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OJ Shellfish

Research, Vol. 17.



1. 159-163.



F E el L

Department of Biochemi try

Genetics and 11I1/1111110/0g)'
Faculty of cience
University of Vigo. Spain
"Department of Biochemistry and Molecular Biology
Faculty of Phanuacy
University of Santiago de Compostela, Spain
A different glucose and glucose--phosphare
inhibition 01' glycogen phosphorylase frorn Mvtilus rnantle relative lO those
01' vertebrates has been demonstrated.
Glucosc and glucose--phosphate
do not inhibir the a forrn (pho phorylated form), but they are
cornpetitive inhibitors 1'01' the substrate glucose-l-phosphate
in the b form (unphosphorylated forrn), with Ki values of 190 and 0.2-0.3
mM. respectively. The resulis cast doubt on the role 01' glucose a, a regulator and sugge t that gluco e-6-pho phate is a major regulator
01' glycogen phosphorylase activity in Myli/lIs
mantle tissue because 01' its special relalion. hip with gameiogenesis.



glycogcn phosphorylase.





form) i activated by MP. LMP and high levels of inorganic

phosphate. and i inhibited by ATP, ADP, G6P and UDPG (Dombrdi 1981). Gluco e also how an inhibitory regulation synergisti !O inhibition exerted by purine derivative (Cori and Cori
1940. Ka vin ky el al. 1978, Carabaza 1992). The binding of G6P
10 the enzyrne cau e the inhibition of enzyrnatic activity through a
ncgative heterotropic effect on the AMP binding (Wang el al. 1970).
The concentration of circulating gluco e has long been con idered respon ible for the regulation of glucose homeo tasi by the
liver. Liver pho phorylase a, as the glucose receptor, i largely
re pon ible for the overall control of hepatic glycogen metaboli m
(Kasvinsky el al. 1978). Gluco e bind to glycogen pho phorylase,
rendering it a better sub trate for pho phoryla e pho phata e.
Then, glycogen phosphorylase is dephosphorylated and inactivated. Glucose is lile molecule in liver tis ue that trigger the
inactivation of glycogen pho phoryla e.
Alth ugh rnantle ti sue in Mytilus rnay be considered analogous to the liver in vertebrate ,a
a storage organ of glucidic
re. erves in the form of glycogen, its physiological function may
differ. o the aciivity and regulation of the glycogenolitic pathways in the mantle tissue of thi organi m doe not appear to serve
solely as a ource of gluco e lO be u ed intracellularly via the
glu litic route and the enzymatic
y tems involved seem lO
present a characteri tic function different from that of vertebrales .
Jt i. known that glycogen i the major energy fuel in the mantle
of bivalve mollu e (Zandee el al. 1980) and that an evident relationship between it degradation and gametogenic developmenl
exi 1 (Gabbott 1975. Bayne el al. 1982). During this developmenl
there i a clear coincidence between the increase of saturated fatty
acid and the decrease of the glycogen contained in the mantle
before pawning (Zandee et al. 1980). A key enzyme in the glycogenolytic pathways i the glycogen phosphorylase, present in the
Mytilus galloprovincialis mantle in IWO forms, a and b (San Juan
el al. 1991, an Juan el al. 1995). In this paper, we de cribe the
different propertie relative ro glycolitic metabolite ' regulation of
Mytilus mantle glycogen phosphoryla e, which is probably because of their differeru physiological role.

