Alcorn_1978_Biochimica-et-Biophysica-Acta-(BBA)---Biomembranes

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361

Biochimica e t Biophysica Acta, 510 (1978) 361--371

© Elsevier/North-Holland Biomedical Press

BBA 78061

A K I N E T I C A N A L Y S I S O F D - X Y L O S E T R A N S P O R T I N RHODOTOR ULA

GLUTINIS

MARY E. ALCORN and CHARLES C. GRIFFIN *

Hughes Laboratories, Department of Chemistry, Miami University, Oxford, Ohio 45056

(U.S.A.)

(Received November 23rd, 1977)

S u m m a r y

T h e k i n e ti c s o f D - x y l o se t r a n s p o r t w e r e s t u d i e d in Rhodotorula glutinis.

A n a l y s i s o f t h e s a t u r a t i o n i s o t h e r m r e v e a l e d t h e p r e s e n c e o f a t l e as t t w o c ar -

r i er s f o r D - x y l o s e in t h e Rhodotorula p l a s m a m e m b r a n e . T h e s e t w o c a r ri er s

e x h i b i t e d K m v a l u es d i f fe r in g b y m o r e t h a n a n o r d e r d f m a g n i t u d e . T h e lo w -

K m c a r r i er w a s r e p r e s s e d i n r a p i d l y g r o w i n g ce ll s a n d d e r e p r e s s e d b y s t a rv a t i o n

o f t h e c e l l s .S e v e r al h e x o s e s w e r e o b s e r v e d t o i n h i b i t D - x y l o s e t r a n s p o r t . I n t h e s tu d i e s

r e p o r t e d h e r e , t h e i n h i b i ti o n s p r o d u c e d b y D - g a la c to s e a n d 2 - d e o x y - D - g l u c o s e

w e r e e x a m i n e d i n s o m e d e ta i l in o r d e r t o d e f i n e t h e i n t e r a c t i o n s o f t h e s e s u g a rs

w i t h t h e D - x y l o s e c a rr ie rs . 2 - D e o x y - D - g l u co s e c o m p e t i t i v e l y i n h i b i te d b o t h o f

t h e D - x y l o s e c a rr ie r s. I n c o n t r a s t , o n l y t h e l ow - K i n c a r ri e r w a s c o m p e t i t i v e l y

i n h i b i t e d b y D - g a l a c t o s e .

I n t r o d u c t i o n

T h e r e d y e a s t Rh. glutinis h a s b e e n r e p o r t e d t o a c c u m u l a t e a w i d e v a r i e t y o f

m e t a b o l i z a b l e a n d n o n m e t a b o l i z a b l e m o n o s a c c h a r i d e s a g ai n st c o n s i d e r a b l e

c o n c e n t r a t i o n g r a d i en t s [ 1 ] . O n e o f t h e i n t e r es t in g f e a t u r e s o f t h is s y s t e m is

t h a t i t is c o m p l e t e l y i n h i b i t e d b y m e t a b o l i c i n h i b i to r s a n d u n c o u p l e r s o f o x i d a -

t iv e p h o s p h o r y l a t i o n ; a t t e m p t s t o d e m o n s t r a t e a p a ss iv e , f a c i l i ta t e d d i f fu s i o n i n

t h e a b s e n c e o f m e t a b o l i s m h a v e b e e n u n s u c c e s s f u l [ 2 , 3 ] . P r i or t o a n e x a m i n a -

t i o n o f t h e m e c h a n i s m o f e n e r g y t r a n s d u c t i o n i n t h e c o n c e n t r a t i v e t r a n s p o r t o f

s u g a r s b y Rhodotorula, i t w a s d e c i d e d t o c h a r a c t e r i z e t h e c a r r i er ( s ) i n v o lv e d .

T h e n u m b e r o f c a r b o h y d r a t e c a r ri er s i n t h e Rhodotorula p la s m a m e m b r a n eh a s n o t b e e n e s t a b l i s h e d . M u t u a l i n h i b i t i o n s b e t w e e n s u g a r p a i rs , s u c h a s

D - a r a b i n o s e a n d D - g l u c o se [ 1 ] , D - x y l o s e a n d D - g lu c o s e [ 4 ] , a n d D - x y l o s e a n d

* To whom correspondence should be addressed.



