Planta

Planta (1981) 152:346-351

9 Springer-Verlag 1981

Stimulation of membrane-associated polysaccharide synthetases by a membrane potential in developing cotton fibers Antony Bacic and Deborah P. Delmer* MSU-DOE Plant Research Laboratory, Michigan State University, East Lansing, MI 48824, USA

Conditions which induce a transmembrane electrical potential, positive with respect to the inside of membrane vesicles, result in a substantial (4-12fold) stimulation of the activity of membrane-associated fl-glucan synthetases in a membrane preparation derived from the developing cotton (Gossypium hirsuturn L.) fiber. Induction of electrical potentials which are negative with respect to the inside of the membrane vesicle results in little or no stimulation of flglucan synthesis. Those products whose synthesis is stimulated are mainly fl-l,3-glucan, but there is also a considerable increase in fl-l,4-glucan. No e-l,4-glucan (starch) was detected in the reaction products. A transmembrane pH gradient was found to have no effect on fl-glucan synthesis. The results indicate that a transmembrane electrical potential can influence, either directly or indirectly, the activity of membrane-associated polysaccharide synthetases.

Abstract.

Cellulose s y n t h e t a s e - fl-l,3-Glucan synthetase - Fibers - Gossypium - Membrane potential - Polysaccharide synthesis. Key words:

Introduction

The concept that a transmembrane potential is required for the biosynthesis of fi-glucan(s) in higher plants evolved from our studies on the assembly of plant cell-wall polysaccharides, in particular cellulose, in the developing cotton fiber. Although cellulose (fl1,4-glucan) is an integral structural component of the plant cell wall, our understanding of its biosynthetic pathway has been hampered by an inability to demon* To whom reprint requests should be addressed Abbreviations: UDP-glucose = uridine-5'-diphosphoglucose; PEG=polyethylene glycol; BTP=bistrispropane (1,3-bis[tris(hydroxymethyl)methylamino]propane); MES= 2(N-morpholino)ethanesulfonicacid; VAL= valinomycin

0032-0935/81/0152/0346/$01.20

strate convincingly in-vitro synthesis. Recent studies from our laboratory, designed to trace the path of carbon into cellulose in vivo, offer strong, albeit indirect, evidence supporting a role for uridine-5'-diphosphoglucose (UDP-glucose) as the precursor to secondary cell-wall cellulose in the developing cotton fiber (Carpita and Delmer 1981). Good evidence exists that UDP-glucose is the precursor to cellulose in the bacterium Acetobacter (Glaser 1958; Colvin 1972) and it is generally assumed to be a likely precursor in algae (Hopp et al. 1978) and other higher plants (see reviews by Delmer 1977; Robinson 1977). In spite of this, repeated attempts to achieve convincing invitro synthesis, in plant systems, with radioactively labelled UDP-glucose as a substrate have met with failure (Delmer 1977). Using crude membrane preparations derived from cotton fibers, we have instead observed substantial incorporation of glucose from UDP-[lgC]glucose into a fl-l,3-glucan (Delmer et al. 1977; Heiniger and Delmer 1977). This fi-glucan, commonly called callose, is often produced by plants as a wounding response, although in the cotton fiber it is also found as a natural cell-wall constituent being deposited concomitantly with the onset of deposition of secondary wall cellulose (Maltby et al. 1979). Such a UDP-glucose:fl-l,3-glucan synthetase has been widely reported in plants (Delmer 1977; Robinson 1977), and it serves as one of the markers for plasma membrane (Anderson and Ray 1978). Putative cellulose-synthetase complexes have been observed by freeze-etch techniques on the plasma membrane of algae (Brown and Montezinos 1976; Giddings et al. 1980) and higher plants (Mueller and Brown 1980). The apparent lability of such a cellulose-synthetase complex indicates that the cell may need to be intact for such a complex to be functional. This supposition is further supported by our observation that the in-vivo rate of cellulose biosynthesis, from supplied [l~C]glucose, drops to near zero when

