cellfractions ofS. mitior (3, 33, 34). Only one phos-phorylase was found among these (35). Al-though the enzyme was not inducible by malt-ose, it was called maltodextrin phosphorylaseon the basis of its specificity pattern. The pres-

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282 PULKOWNIK AND WALKER

ent work was prompted by the finding of aphosphorylase activity in the pellet obtainedafter centrifuging the disrupted cells. The highactivity of this enzyme towards endogenous gly-cogen indicated the possibility that it was atrue glycogen phosphorylase.

In this paper we report the isolation of thisparticulate phosphorylase, compare its proper-ties with those of maltodextrin phosphorylase,and discuss the significance of its location inthe cell in relation to its function.

MATERIALS AND METHODSMaterials. Rabbit-liver (RL) glycogen (type Ill)

and dextrin were purchased from Sigma ChemicalCo. (St. Louis, Mo.) and Thomas Kerfoot (Bardsley,Lancashire, England), respectively. Waxy maizestarch (amylopectin) and its 8-amylase ((3-) limitdextrin were prepared as described by Walker (33);the preparation of S. mitior glycogen has also beendescribed previously (35). Phosphorylase (O-) limitdextrin of amylopectin was a gift from E. Y. C. Lee.Maltodextrins (maltose, G2; maltotriose, G3; malto-tetraose, G4; ... ) were prepared from an acid hy-drolysate of amylose as described by Whelan et al.(37) and purified further by paper chromatography.a-D-Glucopyranosyl phosphate (G1-P) was isolatedas a potassium salt from the products of digestion ofstarch by potato juice and contained no primer forphosphorylase. All other chemicals were analyticalreagent grade.The preparation of S. mitior maltodextrin phos-

phorylase has been described elsewhere (35).Organisms. S. mitior, strain S3, isolated from

human dental plaque, was obtained from R. J. Gib-bons; strain 439 was supplied by the Department ofBacteriology, University of Melbourne (Melbourne,Australia); strain RB1633 was obtained from G. Col-man. For storage, the cells were freeze-dried.Medium. The medium used for all routine growth

contained (per liter): Trypticase (BBL), 20 g; yeastextract (Difco), 5 g; K2HPO4, 4 g; KH2PO4, 1 g; NaCl,2 g; and glucose, 20 g; unless otherwise indicated.Growth conditions. Cells were grown routinely in

a fermenter (MF-114, New Brunswick Scientific Co.,New Brunswick, N.J.) for 16 h. The pH of the me-dium in the fermenter was adjusted to pH 6.0 withlactic acid prior to inoculation. The pH was main-tained by automatic addition of NaOH. Growth wasanaerobic (N2 + CO2 [95:5]) at 37 C.

Cells were harvested by centrifugation at 4 C,washed twice with 67 mM sodium phosphate buffer(pH 7.1), and suspended in 34 mM sodium phos-phate-25 mM sodium citrate buffer (pH 6.8).

Preparation of cell fractions. All subsequentwork was carried out at 0 to 4 C. The cells weredisintegrated by shaking with glass beads (0.10 to0.11 mm in diameter, Glasperlen, B. Braun) in a cellhomogenizer (MSK, B. Braun, Melsunger, WestGermany). Equal volumes of beads and cell suspen-sion (25 ml of each) were shaken at 2 C for 3 min;more than 90% of the cells were disrupted duringthis period. The beads were filtered off and washedwith 25 ml of 50 mM sodium citrate buffer (pH 6.5).

The filtrates were centrifuged at 34,800 x g for 10min to yield a soluble cell fraction and a pellet. Theformer was dialyzed against 50 mM sodium citratebuffer (pH 6.5) and used as a source of crude malto-dextrin phosphorylase. The pellet was washed twicewith the same buffer (preparatory washing) andhomogenized using a Teflon pestle tissue grinder.The resulting suspension (the particulate cell frac-tion) contained 1 to 3 mg of glucan and 10 to 100 mgof protein per ml of suspension. It was stored at-20C and rehomogenized after thawing. When amore concentrated preparation was required, thesuspension was centrifuged at 34,800 x g for 10 minand the pellet was resuspended in a smaller volume.This resulted in a complete recovery of both glucanand phosphorylase activity. A similar procedure wasused when a change of buffer was required.Enzyme assays. All enzyme assays were per-

formed under toluene vapor. The phosphorylase ac-tivity was measured in both the synthetic and deg-radative directions by assay I and assay II, respec-tively. All assays were carried out at 35 C. Assay Idigest mixture contained (in 1.0 ml): G1-P, 32 ,umol;CuS04, 0.6 gmol; primer, 2.5 mg; sodium citratebuffer (pH 6.5), 19 ,umol; and enzyme. Aliquots (0.2ml) were withdrawn from the digest during thecourse of the reaction and used for estimation ofinorganic phosphate (Pi) (1) after deproteinizationwith 0.8% (wt/vol) trichloroacetic acid. The additionof CuSO4 and the protein precipitation step wereomitted in the assays of purified enzymes. Primerwas omitted from the assay of particulate phospho-rylase because enzyme in this fraction was fullyactive in its absence. It was established that therelease of Pi was not due to the action of phospha-tase, as indicated by the absence of glucose in themixture. Assay II digest mixture contained (in 1.0ml): substrate, 2.5 mg; CuSO4, 0.6 ,umol; sodiumphosphate buffer (pH 6.5), 160 ,umol; and enzyme.The release of G1-P in aliquots (0.5 ml) of the digestswas estimated by the method of Liddle and Manners(22). The excess Pi was removed from the samples byprecipitation as a magnesium ammonium complex.The G1-P content was then determined as the differ-ence in Pi before and after a 7-min hydrolysis in theperchloric acid mixture used in the Allen estimation(1).Maltohexaose (G6) or RL glycogen were used rou-