362

L-xylose [1], have been presented as evidence that these sugars utilize the same

carrier. On the basis of countertransport studies, HSfer [5] suggested that pen-

toses and hexoses shared a common carrier in this organism. In 1976, however,

Janda and coworkers [6] reported that, although D-xylose and D-galactose were

translocated by the same carrier, D-fructose employed a different carrier, andD-glucose appeared to interact with all of the carriers in the membrane. How-

ever, none of the studies above presented saturation isotherms for the carbo-

hydrate being transported nor did they provide any evidence that the observed

intersugar inhibitions were indeed compet itive.

The purpose of the present communication is to present a detailed kinetic

analysis of D-xylose transport by Rh. glutinis and to describe the modes ofinhibiti on of this process by two hexoses, 2-deoxy-D-glucose and D-galactose.

Methods

Growth and preparation of cells. Rh. glutinis (ATCC 26194) was grown at

30°C in a medium containing per 1:1.0 g K2HPO4, 0.66 g of NH4NO3, 0.5 g of

NaC1, 1.0 g of MgSO4 • 7 H20, 0.33 g of yeast extract (Difco), 0.05 g of FeC13 .

6 H20, 0.31 g of CaC12 • 2 H20 and 25 g of glucose [1]. The pH of the medium

(wit hout glucose) was adjusted to 5.5 before autoclaving. Five-hundred ml

flasks containing 150 ml medium were inoculated with 5 ml of a 24-h culture

and incubated in a water-bath shaker for 10 h (mid-log phase). Cells were har-

vested by centrifugation and washed 3 times with 0.1 M KH2PO4 at room tem-

peratu re. The washed cell suspensions (5% wet wei ght/ volu me in 0.1 MKH2PO4) were vigorously aerated on a magnetic stirrer for 1.5 h at room tem-

perature, then packed in ice and maintained with gentle stirring at 0°C.

Transport assays. D-Xylose transport was measured by incubating cell sus-

pensions with D-[14C]xylose in a water-bath shaker at 30°C. Uptake was

initiated by the rapid additi on of 1.0 ml of cell suspension (preincubated at

30°C for 5 min) to an equal volume of 0.1 M KH2PO4 contain ing labeled

D-xylose and other ingredients where indicated. At various time intervals,

0.5-ml samples were withdrawn and filtered through a cold prefilter

(AP2502500, Millipore)/glass-fiber filter disc (934AH, Reeve Angel) combina-

tio n. The collect ed cells and the filters were washed with 4.0 ml of ice-cold 0.1 MKH2PO4 and transferred to scintillation vials along with 1.5 ml of 60% (v/v)

ethanol and 10 ml of scintillation fluid (Aquasol II, New England Nuclear).

Samples were counted in a Beckman Liquid Scintillation Spectrometer (LS-

100c).Data analysis. Initial velocities of D-xylose transport were estimated from the

differences in intracellular D-xylose concen trat ion betwee n samples taken after

1 and 4 m in of incu bati on. The progress curves were reasonably linear over thistime interval and the calculated initial velocities were directly proportional to

cell concentration. The absolute rates of transport, calculated on a dry weight

basis, varied somewhat with different cell suspensions. When saturation datafrom different experiments were combined, the velocities were normalized to a

single, reference experiment. For each experiment the ratio of the observed

velocity to the reference velocity was calculated at each concentration of

D-xylose and the average of these values was employed as the normalization



363

f a c t o r . E q u a t i o n s f o r th e s a t u r a t i o n i s o t h e r m w e r e f i t te d t o t h e d a t a s e t b y

u n w e i g h t e d , n o n - l i n e a r re g r e ss i o n t e c h n iq u e s . C o m p u t e r p ro g r a m s w e r e

m o d e l l e d a f t e r th o s e d e s c r i b e d b y C l el an d [ 7 ] .

Materials. D - [ U - 1 4 C ] x y lo s e ( 2 . 9 2 m C i / m m o l ) w a s o b t a i n e d f r o m A m e r s h a m

C o r p . , A r l i n g t o n H e i g h t s , I l li n oi s. C y c l o h e x i m i d e a n d n o n - r a d i o a c t i v e s u g ar sw e r e p r o d u c t s o f S i g m a C h e m i c a l C o . , S t . L o u i s , M o . A l l o t h e r c h e m i c a l s w e r e

o f t h e h i g h e s t p u r i t y c o m m e r c i a l l y a v a i la b l e.