A. Bacic and D.P. Delmer: Membrane potentials and fl-glucan synthesis the fiber cells are cut (once) with scissors. H o w e v e r , if the fibers a r e cut a n d i n c u b a t e d in the presence o f p o l y e t h y l e n e glycol (PEG)-4,000 a l m o s t n o r m a l rates o f cellulose synthesis c a n be a t t a i n e d in a p p r o x i m a t e l y h a l f the fibers (the o t h e r h a l f r e m a i n i n g c o m pletely inactive) ( C a r p i t a a n d D e l m e r 1980). The p r o tection was shown to be the result o f the resealing a c t i o n o f P E G - 4 0 0 0 on the p l a s m a m e m b r a n e o f the fiber a n d n o t exclusively to p r o t e c t i o n o f the energyg e n e r a t i n g systems for p o l y s a c c h a r i d e biosynthesis. These results led us to p r o p o s e t h a t a t r a n s m e m b r a n e p o t e n t i a l m a y be r e q u i r e d to m a i n t a i n an active cellul o s e - s y n t h e t a s e c o m p l e x ( C a r p i t a a n d D e l m e r 1980). Such a c o n c e p t is consistent with o b s e r v a t i o n s obt a i n e d by w o r k e r s in u n r e l a t e d fields which indicate t h a t the fluidity o f m e m b r a n e s is influenced by such p o t e n t i a l s (Lelkes 1979) a n d t h a t the susceptibility o f m e m b r a n e s to d e t e r g e n t s is e n h a n c e d when the cells are in a n energized state ( K o m o r et al. 1979). T h u s p e r t u r b a t i o n s o f m e m b r a n e structure m i g h t be expected to influence the c o n f o r m a t i o n a n d activity o f m e m b r a n e b o u n d enzymes. Since such a m e m b r a n e p o t e n t i a l w o u l d be largely d e s t r o y e d d u r i n g cell d i s r u p t i o n , we decided to test o u r h y p o t h e s i s by i n d u c i n g artificial p o t e n t i a l s in m e m b r a n e vesicles i s o l a t e d f r o m c o t t o n fibers a n d to s t u d y the effect(s) o f such p o t e n t i a l s on the synthesis of/3-glucan(s) f r o m UDP-[14C]glucose.

Material and methods Plant material. Seeds of cotton (Gossypium hirsutum L. cv. Acala SJ-1) were obtained from Hubert Cooper, Jr., U.S. Department of Agriculture, U.S. Cotton Research Station, ShaRer, Cal., and were planted and grown in growth chambers under the conditions described in Delmer et al. (1977).

Preparation of the particulate fraction. Cotton bolls were harvested at 16-20 d postanthesis, corresponding to the onset of deposition of secondary wall cellulose and fi-l,3-glucan in the fibers. The fibers were removed from the ovule with forceps and homogenized (mortar and pestle) in 10 mM bistrispropane - 2(N-morpholino) ethanesulfonic acid (BTP-MES) buffer containing 5 mM dithiothreitol (DTT) and PEG-4000 (0.06 molal) at pH 7.2. The homogenate was centrifuged for 10 rain at 700 g to remove cell walls and cell debris. The supernatant was recentrifuged at 20,000 g for 30 min. The resulting particulate fraction was resuspended (100250 gg protein/ml) in 10 mM BTP-MES buffer containing I mM DTT, 3 mM MgSO4, and PEG-4000 (0~06molal) at pH 7.2. All procedures were performed at 4~ This, unfractionated, 70020,000 g membrane fraction was used in all subsequent experiments. Protein was measured by the procedure of Bradford (1976).

Assay conditions Jbr fi-glucan biosynthesis. Reaction mixtures typically contained 140-200 gg membrane protein, 0.1 mM unlabelled UDP-glucose and UDP-[U-lgC]glucose (3.7 KBq = 1 gCi/gmol) in a final volume of i ml. Unless otherwise stated, incubations were for 15 min at 25~ C, cation ionophore (valinomycin and nigericin) concentrations were 5 gM, proton ionophore (SF-6847) 3 gM, and anion and cation concentrations 50 mM. The rate of incorporation

347

of glucose from UDP-[U-14C]glucose into polymeric products remained linear for a period of 20 min. Reactions were terminated by heating in a boiling water bath (2-3 min).