tinely as substrates in these assays. When an en-zyme preparation was assayed independently withboth these substrates under standard conditions, thetwo activities were expressed as a ratio, G6/Gly. ADEX/GLY ratio was obtained similarly when G6was replaced by an equal weight of dextrine (a mix-ture of maltodextrins). As activities obtained withdextrine were only 50% of those with G6, DEX/GLY= 0.5 G6/Gly.

Units. One enzyme unit represented the release of1 gmol of a product per minute under standardassay conditions. Unless otherwise specified theunits were determined in the synthetic reaction.Units of the particulate activity were calculatedfrom the results obtained in the absence of addedprimer, whereas G6 primer was used with the puri-fied enzymes.

Analytical procedures. Protein content of particu-

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S. MITIOR a-1,4-GLUCAN PHOSPHORYLASE 283

late fractions was determined by the micro-Kjeldahlmethod (23), whereas protein in the soluble prepara-tions was estimated using Hartree's modification ofthe Folin-Ciocalteu procedure (17). Crystalline bo-vine serum albumin, fraction V (Sigma ChemicalCo.), was used as the standard. Estimation of totalhexose content of polysaccharide solutions was de-termined by the primary cysteine-sulphuric acidmethod (9). Glucan content of cell fractions wasestimated after acid hydrolysis in 1.5 N H2SO4 (3 h,100 C) by the glucose oxidase-peroxidase method (7).

Iodine stain was prepared as follows. A samplecontaining 100 &g of glucan was treated with 0.1 mlof iodine reagent (0.2% I2 in 2% KI) and diluted to 2ml. The intensity of the colored complex was mea-sured as absorbance at 490 nm.

Sugars were separated by descending paper chro-matography on Whatman no. 3 MM paper by irriga-tion with ethyl acetate-pyridine-water (10:4:3, vol/vol/vol). The papers were developed with Ag NO3-NaOH reagent (32).Enzyme purification. A portion of the particulate

fraction was diluted fivefold in a digest containing160 mM sodium phosphate-10 mM sodium citrate-0.1% (wt/vol) Triton X-100 buffer (pH 6.7); the glu-can content of the digest was approximately 0.3 mg/ml. The mixture was incubated for 16 h at 35 C andthen centrifuged at 1,800 x g for 20 min. The super-natant fluid was retained, and the pellet waswashed with 1/3 the original volume of 50 mM so-

dium citrate buffer (pH 6.5). The washed pellet ishenceforth referred to as the "residual particulatefraction." All subsequent operations were carriedout at 2 C. The washings and the supernatant fluidwere combined (extracted enzyme) and dialyzedagainst 10 mM sodium phosphate buffer (pH 6.7).The dialyzed solution was stirred into a slurry ofdiethylaminoethyl (DEAE)-Sephadex A-50 (Phar-macia, Uppsala, Sweden) preequilibrated in thesame buffer. After 30 min, the gel was washed withthe above buffer containing 0.1 M NaCl until thewashings were clear. The gel was then packed into aglass column and eluted with a gradient of NaClformed by the continuous addition of 0.6 M NaCl-10mM sodium phosphate buffer (pH 6.5) to 250 ml of0.1 M NaCl-10 mM sodium phosphate buffer (pH6.5).

RESULTSEffect of growth conditions on phosphoryl-

ase activity of cell fractions. The distributionof phosphorylase activity among the cell frac-tions of S. mitior S3 is shown in Table 1; thispattern was obtained repeatedly. The resultsindicate that one-third of the total phosphoryl-ase activity and less than 10% of the endoge-nous glucan were firmly bound in the particu-late fraction.The highest levels of phosphorylase activity

in both the cell fractions were reached at thepoint ofmaximum accumulation of glycogen bythe cells. This occurred in the early stationaryphase of growth (16 h after inoculation).

Phosphorylase activities in the cell fractions

TABLz 1. Distribution ofphosphorylase activity andendogenous glucan in the cell fractions ofS. mitiora

Cell fraction Phosphory- Glucanlaseb

Soluble 67 62Particulate 33 8Preparatory washingsc 0 30

a Distribution is expressed as the percentage oftotal cell content.

b Activities were determined by assay I with gly-cogen primer.

c As defined in the text.

of strains differing in their capacity to storeglycogen were found to vary with glycogen stor-age (Table 2). The particulate phosphorylasewas far more dependent on the amount of glyco-gen stored by the cells than the soluble enzyme.