Re s u l t s

I n i ti a l s t u d i e s o f D - x y l o s e t r a n s p o r t i n Rh. glutinis e x h i b i t e d a v a r i a b i l it y t h a t

p r e c l u d e d a n y a t t e m p t s a t a d e t a i l e d k i n e ti c a n a l ys is o f t h e t r a n s p o r t p r o c e s s .

M o s t o f t h is v a r i a b i l it y w a s t r a c e d t o t w o f a c t o r s : ( a ) d i f f i c u l t y i n a c h i e v in g

r e p r o d u c i b l e f i lt r at i o n r a t es i n t h e t r a n s p o r t a s sa y a n d ( b ) t i m e - d e p e n d e n t

c h a n g e s i n t h e t r a n s p o r t a c t i v i t y o f t h e c e ll s u s p e n s i o n s . T h e f ir s t p r o b l e m

a r o s e f r o m t h e u s e o f a m e m b r a n e f i lt e r ( M i l li p o re , 0 . 4 5 ~ m p o r o s i t y ) in t h e

t r a n s p o r t a s s a y s . Rhodotorula s u s p e n s i o n s f r e q u e n t l y c l o g g ed th i s t y p e o f

f il te r . R e p l a c e m e n t o f t h e m e m b r a n e f i lt e r w i t h a p r e f i lt e r /g l a s s - f ib e r f i lt e r

c o m b i n a t i o n p r o v i d e d f o r r e t e n t i o n o f a ll o f t h e c e ll s w h i l e a l l o w i n g r a p i d ,

r e p r o d u c i b l e r a t e s o f fi l tr a t i o n . T h e s e c o n d f a c t o r c o n t r i b u t i n g t o t h e v a ri ab i l-

i t y o f re s u lt s w a s f o u n d t o b e t h e w a y i n w h i c h c e lls w e r e m a i n t a in e d a f t e r t h e y

w e r e h a r v e s t e d . F ig . 1 d e m o n s t r a t e s t h a t t h e r a te o f D - x y lo s e t r a n s p o r t b y

w a s h e d c ell s u s p e n s i o n s in c r ea s e s d u r i n g a e r a t io n a t r o o m t e m p e r a t u r e f o r

5 0 0

4 0 0

1 0 0 - - ) ,(

i o 6 o , ' oTIM E ( r a in )

300

0

2 0 0

Fig . 1 . T ime-cour se o f ac t iva t ion o f D-xy lose t ranspor t dur ing s t arva t ion o f Rh. glutinis. Cells were har-

ves ted a n d w a s h e d a t 0°C. At zero t ime, cel l s u s p e n s i o n s w e r e s u b j e c t e d t o v i g o r o u s a e r a t i o n a t ro o m

t e m p e r a t u r e i n t h e p r e s e n c e (×) and absence ($ ) o f 100 ~g /ml o f c y c l o h e x i m i d e . T r a n s p o r t was asssayed

a t 30°C in t h e p r e s e n c e of 1 .0 mM n-xy lose . In i t i a l ve loc i t i es were es t imated f rom t h e d i f f e r e n c e s i n i n t r a -

c e l l u l a r D-xylose c o n c e n t r a t i o n b e t w e e n s a m p l e s t a k e n a f t e r 1 and 4 rain of i n c u b a t i o n .



3 6 4

25

20 . / ' /

15

, o / , .5

o s '0 1;0 1;0 2;0

[ D -XYLOSE] m M

Fig . 2 . Satura t ion iso therm for D-xylose t ranspor t by Rhodotoru la . The curve s are from non-linear regres-

sion f its to one-carr ier ( . . . . . ) and two-c arrie r ( ) mode ls for the tra nspo rt process. Velocities are

express ed in /~mol • rain -1 • g-1 (dry w eight) . Init ial velociti es were es tim ate d fr om the differ ences in intra-

cel lu lar D-xylose concentra t ion between samples taken af ter 1 and 4 ra in of incubat ion .