Product analysis. Cellulose was added as a carrier (approx. 5 rag) to the boiled reaction mixtures and the insoluble material washed sequentially with water (four times) and chloroform:methanol:water (10:10:3, by vol.) (twice, for 15rain each at 37~ C). The lipid-free residue was then treated (I h) with acetic-nitric reagent (Updegraph 1969) at 100~ C. Essentially all of the glucan product was solubilized by this treatment. Alternatively, reactions were terminated by the addition of absolute ethanol (four volumes), alIowed to stand overnight at 4~ C, and then washed (four times) with 70% (v/v) ethanol. The lipid-free 70% (v]v) ethanol-insoluble residue was analysed for polysaccharide linkage composition by methylation analysis as described in Maltby et al. (1979). No cellulose carrier was added to the boiled reaction mixtures prior to methylation analysis. Chemicals. Valinomycin, BTP, MES, DTT, and e-amylase (Type IA) from porcine pancreas were obtained from Sigma Chemical Co. (St. Louis, Mo., USA). PEG-4000 was purchased from the J.T. Baker Chemical Co. (Phillipsburg, N.J., USA). Nigericin was from Eli Lilly & Co. (Indianapolis, Ind., USA) and SF-6847 was from Sumitomo Chemical Co. (Osaka, Japan). UDP-[U-14C]glucose (specific radioactivity 11.2 GBq-303 mCi/mmol) was pro-chased from ICN Pharmaceuticals (Cleveland, O., USA). All other chemicals used were analytical grade reagents. Results and discussion Effect o f transmembrane potentials on glucan synthesis. T r a n s m e m b r a n e p o t e n t i a l s are i n d u c e d in m e m b r a n e vesicles by establishing artificial diffusion p o t e n t i a l s with c o m b i n a t i o n s o f anions a n d c a t i o n s h a v i n g different p e r m e a b i l i t y co-efficients. A p o t e n t i a l , positive with respect to the inside o f the vesicles, is e s t a b l i s h e d by s u s p e n d i n g vesicles in 50 m M K + in the presence o f the K +-specific i o n o p h o r e v a l i n o m y c i n ( V A L ) a n d t h e relatively i m p e r m e a n t c o u n t e r ion 2 ( N - m o r p h o l i n o ) e t h a n e s u l f o n i c acid ( M E S ; see K a s a i a n d K o m e n t a n i 1979). V a l i n o m y c i n c a n t r a n s p o r t K ions across the vesicle m e m b r a n e , d o w n t h e c o n c e n t r a t i o n gradient, t h e r e b y establishing a net positive (inside) potential. P o t e n t i a l s which are negative with respect to the inside can be e s t a b l i s h e d b y s u s p e n d i n g vesicles in 50 m M nitric acid which has been a d j u s t e d to p H 7.2 with b i s t r i s p r o p a n e (BTP). T h e n i t r a t e a n i o n is c o n s i d e r e d to be relatively p e r m e a n t to m e m b r a n e s (Beck a n d S a c k t o r 1975) w h e r e a s the BTP, being a relatively large o r g a n i c cation, s h o u l d be c o n s i d e r a b l y less p e r m e a n t . The effects o f i n d u c i n g such artificial diffusion p o t e n t i a l s in a m e m b r a n e p r e p a r a t i o n f r o m d e v e l o p ing c o t t o n fibers on /~-[14C]glucan synthesis, f r o m s u p p l i e d UDP-[14C]glucose, a r e s u m m a r i z e d in Fig. 1. I n d u c t i o n o f a negative (inside) p o t e n t i a l results in o n l y a slight s t i m u l a t i o n o f fl-glucan synthesis. In c o n t r a s t , a positive (inside) p o t e n t i a l results in a s u b s t a n t i a l e n h a n c e m e n t o f activity. The level o f stim-

348

A. Bacic and D.P. Delmer: Membrane potentials and/Lglucan synthesis 6-

m

o

6-

--

2-

--I

x

o

2-

E O

on

F1

CONTROL K-ME$

F1

VAL

n

K-MES/VAL BTP/NO 3

(+) (-1 Fig. 1. Effect of membrane potentials on /Lglucan synthesis from UDP-[U-~4C)glucose in the 700 20,000 g membrane preparation from developing cotton fibers. Transmembrane potentials (positive : K-MES-VAL ; negative: BTP-NO 3) were induced as described in Material and Methods. Data are presented as radioactivity (cpm) incorporated into acetic acid-nitric acid-digestible material/mg protein E