Glucose content of the growth medium isknown to affect glycogen storage in S3 cells(13). Our work indicated that, when the glucoselevel of the growth medium was lowered to0.1%, the phosphorylase activities of the solubleand particulate fractions were reduced to 50and 14% of the normal levels, respectively. Theparticulate cell fraction obtained from cellsgrown in the absence of glucose contained onlya trace of phosphorylase activity (0.5 ,umol ofP,/h per liter of culture), whereas the glucanwas low and variable. When the soluble frac-tion obtained from these cells was fractionatedon a DEAE-Sephadex A-50 column, a singlepeak of phosphorylase activity was eluted with0.38 M NaCl. The DEX/GLY ratios for fractionsthroughout the peak were identical (= 10), in-dicating the absence of two phosphorylaseswith different substrate specificities. By apply-ing a similar technique, Chen and Segel wereable to detect the presence ofglycogen and mal-todextrin phosphorylases in the cell extracts ofE. coli cells (5).

Properties of the particulate phosphoryl-ase. The gradual increase in the absorbance ofthe iodine-glycogen complex that accompaniedthe release of Pi during the incubation of partic-ulate fraction with G1-P (Fig. 1) establishedthat the endogenous glucan was a primer forthe phosphorylase. Since branching enzyme(assayed as in reference 34) was also present inthe particulate fraction, there was no increasein Xmax of the iodine-glycogen complex (37) dur-ing the course of the reaction.The endogenous glucan was a very efficient

primer for the particulate phosphorylase. Theaddition of either RL or S. mitior glycogen (2.5mg/ml) to the assay digest did not affect theactivity of the enzyme, whereas the addition ofan equal weight of G6 resulted in only 20 to 30%activation. The ratio G6/Gly was therefore usu-

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ally close to unity, in spite of the fact that theconcentration of the added primers was in 10-fold excess over the endogenous glucan. In con-trast, the effectiveness of the latter as a sub-

strate for phosphorolysis was relatively poor(Table 3) and variable. The addition of exoge-nous substrates always resulted in further in-creases in activity.The activity of the particulate phosphorylase

TABLE 2. Distribution ofphosphorylase activity inthe cell fractions ofS. mitior strains varying in their

capacity to store glycogena

Phosphorylase activity"Strain (U/mg)c Glycogen stor-aged

Soluble Particulate

S3 0.32 0.04 High439 0.21 0.005 MediumRB1633 0.11 0.001 Low

a Cell fractions were prepared as described in thetext.

b Activity was determined in assay I with dex-trine primer for the soluble fraction and glycogenprimer for the particulate fraction.

c Micromoles of P, released per minute per milli-gram of protein.

d From Walker et al. (35).

E

tO_aSIL

In

10 - 10 Z

s / , os o 4I I 0 0

O 6 12 18 24

TIME (h)

FIG. 1. Action of the particulate phosphorylase on

endogenous glucan. The progress of the reaction wasmeasured by the release ofPi (i) and the increase iniodine stain (U) in assay I digest containing theparticulate fraction (0.6 mg ofglucan and 78 mU ofdigest per ml).

J. BACTErOL.

was proportional to the volume of the enzyme,but the rate of initial reaction was not linearand increased with time. This unproportionalincrease is probably due to the increase in thenumber of side chains resulting from the actionof branching enzyme on the chains elongatedby the initial action of phosphorylase. The con-ditions of pH and temperature optimum foractivity are shown in Table 4. Of the variousbuffers tested, only sodium citrate and sodiumethylenediaminetetraacetate were found suita-ble (Table 5). Citrate activated the enzyme atconcentrations up to 20 mM, but became inhibi-tory at higher concentrations (Fig. 2). The

greater activity obtained in tris(hydroxy-methyl)aminomethane (Tris)-maleate as com-

pared with Tris-hydrochloride suggested a re-quirement for chelators.

The activity ofparticulate phosphorylase wasincreased 30% by the addition of 0.1% Triton X-100 and 10% by 10mM MgSO, and adenosine 5'-monophosphate (6 mM); 10 mM NaF inhibitedthe activity by 10%. Substantial increases inthe activity of the enzyme were produced byfreezing and thawing. The first cycle produced36% increase in activity; four subsequent cyclesresulted in an additional increase of 20%. In-creases in the particulate activity were alsoobtained if the cells were frozen before disrup-tion.

Purification of the particulate phosphoryl-ase. The phosphorylase activity could not beextracted from the particulate fraction by re-

peated washing with buffer or by incubationwith RL glycogen solution. Treatment of thefraction with either crude salivary a-amylaseor glucoamylase for several hours did not solu-bilize the enzyme, despite the fact that endoge-nous glucan was fully hydrolyzed and solubi-lized. Incubation with Cytophaga extract (BDHChemicals, Poole, England), which was used asa source of glycogen debranching enzyme, iso-amylase (15), resulted in 80%o hydrolysis of theendogenous glucan and an apparent loss of all

TABLE 3. Apparent changes in substrate specificity during the purification ofparticulate phosphorylase

Synthesis PhosphorolysisCell fraction

G6° Gly' G6/Gly GO' Gly' G6/Gly

Soluble 40.0 5.8Particulate 35.7 27.6 1.3" 6.6 0.3 22.0"Residual particulate 34.3 29.2 1.2" 5.9 0.05 125.0Extracted 2.3b 7.3Purified 80.0 3.5Maltodextrin phos- 22.0 5.3phorylasea Activities were determined by standard methods with both G6 and glycogen and are expressed in

micromoles of product per hour per milliliter of untreated particulate fraction.b Endogenous glucan is also a substrate.


