Carbohydrare Q

Research,

Ekevier

Scienufic

55 ( 1977)

Publlshlng

COMPARlSON

OF

I93-7-02

Company.

THE

Amsterdsm

ACTION

GLUCOSYLTRANSFERASE

OF

- PrInted

m Belgwm

AND

GLUCOAhl1’LASE

ON D-GLUCOSE,

MALTOSE,

AN-D

MALTO-OLIGOSACCHARIDES JOHN H.

OKAD%*. AF~D L. Scorr

PXZUR. AU~TRA CEPLIRE. SHIGETAU

F~RSBEKG

The Penn.r I IL an~u .Slaie L~n~wrsq

, Dn~wmr.~ A.) (Recci\rd August 23rd. 1976: sccrpted ior publ~~~lron In r;vissd ioim, NoiembLr I%h. Department

of B~ochenrrstr~~ and Biophysics, Parh. Penns~ iranlo 1650.? (LI. S.

19761

ABSTRACT

The action

patterns

ofglucoamylase

on

I - “C]glucose,

(t rsnsglucosylase) saccharides

(labeled

been iowstigated

Under

D-[

at position

into

33%

\\ith

were

ijomalrose

I or the ~-glucose

(~-&x-D-~~uco~J

pyranosyl-r-D-glucose).

from little,

oligosaccharides

D-plucosyl

groups

if any, such activity

recilniques. of

produced

hs\e

~-glucose

ar;d

and 5.7 and

by both

enzymes

panose (O-r-D-elucopyranos~l

glucosyltransferase of higher

end)

I .9 9 L H ith glucoomyla~c,

ranosyl-r-D-glucose),

The

malto-oligosnwharidcs

a-( I +6)-linked

was 2.2 and

of conversIon

I +l)-ic-G-glucosc),and

(I -+6)-0-lr-D-glucopyranosyl-(

[I-“C]mslto-oligo-

at the rcduclng

and oligosaccharide-mappin?

The major

glucosgltransferase.

and

group

the extent

of the experiments.

new oligosaccharidss

and glucosyltransferase

[I-“C]malrose,

by paper-chromarographic

the conditions

oi maltose

(smylo~lucosidase)

ni,oerose(3-O-r-o-gluco-

also

molecular

synthesized

weight

at the non-reducing

oligosaccharldej

to yield compounds

ends. Glucoamylase

hsvlng exhlblred

on malto-oligosaccharidss.

IN-I-RoDLlCTloN

The

development ~-glucose

srqstalline advance the

a fungal

starch

hydrolyzing imporfarlt process

findings

a-( I -+I)-

thar

first,

(transglucosylase: speciesa,

the

were

the

process

syrups

from

glucoamylase’ into

and

the

commercial

starch industry.

because

pertinent that

to the

EC

bonds

development

by the fungus,

of

technological

This process is based on

the enzyme

_elucoamylase

use of mutation

production

IS 3 major

a-( I --+6)-D-glucosidic

also produced

the successful

for

(amyloglucosidase;

D-glucose’

especially

demonjrration

EC 3.4.1.24)

and second,

enzymic

in the starch \+e:t-milling

enzyme,

quantitatibely

both were

the

high-glucose

of the past decade

use of

converts

of

and

and

321.3)

of starch3. of

*Present address. The Osaka hlun~c~psl Technical IqsIltu[r, Oj>hJ, Japan.

of Two

[he enzymic

glucosyltransferase

are distinct

techniques

that

is capable

to obtain

molecular strains

of

J. f-f. PAZUE. A. CEPURJZ. S. OWA.

!9J f?rnsl

that produce

little,

in snqmic

unilzjirnbfs

if 311~. ,olucosylrransferase~~5,

conversion

of starch

ha\ iog 51-D-( I +6)

olig~saccharides GIuco3mylsse

itself. \\hile

almost

linkages

The _gfucosylrransfx.w

as this enzyme

that accumulnte

of synthetic

devoid

L. S. FORSBERG

effects

is

the jjnrhesis

of

in the enzymolyutesJ*7*“.

sctivity’,

under

some conditions

rhe j)nthcG of a-( I +6)-D-~ILICOSJ~ oligosaccbarides by reversion” *’ (recomb~nsrion of free D-glucoje) or bp glycosylation”.’ ’ (cwhnnge of D-glucosyl may

~KXL

groups)

reactions.

