Laboratoire de Mycologie associe au C.N.R.S., Departement de Biologie Vegetale, Universi­te Claude Bernard, Lyon T, 69621 Villeurbanne, France.

Digitonin Solubilization and Protease Stimulation of B Gluean Synthetases of Saprolegnia

MICHEL FEVRE

With 6 figures

Received April 27, 1979 . Accepted June 14, 1979

Summary

,8-glucan synthetases from particulate preparations of Saprolegnia monoica were partial­ly solubilized by digitonin treatment. The enzymes which were still associated with the membrane fraction were increased in total and specific activities. Glucan synthetases as­sayed at low substrate concentration in the presence of Mg Cl2 were inhibited by trypsin. On the contrary, enzymes assayed at high substrate concentration in the absence of Mg Cl2

were stimulated by trypsin but inhibited by other proteases and by a trypsin inhibitor. Ac­tivation followed by inactivation of the syntbetases was observed in the presence of high tryp~in concentration.

Regulation of glucan synthetase activities is discussed in relation to hyphal morphogene-SIS.

Key words: glucan synthetases, trypsin, Saprolegnia, hyphal morphogenesis.

Introduction

Particulate fractions and cell walls of the fungus Saprolegnia monoica are able to transfer labelled glucose from UDPG to an unknown acceptor, leading to the synthesis of complex radioactive glucans (FEVRE and DUMAS, 1977).

Several enzymes must be involved in cell wall synthesis because the wall contains two major polysaccharides: cellulose (1-4 fJ linked glucose) and a glucan (1-3 fJ glucose polymer containing 1-6 fJ glycosidic linkages) (BARTNICKI-GARCIA, 1968).

(1-3) fJ glucan synthesis activities have been demonstrated in Neurospora (MISH­RA and TATUM, 1972) Phytophthora (WANG and BARTNICKI-GARCIA, 1976) and Saccharomyces (LOPEZ-RoMERO and RUlZ-HERRERA, 1977). In Saprolegnia, hot al­kali insoluble glucans can be hydrolyzed by a purified cellulase. A spOt with an Rglr. similar to cellobiose can be detected on paper chromatograms. As in higher plants (TSAI and HASSID, 1973; VAN DER WOUDE et al., 1974), cellobiose and low MgCl2 concentrations stimulate enzyme activities and modify the quality of the radioactive glucans (FEVRE and DUMAS, 1977).

Z. PJlanzenphysiol. Bd. 95. S. 129-140. 1979.

130 MICHEL FEVRE

In chitinous fungi, chitin synthetase is present in the cell under a zymogenic form which can be activated by moderate proteolysis (CABIB and FARKAS, 1971; HASILIK, 1974; Rurz-HERRERA and BARTNICKI-GARCIA, 1976). In Saprolegnia, a thermolabile compound of the soluble fraction inactivates particulate synthetases (FEVRE and DUMAS, 1977).

In order to understand the regulation of glucan synthetases and the possible im­plication in their morphogenic roles, in this paper we describe the effect of digito­nin and proteases on these enzymes.

Material and Methods

Culture methods

Saprolegnia monoica Pringsheim no 539 67 Dick was obtained from C.B.S. Baarn, The Netherlands and maintained on a wheat flour medium. Mycelial disks (4 mm diam.) from plate cultures were inoculated in 100 ml of the liquid medium of MACHLIS (1953) in 250 ml Erlenmeyer flasks and the cultures were incubated at 23 DC for 4 days. The mycelium was harvested by filtration through paper in a Buchner funnel and washed with cold distil­led water.

Preparation of the particulate fraction enzyme

Fresh mycelia were mixed and ground in buffer (0.01 M Tris/HCI, 0.5 M sorbitol, 0.001 M EDTA, pH 7.2) containing 10f0 (w/v) dithiothreitol with a Virtis homogenizer for three periods of 30 S at low speed at 4 DC. The cell walls were discarded after centrifuga­tion at 1000 g for 10 min and the particulate fraction was obtained after centrifugation of the supernatant at 48,000 g for 30 min.

