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a-L-Fucosidase from

a

Soil Bacterium

K. MORTENSSON-EGNUND, R. SCHbYEN, C. HOWE, L. T. LEE, AND A. HARBOE

Virus Department B, National InstituteofPublicHealth, Oslo,Norway,andDepartmentofMicrobiology,

Columbia University, College of PhysiciansandSurgeons, New York,New York 10032

Received forpublication31December1968

Intracellular glycosidases were measured in cell-free extracts obtainedby

ultra-sonicdisruption of a gram-negative soil coccobacillus (Chase, 1938). From these extracts, a-L-fucosidase was purified about 120-fold by saltingout with

(NH4)2SO4,

ionexchange chromatography, and gel filtration. Theapproximate molecular weight of theenzyme was 50,000; its pH optimum was5. Theenzymewas inhibited by

L-fucose and split this sugar from a purified acid mucopolysaccharide from chicken

chorioallantoic fluid. The acidmucopolysaccharideis identical with a component

(hostantigen) of the hemagglutinin of influenza virus.Its antigenic reactivity is

altered by cell-tree extracts ofthe bacterium, in which the responsible enzyme is

thoughttobe ana-L-fucosidase.

A sulfated mucopolysaccharide derived from chicken chorioallantoic fluidhas been shown to be identical with aportion of the hemagglutinin

of egg-grown influenza virus (9-11). This

rela-tionship was demonstrated by the ability of the soluble mucopolysaccharide

[host

factor (HF)] to block the anti-hemagglutinin (antibody) for

influenzavirus producedinrabbits in response to theinjection ofnormalchorioallantoicmembrane. Purified HFcontains sugar constituents charac-teristic of blood group substances, notably hexosamine, galactose, and fucose (10). It was

thought, therefore, that enzymes known to de-grade blood group substances might be useful in further elucidating the structure of HF. One sourceof such enzymes was an unclassified cocco-bacillus originally isolated from soil by Chase

(2) andshownbyhimtodecompose blood group substances. Subsequent studies with this

bac-terium, by Gilmore and Howe (8, 8a), showed that cell-freeextracts (CFE) degradedA, B, and

0(H) substances down to diffusible oligosac-charides and monosacoligosac-charides. CFE from the

Chase organism have recently been found also

to destroy the serological activity of HF on the intact virion and in purified water-soluble form. Evidence to be reported separately supports the hypothesis that the enzyme responsible is an a-L-fucosidase. The present paper concerns at-tempts at separation of this enzyme from other glycosidases present in the crude CFE of the soil bacterium.

MATERIALS AND METHODS

Chemicals. p-Nitrophenyl-a-L-fucopyranoside, not

commerciallyavailablewhen we started the

investiga-tions,wassynthesizedaccordingto amethod described

byConchie andLevvy (4). Later, this substrate was obtained fromKoch-Light Laboratories Ltd., along

withp-nitrophenylglycosides of thefollowingsugars:

,B-L-fucopyranose, 2-acetamido-2-deoxy-,f-D-galacto-pyranose,2-acetamido-2-deoxy-f-D-glucopyranose, a-and 3-D-galactopyranose. Methyl-a-L-fucopyranoside

and methyl-,j-L-fucopyranoside were synthesized by

standard methods(19).

L-Fuconolactoneswereobtainedbybromine

oxida-tion(12) of L-fucose.Twocrystallinereactionproducts

wereisolated and characterizedby meltingpoint and

byinfraredspectrophotometricanalysis.Theprincipal

product wasthe known -y-lactone (1), which had a

melting point of104 to 104Candacarbonyl

absorp-tion at 1,750 cm-'. The other product, which isnot

described in the literature, had a melting point of

145 to 148 C and a carbonyl absorption at 1,717 cm-';itwasthoughtpossiblytobe the 4-lactone.

Ammonium sulfate (special enzyme grade) and

streptomycin sulfate were obtained from Mann

Re-search Laboratories Inc.; sucrose was from Difco;

andp-(hydroxy)mercuribenzoate (sodium salt),

a-D-fucose, a-L-fucOse, alkaline phosphatase type III-S

(E.coli),andhorseradishperoxidase(type VIapprox.