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TABLE 4. Conditions for optimal activity ofS. mitiorphosphorylasesa

Enzyme pH Temp,era-ture (C)

Crude particulate 6.0 54cPurified 7.0_7.5d 54cMaltodextrin phos- 6.0-7.0 40-50phorylaseea Assay I digests with G6 primer were used." Determined in 66 mM sodium citrate buffer.c Determined in sodium citrate buffer, pH 6.5.d Determined in 66 mM sodium citrate and 22 mM

Tris-maleate buffers.e From Walker et al. (35).

TABLE 5. Effect of buffers on the activities of S.mitior phosphorylasesa

Particulate phospho-rylase Maltodex-

Buffer trin phos-Crude Ex- Puri- phorylasetracted fied

Sodium citrate 100 100 100 100Tris-hydrochoride 18 98Tris-maleate 33 90Sodium cacodylate 5 89 100Sodium (3-glycerophos- 5 10 75 97phate

Sodium ethylenedia- 118 105minetetraacetatea Buffers were compared at 20 mM (pH 6.5) in assay I

digests with G6 primer. Activities are expressed relative tothose in citrate buffer.

phosphorylase activity from the particulatefraction; the turbidity of the incubation mix-ture was also decreased. This method for solubi-lizing phosphorylase was not developed furtherbecause a component of Cytophaga extract in-terfered with both assays of phosphorylase ac-tivity.Although the conditions for extraction de-

scribed above resulted in maximum yield, noone individual component was essential. Forexample, the omission of either phosphate ordetergent lowered the yield by 30%, whereas 10mM citrate could be replaced by 5 mM sodiumethylenediaminetetraacetate without any no-ticeable effect. The yield of the extracted phos-phorylase reached a maximum after 6 h of incu-bation and remained constant thereafter. Theextraction process was pH dependent; a pHvalue of 6.5 was optimal.A single incubation removed all the extracta-

ble enzyme. Reincubation of the residual pelletdid not extract more enzyme despite the factthat the pellet still contained considerable ac-tivity that frequently registered little or no losscompared with the original (Table 3). There-fore, the degree of extraction was determinedby assaying the extracted material in the syn-

thetic direction with dextrine primer. Approxi-mately 50% of the endogenous glucan was ex-tracted by this procedure. The remaining glu-can did not stain with iodine and could not bephosphorolysed further by the associated en-zyme. It was a far more efficient primer thanany added glucans.Subsequent ion-exchange chromatography of

the extracted enzyme (Fig. 3) resulted in fullrecovery of phosphorylase activity, with 99% oftotal protein removed. The preparation stillcontained a trace of glucan (15 ug/U), whichwas inactive as a substrate in both the syn-thetic and phosphorolytic reactions. Trace con-tamination by branching enzyme could only bedetected after incubation for 16 h. Column flowrates obtained in this experiment were verypoor. More rapid rates (15 ml/h) were obtainedwhen Triton X-100 was present throughout thepurification. This was probably due to a moreefficient removal of inactive material duringbatchwise washing of the gel. However, TritonX-100 interfered with the determination of en-zymic activity, and its removal at the end of thepurification was very difficult. Therefore, werecommend that the detergent be retained dur-ing the batchwise washing of the gel and re-moved by repeated washing with 0.1 M NaCl-10mM sodium phosphate buffer (pH 6.7) beforethe gel is packed into the column.A summary of a typical purification appears

in Table 6. The activity with respect to G6showed a moderate degree of purification forboth the synthetic and the degradative reac-tions, whereas with glycogen substrate the re-sults imply purification of the enzyme withphosphorolytic function. Although changes in

100

so

60

c 40

20~

SODIUm CITRATE (.M)

FIG. 2. Effect of sodium citrate concentration onthe activities ofS. mitior phosphorylases. The activi-ties ofparticulate (-) and purified (0) phosphoryl-ases and of maltodextrin phosphorylase (A) weredetermined at various concentrations of sodium cit-rate buffer (pH 6.5) in assay I digests and are ex-pressed relative to those in 20 mM citrate (= 100).

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Ez~~~~~~~~°52C Zo 40 50 60~~~~~~~~~~~~~~~~~~~~~

C00 1,

0

FRACTION NUMBER

FIG. 3. Purification of the extracted phosphorylase by chromatography on DEAE-Sephadex A-50. Theenzyme was extracted from the particulate fraction (containing 18 U ofphosphorylase activity) and treatedwith DEAE-Sephadex (dry weight, 7 g) as described. The gel column (50 by 1.9 cm) was eluted with agradient ofNaCl in 10 mM sodium phosphate buffer (pH 6.5) at the rate of3 ml/h. Activity in the fractions (6ml) was determined by assay II. The peak ofphosphorylase activity was eluted with 0.39 M NaCl; fractions 48to 54 were pooled, dialyzed against 50 mM sodium citrate buffer (pH 6.5), and concentrated by ultrafiltrationusing a UM 20-E membrane (Amicon Corp., Lexington, Mass.). The activity ofthe final preparation was 1.29U/ml. Symbols: 0, phosphorylase activity; El, absorbance at 280 nm; A, molarity of NaCl.