Digitonin treatment The particulate fraction (pellet 48,000 g 30 min) was resuspended in extraction buffer

without sorbitol. One ml of the original fraction was mixed with 1 ml of digitonin solution (20 mg ml-1 in extraction buffer without sorbitol). The extract was kept at 4 DC during 45 min and centrifuged at 48,000 g for 30 min. The supernatant (2 ml) was used as the solubi­lized fraction. The pellet was resuspended in 1 ml of extraction buffer without sorbitol.

(J glucan synthetases assays The complete assay mixture (total vol 300,u1) contained 0.3 nM UDP-D-[14C] glu­

cose (306 ,uCi ,uM-l Radiochemical Centre, Amersham); 400 nM UDP-D-[12C] glucose; 10 ,uM phosphate buffer pH 6; 0.1 ,uM dithiothreitol; 4 ,uM Mg C12; 2 ,uM cellobiose and 50 to 100 ,ul of freshly isolated enzymes. The mixture was incubated at 27 DC for 30 to 60 min. Reactions were terminated by heating the tubes for 2 min in a boiling water bath. Af­ter addition of Whatman cellulose (10 to 20 mg) and 2 ml water, the water insoluble poly­mers were collected by filtering the mixture through a Whatman GF/C filter (2.4 cm). The residue on the filter was washed with 40 ml water followed by 20 ml methanol/chloroform (2 : 1 v/v) and 40 ml ethanol. Filters and residues were placed in 2.5 ml scintillation fluid containing 4 g 2,5-diphenyloxazole (PPO) and 0.1 g 1,4-di-2-(5-phenyloxazolyl)-benzene (POPOP)/l toluene.

Action of proteases and their inhibitors Solutions of acid protease (Miles Lab.), trypsin, pepsin, papain (Boehringer), trypsin in­

hibitor (Boehringer) and phenyl pyruvic acid (Sigma) were made in the buffer of the enzy-

Z. Pflanzenphysiol. Bd. 95. S. 129-140. 1979.

fJ-glucan synthetases of Saprolegnia 131

matic assay. 100,u1 solution was added to the standard assay mixture, 100,u1 buffer was used for the control.

Analysis of 14C labelled polysaccharides

The assay mixtures were fractionated into products of different solubility. Powdered Whatman cellulose (20 mg) was added to assay mixtures which were then centrifuged at 25,000 g for 20 min.

Each pellet was extracted once with water (1 ml) at 100°C for 5 min and once with cold water (1 ml). These two extracts were combined to give the hot water soluble fraction. The residue was extracted with M NaOH (0.25 ml) at 100°C for 5 min, then with cold water (0.75 ml). These extracts constituted the hot alkali soluble fraction. The pellet was the hot alkali insoluble fraction.

Results

Effects of digitonin treatment

In mycelial extracts, enzymatic activity is mainly associated with the particulate fraction. By digitonin treatment, the total enzymatic activity is increased and enzy­mes are released in the supernatant. A particulate preparation was divided in two parts. One part was treated with digitonin at the final concentration of 10 mg ml-1

during 45 min at 4°C. The other was kept at 4 °C and used as the control. The supernatant obtained from the treated sample exhibited glucan synthetases activity but no activity was found in the supernatant of the control. Moreover, the specific activity and the total activity of the treated pellet were increased 4 and 2 times, respectively. During several experiments, the stimulatory action of digitonin ranged from 2.5 to 4.6.

In green plants, low UDPG and high MgCl2 concentrations enhance (1-4) fJ glucan synthesis and inhibit the synthesis of (1-3) fJ polysaccharides (TSAI and HASSID, 1973). In Saprolegnia, the enzyme activity is affected by the presence of MgCl2 and reduced when high salt concentrations are used (FEVRE and DUMAS, 1977). The effect of digitonin treatment was estimated with or without the presen­ce of MgCI2• Table 1 shows (1) the diminution of total activity by MgCl2 (2) the stimulatory effect of digitonin both in the presence (X4) and in the absence (X1.S) of MgCI2•

Table 1: Effects of digitonin and MgCl2 on glucan synthetases activities. One ml of the original particulate fraction was mixed with one ml of digitonin (20 mg· -rol-I ).