1.5 h. Increases in uptake rate were also observed with cells stirring in arefrigerator at 4--6°C, but not when cell suspensions were packed in ice andgently stirred at 0°C. Aerated cells, subsequently packed in ice, maintained

their higher rates of transport, unchanged, for several hours. Cycloheximideprevented the increase in transport activity during aeration without signifi-cantly affecting the basal rate of D-xylose uptake (Fig. 1). Unless otherwisenoted, the studies reported below employed cell suspensions which had beenactivated by aeration and stored in ice.

The saturation isotherm for D-xylose uptake by Rh . g lu t in i s is shown inFig. 2. This figure represents a composi te of results from a dozen separateexperiments with different cell suspensions. Fig. 2 contains 105 data pointscovering an 81-fold range of D-xylose concentrations. The data were tested forconsistency with the following models by unweighted, non-linear regressions:

( i ) Mo d e l I , a Mich ae l i s - Men te n f u n c t io n : r ep r esen t in g a s in g le , sa tu r ab l e ca r r i e r

v l • I S ]v ( i )

K m + IS ]



3 6 5

( i i ) M o d e l I I , a M i c h a e l i s - M e n t e n f u n c t i o n p l u s a l i n e a r t e r m : r e p r e s e n t i n g a s i n g l e c a r r i e r

a n d a n o n - s a t u r a b l e d i f f u s i o n p r o c e s s ( ' l e a k ' )

Y l • [ s ]v - + k . [ s ] ( 2 )

K m l + [ S ]

( i i i ) M o d e l I I I , a s u m o f t w o M i c h a e l i s -M e n t e n f u n c t i o n s : r e p r e s e n t i n g t w o s a t u r a b l e c a r r i e r s

i n t h e m e m b r a n e

V 1 " [ S ] V 2 - [ S ]

U - Kml + [S] + (3)K m 2 + [ S ]

w h e r e V , a n d V 2 ar e a p p a r e n t m a x i m u m r a te s o f t r an s po r t K i n1 a n d Ki n 2 a re

kinetic (Michaelis) constants an d k is a first-order rate constant. T h e ab ov e

mo de ls wer e also considered by Atkins and Gar dne r [8] in their analysis of

intestinal transport kinetics. Wi th all three equations, c on ve rg en ce w as attained

fr om several different initial para mete r estimates. Th e conve rged values of the

constants an d the variances for the overall fits are presented in Tabl e I. T h e

data w ere judg ed to be inconsistent with Mo de l I on the basis of high variance

an d visual inspection of its inability to fit the data in Fig. 2. Ev en th ou gh a

wi de range of concentrations wa s em pl oy ed in this study the saturation data

alone did no t allow a clear distinction be tw ee n Mo de ls II an d III. Th e kinetic

c o ns t an t s, K m l a n d K i n 2 , f r o m M o d e l III w e r e l es s- de fi ne d t h a n t h e K m l a n d

k f r o m M o d e l If, b u t t h e va r ia n ce w a s s o m e w h a t s m al l er f or t h e f o r m e r m o d e l

(Table I). Mo d el III wa s selected as the better representation o f the D-xylose

transport sy st em in this org ani sm largely on the basis of its ability to acco unt

for the specificity exhibited by the transport sy ste m in the inhibition studies

described below.

Several hexoses we re ex am in ed for their ability to inhibit D-xylose (2 raM )

transport. D-Glucose, D-fructose, D-galactose an d 2-deoxy-D-glucose we re fo un d

to be inhibitory while L-r ham no se wa s only slightly inhibitory even at a con-

centration of 10 0 raM. D-Galactose an d 2-deoxy-D-glucose pr od uc ed significant

inhibition at relatively lo w concentrations an d we re selected for further study.