TABLE 6. Summary ofpurification of S. mitior particulate phosphorylaseParticulate Purified

ReactionSubstrate Yield W ~~~~~~~~~~~~~PurificationReaction Substrate Total act Sp act (U/ Total act Sp act (U/ Yield (%) (-fold)(U)a mg)b (U)a mg)b

Synthesis G6 23.8 0.05 7.2 6.3 30 122Glycogen 18.3 0.04 0.08 0.07 0.5 1.9

Phosphorolysis G6 44.0 0.01 1.57 1.38 36 145Glycogen 0.21 0.005 0.46 0.4 222 890

a Expressed as micromoles of product per minute.b Expressed as micromoles of product per minute per milligram of protein.

G6/Gly ratios for phosphorolytic reaction alsosuggest that a glycogen phosphorylase hadbeen purified, the opposite must be concludedfrom the trend of synthetic G6/Gly (Table 3).This apparent discrepancy arises from the factthat in some fractions the endogenous glucanwas the preferred substrate, thereby invalidat-ing G6/Gly as a determinant ofenzyme specific-ity. The large change in the phosphorolytic G6/Gly of the particulate fraction after extractionwas apparently caused by the change in actionon glycogen only (Table 3). This is best ex-plained in terms of the poor ability of the partic-ulate enzyme to act on added glycogen. Thisinability was probably masked prior to the ex-traction by the action of the enzyme on endoge-nous glucan. On the other hand, activity to-wards G6 did not change appreciably after ex-traction, indicating that G6 was a true sub-strate.

Properties of the purified phosphorylase.The purified enzyme lost activity at the rate of40% per week at 2 C. It did not sediment aftercentrifugation at 104,000 x g for 1 h and could

therefore be considered fully soluble (6). Thesynthetic activity of the enzyme was linear forat least 60 min and in the presence of up to 34mU/ml. Although the temperature optimumfor activity remained the same, the pH opti-mum was raised after purification (Table 4).The enzyme was less selective with respect tobuffers (Table 5), although it exhibited thesame concentration-dependent effect with so-dium citrate as the crude enzyme (Fig. 2). So-dium chloride (0.1 M) activated the purifiedphosphorylase by 67%, but neither NaF noradenosine 5'-monophosphate (10 mM) had anyeffect on activity.We tested the adsorption of enzyme to var-

ious glucans using the following systems: (i) 10mg of RL glycogen plus 11 mU of enzyme; (ii)1% (wt/vol) defatted maize starch granules plus20 mU of enzyme; (iii) boiled residual particu-late fraction containing 4.8 mg ofglucan plus 27mU of enzyme per ml; (iv) concentrated anddialyzed eluate from DEAE-Sephadex chroma-tography of extracted enzyme containing 200,ug of glucan plus 10 mU ofenzyme per ml. The

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mixtures were incubated for 3 h at 2 C and thencentrifuged at 104,000 x g for 1 h to removeglucans. In each case the enzyme activity wasfully recovered in the supernatant fluid.

Substrate specificity. It became apparentthat the purified enzyme and maltodextrinphosphorylase of S. mitior were very similar;the latter was therefore included in the specific-ity studies.

Michaelis constants (K,,,) were obtained froma Lineweaver-Burk plot of initial velocitiesagainst substrate concentrations. The concen-tration ofglycogen was expressed as molarity ofthe nonreducing ends. In the synthetic reac-

tion, the value of K.. for glycogen was 21 mM(40 mg/ml), and for G6 the value was 3.0 mM.The K,,, for glycogen in the direction of degrada-tion was 5.0 mM (9.5 mg/ml). The Km values ofmaltodextrin phosphorylase were closely simi-lar- 1.5 mM for G6 and 14 mM for glycogen(35).Various branched a-1,4-glucans were com-

pared as primers for the two phosphorylases(Table 7). Except for the action on 8-limit dex-trin of amylopectin, the two enzymes respondedsimilarly to the primers tested. Amylopectinand its limit dextrins were better primers thanglycogen. On the completion of this experimentthe remainder of the digests were incubated for16 h at 35 C. In all cases the iodine stain of thedigest retained the deep-blue color of the stainwith Xm<ix at 580 nm. This indicated that tracesof branching enzyme did not interfere with thespecificity determination.The priming efficiency of maltodextrins was

also tested (Table 8). Maltotetraose (G4) wasthe first efficient primer of the series for both S.mitior phosphorylases, and the priming effi-ciency increased with the chain length. Onlythe purified enzyme could use maltose as aprimer (at 4% the rate of G4), whereas malto-

TABLE 7. Polysaccharides as primers for S. mitiorphosphorylases0

Purified Maltodextrinphosphorylase phosphorylase

Primer

Actb Relative Act Relativeactr acte

RL glycogen 0.24 1.0 0.07 1.0S. mitior glycogen 0.16 0.7 0.04 0.6Amylopectin (Ap) 1.37 5.6 0.34 4.9Ap #-limit dextrin 0.56 2.3 0.02 0.3Ap +-limit dextrin 0.49 2.0 0.15 2.2

a Primers were compared in assay I digests at 2.5 mg/mland with either 51 mU of maltodextrin phosphorylase perml or 0.32 U of purified phosphorylase per ml.

bExpressed as micromoles of P, per hour per milliliter ofdigest.

I Expressed relative to activity with RL glycogen.