After standing 45 min at 4°C, the mixture was centrifuged (48,000 g for 30 min). The super­natant (2 ml) was saved and the pellet resuspended in the original volume (1 ml) of extraction buffer. Enzyme activity was assayed in the presence of MgCI9 •

Original fraction (1 ml) Treated pellet (1 m!) Solubilized fraction (2 ml)

Total activity (cpm) - MgCl2 + MgCl2

235,740 290,420

68,500

33,260 88,580 41,520

Z. Pflanzenphysiol. Bd. 95. S. 129-140. 1979.

132 MICHEL FEVRE

The difference of MgCl2 action on the various enzyme preparations (original fraction, digitonin treated pellet and solubilized fraction) indicates that several en­zymes more or less sensitive to MgCl2 are implicated in synthesis activity. Howe­ver, enzyme activity in the presence of MgCl2 at low substrate concentrations (0.3 n M UDPG) seems to be more easily solubilizable. The activity of the soluble fraction represented twice the activity of the original fraction (405,400 cpm instead of 193,400 cpm). On the contrary, activities of the same fraction assayed at high substrate concentration (400 n M UDPG) in the absence of MgCl2 were more im­portant in the original fraction (679,800 cpm instead of 412,400 cpm).

Thus, by digitonin treatment, enzymes are solubilized because an important acti­vity is recovered in the soluble fraction. Moreover enzyme activities are unmasked as in each case activities of treated pellets are always higher than that of the origi­nal fraction.

Effects of proteases on glucan synthetases activities

The enzymatic activities of particulate fractions were estimated in the presence of various concentrations of several proteases. At low concentrations, all of them inhibited glucan synthetases activity. At high concentrations, pepsin, papain, and acid protease still inhibited enzyme activity but at different degrees. On the other hand, trypsin increased activity as the concentration became higher (fig. 1).

When 2 mg of trypsin (100 Ill) were added to the enzyme assay (300 Ill), a two­fold stimulation was observed. Trypsin previously incubated at 100°C had only a

relative aetiv i ty

0,001 0,01 0,05 0,1 0,5 mg

Fig. 1: Effect of proteases on the glucan synthetases activity of the particulate fraction. The indicated amount of proteases were added to the assay mixtures. The enzymatic activi­ty is expressed as a percent of the control (without protease). Pepsin (.); Acid protease (.); Papain Ce); Trypsin CD).

Z. Pflanzenphysiol. Bd. 95. S. 129-140. 1979,

j1-glucan synthetases of Saprolegnia 133

slight stimulatory effect. Phenyl pyruvic acid, a protease inhibitor showed an ac­tion spectrum inverse to that of trypsin; with increasing concentrations an impor­tant inhibition occurred (fig. 2).

relative activity

200

// ,//,,0

100 ------___ . ___ . _ ".,..-' ._._.-:a::--.-............. _ ....... __ ... -----• .,.." .... ----------------~----------- ---~--~ ...........

.............. ---., ..........

......... ---... 0.001 0.01 0.05 0.1 0.25 0.5 2 mg

Fig. 2: Effect of trypsin, boiled trypsin and phenyl pyruvic acid on glucan synthetases acti­vity of the particulate fraction. The indicated amount of proteases or protease inhibitor were added to the assay mixtures. The enzymatic activity is expressed as a percent of the control. Trypsin (.&); Boiled trypsin (e); Phenyl pyruvic acid (.).

Effects of trypsin and trypsin inhibitor on digitonin treated preparations

The effect of trypsin on the enzyme preparations obtained by digitonin treat­ment was studied.

When assayed at high UDPG concentration in the absence of MgCb, stimulations of activity were observed. The increase of glucan synthetases activities of the digi­tonin treated pellet and the original particulate fraction were respectively 145 and 250 Ofo of their control. The solubilized enzyme showed a more important stimula­tion and the activity was 31

/ 2 times greater than its control (fig. 3).

When assayed at low UDPG concentration (0.3 n M) in the presence of MgCI2,

enzyme activities were inhibited. The patterns of inhibition were the same for the 3 sources of enzymes and at the higher protease dose used, enzymatic activities repre­sented only 20 Ofo of their control (fig. 3).

Depending on the assay conditions, the difference of trypsin actions confirms that several enzymes are involved in glucan synthesis as previously suggested by di­gitonin solubilization.