The glycogen pho phoryla e can be regulated by eo aleru

rnodifcation (pho phorylation-depho phorylation) and by allosteric rnodulation exerted al ihe molecular level by various phy iological factor acting through conformati nal change of the enzyrne. These factor can aCl as po itive and negative effector or
inhibitors (Fischer et al. 1971. Graves and Wang 1972. Bu. by and
Radda 1976. Kreb and Beavo 1979. Krebs 1981. Fi cher 19 3.
Madsen 1986).
Four separate ligand binding sites are localized in glycogen
phosphorylase of vertebral e . The active ite, which bind. the substrate . p or glucose-I-phosphate
(G 1P) inducing a a tive conformation R (Withers el al. 1982) and the nonredu ing terminal glucosyl unit of glycogen. as well as glucose and uridin-5'diphosphoglucose (UDPG). inducing an inactive conforrnauon T
(Sprang el al. 1982, Mad en el al. 1983). The nucleotide ite or
AMP allosteric site, consisiing 01' three different ub ite . namely
the pho phoryl group, the ribosyl residue and the purine ba ebinding loci. This site represent the primary binding site of allosteric activators: AMP and LMP as well as inhibitors:
and gluco e-6-pho phate (G6P) (Johns n el al. 1979, Johns n el al.
1987). Furtherrnore, the ub. trates (i.e .. G 1P and Pi.
nucleoside analogues,
r even dinucleotides are all capable of
. econdary binding to the nucleotide 1 cus. ATP. ADP. and G6P
exert iheir effe 1 through AMP expul ion (Dombrdi 1981. Morange el al. 1976. Eguchi el al. 1977). The nucleo ide (or inhibitor)
ite bind nucleosides fir 1 of all: i.e., adeno ine, ino ine. etc ..
purine ba e analogue (i.e., caffeine and theophyline and dinu leotides). furthermore. it al o repre ents the econdary binding . ite
of nucleotide (Ka vin ky el al. 1978. John on el al. 1979). And
the glycogen storage ite (Dombrdi 19 l. ewgard el al. 19 9,
Johnson el al. 1989). In vertebrate , the unpho phorylated form (b

Correspondence 10 F. San Juan errano. Departamento de Biologfa Fundamental. Facultad de Ciencia .. Uni er idad de Vigo. Apdo. 784. 36200
Vigo. Spain.








Sea rnu sel were supplied by a purification plant in Villagarca

de Arosa (Galieia,
Spain). Mussel with helllength between
7 and 10 cm were elected for the experiment . They were transponed lO the laboratory within 1 hr of collection. Their rnantle
were separated and lo red at -30C until u e.

The ubstrates, enzyme. and eoenzymes were obtained from

Sigrna Chernical Company (St. Louis. MO) and Boehringer Mannheim (Mannhei m , Gerrnany).
Mag ne s iu rn acet ate , t r ishydroxyrnethyl aminornethane, irnidazole and other ehemieals and
salts were purehased from Merk (Darrnstadt, FRG).

o/ Phosphorylase


Phosphorylase b extraer was prepared frorn rnantle tissue

hornogenized in 2 volumes (w/v) of ice eold 40 mM tris-acetate
buffer pH 7.0, eontaining EOTA, imidazole and 2-mereaptoethanol in a concentration of 5 mM and 0.12 M KCI. For preparation of pho phorylase a extraer, a hornogenate in the same eonditions de eribed above. but without EOTA, wa incubated with 2
mM ATP, MgCI2 and CaCI2. In thi medium. endogenou phophoryla e kina e tran forms the pho phoryla e b lO its a form ( an
Juan el al. 1995). Both homogenate . coruaining b and a phosphoryla e forms respectively. were centrifuged at 150400 x ga, for 20
rnin and the pellet was di earded. The upernatant were dialyzed
again l 30 vol of extraction buffer containing 10 mM aF and
without KCI.
EIIlJ!me Determinations

Glyeogen pho phorylase aetivity wa mea ured spectrophotornetrically in the direetion of glyeogen breakdown, a de eribed in
San Juan el al. (1991). The reaction mixture for phosphoryla e b
contained 40 mM tris-acetate buffer pH 7.0, 5 rnM irnidazole, 2
mM EOTA, lA mM 2-ereaptoelhanol. 5 mM acetate-Mg, 5 ~M
0.6 mM
AOP, 80 mM pho phate
buffer, pH 7.0, 1.6 mM AMP, 16 mg/mL glycogen, 0.5 I.U. of
glueo e-6-phosphate dehydrogena e. 0.1 J.U. of pho phoglucornula e. and enzyme preparation in a total volume of 1.0 mL. The
reaction mixture for pho phoryla. e a lacked EOTA and AMP. and
the glycogen and pho phate buffer concentrations were
and 30 mM, re pectively.
Pho phoryla e activity wa as ayed in the direction of glycogen
ynthesi by mea uring the relea e of P, from G 1P. The reaetion
rnixture for the pho phoryla e b eontained 40 mM tri -a etate
buffer, pH 7.0.2 mM EOT A, .2 mg/mL glycogen, 1.6 mM AMP,
00 mM G-I-P, and enzyme preparation in a total volume of I mL.
The reaetion mixture for pho phoryla e a laeked EOTA and AMP.
and the G 1P eoneentration wa 50 mM. After incubation al 30C
for 45 mino the reaction was topped by the addition of 0.1 mL of
20% (w/v) SOSo After centrifuging, P, in the upernatant was
determined by the method of Saheki et al. (1985).
Dala Analysis

