FEMS Microbiology Letters 83 (1991) 173-178 © 1991 Federation of European Microbiological Societies 0378-1097/91/$03.50 Published by Elsevier ADONIS 037810979100433S

173

FEMSLE 04626

Stability of chitin synthetase in cell-free preparations of a wild-type strain and a 'slime' variant of Neurospora crassa

Glor ia Gim6nez , Dan ie l Go za lb o and Jose P. Mar t l n ez

Secci6n de Microbiologfa, Facultad de Farmacia, Universitat de Valencia, Valkncia, Spain

Received 27 June 199l Revision received 12 July 1991

Accepted 14 July 1991

Key words: Chitin synthetase; Enzymatic stability; Chitinase cell-free extracts; Gel filtration; Neurospora crassa

1. SUMMARY 2. INTRODUCTION

Chitin synthetase activity in cell-free prepara- tions from a wild-type strain and a 'slime' variant of Neurospora crassa was monitored over many days in samples stored at 0 ° C. Total activity in whole-cell-free extracts and low-speed super- natants from both organisms was very unstable, losing more than 90% of the initial activity on storage at 0°C for 96 h. Chitin synthetase detec- tion was not masked by chitinase activity present in the preparations. Gel-filtration chromatogra- phy of these preparations increased the stability of the activity from the 'slime' variant, whereas removal of particulate structures by high-speed centrifugation stabilized the chitin synthetase ac- tivity in the supernatant, particularly in the wild type. These results suggest that factor(s) involved in the regulation of chitin synthetase may be differentially located or altered in 'slime' cells.

Correspondence to: J.P, Martlnez, Secci6n de Microbiolog~a, Facultad de Farmacia, Universitat de Valencia, Avda. Blasco Ibfifiez, 13, 46010-Valencia, Spain.

Chitin synthetase (E.C. 2.4.1.16) is essential for the biosynthesis of the walls of most fungi [1]. Spatial control of chitin synthesis is believed to occur by compartmentation of chitin synthetase in chitosomes, microvesicles present in many dif- ferent chitinous fungi, including Neurospora

crassa [2-4]. These microvesicles transport the zymogenic enzyme to its final destination at spe- cific sites of the cell surface, where chitin mi- crofibrils are synthesized and assembled.

Wild-type N. crassa grows as a mycelium with typical hyphal walls, but the 'slime' variant [5] lacks cell walls due to its incapacity to synthesize both glucans and chitin [6]. 'Slime' cells have undetectable levels of glucan synthetase [6], al- though they contain normal levels of chitin syn- thetase. Different studies have revealed that chitin synthetases from both N. crassa strains have similar catalytic and kinetic properties [7], zymogenic character [8,9], and are mainly located in chitosomes [4,8-10], and that UDP-N- acetylglucosamine, the precursor for chitin syn-

174

thesis, is present at roughly equal concentration in the cytosol of both 'slime' and wild-type strains [6,11]. Nevertheless, no explanation for the inabil- ity of 'slime' strains to make chitin has been reported yet. Hence, in this paper we have inves- tigated the stability of chitin synthetase in cell-free preparations from N. crassa wild-type and 'slime' strains, in order to obtain information that might account for the absence of chitin synthesis in the 'slime' cells.

3. MATERIALS AND METHODS

3.1. Strains and culture conditions N. crassa wall-less 'slime' variant FGSC 1118

(fz, sq, os-1) and the wild-type strain FGSC 988 (St. Lawrence 74-OR8-1a) were used. Cells were grown as described previously [10,11].

3.2. Preparation of cell-free extracts Wild-type and 'slime' cells were harvested re-

spectively by filtration through Whatman No. 1 paper or by centrifugation (600-700 × g, 10 min) as already reported [11], washed with 50 mM Tris. HC1 buffer (pH 8.2) containing 10 mM MgCI 2 and 1 mM E G T A (homogenization buffer), independently resuspended in appropri- ate volumes (15-20 ml) of the same buffer solu- tion in Duran bottles, mixed with an equal vol- ume of glass beads (0.45-0.5-mm diameter), and shaken for 45-60 s in a Braun MSK homogenizer cooled with liquid CO 2. Crude cell-free extracts (CFEs) were removed by decanting and cen- trifuged at 1000 x g for 10 min to sediment the cell-wall fraction and large-cell debris. The result- ing supernatant fluids (SI) were subsequently centrifuged at 54000 × g for 45 rain in a Beck- man type 30 rotor. The 54000 × g supernatant ($54) contained the 'mini-organelle' population of the cells (ribosomes, miscellaneous enzyme particles such as fatty acid synthetase, and mi- crovesicles including chitosomes) together with soluble components of the cytoplasm [12]. Sam- ples (1.5 ml) of the CFE, S1 and $54 were filtered through a Bio-Gel A-5 m column (1.5 x 5 cm; 5-ml gel-bed volume) and the void volume (ap- prox. 1.5 ml) containing the chitin synthetase

activity was recovered (CFEbg, Slbg and S54bg preparations).

3.3. Chitin synthetase and chitinase assays Chitin synthetase was assayed by following the

incorporation of N-acetyl-D-glucosamine (GIcN- Ac) into chitin as previously described [10,13]. Zymogenic chitin synthetase was activated with trypsin (final concentration, 10 p.g/ml). Activity was expressed in units, one unit being the amount of enzyme that catalyzes the incorporation into chitin of 1 nmol of GlcNAc per rain.