TABLE 8. Maltodextrins as primers for S. mitiorphosphorylasesa

Chain . phosphorla Maltodextrin phospho-length rylaseof mal-todex- Actb Relative Actb Relativetrin Ac" actc ct actc

2 0.05 0.04 0 03 0.11 0.09 0.04 0.044 1.16 1.00 0.93 1.005 2.13 1.84 1.5d6 2.80 2.42 2.2d7 3.02 2.62 2.5da Primers were compared at 1 mM in assay I

digests with either 81 mU of purified enzyme per mlor 52 mU of maltodextrin phosphorylase per ml.

b Expressed as micromoles of Pi per hour per mil-lilter of digest.

c Activities relative to that of G4.d Unpublished data of G. J. Walker and A. Lav-

rova.

triose was the smallest substrate used by mal-todextrin phosphorylase. This difference inspecificity accounts for /3-limit dextrin of amy-lopectin being a better primer for the purifiedphosphorylase. As a result of its derivation thedextrin should contain equal number of malto-syl and maltotriosyl side chains. All of theseshould be suitable primers for the purifiedphosphorylase, whereas maltodextrin phospho-rylase could use only the maltotriosyl chainends.

Several reports of priming by maltose werelater shown to be caused by impurities in eitherthe enzyme or substrate preparations (20). Wecould find no evidence of primer for phosphoryl-ase in our G1-P preparation over an incubationperiod of 16 h. Maltose was chromatographi-cally pure. Maltose was also incubated withenzyme in the absence of G1-P for 16 h at 35 C,and the products were examined by paper chro-matography. No products higher than maltosewere detected, even though 300 jig of the sugarwas chromatographed. Therefore maltose is aprimer for the purified phosphorylase.The endogenous glucan of the particulate cell

fraction was also tested as a substrate in thephosphorolysis reaction using the two enzymes.A portion of particulate fraction that stained atypical reddish-brown glycogen color with io-dine was inactivated by heating and used as asource of glucan in assay II. The two phospho-rylase preparations were tested previously inphosphorolysis digests with RL glycogen so thatequal numbers of units with respect to thatglycogen were used in this experiment (2.6 mU/ml). The rate of phosphorolysis of the particu-late glucan by maltodextrin phosphorylase and

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purified phosphorylase was 18 and 26%, respec-tively, relative to that of the RL glycogen(100%).

DISCUSSIONThe foregoing results pose the interesting

question of why the purification of what ap-peared to be a glycogen phosphorylase yieldedan enzyme so similar to the second phosphoryl-ase produced by the cells. The only differencesin properties between the two enzymes were inthe temperature optima for activity and in theability to act on maltose.The effect of growth conditions on the activ-

ity of the particulate phosphorylase indicatedthat it was dependent on the cell glycogen lev-els. This suggested that the enzyme might beinvolved in a glycogen-protein complex. Leloirand Goldemberg reported that the incubation ofa particulate liver glycogen complex, contain-ing glycogen synthetase, with a solution of gly-cogen of low molecular weight resulted in therelease of synthetase activity into solution (21).This method proved unsuccessful in solubi-lizing S. mitior particulate phosphorylase. De-pletion of the liver cell glycogen by starvationresulted in solubilization of phosphorylase ac-tivity (30). Our attempts to detect a glycogenphosphorylase activity as distinct from malto-dextrin phosphorylase in the soluble cell frac-tions of S. mitior cells grown under conditionsresulting in minimal glycogen synthesis wereunsuccessful. Furthermore, once the particu-late enzyme had been purified it no longer ad-sorbed to branched a-1,4-glucans.Treatment of S. mitior particulate fraction

with a-amylase or glucoamylase indicated thatthe binding of phosphorylase was largely unaf-fected by the removal of endogenous glucan.Consequently, the solubilization of phosphoryl-ase activity by the Cytophaga preparation wasdue more to the contaminating lipolytic andchitinase activities (BDH product informationsheet 610D/1.D/037) than to isoamylase. Thisimplied the involvement of membranes in theglycogen phosphorylase complex.

Electron microscope studies of S. mitior cellsrevealed that glycogen synthesis occurred nearthe cell membrane (2). Since the last moleculesof glucose incorporated into glycogen were thefirst to be released during degradation (12), it isreasonable that phosphorylase should be inclose association with the membrane. The im-portance of a protein-glycogen membrane com-plex as a functional unit has been emphasizedrecently in a report describing the regulation ofglycogen phosphorylase in a muscle glycogenparticle. The results obtained with this prepa-

ration bore a greater resemblance to the in vivosituation than if a mixture of components hadbeen used (10).The involvement of membranes in the bind-

ing of S. mitior phosphorylase was also sub-stantiated by the changes in properties of theenzyme caused by purification. These changesoccurred only after the enzyme was fully solubi-lized and are typical of those recorded for mem-brane-bound enzymes: increases in recoveryafter purification, negligible losses of activityfrom the residual pellet, and changes in kineticand physical properties (6, 18, 29). Suchchanges have been explained in terms of transi-tion from a restricted micro-environment of themembrane to the soluble state (6).