D-glucose.

msltow.

of obtaining w11h the

data

mapping’

has been re-io\v;ti,oated

purposes.

from

the

mrchaolsms

of sqnthejis

parallel

same

3 has been especially

on the rewlon

rl_~~e@//trs niger on ’ 4C-labeled

from

on the estent

For comparative

~lucos~ltransferase

saccharide

of _glucoamylase

and mslto-oli~osaccbaridcs

qusnlitaii\e

lhcse subwares.

mation

The action

experrments

organism.

baluable

baw

The

in our

of the giucoamqlase

\\itb

the v&v

of oligoxxcharidzs technique

studies,

from

been conducted of

olrgo-

aad new infor-

and the giucosqltransferaje

h3s been obtsincd. RESL LTS AND

DISCUSSION

PhotoFrspbs rrsnsferaie

condirions.

Lhc test

with

of rad1oauto~ram~

and glucoamylsse ~>nthesij

olr~osaccharldrs

vslUcs

on

converted smounts

paper

chromstogrclms

maltose

rapidly

into

\xere

The

used

for

but

l5,3

5

0

rapidly

1.5

structural

and,

,nlucosyl-

that,

into

under

~-glucose

predominan[t~

36’

charscrerization

in the current

identification.

this enz)mc

I ).

-

0

malroje

of oligosacchandes,

e;lrler’J

~-glucose.

I Fig.

wirh

I. it may be noted

and panose [O-r-D-glucopgrclnosyl-

I --!)-r-~-glucose].

had been achlP;ed

of oligojaccbarides

PANOSE

iI___^_

of Jar-se amounts

(6-0-r-D-giucopvrsnojvl-r-o-_elucose)

(I -+6)-0-r-D-glucop:lr3nosyl-( thwz

of [I- ‘-QYIaJtose

in Fig.

ihz glucosyltransiirasr:era+zconverted

rbe concurrent

Isomaltose

of dtgests

are reproduced

Glucoamjlase iyntheslzed

of

sludi?s,

only

R,

aljo small

. ACTIGN

PAI-TTRW

OF GL~JCO.&~IYLASE

Quantitative produced

from

culations 9W0

from

data

disappzarsd

~lucosyltransferase

\\as conberted

shov:ed

that

there

\!erc

and panow) of maltojr.

‘1 h on incubation _

In Lhe glucosyltmnsferase

rhe ratio

digest con;+arcd

by the LWO cnqmej. of lsomaltose

I .S, rejpecti\rly.

dlKcrences

in amoonrs

c,f ollgosaccharidcs

In reaction:

mechanlstn~

compounds

~1 nthcsized

initially

\\ith

the enz.;mes.

It has bwn

reported

yield oligowcchsrides with

non-purilied

amjlnse’-’

yielding

can function

thereby

Il\ct

at dlfkrent

alfecting

purified

of recombinntion

of products 12

Table

ijomaltose

in digests II. (RF

reactions of

D-[

Both enzymes value.

0.40)

the

in

is FoSslbk

of D-glucox

glucosyltranskraw may be calculared

I- “C]glucose

ivlth

do indeed carslyzc and

24-h

nigdrose’

reflects that

his wzondary

the

acceptor-

to psnox.

of D- glucose (re\rrsron of

stases of

In the glucosyl-

bhr‘reas

the r;ltlo of isomalrose

preparations

data rllso

in this rcltlo and the

It

raw

of the

digest only

by the I\~CI cnzymcs

enqmcs.