Q

,o

Q 2

,b 2'0 3b go sb do 73 8b 9'o ,;o [K+]mM

Fig. 2. Stimulation of fi-glucan synthesis from UDP-[U-*4C]glucose in the 700-20,000 g membrane fraction from developing cotton fibers as a function of K + concentration. Positive (inside) potentials were induced by incubating vesicles in the presence of K-MES-VAL at the K + concentrations indicated by appropriate dilution of a stock solution of 500 mM KOH/500 mM MES at pH 7.2. Data are presented as radioactivity (cpm) incorporated into acetic acidnitric acid-digestible material/mg protein u l a t i o n varies c o n s i d e r a b l y a m o n g p r e p a r a t i o n s ( 4 12 fold over c o n t r o l i n c u b a t i o n ) 1 T h e c o n s i d e r a b l y s m a l l e r s t i m u l a t i o n o b s e r v e d with 50 m M K - M E S , in the a b s e n c e o f V A L , c o u l d be the c o n s e q u e n c e o f the f o r m a t i o n o f a small p o t e n t i a l resulting f r o m the differential p e r m e a b i l i t y c h a r a c t e r i s t i c s o f K § a n d M E S . T h e s t i m u l a t i o n o f fi-glucan synthesis by V A L a l o n e is n o t u n d e r s t o o d b u t c o u l d be the result o f a p e r t u r b a t i o n o f m e m b r a n e structure c a u s e d b y the i n s e r t i o n o f the i o n o p h o r e into the m e m b r a n e . Stimul a t i o n in the presence o f K - M E S / V A L d e p e n d s on 1 We have also found that fi-glucan synthesis in membrane vesicles isolated from soybean protoplasts is also stimulated, although to a lesser extent than in vesicles from cotton fibers (1.5-2 fold), by a positive (inside) potential (Klein and Delmer, unpublished results)

O

-A

~

_.1

.~

-.1

~

--I

5

-.1

5

_1

o~

Fig. 3. Effect of varying the K+-anion combination on ~-glucan synthesis from UDP-[U-~4C]glucose in the 700-20,000 g membrane

preparation from developing cotton fibers. Positive (inside) potentials were induced by preincubating vesicles in the presence of K+-anion/VAL for 5 min, prior to the addition of 0.1 mM UDP[U-14C]glucose. Following the addition of UDP-[U-14C]glucose, reactions were allowed to proceed for 15 min at 25~ and the products analyzed as described in Material and Methods. K +-anion combinations were prepared by appropriate dilution of the following stock solutions to give a final K+-concentration of 50 mM: 500 mM KSCN, 500 mM KNOa, 500 mM KC1, 250 mM K2SO4. All solution were adjusted to pH 7.2 with BTP. K-MES was prepared as described in Fig. 2. Data are presented as radioactivity (cpm) incorporated into acetic acid-nitric acid-digestible material/ mg protein the c o n c e n t r a t i o n o f K ions, r e a c h i n g a m a x i m u m between 50 a n d 70 m M K + (Fig. 2). I f the o b s e r v e d s t i m u l a t i o n is the result o f a transm e m b r a n e electrical p o t e n t i a l , t h e n r e p l a c i n g the relatively i m p e r m e a n t M E S a n i o n w i t h m o r e p e r m e a n t a n i o n s s h o u l d d i m i n i s h the s t i m u l a t i o n since the m o v e m e n t o f the m o r e p e r m e a n t a n i o n s a c r o s s the m e m b r a n e s h o u l d effectively dissipate the p o t e n t i a l . T h e results s h o w n in Fig. 3 s u p p o r t this concept, since little or no s t i m u l a t i o n o f / ~ - g l u c a n synthesis is o b served when relatively p e r m e a n t a n i o n s such as S C N o r N O ~ are s u b s t i t u t e d for M E S . In c o n t r a s t , C1o r S O l - , which are c o n s i d e r e d to be relatively i m p e r m e a n t a n i o n s (Beck a n d S a c k t o r 1975), give s t i m u l a tions c o m p a r a b l e to t h a t o b s e r v e d with M E S . These results s t r o n g l y i n d i c a t e t h a t the s t i m u l a t i o n o b s e r v e d is a result o f the presence o f a t r a n s m e m b r a n e electrical p o t e n t i a l r a t h e r t h a n a direct effect o f K ions s u p p l i e d to the inside o f t h e m e m b r a n e vesicles. W e have n o t yet a t t e m p t e d a direct m e a s u r e m e n t o f the i n d u c e d p o t e n t i a l since the m e m b r a n e fraction utilized in this study (700-20,000 g) is c o m p r i s e d o f a m i x t u r e o f cellular m e m b r a n e s .