Therefore, the low activity of the particulatephosphorylase towards glycogen was probablydue to the fact that a large substrate could notbind to an enzyme in a restricted conformation.A smaller substrate, such as maltohexaose,would not experience the same difficulty. Therapid action of the enzyme on the endogenousglucan indicated that the latter was bound in asterically favorable manner. Thus, low concen-trations of the endogenous glucan (0.5 mg/ml)elicited maximal activity, whereas high levels(40 mg/ml) of added glycogen were required forhalf-saturation of the enzyme after solubiliza-tion. This implies that solubilization produceda change in the conformation of the catalyticsite of the enzyme. Such a change could alsoexplain the priming by maltose.We therefore suggest that only one phospho-

rylase is elaborated by S. mitior cells and thatthe properties of this enzyme are determined byits binding state which, in turn, depends on thelevels of glycogen in the cell. It then becomesclear why under the conditions of starvationonly a single phosphorylase was detected in thecells.We observed that the activity of the particu-

late phosphorylase in fractions derived fromcells grown in 0.1% glucose was low. Yet thesecells could accumulate as much glycogen in thelog phase of growth as the cells grown in excessglucose (13). Since this glycogen was rapidlydegraded as soon as the exogenous glucose wasdepleted, the glycogen phosphorylase musthave been very active at that stage. These twoapparently conflicting observations can only beexplained in terms of a shift in equilibriumbetween bound and soluble forms of the enzymeafter the glycogen had been degraded. The si-multaneous appearance of glycogen and thephosphorylase activity in both cell fractionsduring cell growth also supports this theory. InE. coli, on the other hand, glycogen phospho-rylase activity reached a peak in the stationary

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phase of growth, whereas maltodextrin phos-phorylase was maximal in the log phase (4).Thus, the phosphorylase in S. mitior does not

conform to the "two-phosphorylase" theory sug-gested for other bacteria (24), especially sincethe soluble enzyme was not inducible by mal-tose.No other phosphorylase besides S. mitior en-

zyme is primed by maltose; the primer with theshortest chain length was either maltotriose(10, 36) or maltotetraose (14). The enzyme re-

sembles plant phosphorylases in its preferencefor amylopectin over glycogen primers. In fact,Km values for the phosphorolysis of glycogen byS. mitior enzyme and corn phosphorylase (20)were identical. Nevertheless, the reserve mate-rial in S. mitior is glycogen and not starch.The substrate specificity of S. mitior phos-

phorylase was in direct contrast to that of rab-bit muscle glycogen phosphorylase, whichshowed low affinity for maltopentaose (28). S.mitior phosphorylase was characterized by thehighest affinity constants for glycogen in thesynthetic reaction. The Km values for glycogenphosphorylase from E. coli and N. crassa were

6.7 mg/ml and 9 mg/ml, respectively (5, 27). Onthe other hand, the Km values of S. salivariusglycogen phosphorylase in both synthetic anddegradative reactions were very similar tothose of S. mitior enzyme (19).A degree of similarity between the phospho-

rylases ofS. mitior and S. salivarius was to beexpected, as these organisms share the same

ecological niche. The Km values could reflectthe low metabolic rate of oral organisms (19).However, the conventional kinetic constants ofthe solubilized enzyme become irrelevant inrelation to the rate of glycogen degradation ifthis process is carried out in the particulatephase where the substrate and the enzyme are

already in close proximity. If the major factordetermining the properties of S. mitior phos-phorylase is the physical state of the enzyme,then the binding process may well provide themeans for cellular control of glycogen metabo-lism.

ACKNOWLEDGMENTThis investigation was supported by a grant from the

Dental Board of New South Wales.

LITERATURE CITED

1. Allen, R. J. L. 1940. The estimation of phosphorus.Biochem. J. 34:858-865.

2. Berman, K. S., R. J. Gibbons, and J. Nalbandian. 1967.Localization of intracellular polysaccharide granulesin Streptococcus mitis. Arch. Oral Biol. 12:1133-1138.

3. Builder, J. E., and G. J. Walker. 1970. Metabolism ofthe reserve polysaccharide of Streptococcus mitis.

Properties of glycogen synthetase. Carbohydr. Res.14:35-51.

4. Chen, G. S., and I. H. Segel. 1968. kEcherichia colipolyglucose phosphorylases. Arch. Biochem. Bio-phys. 127:164-174.

5. Chen, G. S., and I. H. Segel. 1968. Purification andproperties of glycogen phosphorylase from Esche-richia coli. Arch. Biochem. Biophys. 127:175-186.

6. Coleman, R. 1973. Membrane-bound enzymes andmembrane ultrastructure. Biochim. Biophys. Acta300:1-30.

7. Dahlqvist, A. 1961. Determination of maltase and iso-maltase activities with a glucose oxidase reagent.Biochem. J. 8&0547-551.

8. Dawes, E. A., and D. W. Ribbons. 1964. Some aspects ofthe endogenous metabolism of bacteria. Bacteriol.Rev. 28:126-149.

9. Diwche, Z., L. B. Shettles, and M. Osnos. 1949. Newspecific color reactions of hexoses and spectrophome-tric micromethods for their determination. Arch. Bio-chem. 22:169-184.

10. Fischer, E. H., A. Pocker, and J. C. Saari. 1970. Thestructure, function and control of glycogen phospho-rylase, p. 23-68. In P. N. Campbell and F. Dickens(ed.), Essays in biochemistry, vol. 6. Academic PressInc., London.

11. Frank, R. M., and A. Brendel. 1966. Ultrastructure ofthe approximal dental plaque and the underlyingnormal and carious enamel. Arch. Oral Biol. 11:883-912.