may occur when high concentrations

SUC!I data are presented reactions,

synthesized the

33”b

in the initial

digest,

ober

oligosaccharidcs

was 0.37

The \3rlxtion

thar ;L recombination

and

‘. The extent

on the radioactivity

for

major

ihus,

to panose

1. Cal-

with either

The quantltatke

of the t\io

to 3.5 In the glucoamylass

the ratios were 0.73 and

digest,

but in the glucoamylase

in the ratio

synthesized

in Table

of the e\pernnents. of the substrate

into ollgosaccharides.

dlfferenccs

and oligosaccharides

are presented

under the conditions

ditixznccs mo!eculcs

enzymes

into new olisosaccharides.

enzymoly5l5 trsnsferase

during

was converted

(isomnlrose

samples,

that,

or glucoamylase.

I .9‘:0 of the maltose

of the D-glucose

by the two

these data showed

195

GI UCOSYLTRANSFME

on the radioactivities

[I-“C]maltose

of the maltose

maltose

AND

reaction)

to

are incubated or from

@ucothe data

these enzymc~. recombination

5 (3-0-Z-D&CO-

_I. H. PAZLrR,

196

R, WIIJC, 0.5-I) itis the

pyr3flos}1-~-D-glUCojC;

v,cre ohtaincd

from experiments

D-gIUCc75S)

long

ancl

in commercrai ~3s con\er[rd con\wted ihai

incubarion-periods

On

CEPURE,

S. Of&DA,

into olizosaccharides

of subsrrate

(96 h) \%cre employed,

by @ucoamjlase.

produced

about

L. S. FORSBERG

major products. The data in Table II

high concentrations

fi\e

of

done

5.7’?:, of the D-glucose but

only

2.2?b

was

note IS the observation

Of jp. from D-=glucose. In this connection, the studies on highly rextite ,olucos~l of Hchre t;l al.’ ’ ” OR the action of @lucosmylsse compounds or on pure anomw of slucojr should be mentioned. These investigators tronjferase

identified

and

:lucoam\lsie

msltosc

35

3

in our e\pcrimenti. nnomers conditions In

of

product

maltose

D-SlUCOSC

with

do elect

(Jf

~35

3zrion

wersion

of

glucosm)-lass

nor dztectsblc

glucosm>law.

reactions

on thcx

in digests

synthesize

and

substrates.

of a mlkture

Ho\\s\cr, of the two

because of differences

perhps

i?

the

used. order

to ohtnln

glucosyltrsnsftirase

[I -’ ‘C]malto-olizosaccharides oligosaccharide

additional

information

and Elucosmylssz, maps

bj

\vcre obtained

the

the acrion

on

the

reaction

mechsnijms

of the c\~o eai~r~es

ollgojaccharide b> the methods

mapping described

\+aj studied

procedure’

for on

3S 7Ie

in rhe experimental

arim. and typical maps ar? reproduccld in Fig. 2. The results in this Fgure show that plucoam~lclsc ~lelded only bydrol)*tic products (RF talues higher than the RF values

9CTION

PATTERN

OF GLLICOAMYLASE

of the substrates), DS hydrolytic lower

produced

from

maltose.

cstalJzes

and new ollgojaccherides malr3heusose.

transfer-rcacrions of higher

not

molecular

only

synthetic

compounds

It will be noted

and

197

\\ith

that

isomaltose

3s

products

possessing

R,

dell

values

and panose

were

\%erc produced

from

Quite

rhe glucosyltrans-

clearly.

maltose.

but

mnltorriosc,

also

lrirh

malto-

\telphr.

_.___-

I

CL’L’C.@SE KALLTrJSE

Fe

*

-

,hWLTVfE TRAOSE

-

IIN_T~=‘ENTAO~E f~cLFHE.C4ljsE

-

I

The pniterns from

of Isbeled

one less

~-glucose on

group

group

strllninz

on the orher linear

hand.