Z. Pjlanzenphysiol. Bd. 95. S. 129-140. 1979.

134 MICHEL FEVRE

relative activity

300

200

1 00 l_~o=="o=="o=="o=="-

--~~~~~:::::::::~::::::~:::~:O:::lf==O~===~~'~" 0.01 0.05 0,1 0,25 o.s 2 mg

Fig. 3: Effect of trypsin on glucan synthetases activity of the particulate fraction and digi­tonin treated preparations. Enzyme preparations were obtained as described in Table 1. The indicated amounts of protease were added to the assay mixtures. The enzymatic activity of each fraction is expressed as a percent of their respective control. Enzymatic assays were done at two substrate concentrations, 400 nM UDPG (solid curves), 0.3 nM UDPG (broken curves). Original particulate fraction (.); Digitonin treated pellet (e); Solubilized enzyme (.).

Using a digitonin solubilized fraction as the enzyme source, trypsin was added to the assay mixture at the beginning or at the end of incubation. When trypsin reac­ted from the beginning, inhibition and stimulation of enzyme activities were obser­ved depending on the concentrations used. On the other hand, trypsin added at the end of enzymatic reactions had no effect. These observations demonstrate that the increase of glucan synthesis is due to the enzymatic reaction of trypsin and is not due to a hypothetic fixation of radioactivity by the protease.

The effect of a trypsin inhibitor on the synthetases activities of each fraction was studied. A similar pattern of action was observed on the three different enzyme preparations. Enzyme activities were inhibited but inhibitions provoked at higher concentrations, were never superior to 30 % of their respective controls (fig. 4). When trypsin inhibitor was added to the assay mixture containing trypsin, the sti­mulation provoked by the protease was not observed.

Effect of trypsin as a function of time

The effect of trypsin was studied as a function of time. This protease was added at the beginning of the enzymatic reactions which were stopped at various times of

Z. Pjlanzenphysiol. Bd. 95. S. 129-140. 1979.

fl-glucan synthetases of Saprolegnia 135

relative activity

L-_' -.-._.~~. ~. ~"'=-~=-;;i?::='=- ~ ------ ......... , .... 100 ~ ---------- "

.~. .... ..... ~ ---..,

--.... ~~;~-A

50

0.001 0.01 0.05 0.1 0.25 0.5 2 m 9

Fig. 4: Effect of a trypsin inhibitor on glucan synthetases activity of the particulate frac­tion and digitonin treated preparations. Enzyme preparations were obtained as described in TabLe 1. The indicated amounts of inhibitor were added to the assay mixtures (400 nM UDPG). The enzymatic activity is expressed in percent of the control. Original particulate fraction (.); Digitonin treated pellet (e); Solubilized enzyme (.A).

incubation. With the higher trypsin concentration, enzymatic activity was stimula­ted from the beginning of incubation but a decrease in activity was observed for longer periods (after 90 min). With the lower trypsin concentration, inhibition oc­curred at the beginning but when the incubation time was extended to one hour, enzyme activity became higher than the control (fig. 5).

In other experiments, trypsin was pre-incubated with the enzyme during various periods, then the reaction (one hour incubation) was started by the addition of sub­strate and activators (fig. 6).

Activities of the original particulate fraction (pre-incubated with buffer) decrea­sed during the course of these pre-incubations. With a low trypsin dose, the enzy­matic activities were stimulated even after a short time of pre-incubation. The sti­mulation increased then stabilized after 90 min. With a high trypsin concentration, maximum stimulation was obtained after 30 min pre-incubation. At longer periods, the synthesis activity decreased and was even lower than that observed in the pre­sence of the low trypsin concentration after 180 min of pre-incubation (fig. 6).

As the same amount of enzyme was used in each assay, the inhibition observed cannot be due to the action of hydro lases able to act on the synthetized polymers but is more probably due to the protease inactivation of the synthetases.

Z. P/lanzenphysiol. Bd. 95. S. 129-140. 1979.

136 MICHEL FEVRE

40

30

cpm .103

I , 0/ , , ,

/ / / / /

/ I

I

/ I

/

//"e­

/

/ . -----.... -

20 ; .... --.:-=~ / ,,/ .... ---: /---: ~~-;.

: / . .-I ~/./ : / rI / ~/ /

10 : I . /' / /. ,i / / . :, ./ /' / 1/, /, III

30 60 90

Incubation time

120 mn

Fig. 5: Trypsin effect as a function of time on the glucan synthetases activity of the parti­culate fraction. Trypsin solutions (100 ,ul) were added to the assay mixtures. Enzymatic reactions were incubated during various times. Control ( .. ); Trypsin (500 ,ug) (e); Trypsin (10 ,ug) (.).