12. Gibbons, R. J. 1964. Metabolism of intracellular poly-saccharide by Streptococcus mitis and its relation toinducible enzyme formation. J. Bacteriol. 87:1512-1520.

13. Gibbons, R. J., and B. Kapsimalis. 1963. Synthesis ofintracellular iodophilic polysaccharide by Streptococ-cus mitis. Arch. Oral Biol. 8:319-329.

14. Giri, N. Y., and D. French. 1971. Pseudo-priming ofEscherichia coli maltodextrin phosphorylase by 63-a-D-glucopyranosyl maltotriose. Arch. Biochem. Bio-phys. 108:505-510.

15. Gunja-Smith, Z., J. J. Marshall, E. E. Smith, and W. J.Whelan. 1970. A glycogen-debranching enzyme fromCytophaga. FEBS Lett. 12:96-100.

16. Hamilton, I. R. 1968. Synthesis and degradation ofintracellular polyglucose in Streptococcus salivarius.Can. J. Microbiol. 14:65-77.

17. Hartree, E. F. 1972. Determination of protein: a modifi-cation of the Lowry method that gives a linear photo-metric response. Anal. Biochem. 48:422-427.

18. Houslay, M. D., and K. F. Tipton. 1975. Rat livermitochondrial monoamine oxidase. A change in thereaction metabolism on solubilization. Biochem. J.145:311-321.

19. Khandelwal, R. L., T. N. Spearman, and I. R. Hamil-ton. 1973. Purification and properties of glycogenphosphorylase from Streptococcus salivarius. Arch.Biochem. Biophys. 154:295-305.

20. Lee, E. Y. C., and J. J. Braun. 1973. Purification andproperties of a-1,4-glucan phosphorylase from sweetcorn. Ann. N.Y. Acad. Sci. 210:115-128.

21. Leloir, L. F., and S. H. Goldemberg. 1960. Synthesis ofglycogen from uridine diphosphate glucose in liver. J.Biol. Chem. 235:919-923.

22. Liddle, A. M., and D. J. Manners. 1957. a-1,4-Glucos-ans. VIII. Multiple-branching in glycogen and amylo-pectin. J. Chem. Soc., p. 4708-4711.

23. McKenzie, H. A., and H. S. Wallace. 1954. The Kjeldahldetermination of nitrogen: a critical study of diges-tion conditions-temperature, catalyst, and oxidizingagent. Aust. J. Chem. 7:55-70.

24. Palmer, T. N., G. Wbber,id W. J. Whelan. 1973. Thepathways of exogeaous and endogenous carbohydrateutilizatjinnwsKEcherichia coli: a dual function for the

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enzymes of the maltose operon. Eur. J. Biochem.39:601-612.

25. Ryman, B. E., and W. J. Whelan. 1971. New aspect ofglycogen metabolism, p. 285443. In F. F. Nord (ed.),Advances in enzymology, vol. 34. Interscience Pub-lishers, New York. i

26. Schwartz, M., and M. Hofnung. 1967. Maltodextrinphosphorylase of Escherichia coli. Eur. J. Biochem.2:132-145.

27. Shepherd, D., and I. H. Segel. 1969. Glycogen phospho-rylase ofNeurospora crassa. Arch. Biochem. Biophys.131:609-620.

28. Smith, E. E. 1971. On the affinity of rabbit-muscleglycogen phosphorylase for highly branched macro-molecular substrates. Arch. Biochem. Biophys.146:380-390.

29. Srinivasan, R., A. Karczmar, and J. Bernsohn. 1972.Activation of acetylcholinesterase by Triton X-100.Biochim. Biophys. Acta 284:349-354.

30. Tata, J. R. 1964. Subcellular redistribution of liver a-

glucan phosphorylase during alterations in glycogencontent. Biochem. J. 90:284-292.

31. Tellez-In6n, M. T., and H. N. Torres. 1970. Intercon-vertible forms of glycogen phosphorylase in Neuro-

spora crassa. Proc. Natl. Acad. Sci. U.S.A. 66:459-463.

32. Trevelyan, W. E., D. P. Procter, and J. S. Harrison.1950. Detection of sugars on paper chromatograms.Nature (London) 166:444-445.

33. Walker, G. J. 1966. Metabolism of the reserve polysac-charide of Streptococcus mitis. Properties of a trans-glucosylase. Biochem. J. 101:861-872.

34. Walker, G. J., and J. E. Builder. 1971. Metabolism ofthe reserve polysaccharide of Streptococcus mitis.Properties of branching enzyme, and its effect on theactivity of glycogen synthetase. Eur. J. Biochem.20:14-21.

35. Walker, G. J., M. C. Lukas, and A. Lavrova. 1969.Metabolism of the reserve polysaccharide of Strepto-coccus mitis. Properties of maltodextrin phosphoryl-ase. Carbohydr. Res. 9:381-395.

36. Whelan, W. J., and J. M. Bailey. 1954. The actionpattern of potato phosphorylase. Biochem. J. 58:560-569.

37. Whelan, W. J., J. M. Bailey, and P. J. C. Roberts. 1953.The mechanism of carbohydrase action. 1. The prepa-ration and properties of maltodextrin substrates. J.Chem. Soc., p. 1293-1298.

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