T he Di xo n [9] pl ot for 2<ieoxy-D-glucose inhibition of D-xylose transport

is s h o w n in Fig. 3. T h e curvature of the graph suggested a parabolic inhibition,

T A B L E I

C O M P U T E D P A R A M E T E R S F R O M T H E S A T U R A T I O N I S O T H E R M F O R D - X Y L O S E

T h e p a r a m e t e r s w e r e o b t a i n e d f r o m u n w e i g h t e d , n o n -l i n ea r re g re s si o n a n a l ys e s of 1 0 5 d a t a p oi n ts . T h e

t h r e e m o d e l s c o n s i d e r e d w e r e : I, M i c h a e l i s - M e n t e n f u n c t i o n ; II, M i c h a e l i s - M e n t e n e q u a t i o n p l u s a l in ea r

t e r m ; I II , s u m o f t w o M i c h a e l i s - M e n t e n e q u a t i o n s . V i s e x p r e s s e d i n / ~ m o l • r n i n I • g - 1 ( d r y w e i g h t ) , K m i n

r a M , a n d k i n # I • r a i n I • g - 1 ( d r y w e i g h t ) . P a r a m e t e r s a r e g i v e n ± S . E . T h e v a r i a n c e i s 2: ( V o b s - -V c a l c ) 2/

( 1 0 5 m i n u s t h e n u m b e r o f p a r a m e t e r s e v al ua te d) .

P a r a m e t e r M o d e l I M o d e l I I M o d e l I II

V 1 3 1 ± 1 1 9 -+ 1 13 -+ 2V 2 - - - - 3 3 ± 4

K i n 1 2 . 5 ± 0 . 2 0 . 9 7 -+ 0 . 0 9 0 . 5 6 ± 0 . 1 3

K i n 2 - - - - 1 8 ± 6

k - - 6 6 0 -+ 4 0 - -

V a r i a n c e 3 . 2 1 . 5 1 . 3



3 6 6

0.4

0.3

1

F o.2

0.1

, , L , ' 0 10 0.2 0.4 6

[ 2 - d e o x y - D - G l u c o s e ] mM

F i g . 3 . D i x o n p l o t o f t h e i n h i b i t i o n o f D - x y l o s e t r a n s p o r t b y 2 - d e o x y - D - g l u e o s e . T h e c o n c e n t r a t i o n o f

D - x y l o s e w a s 1 . 0 r a M . T h e s o l id c u rv e w a s g e n e r a t e d f r o m E q n . 4 f o r c o m p e t i t i v e i n h i b i t i o n o f a t w o -

c a r r i e r s y s t e m ( M o d e l h i ) . V e l o c i t i e s a x e e x p r e s s e d i n t ~ m o l • r a i n - I • g - 1 ( d r y w e i g h t ) . I n i t i a l v e l o c i t i e sw e r e e s t i m a t e d f r o m t h e d i f f e r e n c e s in i n t r ac e l lu l a x D - x y l o s e c o n c e n t r a t i o n s b e t w e e n s a m p l e s t a k e n a f t e r

1 a n d 4 r a i n o f i n c u b a t i o n .

probably involving two molecules of inhibitor, and the data were consistent

with inhibition of both carriers (Model III) of the transport system. The inhibi-

tion was further analyzed by examining the initial rates of transport as a func-

tion of the concentration of D-xylose in the presence and absence of 0.2 mM

and 0.4 mM 2-deoxy-D-glucose. These data are presented in double-reciprocal

for m [10] in Fig. 4. The presence of at least two carriers for D-xylose is seen inthe distinct curvature of the plots. A strict analysis of the data by non-linear

regression comparisons of the fits to equations for competitive, uncompeti-

tive and noncompetitive inhibitions was not feasible because of the limited

amount of data (68 points) and the large number of parameters in the appro-priate rate equations (e.g. Eqn. 4). Convergence of the data near the recipro-

cal velocity axis in Fig. 4 strongly suggested that the inhibition was competi-

tive and the lines in both Figs. 3 and 4 were generated from Eqn. 4 with values

for V~, V2, Kml, and Km2 from Table I, and g i l = 0.11 mM, Ki2 = 0.32 mM,K i i I = 0.17 mM 2 and K i i 2 = 0.48 mM 2. Eqn. 4 describes a (non-linear) compe-

titive inhibition of both the high- and low-Km carriers for D-xylose (ModelIH).

v l • [ s ] v2' [Sl

V = K . [ 1 + - [ I ] + [ I ] 2 ~ ? I S ~ + - ( [ I ] [ I ] 2 ~ +

m l " ~ K i ~ l K i l l ~ l l K m 2 " 1 + K i + K i i 2 ] [ S ]

( 4 )