D-,~lUccSe

from

yielded

occurs.

all The

on oligosaccharidsj sivlglr-chain The

hydrolytic

of

from

a “C-labeled reducing

products

lsbelins

indicares

sugars.

that \kere about

mechanism

enccunter

the substrate

yielded,

of action

rhc enzyme

of enzyme

molecuk

unrll

as rhe initial

oligossccharide

ss ~iell 33 non-labeled for

lo.’ 3. In this mechanism, per single

[I - ’ 'Cjmalto-oligosacchrrides

2). Ths glucoamylnse

oligosaccharidsj.

with a multichain

the substrate

~-glucose

produced

(Fig.

than the substraw.

polymers

shortens

prodtic1s

markedly

the chromatogrom

result is in accord

grcsG\ely

glucojc,

having

which

was

Glucosylrransferase. equally

labeled.

The

for glucoamylase removes

and

one

substrate.

complete

on

glucose

and

l:onverslon

pattern

in the products of glucosyltransferase acfs predomlnantlq rhnt the glucosyltransfemse

prointo

action by a

mechnnlsm. action

maltoheptnose new

dlKer

the Indlvldual

dercctnbls former

a*

-

by the Tao enzymes product

**4@

f

-

fXILTOTi;lOSE

!

yielded

some of the synthetic

maltopsnraoje.

oligosacchsrides

3

with

those of the substrates.

maltotetraose, fcrase

but that the glucos)ltrnnsferase

products,

than

AND GLUCOSYLTZANSFERASE

producis.

of glucosyltrnnsfersse

with

the objective

Chromatoprams

~a5

also tested

on pure

of obtsinin, 0 information sho\ving

the

products

malfotetraose

on the structure from

both

and of the

substrates

are

J. H. PAZIJR.

198 reproduced R,

III Fig.

values

lower

bydrolyk!

results

those

and synthetic

products

the paper chromarogmm

products

were present.

comparison

in

as the hqdrolgtic

converted maltose

into and

maltose,

Holie\er.

D-~~ucosrt.

than

the z-( I-4)

completely

the

on the compounds

utilizing

digest,

other

rhr:

h> and

and

having

digest.

maltohexaose

trere into

with

,olucoamylase

in the two dlgcsts Inlo

the .z-(I 46)

some compounds

substrate,

were converted

prepared

al1 of the oligosaccharides hydrollzzs

as

only

In this digest Here

in the maltoheptnose

maltoterraose maps

hydrolyzed

unreacted

compounds

and msltoheptaose

oligosaccharidz

linkage’.“‘,

oew

beta amylase and glucoamylase

of the products

and maltopentaose

conbsrrrd

eight

was obtained

that bptn amylase

specifically

All

amyiase:

a3 this enzyme

were detectable digest,

to th.?se of reference compounds”.”

product.

Howe\cr,

The

havin_g

In addition.

Ilnkn_ee at 3 lo\xer rare

a-( I-+6)

linhapcs

\tere not

hydrolyzed.

In vie\r of the action on glucosyi

oligossccharides.

must contain

z-( I-6)

linked

some of the compounds D-glucose

maltotetraose

in

maps shelved

by lxta

maltorriosc.

shoi\ed rhat this enqmc

new compoundI.

maltoterraoje

h>drolzed

produced.

digest. thirteen

g method,

one compound

were

indeed

3). and

the producti

>ieldln_e maltose

res;stan t to beta nmylolysis.

L. S. FORSBERG

that new oli_eosncchnrides

were

ir,formation

The oIIgosaccharide the

S. OKADA.

havrng R, values higher than those of the substrates

Structural

of RF values of

enzymeI@lucose next lo~lesr

199

GLLICOSYLTRkNSFEME

maltotrtraosz

was verified

mass spectrometry. digest yielded

AND

Such

rates

for

transfer

ne\i

of malto-ollgosaccharidzs

oligoby this

enz) me. t\PERlblEhThL

:I&-r.

.\/ur~v-/u/~

-

(DlAL\‘hl

E. Lot

highI>

Glucoam)

pure slate

earlier’

sedlmenration

beha\lor.

resins

a

enzyme 30

ilaj

chrornatographic

centrifuge

patterns

and

of

WaIlerstein

per

Laborarork

D-[ I - “C]GIucoje Solutions

of the labeled

authentic

Typical window

residue

counter. from

ai

for maltotriose.

p,slrern;

slraln

anl)la~e

when examined

from

ton-

contalned

of

-l.cprrgi/lu~

rmpr

publwxtlon’.