Effect of digitonin and trypsin on the nature of synthetized polysaccharides

Radioactive polysaccharides can be fractionated according to their solubility properties into hot water soluble (HWS) hot alkali soluble (HAS) and hot alkali insoluble (HAl) glucans. In order to precise the actions of digitonin and trypsin, enzymatic reactions were scaled up 3 times, 1.25 mg/mJ-! trypsin concentration was used and 14C labelled glucans were fractionated. (Table 2).

In the absence of MgC12, HAS represented the main products synthetized by the original particulate fraction. After digitonin treatment HAS were still the main fractions and their relative proportion had even increased in both treated pellet and solubilized fraction. On the other hand, after digitonin action, the rate of HAl produced was very low and reduced compared to that obtained with the original fraction. Trypsin treatment of each enzymatic source resulted in an important rise

z. Pflanzenphysiol. Bd. 95. S. 129-140.1979.

/1-glucan synthetases of Saprolegnia 137

10

30 60 90 120 mn 160

Pre·incubation time

Fig. 6: Effect of pre-incubation of trypsin with enzyme on the glucan synthetases activity of the particulate fraction. Trypsin solutions (100,u1) were mixed with the enzyme prepara­tions and incubated at 27°C during various times, then reactions (1 h) were started by the addition of substrate and activators. Control (A); Trypsin (500 ,ug) (e); Trypsin (10 ,ug) (.).

of HAS and a slight stimulation of HAL HWS of the original fraction and solubi­lized enzyme were unaffected by the protease but inhibited in the treated pellet.

In the presence of MgCl2, the different glucans synthetized by the original fraction had nearly the same importance. After digitonin treatment, HAl and HAS were increased: HAS becoming the major products in both the treated pellet and the solubilized enzyme.

Following trypsin treatment, HAS were increased in the original fraction and treated pellet. HAl were also stimulated and they became the main glucans of the solubilized fraction. As previously shown in the absence of MgCl2, HWS were not affected by trypsin action.

Z. PjlanzenphysioL Bd. 95. S. 129-140. 1979.

138 MICHEL FEVRE

Table 2: Effect of trypsin and MgCl2 on the solubility of radioactive polysaccharide synthet­ized by the original particulate fraction, digitonin treated pellet and solubilized fraction. The different enzyme sources were prepared as described in table 1. Hot water soluble (HWS), hot alkali soluble (HAS) and hot alkali insoluble (HAl) glucans were obtained as described in methods. Activities expressed in dpm were determined in rhe presence or absence of MgCl2 and trypsin and at the higher substrate concentration.

-MgCI2 + MgCl2 - Trypsin + Trypsin - Trypsin + Trypsin dpm Ofo dpm Ofo dpm Ofo dpm Ofo

Original fraction 113,280 100 184,310 100 52,720 100 61,330 100 HWS 22,600 20 24,790 13.4 16,260 30.8 14,730 24 HAS 63,680 56.2 126,750 68.8 17,860 33.9 29,980 48.9 HAl 27,000 23.8 32,770 17.8 18,600 35.3 16,620 27.1 T reared pellet 141,380 100 211,420 100 74,580 100 136,300 100 HWS 21,010 41.9 13,330 6.3 15,850 21.3 14,930 11 HAS 117,020 82.7 189,620 89.7 35,500 47.6 79,070 58 HAl 3,350 2.4 8,470 4 23,230 31.1 42,300 31 Solubilized enzyme 50,600 100 66,460 100 58,400 100 69,630 100 HWS 14,080 27.8 15,140 22.8 13,110 22.4 13,920 20 HAS 34,000 67.2 47,650 71.7 26,940 46.2 18,060 25.9 HAl 2,520 5 3,670 5.5 18,350 31.4 37,650 54.1

Discussion

When cytoplasmic particles were treated with digitonin, glucan synthetases activities were recovered in the soluble fraction (supernatant 48,000 g 30 min). This compound has also been used to solubilize other cell wall polysaccharide synthetases in green plants (LIU and HASSID, 1970; TSAI, 1975) and fungi (GOODAY and DE RoussET HALL, 1975). After digitonin treatment, enzyme activity still associated with cytoplasmic particles was increased and the total activity recovered (treated pellet + supernatant) was always superior to that of the original fraction. Enzymes acting in the presence of MgCl2 were solubilized at a higher rate than MgCl2 sensitive synthetases, and activities in the soluble fraction were always superior to that of the original particulate fraction. Thus digitonin in releasing soluble enzymes also unmasked activities still bound to cytoplasmic membranes.