1.0

367

0 .8

l l v

0 6B

I

0.4

0. 2

0 ' 1 : 0 2 : 0 3 : 0 , i 0 ~ 0

I / / [ D ' X Y L O S E ] m M "~

F ig . 4 . L i n e w e a v e r - B u r k p l o t o f t h e u p t a k e o f D - x y l o s e ( A ) a n d i ts i nh ib it io n b y 0 . 2 m M ( B ) a n d 0 . 4 m M

( C ) 2 - d e o x y - D - g l u c o s e . T h e s o li d c u r v e s w e r e g e n e r a t e d f r o m E q n . 4 f o r n o n- l in e ar c o m p e t i t i v e i n hi b it i on

of a two-carrier sy st em (M od el III). Velocities are ex pres sed in /~rnol • rnin-t, g-1 (dry weight). Initial

v e lo c it i es w e r e e s t i m a t e d f r o m t h e d if f er e nc e s i n i n tx a ce l lu l ar D - x y l o s e c o n c e n t r a t i o n b e t w e e n s a m p l e s

t a k e n a f te r 1 a n d 4 r a in o f i n c u b a t i o n .

0 8

0. 6

1/ y

0 . 4

0 . 2

o ' 2 ; ' 4 ' 0 ' s ; ' 8 ' o ' 1 ; o

[ D - G A L A C T O S E ] m M

F i g. 5 . D i x o n p l o t o f t h e i n hi b it i on o f D - x y l o s e t r a n s p o r t b y D - g a l ac t os e . T h e c o n c e n t r a t i o n o f D - x y l o s e

wa s 1.0 r aM . Velocities are ex pres sed in #to ol • rain I • f t (dry weight). Initial velocities we re esti mate d

f r o m t h e di ff er en ce s i n i nt ra ce Uu la r D - x y l o s e c o n c e n t r a t i o n b e t w e e n s a m p l e s t a k e n a f t er 1 a n d 4 m i n o f

i n c u b a t i o n .



368

The mode of inhibition of D-xylose transport by D-galactose was signifi-

cantly different from that observed with 2-deoxy-D-glucose. The non-linear

Dixon plot in Fig. 5 indicates th at the two carriers differ ed considerably in

their sensitivities to inhibition by D-galactose and very little inhibition was

observed at high concentrations of D-xylose. Data from a representative experi-ment in the presence and absence of 2.5 mM and 5.0 mM D-galactose are

presented in double-reciprocal form in Fig. 6. The lines in Fig. 6 were generated

from Eqn. 5 which assumes that only the low-Kin carrier was comp etit ivel y

inhibited by D-galactose. Values for the parameters were obtained from Table I

and Kil 0.6 mM was used.

v, .[S] v2 • [Sl= . . . . . . . . . . . . . . .

K m l • ~ / 1 + [S] Km2 + [S]

Kill

In order to furt her characterize the activation of transport p roduced by aera-

tion (starvation) of the cell suspensions (Fig. 1), a comparison of the ability of

hexoses to inhibit D-xylose transport was made between cells which had been

aerated in the usual way and those which had been harvested, washed and

maintai ned at O°C. Table II demonstra tes that the percent inhibition produced

11v

1.0 • C

08

B

06 • • •

0 . 4

A

O 2

i I I I

0 1 0 2 0 3 0 4 0

1 / [ D - X Y L O S Ic] m M "~

F i g . 6 . L i n e w e a v e r - B u r k p l o t o f t h e u p t a k e o f D - x y l o s e ( A ) a n d it s i n h i b i t i o n b y 2 . 5 m M ( B ) a n d 5 . 0 m M

( C ) D - g a l a c t o s e . T h e s o l i d c u r v e s w er e g e n e r a t e d f r o m E q n . 5 f o r l i n e a r c o m p e t i t i v e i n h i b i t i o n o f t h e l o w-

K m ca r r i e r i n a two-c a r r i e r sys t em (Mode l I I I ) . V e loc i t i e s axe expressed in t zmol - r a in -1 • gq (d ry we igh t ) .

I n i t i a l v e l o c i t i e s w e r e e s t i m a t e d f r o m t h e d i f f e r e n c e s i n i n t r a c e l l u l a r D - x y l o s e c o n c e n t r a t i o n s b e t w e e n

s a m p l e s t a k e n a f t e r 1 a n d 4 r a i n o f i n c u b a t i o n .