Cllrrs-

homo~enelly

In the

expcrlments

wa$

the label from

conrslned

purchnscJ

from

the

end were by

c.p.m.

prepared

uillizing

for

from

Bacillus

as measured D-glucose,

f;a\ing

I of the supplIers. and

RF. ~alucs

labeled

at C-I

D-[I-“C]glucose

tmc~rutls

for

3.

endmal:ose,

hlxltohepraose

as previously

of

oi and

amylase’

by a Geiger-hluller

10,700 c.p.m.

for maltotetraose.

(cyciomaltoheptaose)

31 posItIon commercial

by p:lpcr chroma:o,oraphy

component

[I - I ~C]hlalto-ollgosrlccharldej

and 8,100 c.p.m.

cgclohsptaamylose

denstty-gradlent

elperlments

lndlcsted

puxhascd

radroactlie

the reducing

39,900

same

(haking

of these compounds.

v.ere:

in hav?

on starch as the sobstrare’. the

in the current

end) \\ere

maltose.

46513)

for purity

N.1’.

(cyclomsltohcwosr)

radioactivities

13,800 c.p.m. prepared

and

clution

In an earlier

Beta

[I-“C]msltosc

a sing!e.

patterns.

and

strips

emplojcd solution-.

compound-.

yielded

c)clohe.\nnmylo~e

0i

31 the rcduclng

D-gllJCOSZ

the ~-glucose

sample ml

and

residue

rzdtoautographv.

a5 dsjcrltwd

iWe\\ t’orh,

D-glucose

from

paper-electrophoretlc

The

activiry

as ussa)ed

ID

Crtterla

u>cd In III? current

also Isolated

procedure,

preparation.

Unil;

migration.

of 4 sp,wgi/lu~

rlltract

Elhhart.

of ultraccnrrlfuze

solution

per ml of solution.

Glucosyltransferasc

Inc..

on DEAE-cellulose’.

elcctrophorerx enzyme

from dn enzyme

Laboratories,

and consisted

I.”

The

900 uolts oi actlvitj b>

hlrles

by chromJto?raphy

bssn prejcnrcd exchange

last3 itas isolated

F S! 50.

described’

was 3.

200

J. H.

and purlficd a\silablc

by preparative,

of Science. Prague. Chemicsl

Co..

purchssed

from

various

50/A

bias removed

ch,oms!oFram. samples

of

dc\rlopcd

sol\ent.

on

wiih

rcproducrd

rn Fig.

chromatogram,

transferase

k;-r3y

irere

v.vre

products

tilm

of

on a paper

by volume)

the

rnlxrure

for

cbromatogrsms

\%ere

by three 3xents

of the

\v3s obtnlneil

fog Z-l- to -M-h

placed of

The

bq placing

periods

the dried

and subsequemlj

by using [I-“C]malrose

\\a5 performed

solution

of the mnllosc

of the r.adlosutonrams

sufficiently

3rc recorded

in Tab’s

of isomaltosr

and

been reported

earlier’

panose

af

En_rmo/.rsic

from I

in

3s the enzyme.

dijsppeared

in a

of the INO dtgcjts ar?

35 products

each

on

other

hy plncing

the

paper

the G.-hl.

tube

g to the desired compound.

msltose

h!

gluctiamylasc

of the rsdioacti\r

I&ntiFication

to reference

from

r3dloacti\ltiej

corrcspondin

compounds.

31;d

of gfucos~ltransfers~r

$ucosylis bajlcd

producrs

The structurcll sction

for

Values

characterization on maltose

had

’ D-[ /- ’ ‘c]ghcow.

-

trith sn equnl volume

sample

.>f the g lucoomylsse

AnalJsej

of the digests 91 b;IrIous

prccedlng

sectIon.