The inhibitory effect eau ed by everal concentration

of gluco e and G6P wa determined by a rnean of provoked modifications in the kinetic constants of glyeogen pho phoryla e in equilibrium. in relation to its ub trates. The inhibitor aetion meeha-




ni m wa deterrnined u ing Lineweaver-Burk double reeiprocal

and Hill plots. The inhibition constants were calculated from the
representation of double reeiproeal (-I/Kmap = -l/Km (1 + [IV
K,). When the pre. enee of an inhibitor di places the repre en lations 01' double reeiproeal from linearity, the repre entation of IIV
a oppo ed lO 1/[ )2 was used. In this rnanner, the illu tration is
linearized and the inhibition con tant may be calculated in these
ea. es, a. in tho: e de eribed above (Segel 1993).
Kinetic pararneters (Km or Scu and VmuX>were ealculated u ing
thc "Graf'it program (Erithacu Software Ltd .. Sraines, U.K.).
Result are given as mean EM. Eaeh date corre pond to rneans
of 4-5 determination
and regre sion coefficient of LineweaverBurk, and Hill plOIS were 0.96-0.99.

Because 01' the method used lO rneasure pho phorylase activity

in the direction of glycogen brcakdown, only the effect produced
by the gluco e and the UOPG eould be examined. Neither of the
lWO rnolecules present inhibitory effects on the enzyme (Iorrns a
and b) of Mytilus in this sense.
Glyeogen pho phoryla e (a-lA glyeogen, ortho-pho phate glueosyltransfera e E.e. 204.1.1) eatalyze the degradation of glyeogen lo glucose-t-phosphate.
Thi reaction i rever ible. but in the
cell the ynthe i of G 1P trongly favor the high Pi concentration . In vitro, the ratio [Pi]/[G 1P)that determined the equilibrium
con tant can be rnodified (i.e., inereasing the G 1P al saturating
concentration, 800 mM), di plaeing the reaetion in reverse direeiion. BUl the enzyme affinity for the ubstrates and effector and
the interactions between them are the ame. and they can be studicd in both direcLion .
In the rever e reaetion direction ( ynthesis of glyeogen), UOPG
neither showed any inhibitory effect, wherea the glueo e and G6P
present an evideru inhibition of form b relative to the G IP ubtrate, but they do n 1 affe t the activity of form a, whieh G6P even
seem lO actvate mildly (Fig . l. 2). Kinetic parameters of this
enzyme were determined using aliquots of the purified enzyme
(esscntially a San Juan el al. 1991) and u ing crude li ue extract,
before purification, giving identical result . Although glyeogen
pho phoryla. e reaction wa not rneasured in physiological direction, the re ults of this srudy show the Mytilus enzyrne affinity for
the e inhibiton and their effects on the conformational changes of
moleeular tructure, which affect to catalitic ite and its affinity for
all ub trate.
In relation t form b of the Mytilus enzyme, both moleeule
(glucose and G6P) follow a cornpetitive type pattern, only affecting the affinity of the enzyme by the G 1P ub trate. G6P increa es
the Km in an order of magnitude from 180 mM to 1.8 M when il
increa e conceniration from O lO 1.5 mM. The Ki obtained for lhi
inhibitor i between 0.2-0.3 mM (Fig. 3). a value similar to that
obtained in the adductor mu eJe of Pecten (Hata et al. 1987). Al o,
the glueo e produce a far lower increa e of Km for the G IP (up
to 217 mM) when it
n entration range from O to 20 mM,
alculating a Ki of 190 mM (Fig. 4). Both in the presenee of G6P
and of gluco e, an increa e in the Hill Index for the G I P frorn
values approaehing 1 10 value of 1.5 and 1.9. re pectively i
ob erved (Figs. 3. 4 insets).
The behavior of both forrn of enzyme in the mantle tis ue of
Mytitus in relation LO the e two inhibitor differ notably from
what is de cribed in the bibliography, particularly a. regard form
a and inhibition by glueose of forrn b.