According to the enzymes involved in glucan synthesis, trypsin had different effects. Glucan synthetases assayed at high substrate concentration which promotes 1-3 fJ glucan synthesis (TSAI and HAS SID, 1973; VAN DER WOUDE et aI., 1974; RAYMOND et aI., 1978) were stimulated by trypsin and inhibited by other proteases (pepsin, papain, acid protease). When enzymes were assayed at low substrate concentration in the presence of MgCl2 which enhance 1-4 fJ glue an synthesis (TSAI and HASSID, 1973; VAN DER WOUDE et aI., 1974) synthetases were inhibited by trypsin.

z. Pflanzenphysiol. Bd. 95. S. 129-140. 1979.

p-glucan synthetases of Saprolegnia 139

Digitonin solubilization and trypsin treatment indicate that enzymes producing 1-3 fJ and 1-4 fJ glucans are present under different forms and can be submitted to different regulation mechanisms.

Proteolytic activation of chitin synthetase has been described in yeast (CABIB, 1975) dimorphic fungi (RUIZ-HERRERA and BARTNICKI-GARCIA, 1976) and filamentous fungi (LoPEz-RoMERO and RUIZ-HERRERA, 1976). But data concerning fJ glue an synthetases are rare. A correlation exists between the intracellular level and 1-3 fJ glucan synthetase activities of Phytophthora (WANG and BARTNICKI-GARCIA, 1976). Recently, CHAO and MACLACHLAN (1978) have shown that Pisum 1-4 fJ glucan synthetases were inactivated by a heat labile component which is probably a protease.

Our results suggest that an important part of the glucan synthetates (probably f3 1-3 glucan synthetases) exists in a form which can be activated by proteolysis; on the contrary, 1-4 f3 glucan synthetases could be inactivated by proteases. Maximum activation was obtained for 90 min of contact between enzyme and trypsin (1.25 mg' mt-l). Above that time inactivation of glucan synthetases occurred. Hot alkali insoluble and mainly hot alkali soluble glucans were increased after trypsin treatment. High trypsin concentrations (1 to 3.3 mg . ml-1

) were needed to obtain maximum stimulation, this may indicate the lack of specificity of trypsin. However such concentrations of protease (rennilase) have been used to activate chitin synthetase (RUIZ-HERRERA et al., 1977).

Trypsin stimulation does not seem to be due to a proteolysis of cytoplasmic structures and consequently a release of enzymes, as maximal activation is obtained on the digitonin solubilized fraction and the behaviour of enzymes varies according to the substrate concentration. Moreover, trypsin only stimulates synthesis of certain glucan fractions.

The regulation of glucan synthetases could have morphogenetic implications in hyphal morphogenesis and cell wall growth (FEVRE, 1979). Previously we have shown that the soluble fraction of mycelial extracts contains a synthetase inactivator (FEVRE and DUMAS, 1977). Cell wall bound proteases have been localized at the branching sites and septa of Neurospora (SUKUMARAN and MAHADEVAN, 1975). At the sites of morphogenesis, synthetases could be stimulated by proteases (activation of a zymogenic form or liberation of primers). Inactivation could happen because of a prolonged proteolysis or by the soluble inhibitor.

References

CABIB, E.: Molecular aspects of yeast morphogenesis. Ann. Rev. of Microb. 29, 191-214 (1975).

CABIB, E. and V. FARKAS: The control of septum morphogenesis: an enzymatic mechanism for the initiation of septum formation. P.N.A.S. 68, 2052-2056 (1971).

CHAO, H. I. and G. A. MACLACHLAN: Soluble factors in Pisum extracts which moderate Pi­sum B glucan synthetase activity. Plant Physiol. 61, 943-948 (1978).