Effects of different ionophores. Is

the s t i m u l a t i o n o f /~-glucan b i o s y n t h e s i s the result o f o n l y an electrical p o t e n t i a l o r does a p H g r a d i e n t also c o n t r i b u t e ? Nigericin is a n i o n o p h o r e which catalyzes the electron e u t r a l e x c h a n g e o f K ions for p r o t o n s ( P r e s s m a n 1976). T h u s the e q u i l i b r i u m a t t a i n e d is electrically

A. Bacic and D.P. Delmer: Membrane potentials and fl-glucan synthesis

349

Table 1. Linkage analysis, by methylation, of the fl-glucans synthesized from UDP-[U-l~C]glucosein the absence (control) and presence (K-MES-VAL)of a membrane potential by a membrane preparation (700-20,000 g) derived from developingcotton fibers. Time of incubation was 15 min. After incubation, the lipid-free 70% ethanol-insoluble products were permethylated, hydrolyzed, reduced, and acetylated. The resulting derivatives were separated in a gas-liquid chromatograph equipped with a stream splitter to collect radioactive peaks. Essentially all of the radioactivity eluted in three peaks which co-eluted with the sugars derived from terminal-, 3-1inked- and 4-1inked glucose

o o.

Linkage

trlIE

~o

Fig. 4. Effect of different ionophores on the incorporation of UDP[U-14C]glucose into fl-glucansin the 700-20,000 g membrane fraction derived from developing cotton fibers. Reaction conditions are as described in Material and Methods (NIG, nigericin; PI, proton ionophore SF-6847). Data are presented as radioactivity (cpm) incorporated into acetic acid-nitric acid-digestible material/ mg protein neutral, but a p H gradient is established. Results presented in Fig. 4 show that the combination of K - M E S and nigericin does not stimulate fl-glucan biosynthesis, indicating that a proton gradient is not involved. Therefore, the small reduction observed when KM E S / V A L stimulation is assayed in the presence of the proton ionophore SF-6847 (Fig. 4) is thought to be a secondary effect of the proton ionophore on the electrical potential, associated with a potentialdriven effect of protons, rather than the establishment of a pH gradient.

Characterization of glucan products. Heiniger and Delmer (1977) have shown that the only polysaccharide synthesized f r o m UDP-glucose in appreciable quantity by isolated cotton fiber membranes is a fl1,3-glucan. Their conditions of membrane isolation and assay differed from those used in this study and the effects of transmembrane potentials were not known at that time. Therefore, it was of interest to determine the nature of the glucan products synthesized in the experiments reported herein. The 70% ethanol-insoluble products, synthesized from U D P [I4C]glucose, were subjected to methylation analysis in order to determine the polysaccharide linkage composition. The resulting permethylated, peracetylated alditols were separated by a gas-liquid chromatograph equipped with a stream splitter to collect radioactive peaks. Label was found in glucose derivatives which co-eluted with standards of terminal-, 3-1inked and 4-1inked glucose (Table 1). The results show that the predominant product, in the absence of a potential, is a glucan containing 3-1inked glucose residues, al-

Terminal-glucose 3-1inked glucose 4-1inked glucose

Radioactivity (cpm) Control

K-MES-VAL

50 4,200 1,020

490 44,320 4,640

though synthesis of4-1inked glucan is also demonstrable. Induction of a positive (inside) potential with K - M E S / V A L results in substantial stimulation of both 3-1inked and 4-1inked glucan. Treated with the acetic-nitric reagent (Updegraph 1969) for 1 h at 100~ less than 1% of the glucan products remain insoluble (results not shown). This procedure has been shown to hydrolyze all cotton-fiber polysaccharides except crystalline cellulose (Updegraph 1969; Maltby et al. 1979; Carpita and Delmer 1980). Cotton fibers do not contain mixed-linkage fl-l,3;1,4-glucan or starch (Maltby et al. 1979) and as a-amylase (porcine pancreas; Type l-A) did not release any labelled material from the newly synthesized products it is concluded that the 4-1inked glucose residues detected by methylation analysis are derived from fi-l,4-glucan and not from starch (~-l,4-glucan). Since this material is largely hydrolyzed by acetic-nitric digestion, it must be concluded that the fi-l,4-glucan does not exist in a highly crystalline state.