.-.. ..







'09 (GIP)""







o. -





1 . ()

Figure 1. Double recprocal plot of initial velocity of pho phorylase a
as funclion of G lP concentration
in the pre ence of several gluco e
concentratons: ( ) O rnM, (e) 10 mM, (6) 20 111M.

G6P i. de. cribed as a typical allo teric inhibitor of form b of

the glycogen phosphoryla e in relati n 10 the AMP with which it
cornpetes for the nucleotide site (Mad en and hecko ky 1967) by
binding 10 the phosphate ubsite (Lorek el al. 19 4). Thi would
explain our results, becau e a le er bind of the AMP lO the nucleotide place would not actvate the bind of the substrate 10 the acti e
ccnter 10 the ame extent as if an inhibir r were la king, thus
causing an evident decrease in affinity. In turn, a. the bind of the

1~' l rn

Figure 30 Double recproca] plotsof initial velocity of phosphorylase b

as function of G I P concenlralion
in the presence of everal G6P concentrations:
( ) O 111M, (e) 0.5 mM. (6) 1.5 mMo Inset: HiII plot,

G 1P with the active center i le strengthened. thi ubstrate may

bind. in a econdary formo 10 the nucleotide place by [he subsite for
the P, (Dornbrdi
19 1), a indicated by the increase in the Hill
Index (Table 1)0
Gluco e i a well kn wn inhibitor of both form of the glycogen pho phoryla e, tabilizing the inactive T conformation by bind
10 the acti e center and redering it a better sub trate for pho phorylase pho phata e (Her 1976, Sprang el al. 1982, Mad en el al.

















~, ~


1 -



,1 F

O. '



Figure 2. Double reciprocal plots of initial velocty of phosphorylasc

as function of G I P concentration
in the presence of several G6P concentrations:
( ) O mM, (e) 005 111M, (6) 1.5 111M.

o. 7

::! 08

1 .




Figure 4. Double reciprocatplotsof initial velocityof phosphorylase b

as funclion of G IP concentration
in the pre ence of several gluco e
( ) O rnM, (e) 10 111M, (6) 20 111M. lnset: HiLl plot.







Modification of kinetic parameters

of glycogen phosphorylase
(a and
b) from Mylilus mantle with respect to substrate G 1P in the
presence of several G6P and glucosc concentrations.

the sarne order.



Vm (1 /mL)



Km (111M)

for both, substrate (G 1P) and inhibitor

The value of both constants,


Km and Ki respectively,

the value of Km

is 95-fold

are within

for the glycogen


srnaller (San Juan et al. 1993).

into account the seasonal coruent of free tissue glucose

regulated regardless 01' the mobilization of glyco01' glucose--phosphate phosphaiase activity in the rnantle tissue of Mytilus, which involves an irnportant
difficulty for the gluco e to be the final expression 01' the glycogenolitic effector system (Cres po 1989). and the high values 01' Ki


gen. the non-existence

[glycogen]: 3.2 I11g/I11L
IAMP): 1.6 111M

0.0 mM
0.5 mM
1.5 111M

0.475 3 x 10--'

182.38 2 x 10-2
296.59 6 x 10-'
1699.0 6 x 10-'

0.415 7 x 10
0.491 5 x 10--'


191.98 6 x 10-'

0.445 3 x 10
0.395 5 x 10



10.0 111M
20.0 mM
1: 3.2 rng/ml,

for this metabolite


the regulating role 01' the glucose

paihway lacks the meaning and importance
de scribed in vertebrate where il is the rnost important physiological inhibitor of phosphorylase (Glin rnann el al. 1970, Stalrnans
and Gevers 1981. Carabaza el al. 1992) as occurs in other invertebrate (in ects) where the regulatory role is in doubt (Applebaurn
in the glycogenolitic

1973. Van Marrewijk



0.0 mM
0.5 mM
1.5 111M


0.123 4 x 100.157 4 x 10--'