R.Z. 3.2) were fromSigma Chemical Co.

Diethyla-minoethyl (DEAE) cellulose (DE-22) was obtained

fromWhatman, CM-Sephadex C-50fromPharmacia,

andBio-Gel P-200 from Bio-Rad Laboratories.

Mcllvaine citric acid-phosphate, consisting of 0.1

M citric acid-0.2 M Na2HPO4, was used in the pH

range 3.0 to 7.8. Buffer solution A was Mcllvaine

buffer, pH 7, diluted 1:10 with distilled water;

buffersolutionBwasMcllvainecitric acidbuffer, pH

7, diluted 1:50 with distilled water. Gomory Tris

buffer, pH7.2, contained 0.2 M

tris(hydroxymethyl)-aminomethane (Tris)-0.2 N HCl. Thesolutions were stored in the cold with toluene to avoid bacterial

contamination. Methylpentose was determined by the method of Dische and Shettles (5).

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,a-L-FUCOSIDASE FROM A SOIL BACTERIUM

Propagation oforganism. The Chase coccobacillus

(unclassified, accessionno. 13949) wasobtainedfrom

theAmerican TypeCultureCollectionand was prop-agated accordingto theprocedures described by Gil-more and Howe (8) in a 0.1% solution of hog gastric mucin in 0.1% (NH4)2SO4 and 0.2% K2HPO4 ad-justed to pH 7.2. For large-scale production slight

modifications wereintroduced.A 40-gamountof hog

gastric mucin (Koch-Light Laboratories Ltd.) was

dissolved in 12 liters of distilled water and dialyzed

against tap water for 48 hr and distilled water for 18 hr. Themucin solution was then clarified by

continuous-flowcentrifugation in aServall centrifuge (12,000 x

g). Volumes of 300 ml of mucin were made up to 1 liter with salt solution [0.1% (NH4)2SO4 and 0.2%

K2HPO4, pH 7.2]. Dry weight determinations

indi-catedthe finalconcentration of mucin to be 0.01%. The medium was autoclaved at 120 C for 30 min.

Theorganismwaspropagatedin 10-literbottles.Each

bottle received aseedinoculumof 150 ml of a

3-day-oldcultureand wasincubatedat 30Cfor 2 to 3 days,

during which time aeration was effected for periods

of 1 hr, spacedat6-hr intervals. The organismswere

harvestedbycentrifugationat roomtemperaturein a

continuous-flow centrifuge (27,000 X g), and the cells

were washed in cold 0.01 Mcitric acidbuffer, pH 7.

Thereafter, all procedures were carried out at 4 C.

Onevolume of sedimented bacteria wasresuspended

in sixvolumes of the same buffer and disrupted by ultrasonicvibrations with aBransonSonifier (model S-125, position5) for 5min.Thedisruptedcells were

centrifugedat 12,000 X gfor 45 min in aServall

high-speedcentrifuge. The sediment wasdiscardedand the

CFE wasfrozen.

Enzyme assays. The glycosidaseswere assayed by

theprocedureofLewyandMcAllan (13),withminor

modifications. Theincubation mixture contained the

following components: 0.45 ml of Mcllvaine citric

acid-phosphate buffer (pH 5), 0.45 ml ofsubstrate (2

mMp-nitrophenyl-a-L-fucopyranoside or 5 mm solu-tions of other p-nitrophenylglycopyranosides) and

0.10 mlof suitably diluted enzyme solution. After 1

hr at 30C, the reaction wasstopped by the addition of3.5 mlof 1 N NaOH.The resultingyellow color,

which wasstable inthe alkalinemedium, was

meas-uredat420 nminaBeckmanBmodel

spectrophotom-eter. The amount of liberated p-nitrophenol was

determined by referenceto acalibrationcurve.