Z. PJlanzenphysiol. Bd. 95. S. 129-140. 1979.

140 MICHEL FEVRE

BARTNICKI-GARCIA, S.: Cell wall chemistry morphogenesis and taxonomy of fungi. Ann. Rev. of Microb. 22, 87-108 (1968).

FEVRE, M.: Glucanase, glucan synthetase and wall growth. In: Wall and Hyphal Growth, ]. H. BURNETT and A. P. ]. TRINCI (Eds.) Cambridge University Press (in press) (1979).

FEVRE, M. and C. DUMAS: fl glucan synthetases from Saprolegnia monoica. ]. of Gen. Mi­crob. 103, 297-301 (1977).

Goo DAY, G. W. and A. DE ROUSSET-HALL: Properties of chitin synthetase from Coprinus cinereus.]. of Gen. Microb. 89, 137-145 (1975).

HASILIK, A.: Inactivation of chitin synthetase in Saccharomyces cerevisiae. Arch. of Microb. 101, 295-301 (1974).

Lm, T. Y. and W. Z. HASSID: Solubilization and partial purification of cellulose synthetase from Phaseolus au reus. J. of Biol. Chern. 245, 1922-1925 (1970).

LOPEz-RoMERo, E. and J. RUIZ-HERRERA: Synthesis of chitin by a particulate preparation from Aspergillus jlavus. Ant. van Leeuw. 42, 261-276 (1976).

- - Biosynthesis of B glucans by cell free extracts from Saccharomyces cerevisiae. Bioch. Bioph. Acta 500, 372-384 (1977).

MACHLIS, L.: Growth and nutrition of water molds in the subgenus Euallomyces. II. Opti­mal composition of the minimal medium. Am.]. of Bot. 40, 449-460 (1953).

MISHRA, N. G. and E. L. TATUM: Effect of L-sorbose on polysaccharide synthetase of Neu­rospora crassa. P.N.A.S. 69, 313-317 (1972).

RAYMOND, Y., G. B. FINCHER, and G. A. MALACHLAN: Tissue slice and Particulate B glucan synthetase activities from Pisum epicotyls. Plant Physiol. 61, 938-942 (1978).

RUIZ-HERRERA, J. and S. BARTNICKI-GARCIA: Proteolytic activation and inactivation of chi­tin synthetase from Mucor rouxii. J. of Gen. Microb. 97, 241-249 (1976).

RUIZ-HERRERA, ]., E. LOPEz-RoMERO, and S. BARTNICKI-GARCIA: Properties of chitin syn­thetase in isolated chitosomes from yeast cells of Mucor rouxii. J. of Biol. Chern. 252, 3338-3343 (1977).

RYDER, N. S. and J. E. PEBERDY: Chitin synthetase in Aspergillus nidulans: Properties and proteolytic activation. ]. of Gen. Microb. 99, 69-76 (1977). .

SUKUMARAN, C. P. and P. R. MAHADEVAN: Localization of enzymes in the cell walls of Neurospora crassa. Ind. J. of Exper. Biol. 13, 127-130 (1975).

TSAI, C. M. and W. Z. HASSID: Substrate activation of fl1-3 glucan synthetase and its ef­fect on the structure of B glucan obtained from UDP-D-glucose and particulate enzyme of oat coleoptiles. Plant Physiol. 51, 998-1001 (1973).

TSAI, C. M.: Isolation of UDP-glucose: fl (1-4) glucan glucosyltransferase from oat and its properties.]. Carb., Nucleos., Nucleot. 2, 419-431 (1975).

VAN DER W:OUDE, W. ]., c. A. LEMBI, D. ]. MORRE, J. 1. KINDINGER, and L. ORDIN: fJ glu­can synthetases of plasma membrane and golgi apparatus from onion stem. Plant Phy­siol. 54, 333-340 (1974).

WANG, M. C. and S. BARTNICKI-GARCIA: Synthesis of fl-1,3 glucan micro fibrils by a cell­free extract from Phytophthora cinnamomi. Arch. of Bioch. and Bioph. 175, 351-354 (1976).

MICHEL FEVRE, Laboratoire de Mycologie associe au C.N.R.S., Departement de Biologie Vegthale, Universite Claude Bernard, Lyon I, 43 boulevard du 11 Novembre 1918, 69621 Villeurbanne, France.

Z. Pjlanzenphysiol. Bd. 95. S. 129-140. 1979.