The rncub3tion digests.

A

of 0.1 ml of 3 60%

I.6 x IO” c.p.m.)

(tot31 rsdicsctifity

the [I- “C]maltose

separated

to measure

formed

in comparrson

mrted

(6:1:3

citrate

A Sample

the enzyme. Subsequent

incubstion

temperature.

of 0. I ml Of

I.

it \+as posjlble

“CJgIucose

3fter

room

buch lh3t 909:

Photorraphj

on h’, values

&[I-

the Pierce

in 0.1~

5olutron.

of rhe enzyme.

of hot sir to Insctibare

e\perlment

on the area of rhe chromatogram radioactlie

from

a sample

cxprrimrnt,

3.5 snd 0. I ml of glucosj ltransfernsc

period.

.4s the products

the

sddltlon

chromatogxm

lvith

A similar

buffer ofp1-i

rncubatron

purchnsed

were

Academy

were rcapent-grade compounds

its’ 1.1 x lO[’ c.p.m.)

of the chromatogram

in contact

the film.

nnd panose

by H. Erhetovs,

0.1 ml of the plucoamylase

2-l h a[

The concentration?; of enzyme, 25-h

In a typical

but}1 alcohol-pgrtdlne-crate!

in

~3s

chemicals

radiosctii

3fter

the and

A rsdioauropram

f hl acet~trl

-

(total

immedlatel}

placed

chromstogram

0.

miwd

1.5. 3. 6. 1.

dsLltransferaje \rere

conducted

in citrate buff&

(96

h) :han

in the bbrth

in the ~-glucose

ior nrgsrose and 0.40 for the rso;naltojc. Under comparable conthe RF r~slue for D-plucosc nas 0.67. nnd for mr-ltose 0.36. Radioscti\it) ralucs for the products rn the drgcsts of D-[I- ‘Yjgiuc ose l\ere obtained 8s already Indicated, snd these values 3rrI recorded in Table II. The isolation snd characdigests

\+ere 0.5-I

ditions.

Ferizstion esrlicr

of nrgerose as a product

Oltgoracd~ari& suited

of enzyme

action

on maltose

has been described

’J for In\estlgatlng

mnlro-oli~osaccharidcs’

maps. -

The oligosaccharlde-mapping procedure is especially the action patterns of various omylases and tranjferajes on ’ ’ “. In this procedure. a sample of SO,YI of [I-“C]malto-

ACTION

PATTERN

OF GLUCOP_MYLASE

oligosarchnride

has

chromatognm butyl

placed

at

one

wzs developed

oligowccbarides room

was

under

become

sprayed

lightly

sprayed

chromstogram

which

The

chromatogram

and used to obtain matogram sugars. employed

stained

in order

to obtain bonds

oligowccharide

of the

nitrate

dilutions

oligosacchsride

Glrtco~~~ltratrsft~rase actiutl

on ttlaIlol~Ira0_w

bath

In order

lase’

dtgejt.

to maltose,

and mnltotriose

hydrolyzed linkage

to

only

~hsrsn~

D-glUCOse

by

the

\iith

spproprlatc

stl\cr

mixture

wz~s daeloped

maltopsntnow

\L;ij

beta gluco-

of malto-

the products

The tinlshed

dtsr5t.

the area of

5 ml of 0.05”0

the reductnp

maltotetrao~e

nitrate

tn Fig. 3.

5 ml of 0.110

A reference

system.

were wafer-

oliposacchsrId+mappi?g

in line with

b> the beta amJlaje.

is hydrolyzed

‘, oligojsccharldrs

to D-glucoje.

for

chromarogram

In the maltoheptaose

digests,

in one direction,

IO locair:

rea,oent’-

The mt\tures

reproduced

of pH 5.0 or \ttth

by three ascents of the soltent sll\er

15

was sprayed

xetate

moltoheptaox

the

0i

Sample5

by 5 aScents of the solvent

stained

used

in 0.05~

D-glucostdic

hydrolyzed

and

chromatogram

of the chromato$rsm

containing

\\lth

of

of

0 I ml of 0 _7\t

IS mln in a bolllng

dewloped

‘5”;