369

T A BL E I I

CO MPA RISO N O F T H E E FFE CT S O N IN H IBIT O RS O N ST A RV E D A N D N O N -ST A RV E D CE L L S

Non-s tarved ce ll s were harves ted , washed and main t a ined a t 0°C. Starved cel ls were harves ted , washed and

v igorous ly aera ted fo r 1 .5 h a t room tempe ra tu r e and then mai n ta in ed a t 0°C.

In h i b i t o r [X y l o s e ]

(raM)

Percen t inh ib i t ion o f D-xy lose t ranspor t

Non-s tarved Starved

0.4 mM 1.94 45 43

2-Deoxy-D-g lucose 0 .97 63 68

0.24 66 69

5.0 mM 1.94 6 43

D-Galac tose 0 .97 30 65

0.24 43 77

by 0.4 mM 2<leoxy-D-glucose was essentially independent of the way in which

the cell suspensions had been prepared. In contrast, the aerated (starved)cells

were much more sensitive to inhibition by D-galactose. These results suggest

that the activation process increased the p roporti on of low-Km carrier in the

cells.

Discussion

Although the carbohydrate transport systems of both Saccharomyces cere-visiae and Rhodotorula glutinis have been extensively studied, the number and

nature of the monosaccharide carriers in these organisms is still controversial

(see review by Barn ett [11]). Careful examin ati on of the literatu re reveals tha t

much of the inf orma tion on the carbo hydrat e carriers in these organisms comes

from saturation studies over a narrow (e.g. refs. 5 and 12) or unspecified (e.g.

refs. 1 and 12) range of concentrations or from inhibition studies in which a

simple competitive relationship is assumed (e.g. refs. 12 and 14) but usually

not demonstrated. The results presented in this paper demonstrate the complex

behavior that is observed when wide ranges of solute concentrations are

employ ed in transp ort kinetic studies.The sa turation isotherm for D-xylose uptake by Rh. glutinis (Fig. 2) exhibi ted

marked curvature in double-reciprocal form. If only a narrow range of D-xylose

concentrations had been examined, the curvature could have been masked by

experimental error. A 2-component system had to be invoked to explain the

data over the range of concentrations employed. Whether the second compo-

nent was a saturable (Model III) or a non-saturable (Model II) process could not

be dete rmine d f rom the saturation data alone. The specificity exhibited by the

second component in the inhibition studies, i.e., inhibition by low concentra-

tions of 2-deoxy-D-glucose compared to little or no inhibition by 10-fold

higher concentrations of D-galactose, suggested that this component of thetransport was indeed carrier-mediated (Model III). The 2 D-xylose carriers

exhibited Michaelis constants differing by a factor of about 30, and the maxi-

mum velocity of the high-Km carrier was about 2.5 times larger than that of the

low-Km system.



370

Both D-xylose transport systems of Rhodotorula appeared to be competi-

tively inhibited by the hexose, 2-deoxy-D-glucose. This type of inhibition is

consistent with a model in which either D-xylose or 2-deoxy-D-glucose can

occupy the same binding sites on the carriers. The inhibition, however, was not

simple. B oth the Dixon plot (Fig. 3) and the Lineweaver-Burk plots (Fig. 4) forthe inhibiti on required the [I] 2 terms in Eqn. 4 for good correla tion between

the data set and the rate equation. Any mechanism represented by Eqn. 4 must

involve at least two molecules of 2-deoxy-D-glucose in the inhibition of each of

the carrier systems. It would be premature to begin to develop such a model

until the transport of 2-deoxy-D-glucose itself has been characterized. The

inhibition data do suggest, however, that Rhodotorula may utilize at least two

carriers, with similar Km values, for the transport of 2-deoxy-D-glucose, and

that the kinetics of transport may exhibit some form of cooperativity. Posi-

tive cooperativity in the uptake of several sugars by Rhodotorula has been

suggested by Janda et al. [6].When D-galactose was tested as an inhibitor of D-xylose transport, the

character of the inhibition was distinct from that observed with 2-deoxy-D-

glucose. The Dixon plot (Fig. 5) indicated a "partial" inhibition, and very little