Mechanism of stimulation of glucan-synthetase activities. The observations described above lead us to conclude that we are dealing with m e m b r a n e vesicles (or at least two compartments with little or no intercommunication) as, otherwise, it is not possible to explain the opposite effects resulting from positive and negative diffusion potentials. Carpita and Delmer (1980) have shown that U D P glucose, supplied to the cotton fiber from the outside, is not accessible to the fi-glucan synthetase(s). This would indicate that the fi-glucan synthetase (s) activity observed here is from that population of vesicles which is inside-out, i.e. with the cytoplasmic surface of the plasma m e m b r a n e exposed. In this regard, it is of interest to note that a diffusion potential which is positive with respect to the inside of the vesicle

350

A. Bacic and D.P. Delmer: Membrane potentials and/?-glucan Synthesis

affords greatest stimulation. Thus, induction of such a potential would mimic the in-vivo situation, i.e. negative inside, since the m e m b r a n e surfaces are reversed. However, we have not yet ruled out the possibility that the stimulation induced by a positive (inside) potential could result f r o m an enhanced movement of the negatively-charged substrate, UDP-glucose, to the interior of a population of right-side-out vesicles, thereby exposing additional latent fi-glucan synthetase(s) activity. Therefore two mechanisms for the action of a transmembrane electrical potential on the activity of fi-glucan synthetase(s) can be envisaged. I f the m e m brane vesicles have retained their in-vivo orientation, i.e. right-side-out, then the induction of a positive (inside) potential m a y facilitate the m o v e m e n t of the negatively charged substrate (UDP-glucose) to the interior of the vesicle thus making the substrate accessible to the active site of the enzyme. However, we have observed that if vesicles w i t h an induced positive (inside) potential are incubated, prior to the addition of UDP-[14C]glucose, with a non-metabolizable nucleotide sugar (guanosine 5'-diphosphoglucose, G D P glucose) at concentrations 10-100-fold greater than that of UDP-glucose, then there is no significant inhibition of the stimulated glucan synthetase(s) activity (data not shown). If the negatively charged nucleotide sugar could move across the m e m b r a n e in response to the established positive (inside) potential, competition for movement across the m e m b r a n e would have been expected. Alternatively, if the m e m b r a n e vesicles are inside-out with respect to their in-vivo orientation then substrate accessibility should not be a limiting factor. Thus the mechanism of potential-induced stimulation of fl-glucan synthetase(s) activity would be that such potentials alter the lipid environment surrounding the enzymes, resulting in a more favorable conformation and-or environment for activity. As previously mentioned, evidence exists which demonstrates that m e m b r a n e structure and fluidity can be influenced by potentials (Lelkes 1979 ; K o m o r et al. 1979). In addition, it has been shown that the ATPase activity in isolated m e m b r a n e vesicles derived from the sarcoplasmic reticulum is influenced by artifically induced diffusion potentials, although it is not yet clear whether the effect could, at least in part, be the result of an intrinsic effect on the protein itself (Dupont 1979). Toci et al. (1980) have shown that " e n e r g i z a t i o n " of E. coli m e m b r a n e vesicles increases the affinity of the lac carrier protein for its substrate, indicating either an effect on increased accessibility of the carrier or a conformational change resulting in increased affinity for the ligand. Taken together, these results provide support for the hypothesis that transmembrane potentials can influence a variety of

membrane-associated carriers or enzymes. However, neither of the two specific suggestions can be excluded at this stage and experiments are currently in progress in an attempt to elucidate the mechanism of action. Conclusions As far as we are aware this is the first report showing that a transmembrane potential can influence, either directly via induced changes in conformation, or indirectly via increased substrate accessibility, the activity of polysaccharide synthetases. Stimulation of activity is the consequence of a transmembrane electrical potential and is not influenced by a p H gradient. The results show greatest stimulation of a fl-l,3-glucan synthetase, but significant stimulation of fi-l,4-glucan synthesis is also observed. Thus these results lend further support to the concept that the existence of a transmembrane potential may be at least one factor which regulates the synthesis of cellulose in plants (Carpita and Delmer 1980). The authors wish to thank Professor N.E. Good (Michigan State University) and Dr. Anita Klein (presently, University of Wisconsin, Madison, USA) for their helpful and critical evaluation of this work. We are grateful to Kathy Johnson and Barbara Mitchell for their technical assistance. This work was supported by the U.S. Department of Energy Contract ~/DE-AC-02-76ER01338.