1.

with

miwd

X chromarogram

\\ere

about

of radloautograms

in Fig.

for

\iere

Stages of enzymolysrs.

solution.

thctn bested

\\a~ prepared.

of a typical

de\eloprhrlnt

were hydrolyzed

hydrolyzed

enzyme.

the reducing

utril llluliOlit~pICic~j~~. -

alcohol-pyrrdtns-water),

~\;is placed

maltotelraow

beta amylasc

and

of 50 111 of the digest After

the chromato_gram

compound
zed

\\rlrr’

and

\\faj drtcd

Sectton.

Indlvldual

Photographs

t’ns olisosaccharidc

at had

to be developed

to raea!

the

are reproduced

bufTer of pH 3.5 and 0.1 ml ofglucoj~ltransferase for 1 h at room

the

stapled.

the chromarogram

solutions

acetate

Incubated

of

30 min

(malto-oligosaccharides)

method”

of

of

the

In some Instances

chromatogrsm

of

of rhc oli,oosxcharidtts.

solution

for

a q4tnder.

In a precedtng

0.1 ml

IO”;

a solutton

system.

maps at several

with enzynle

maps lor thz two enqmes

a

In

solvent

compounds

3~ descrtbcd

several

results Lvers obtatnrd

of the glucosidic

roiled

at the bottom

by the silver

cuperrmcnls,

ir~th

containing

113s maintained

five ascents of the solvent.

most

Salisfactorj

NX

of rekrence

a radioautogram

\\a.~ then In

chromato,oram

untformly

in the foregotng

in line with the other compounds After

of 6:4:3 (by volume)

time the paper strips had drlsd and the ergme

to place a sample

in the second direction.

and

snd the

127 x 25 cm),

by five wxntS

Lrea of the dried

in the second dIrection

desirable

it \sas

a chromatogram

The

during

inactivated.

developed

of

then

study.

temperstuw.

comer

The

201

GLLICOS’iLTRANSFERE

in one direction

alcohol-pyridine-Lbaater.

enzymes

AriD

in the in the

chromatograms products.

hydrolyzed

maltotetraose

Of

the

by the

and mnlto-

and mrtltoheptaow

\\?re

In both dtgests. ~111of the

by the

glucosmylase.

at a lo\\er

rate than

ha\ Ing x-( I -+6) Ilnkagc~

Hoikeever, BS the the I-( I +1)

linhage

were not completely

202 The (Fig.

PXZLIR,

J. ii.

tuo

oligosaccharidss

3) were isolated

\vas merhylsted

bj rhe Hakomori

identified

by g.l.c.

and

I ,I.Wri-O-acet)

RF values

lowest

following

methylated

S. OKADA.

L. S. FORSBERG

in the maltotctraose

digest

‘. Each oligosaccharidc

the procedure described in a

alditol acetates from each compound

mass-spectrometric

ncc~l-2,3.-1,6-tctra-O-methyl~luc~tol.

CEPURE,

paper chromatogTapby2 method’“,

The partially

recent publication”. were

ha\.ing

by preparative,

A.

metbods2’m28

and

were:

I,5di-U-

I.~,6-tri-O-ace~l-~.j,4-tn’-O-methylgind Th c ratio

1-2.3.6-t ri-O-methvlducrtol.

of these

products

was

l:l:3

for the oli:osaccharide having thhlksst RF value and I:[:2 for the oligosacchsride hsvmg the xcond-lowest R, value. blethylation of rhe substrate, maliotcrraose, yielded

only

the tefra-O-methyl

derivative

and

the 1,3,6-tri-O-methyl

derivative,

in

the ratio of 19.

This in\estipation Association,

was supported.

U’ashington.

D.C..

in part, by a grant from the Corn

and 15 authorized

in the Journal series of the P?nnsyl\,snia

Agricultural

for publication Etperiment

Refiners

as paper No. 5159. Station.

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