Suita-ble enzyme and substrate controls were included. A

unit ofenzyme wasdefinedasthe amount thatwould

liberate 1 ,umole of p-nitrophenol from the

p-ni-trophenyl-glycopyranoside per min at 30 C. The

specific activity was expressed as the number of

enzyme units per milligram ofprotein. The protein

determinations were performed according to the

method of Lowry et al. (15) with crystalline serum albumin used as a standard. Alkaline phosphatase

was assayed in a mixture containing 0.45 ml of

p-nitrophenyl-phosphate (4mg/ml),0.45 mlof alkaline

glycinebuffer (pH10.5), and 0.10 ml ofenzyme

solu-tion.After incubationfor 15 min at 30C, thereaction

wasstoppedbyadding3.5ml of1NNaOH, and the

absorbance wasmeasured at420nm.Peroxidasewas

assayed accordingtoamethod developed by

Worth-ington Biochemical Corp., based upon the use of

o-anisidine as the hydrogen donor. The rate ofthe

color development was determined by measurement ofoptical densityat460nmat20 C.

RESULTS

Purification of the enzyme. The

purification

procedures

were carried out at 0 to 4 C. The

frozen,

crude CFE was thawed and mixed with

10% (w/v)

streptomycinsulfate, pH7 (20

ml/100

ml of thawed CFE). After standing for about 30minwith occasionalshaking, the mixturewas

centrifugedandthesediment wasdiscarded. The supernatantfluidwasmade upto33%saturation with solid (NH4)2SO4 (196 g/1,000 ml) and allowed to stand for 14to 16 hr.After

centrifu-gation

at

12,000

X gfor 1 hr,theprecipitatewas

discarded and the supematant fluidwasmade up

to 60% saturation (1,777 g/1,000 ml). After

standingfor another 14to16hr,themixturewas

centrifuged and the supernatant fluid was dis-carded. The redissolved precipitate was exten-sively dialyzed against buffer B to remove the

(NH4)2SO4. This preparation was assayed for enzymeactivity. Specificactivities of the various enzymes in (NH4)2SO4 33 to 60% precipitate

were as follows (102 units per mg of

protein):

f3-L-fucosidase,

0.0; a-L-fucosidase, 2.8;

13-D-galactosidase, 13.8; a-D-galactosidase,

14.2;

N-acetyl-,3-D-galactosaminidase, 57.9; and

N-acetyl-(3-D-glucosaminidase, 118.5.

A column (2.6 by 20 cm) of DE-22 cellulose (Whatman) was equilibrated with buffer B. A 50-mlamountof thedialyzed preparationwas clarified by centrifugation at low speed and

ap-pliedtothe column. BufferBwasusedaseluant. The enzymescameoffjustafter the voidvolume,

and about30%of the activitieswerecollected in the first 100 ml of buffer. This fraction was freeze-dried at once and dissolved in distilled

water, giving a protein concentration of about 10mg/ml.

A column (1.3 by 35 cm) of CM-Sephadex

C-50 (Pharmacia)wasequilibrated with buffer A. A 5-ml sample of enzyme preparation from the DEAE cellulose columns was dialyzed against

buffer A andappliedtothecolumn. The column

wasfirst eluted with about 60 ml of the same

buffer to wash off unabsorbed protein, which represented about 60% of the applied material. Itwasthen eluted with the same buffer contain-ing 0.2 M NaCl. The peak of the enzymesemerged with 0.10 to 0.14 M NaCl in the effluent. The frac-tions were pooled, dialyzed against distilled water,freeze-dried,and dissolved in avery small volume of buffer A, resulting in a decrease of about30% in the total amount of thefucosidase activity.

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Acolumn (1.8 by 30 cm) of Bio-Gel (Bio-Rad Laboratories) P-200, 100 to 200mesh, was equi-librated with buffer A. The concentrated enzyme fractions pooled from the CM-Sephadex columns were applied in a volume of 1 ml containing 20

mg ofprotein. The column was eluted with the

same buffer and the flow rate was adjusted to about 1 ml/hr. Fractions (1.7 ml) werecollected and assayed for protein and enzyme activities. Atypical elution pattern is shown in Fig. 1.