References Anderson, R.L., Ray, P.M. (1978) Labelling of the plasma membrane of pea cells by a surface-localized glucan synthetase. Plant Physiol. 61,723-730 Beck, J.C., Sacktor, B. (1975) Energetics of the Na+-dependent transport of D-glucosein renal brush border membrane vesicles. J. Biol. Chem. 250, 8647 8680 Bradford, M.M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal, Chem. 72, 249-259 Brown, R.M., Jr., Montezinos, D. (1976) Cellulose microfibrils: visualization of biosynthetic and orienting complexesin association with the plasma membrane, Proc. Natl. Acad. Sci. USA 73, 143-147 Carpita, N, C., Delmer, D.P. (1980) Protection of cellulose synthesis in detached cotton fibers by polyethylene glycol. Plant Physiol. 66, 911-916 Carpita, N.C., Delmer, D.P. (1981) Concentration and metabolic turnover of UDP-glucose in developing cotton fibers. J. Biol. Chem. 256, 308 315 Colvin, J.R. (1972) The structure and biosynthesis of cellulose. Crit. Rev. Macromol. Sci. 1, 47-81 Delmer, D.P. (1977) The biosynthesis of cellulose and other plant cell wall polysaccharides. Rec. Adv. Phytochem. 11, 45-77 Delmer, D.P., Heiniger, U., Kulow, C. (1977) UDP-glucose: glucan synthetase in developingcotton fibers. I. Kinetic and physiological properties. Plant Physiol. 59, 713 718 Dupont, Y. (1979) Electrogenic calcium transport in the sarcoplasmic reticulum membrane. In: Cation flux across biomembranes, pp. 141-160, Mukohata, Y., Packer, L.. eds. Academic Press, New York

A. Bacic and D.P. Delmer: Membrane potentials and ]~-glucan synthesis Giddings, T.H., Jr., Brower, D.L., Staehelin, L.A. (1980) Visualization of particle complexes in the plasma membrane of Micrasterias denticulata associated with the formation of cellulose fibrils in primary and secondary cell walls. J. Cell Biol. 84, 327339 Glaser, L. (1958) The synthesis of cellulose in cell free extracts of Acetobacter xylinum. J. Biol. Chem. 232, 627-636 Heiniger, U., Delmer, D.P. (1977) UDP-glucose: glucan synthetase in developing cotton fibers. II. Structure of the reaction prodacts. Plant Physiol. 59, 719-723 Hopp, H.E., Romero, P.A., Daieo, G.R., Pont-Lezica, R. (1978) Synthesis of cellulose precursors. The involvement of lipidlinked sugars. Eur. J. Biochem. 84, 561-571 Kasai, M., Komentani, T. (1979) Ionic permeability of sarcoplasmic reticulum membrane. In: Cation flux across biomembranes, pp 167-177, Mukohata, Y., Packer, E. eds. Academic Press, New York. Komor, E., Weber, H., Tanner, W. (1979) Greatly decreased susceptibility of nonmetabolizing cells towards detergents. Proc. Natl. Acad. Sci. USA 76, 1814 1818

351

Lelkes, P.I. (1979) Potential dependent rigidity changes in lipid membrane vesicles. Biochem. Biophys. Res. Comm. 90, 656 662 Maltby, D., Carpita, N.C., Montezinos, D., Kulow, C., Delmer, D.P. (1979)/~-l,3-Glucan in developing cotton fibers. Structure, localization, and relationship of synthesis to that of secondary wall cellulose. Plant Physiol. 63, 1158-1164 Mueller, S.C., Brown, R.M., Jr. (1980) Evidence for an intramembrahe component associated with a cellulose microfibril synthesizing complex in higher plants. J. Cell Biol. 84, 315-326 Pressman, B.C. (1976) Biological applications of ionophores. Ann. Rev. Biochem. 45, 501-530 Robinson, D.G. (1977) Plant cell wall synthesis. Adv. Bot. Res. 5, 89-151 Toci, R., Belaich, A., Belaich, J-P. (1980) Influence of "energization" on the binding of M protein with p-nitrophenyl c~-Dgalactopyranoside, J. Biol. Chem. 255, 4603-4606 Updegraph, D.M. (1969) Semi-micro determintation of cellulose in biological materials. Anal. Biochem. 32, 420-424 Received 15 January; accepted 13 April 1981

Stimulation of membrane-associated polysaccharide synthetases by a membrane potential in developing cotton fibers.

Conditions which induce a transmembrane electrical potential, positive with respect to the inside of membrane vesicles, result in a substantial (4-12-...
580KB Sizes 0 Downloads 0 Views