1.49 5 x 10
0.89 1 x 10-'
0.81 4 x 10--'




1.34 1 x 10-'

el al. 1988).

the most rnarked

with the regulating

main interaction


of G6P, it


found in the mantle

1990), besides the high phosphoglucormnase


akada 1968) and the non-existence

phosphate pho sphatase

0.112 4 x 10--'


site of the enzyme and its low

Ki. inside the range of concernrations


10.0 mM
20.0 mM

in this work (190 111M), we rnay con-

sider that in the Mytilus mantle,




01' glucose-6-

1989). lead us to attribute

a regu-

Results are expressed as mM (Km - SIl~) and IU/ml SEM (standard error

lating effect on this pathway to this rnolecule, in this lis ue, which
is more important than that of gluco e. as also eern lO occur in
the Iar-body 01' in ects (Applebaum
1973) and in yeasts (Sagardia


el al. 1971).

1983, Carabaza et al. 1992). In ihe case 01' the Mytilus enzyme, we



1 x 10-'

lO recently obtained but, unpublished data, we have

a higher proporrion of phosphorylase b in the rnantle
ti sue of Mytilus which i significantly
correlated with garnetogeAccording

have observed the inhibition

Iorrn a. The inhibition
is clearly a cornpetitive

on forrn b and the noninhibition


exerted by rhis rnolecule on the G 1P bound

type, both binding 10 the same area in the

center and nOI via negative


behave .. As before, the G 1P displaced


by the inhibitor

active center tends to interact with the nucleotide

as G6P
frorn the

site, and consis-

tenily an increase in the Hill Index is observed (Table 1).

A curious fact, consiantly observed in the re ults obtained in

paper. is the greater affinity

of the form phosphorylated

G 1P ubstrate (the Km decrea e between

the sarne lime as it
of the affinity
forrn (Table



decrease four-fold

and mximum


of the non-pho

1). Moreover,

is particularly

becau e the inhibiiory

on the affinity

by the

122 and 2098 times), al

in rerrns


effect of b th

by the G 1P and since this



al limes when the concentration



rnay be derivecl toward


form is described


pr bably needs lo Iransfer the glucidic

re erves in the forrnation

in short periods 01' time.

the activity nor the affinity

upports the hypothe i

ite than
cerner in the pho phorylated forrn of the Mytilus

of a greater interaction
with the active


of form a, this

of the G 1P with the nucleotide

(San Juan 1991).

The inhibitory

do nOI affect either


can be explained


in reaction

by the low affinity

rever. e direction

of Mytilus glycogen

Applebaurn, S. W. & H. M. Schlesinger. 1973. Regulan
body pho sphorylase. Biochem. 1. 135:37-41.
Bayne. B. L., A. Bubel, P. A. Gabbou, D. R. Livingston,

n of Locust falD. M. Lowe &



in vertebrates,


by the substrate



of glycogen degradation by ihese me-


paper. in comparison

may be considered


01' phosphorylitic

cogen in the gametogenic

10 lipidic

regard to this

of high energy dernand as stress

as shown in thi


when it very


a with

which together with its high affinity

The different

of form

of low


re erve depo it

01' garnete . Also, thi

of the independence

10 a different

of glucose

in situations


time (Johnson el al. 1989), as it is the garnetogenesis.

duced. Apart from the fact that these inhibitors


as fundamental

bUI is maintained

respond al situations


the pentose phos-

phate pathway lO form nucleic acids and reductor power required

in ihe biosynthesis of lipids, during garnetogenic develcpment.
This SUppOl1S and justifie
the low affinity of forrn b by the
glucose and its special regulation by G6P. The activity of this

low, al o in the absence of inhibitors (Km

= 180 mlvl), the
arce phy i logical importan e of the glycogen
pho phorylase reaction of Mytilus in this direction rnay be de-


of G6P is al a

mnimum and G6P dehydrogena e activity i at a rnaximum. The e

data suggest that, al particular times, the phosphorolitic pathway 01'

with that described

as an adaptarional consequence
and provides new data on the


in the mobilization

process of bivalve

of gly-


M. . Moore. 1982. Glycogen utili ation and garnetogenesis in Mytilus
edil lis L. Mar. 8iol. Lett. 3: 9-105.
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