inhibition was observed at high concentrations of D-xylose. These observations

suggested tha t only the low-Km syst em was significantly inhibited by D-galac-

tose. Another distinguishing feature of the inhibition studies with this hexose

was a day-to-day variation in the percent inhibition produced by a given con-

centration of the sugar. On the basis of a model in which only one of the two

carriers was inhibited by D-galactose, the observed variations could arise from

differ ences in the relative amou nts of the t wo carriers present in diff eren t sus-

pensions of Rhodotorula. As noted below, the relative amounts of the carriers

may depend on the physiological state of the cells. The effect of that type of

variation would be averaged out in the analysis of the saturation i sotherm, and

the sym me try of Eqn. 4 would make it difficult to de tect in the inhibiti on

studies with 2-deoxy-D-glucose. The data in Fig. 6 are from a representa tive

experiment with D-galactose as the inhibitor. These data correlated reasonably

well with curves generated from Eqn. 5 assuming competitive inhibition of only

the low-Kin carrier. These results suggest that D-xylose and D-galactose may

share a common carrier in the Rhodotorula plasma membrane. A similar con-clusion was reached by Janda et al. [6], although these workers apparently

did not observe the second D-xylose carrier which was not inhibited by D-galac-

tose.

The relative activities of the high- and low-Kin systems and the overall rate

of D-xylose transport appeared to depend on the nutritional state of the

Rhodotorula cells. When cells were harvested during vigorous growth on D-glu-

cose and maintained close to 0°C, their transport activity was low. Starvation(aeration) of the washed cells at room temp erat ure for 1.5 h dramatically

increased their rate of D-xylose transport. A similar stimulation of D-glucose

transport during starvation of Rhodosporidium toruloides (= Rh. glutinis) hasbeen described by Barnett and Sims [15]. Comparison of the inhibitory pro-perties of 2-deoxy-D-glucose and D-galactose on D-xylose transport by starved

and non-starved cells suggests that the increased rate of D-xylose transport

produced by starvation was due to an increased activity of the low-K m system.



3 7 1

The effect of cycloheximide on this process is consistent with a derepression

of protein synthesis during starvation, but the identity of the protein(s) formed

during this period is unknown. The derepression was not limited to cells grown

on D-glucose; we have observed a similar process in D-xylose-grown cells. It

thus appears that transport of D-xylose (and perhaps other sugars as well) byRh . g lu t in i s is effe cte d by at least two carrier systems, one of which is repressed

when cells are growing in the presence of an adequate supply of carbohydrate.

A ck n ow l e d g em e n t s

This work was supported in part by grants from the Faculty Research Com-

mittee of Miami University and the Research Corporation. Taken in part from

an M.S. thesis submitted by M.E.A. to Miami University, Oxford, Ohio.

Re f e r en c e s

1 Koty k, A. and H6fer , M. (1965) Biochim. Biophys. Acta 102, 410- -4 22

2 H6fer , M. and Kot yk, A. (1968) Fol ia Microbiol . 13 ,1 97- -204

3 H6fer , M. (1971) Arc h. Mikxobiol. 80, 50--61

4 H6fer , M. and Dahle, P. (1972) Eur. J. Biochem. 29, 326- -33 2

5 H6fer , M. (1970) J. Membran e Biol. 3, 73--82

6 Janda, S ., Kotyk, A. and Tauchova, R. (1976) Arch. Microbiol . 111,1 51-- 154

7 Cleland, W.W. (1967 ) Adv. Enzy mol. 29, 1--3 2

8 Atkins , G.L. and Gardner , M.L.G. (1977) Biochim. Biophys. Acta 468, 12 7- -14 5

9 Dixon, M. (1953) Biochem. J . 55 ,1 70-- 171

10 Lineweaver, H. and Burk, D. (1934) J. Am. Chem. Soc. 56,6 58 --6 66

11 Barnet t , J .A. (1976) Adv. Carbo hydra te Chem. Biochem. 32, 125- -2~412 CixiUo, V.P. (1968 ) J. Bacteriol . 95,6 03 -- 611

13 Koty k, A. (1967) Folia Microbiol . 12 ,1 21- -131

14 Heredia, C.F., Sols, A. and Dela Fuente, G. (1968) Eur. J. Biochem. 5, 321 --3 29

15 Barnett , J.A. and Sims, A.P. (1976) Arch. Microbiol . 111, 185 --19 2

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