Toobtain a better separation of a-L-fucosidase from the other enzymes, material was rechro-matographed on Bio-Gel P-200under conditions identical to those of the first Bio-Gel step. All fractions containing a-L-fucosidase (peak I in Fig. 1) were pooled, dialyzed, and concentrated by freeze-drying, resulting in some loss of

ac-tivity.Inthiswayit waspossible to obtain some fractions with a-L-fucosidase activity and no detectable a- or ,B-galactosidase activities. How-ever, small amounts of N-acetylhexosaminidase

activity were still present (Fig. 2). The averages of results in several experiments arepresented in Table 1. The total activity was increased after

streptomycin sulfate precipitation, possibly ow-ing toremoval of an inhibitor. The final product

represented a purification of specific a-L-fucosi-dase about 120 times over that of the original

crudeextract (Table1).

Properties of the a-L-fucosidase. Crude

prep-arations of the enzyme were stable at 4 C for several weeks. The enzyme in a purified concen-trated preparation did not show any significant

max 2.6. I ' 1.8 - 1.4 z wI o1.0 0.6 a. 16 20 24 28 32 36 40 44 FRACTION NUMBER

FIG. 1.ElutionpatternfromaBio-Gel P-200 column.

0, N-acetyl-8-D-glucosaminidase; 0,

ci-D-galactosid-ase; U, ,B-D-galactosidase; A, a-L-fucosidase. All

enzyme activities are expressedas optical density at

420nm. 6 z w 0 C4 -Z CL 0 0 x 16 20 24 28 32 36 40 FRACTION NUMBER

Fio. 2. Elution patternfrom rechromatography of peak I in Fig. I on a Bio-Gel P-200 column. 0, N-acetyl-j3-D-glucosaminidase; 0, ce-D-galactosidase; *,

,B-D-galactosidase; A, ca-L-fucosidase;X, ,g ofprotein

per ml. All enzyme activities areexpressedasoptical density at 420 nm.

TABLE 1. Steps inpurification of a-L-fucosidase Step Cell-free extract... Streptomycin sul-fate Supernatant fluid... Ammonium sulfate 33-60% Precipi-tate ... DE-22 cellulose Freeze-dried fractions... CM-Sephadex Fractions in 0.10-0.14M NaCl... Samefractions freeze-dried.... Bio-Gel P-200 Peak I inFig. 1.. Specific activitya 0.88 1.27 2.83 11.16 55.16 41.33 105.16 Vol ml

444

533 105 8 15 0.7 18.7 Total unitsb 28.3 31.1 27.5 8.3 7.0 5.2 4.8 Yield 100 110 97 29 25 18 17

aExpressed as

10'

units of enzyme activity per

milligram ofprotein.

bOne unit = amount liberating 1 ,umole of

p-nitrophenol fromsubstrate per minat30C.

decrease inactivity when tested after2 weeks of

storage at 4C, butdiluted solutions rapidlylost activity. Freezingand thawing causeda 30% loss ofactivity. The effect ofpH on the rate of hy-drolysis of

p-nitrophenyl-a-L-fucopyranoside

is shown in Fig. 3I Maximal enzyme activity was obtained at pH 5. On varying the enzyme

con-centration against a constant amount of

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a-L-FUCOSIDASE FROM A SOIL BACTERIUM 1.3 1.1 E 0 C- a-0 0.9

[

0.7[ 0.5p 0.3 0.1 3 4 5 pH x0 O 30 0 VI o 20. 10 z I CL 0

-jc

6 7 8

FIG. 3. pH optimumofa-L-fucosidase. Assays were performed under standard conditions in citric acid-phosphate buffer; the enzyme activities are expressed

asopticaldensityat420 nm.

strate under standard conditions at pH 5, a

linear relation was demonstrated between the

amount ofchromogen released and theamount

of enzyme added. The effect of substrate

con-centration on the reaction rate at pH 5 was studied. The rate ofhydrolysis as a function of p-nitrophenyl-a-L-fucopyranoside concentration is shown in Fig. 4. The Lineweaver-Burk plot

(14) was found to be linear; the estimated Km

valuewas3.6 X 104M.

Theenzyme wastested forsusceptibilityto

in-hibition by monosaccharides and other

inhibi-tors. Theincubation mixtures had the following

composition: 0.45 ml of citric acid-phosphate

buffer (pH 5), 0.45mlof2 mm p-nitrophenyl-a-L-fucopyranoside, 0.10 ml of enzyme solution, and 0.10 ml ofinhibitor solution. All inhibitor solutions were freshly prepared just before use. After 1 hrat 30 C, the reactionwas stopped by adding 3.4 ml of 1 N NaOH. a-L-Fucose was

found to be a

competitive

inhibitor for the

a-L-fucosidaseasdetermined bythe Lineweaver-Burkplots(Fig. 5).Theenzyme wasalsostrongly inhibited by p-hydroxymercuribenzoate. With p-nitrophenyl-a-L-fucopyranoside, the Ki values for theL-fucoseand the

p-hydroxymercuribenzo-atewereestimatedtobe 2.3 X 10 4Mand7.1 X 10-8 M, respectively. Assays with D-fucose,

L-fucono-y-lactone,

L-fucono-6-lactone,

D-galac-tose, and D-galactono-y-lactone, in

concentra-tions from 5 to 100 mm, showed no

inhibitory

effect. Neither a-methyl- nor

f,-methyl-fucoside

affected the activity oftheenzyme.

The purified influenza virus host antigen, a

fucose-containing

acid mucopolysaccharide (10),

o0.5 1 2

p-NITROPHENYL-Jl-L-FUCOPYRANOSIDE mM (S)

FIG.4. Effect of varying substrate concentration (S) onthe reaction rate (V) at pH 5. Enzyme activity is

ex-pressedas micromoles of p-nitrophenol liberatedper

hour times102. Innergraph (1/Vversus 1/S) is Line-weaver-Burk plot.

2 4 6 8 10 1 2

S [MM]

FIG. 5. Inhibition ofa-fucosidase activity by

L-fucose andp-hydroxymercuribenzoate assayed by the

method of Lineweaver and Burk (13). p-Nitrophenyl-a-L-fucopyranosidewasusedassubstrate.

was dissolved in citric acid-phosphate buffer (pH 5) at a concentration of 1 mg/ml. Enzyme

(from the first Bio-Gel chromatogram, peak I, Fig. 1) was added, and the mixture was incu-batedat30Cfor48 hr.Afterheatingto56 Cfor

10 min to inactivate the enzyme, the reaction

mixture was dialyzed against distilled water for

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18 hr. The methylpentose content of the

non-dialyzable residue was found to be

approxi-mately 20% of that of the untreated

mucopoly-saccharide, asshown by the cysteine-sulfuric acid

reaction (5). The dialysate was concentrated to

dryness and dissolved in about 100 ,uliters of

distilledwater.Thepresence of fucose was

identi-fied by paper chromatography in

ethylacetate-pyridine-water [12:5:4 (21)1 developed with

AgNO3.

Sucrose gradient centrifugation. A Christ

Omega II ultracentrifuge with the swinging bucket

(3 by 5 ml) rotor 9750 was used for sucrose

densitygradientanalyses (18). Samples (0.25 ml)

fromaDEAEcellulose column preparation

con-taining 0.5 to 1.0% protein in Tris buffer were

appliedto agradientof 5 to 20% sucrose (4.6 ml)

in0.05 MTris-hydrochloridebuffer (pH 7.2) and

centrifuged at 4 C for 18 hr at 100,000 X g.

Twenty fractions of approximately 0.25 ml were

collected from each of the tubes by introducing glycerol at constantvelocity from the bottom by

means ofa syringe. This procedure effected

par-tial separation of the different enzymes. Typical results are shown in Fig. 6. Peroxidase

[horse-radish type VI; molecular weight 40,000 (16)]

and alkaline phosphatase [E. coli type III-S;

molecular weight 80,000 (7)] were used as

ref-erence enzymes. The molecular weight of the

a-L-fucosidase calculated on the basis of the ultracentrifuge analyses was approximately

50,000.

DISCUSSION

The presence of a-L-fucosidase in the

cocco-bacillus described by Chase (2) had not

previ-(I) LA c - :3 N < z ._ A 0) 0 K : Vl 0 L. 0 L-A A. 5 10 15 -bot tom FRACTION NUMBER

FIG. 6. Ultracentrifugal analysis in sucrose density

gradient (S to20%). After 18 hrat 100,000 X g;20

fractionswere collected, numberIat thetop. Enzyme activitiesweremeasured accordingtomethods described in thisreportand plottedinarbitrary units.

ously been reported. In addition to the

a-L-fucosidase, this coccobacillus contained several

other glycosidases butwasfree of,B-L-fucosidase.

In crude preparations, thespecific a-L-fucosidase activity was lower than either galactosidase or

N-acetylhexosaminidase activities assayed with corresponding p-nitrophenyl-glycosides as

sub-strates. Among several methods tested for

puri-fication, that described earlier in this report

seemedto berelatively simple and reproducible. Conventional methodswereused,namely salting outwith (NH4)2SO4, ion exchange

chromatogra-phy, and gel filtration. The a-L-fucosidase

finally isolated was 120 times more active than

the crude CFE. The molecular weight was

esti-mated to be approximately 50,000. The

determi-nationof a-L-fucosidase activity as afunction of

pH indicated an optimum ofpH 5; there was

little activity below pH 3.4 or above pH 7.8. ThispH optimumiswithin the rangefound for fucosidases from other sources. Thus,

a-L-fucosidases in homogenates from various

mam-malian tissues were studied by Levvy and McAllan (13). Thehighestactivity wasfound in

rat epididymis and ox liver with pH optima of 6.1 and 5.6, respectively. The same authors

re-ported the visceral hump of the limpet, Patella vulgata, to be a better source of a-L-fucosidase than mammalian tissues. Marnay etal. (17) de-scribedtwo different fucosidases fromthe

diges-tive juice of Helix pomatia, an a-L-fucosidase with a pH optimum of 3.2 anda 3-D-fucosidase

with a pH optimum of5.5. Tanaka et al. (20)

reported the isolation and

purification

of a-L-fucosidase fromabalonelivers.Theirpurification

procedure increased the specific activity about eightfold. Two types of a-L-fucosidase were re-ported, differing in pH optimum and substrate specificity. One had a pH optimum at about 5,

tested with

p-nitrophenyl-a-L-fucopyranoside,

but didnot act on thefucosidiclinkages of

por-cine submaxillary mucin. The other had a pH

optimum at about 2, tested with the synthetic

substrate as well as with

porcine submaxillary

mucin. Wefound only one pHoptimum (pH 5)

withenzyme preparation from the Chase

cocco-bacillus, tested with the

p-nitrophenyl-a-L-fucopyranoside. Our results, however, do not

exclude thepresence ofisoenzymes. Theactivity

of the presentpreparation on

p-nitrophenyl-a-L-fucopyranoside washighly sensitive to

hydroxy-mercuribenzoate, a finding which suggestedthat

sulfhydryl groups mightbe essential foractivity.

The curve shown in Fig. 4 is consistent with

simple Michaelis-Mentel kinetics. L-Fucose

com-petitively inhibited the reaction with p-nitro-phenyl-a-L-fucopyranoside. Surprisingly, the

re-action was not affected by fucono-y-lactone or

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a-L-FUCOSIDASE FROM A SOIL BACTERIUM byfucono-5-lactone, although the transformation

from y to 6 configuration has been reported to

increase the inhibitory power of lactones in certain instances (6). Likewise, methylfucosides were ineffective as inhibitors and were not sus-ceptible to the action of the enzyme. In these respects, our findings accorded with the results reported by Levvy and McAllan (13) regarding the characteristics of mammalian fucosidases.

Our preparations of fucosidase from the coccobacillus of Chase were active against the purified host antigen (HF) of influenza virus hemagglutinin, 80% of the fucose normally con-tained in the acid mucopolysaccharide being rendered dialyzable as the result of enzyme action. Concomitantly, the antigenic specificity of HF was altered. These latter results, to be reported in detail subsequently, suggested that fucose may beinvolved in the structural

configu-rations which determine the antigenic specificity

of the hostantigen. The presentdata, however, are not sufficient to exclude the presence in our preparations ofadditional enzymeswhich might

act in concert with the fucosidase on this and

othersubstrates.

ACKNOWLEDGMENTS

We areindebted to E. Jantzen, Methodology Department, National Institute of Public Health, Oslo, Norway, for the syn-thesis ofp-nitrophenyl-a-L-fucopyranoside.

This investigation was supported by Public Health Service grantAI-03168 from the National Instituteof Allergy and In-fectiousDiseases.

LITERATURE CITED

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3.Conchie,JJ,andG. A. Lewy. 1957. Inhibition of glycosidases byaldonolactonesof corresponding configuration. Biochem. J.65:389-395.

4. Conchie, J.,andG. A. Lewy.1963.Aryl glycopyranosides by theHelferichmethod,p.345-347. In R. L. Whistler and M. L. Wolfrom (ed.), Methods in carbohydrate chemistry, vol. 2. Academic Press Inc., New York.

5. Dische, Z., and L. B. Shettles. 1948. A specific color reaction ofmethylpentoses and a spectrophotometric method for theirdetermination.J.Biol. Chem.175:595-603.

6. Findlay, J.,G. A.Levvy,and C.A. Marsh. 1958. Inhibition ofglycosidases by aldonolactones of corresponding

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8a. Gilmore, T. E., and C. Howe. 1959. An aerobic soil micro-organism which decomposes blood group substances. II. Effect ofcell-freeextracts onblood group substances. J. Bacteriol.78:814-820.

9. Haukenes, G., A. Harboe, and K.Mortensson-Egnund. 1965. A uronicand sialic acid free chick allantoic mucopoly-saccharide sulphate which combines with influenza virus HI-antibody to hostmaterial. I. Purification of the sub-stance.ActaPathol. Microbiol. Scand. 64:534-542. 10. Haukenes, G., A. Harboe, andK. Mortensson-Egnund. 1966.

A uronic and sialic acid free chick allantoic mucopoly-saccharide sulphate which combines with influenza virus HI-antibody to host material. II. Chemical composition. ActaPathol.Microbiol. Scand. 66:510-518.

11. Howe, C., L. T. Lee, A. Harboe, and G. Haukenes. 1967. Immunochemical study ofinfluenza virus and associated host tissuecomponents. J. Immunol. 98:543-557. 12. Isbell, H.S. 1963. Aldonic acids, p. 13-15. In R. L. Whistler

andM. L.Wolfrom (ed.), Methods in carbohydrate chem-istry, vol. 2. AcademicPress Inc., New York.

13. Levvy, G. A., and A. McAllan. 1961. Mammalian fucosidases. 2. a-L-Fucosidase. Biochem. J.80:435-439.

14. Lineweaver, H., and D. Burk. 1934. The determination of enzymedissociation constants. J. Amer. Chem. Soc. 56: 658-666.

15. Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall. 1951. Protein measurementwiththe Folin phenol reagent. J. Biol.Chem.193:265-275.

16.Maehly, A. C. 1955. Plant peroxidase, p. 801-813. In S. P. Colowickand N.0. Kaplan (ed.), Methods inenzymology,

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17. Mamay, A., R. Got, and P. Jarrige. 1964. Fucosidases dusuc digestif d'Helix pomatia.Experientia 15:441.

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R. L.Whistler and M. L. Wolfrom (ed.), Methods in carbohydrate chemistry, vol. 2. Academic Press Inc., New York.

20.Tanaka, K., T. Nakano, S. Noguchi, and W. Pigman. 1968. Purification of a-L-fucosidase of abalone livers. Arch. Biochem.Biophys. 126:624-633.

21.White,B.N., M. R.Shetlar, H. M.Shurley,andJ. A. Schilling.

1965.Incorporation ofD-(I14C) galactosamineintoserum

proteins and tissues of therat. Biochim. Biophys.Acta. 101:259-266. 929 VOL.98, 1969 on January 13, 2021 by guest http://jb.asm.org/ Downloaded from

Figure

TABLE 1. Steps in purification of a-L-fucosidase Step Cell-free extract... Streptomycin  sul-fate Supernatant fluid..........
FIG. 4. Effect of varying substrate concentration (S) on the reaction rate (V) at pH 5
FIG. 6. Ultracentrifugal analysis in sucrose density gradient (S to 20%). After 18 hr at 100,000 X g; 20 fractions were collected, number I at the top

References

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