Chitinase activity was measured against nascent chitin by the procedure of L6pez-Romero et al. [14]. After incubation, the chitin synthetase assay mixtures (0.140 ml) were applied to a strip of Whatman 3 MM paper and irrigated in descend- ing order with isoamyl a lcohol / pyr idine/ water (1 : 1 : 0.8 by vol.) for 26 h. Chromatograms were dried and cut into 1-cm segments and radioactiv- ity was determined by liquid scintillation. This procedure separated the reaction product (chitin), the substrate (UDP-GIcNAc) and two chitin degradation products (GlcNAc and diacetylchito- biose).

4. RESULTS

4.1. Stability of chitin synthetase preparations We determined the stability of the enzyme in

the preparations used as starting material (whole-cell-free extracts, supernatant fluids ob- tained after low- and high-speed centrifugation of cell free extracts or gel-filtration eluates) in the purification of chitosomes [4,9,10,12]. Results shown in Fig. 1 indicate the following: (i) in CFEs from both wild-type and 'slime' strains, activity decreased during storage (chitin synthetase was almost non-detectable after 96 h) and similar behaviour was also observed in the S1 prepara- tions, which d~ not contain cell wall-related ma- terials [10,15-17]; (ii) removal of the membranous fraction from cell-free extracts by high-speed cen- trifugation resulted in a stabilization of chitin synthetase present in the supernatant ($54), this effect being more significant in the wild type (75% of the enzyme remains active after 144 h at

100

1 - F a t _ TIII I ~ so !

:=_ ' ~ 200

I,oo o i1 0

0 24 48 72 96 120 144 0 24 48 72 96 120 144

time(h~ Fj, g. 1. Chitin synthetase activity in different cell free prepara- tions from the wild-type strain (open bars) and the 'sl ime' variant (solid bars) of N. crassa (obtained as described in MATERIALS AND METHODS) Oil prolonged storage at 0°C. Total activity values are expressed as percentage of the initial activi- ties (time 0) detected in each of the chitin synthetase-contain- ing preparations assayed. For the wild-type these values (as units ml i for each sample assayed) were: CFE, 17.9; CFEbg, 9.1; SI, 14.5; Slbg, 9.3; $54, 5.8; S54bg, 1.8. In the case of the 'slime' variant values found were: CFE, 21.7; CFEbg, 11.4; S1,

22.1; Slbg, 12.5; $54, 13.3; S54bg, 6.5.

0-4 °C), than in the 'slime' strain (40% of the activity was detected in this case after 144 h of storage); (iii) removal of soluble material from 'slime' CFE and $1 preparations by gel-filtration chromatography (CFEbg and Slbg preparations; see METHODS) resulted in the stabilization of the chitin synthetase activity eluting in the void vol- ume (60% of the activity was present after 144 h), whereas the stability of the enzyme in the analo- gous preparations from wild type was even lower

175

than that present in their non-filtered counter- parts (the activity was lost in this case after 48 h of storage at 0 -4 °C); (iv) filtration of both $54 preparations through Bio-Gel A-5 m column re- sulted in stabilization of the enzyme which, in addition, became increasingly susceptible to acti- vation in vitro by trypsin during storage; this effect was particularly marked in the wild-type S54bg preparation.

4.2. Chitinase does not mask chitin synthetase de- tection

Since, in the experiments shown in Fig. 1, chitin synthetase activity was detected by measur- ing the amount of polymer synthesized, nascent chitin could have been hydrolyzed by endogenous chitinase(s), thus partly masking detection of

10 ~udp.glcnac ~glcnac Oh

i SLIME • P I B t

1

48h

5 o ~ l ' . " , , , ,

15

10

5

0 [- i

10

96h

~udp,gfcnac ~glcnac

T,, ,,

WiLD,TYPE I ~ l i i

-J•l- L~ I I t

Oh

, ~1[ L , 18 26 340 10 18

DISTANCE FROM ORIGrN(CM} 26

48h

I

96h

34

Fig. 2. Paper chromatography of chitin synthetase assay mix- tures. Standard chitin synthetase assay mixtures (nascent chitin) were incubated for 60 min at 28°C (samples from the CFEs of each N. crassa strain were assayed at the indicated times), applied to chromatographic paper strips and irrigated in descending fashion as described in METHODS. The chro- matographic mobility of s tandard UDP-GIcNAc and GIcNAc samples in chromatograms run in parallel is indicated by

a r r o w s ,

176

chitin synthetase, as has been described [14]. Ac- cordingly, we analyzed the chitin synthetase assay mixtures by paper chromatography to detect the presence of hydrolysis products (diacetyl-chitobi- ose and GlcNAc) resulting from chitinolytic degradation. Results shown in Fig. 2 indicate that there was no such chitinase activity, at least in our experimental conditions. Thus, only two peaks of radioactivity were detected in the chro- matograms, one corresponding to chitin (which does not enter the chromatogram) and the other to the non- inco rpora t ed subst ra te ( U D P - GlcNAc). In addition, the decrease in the amount of chitin detected in the chromatograms was as- sociated with an increase in the amount of sub- strate; this observation also gives support to the contention that chitinase did not mask chitin syn- thetase detection. Results in Fig. 2 are from assays performed using CFE aliquots as enzy- matic samples; similar results (not shown) were obtained when samples from the S1 preparations were used in the chitin synthetase assays, subse- quently analyzed by paper chromatography.

5. DISCUSSION

The inability of the N. crassa 'slime' variant to make a normal cell wall may result from a combi- nation of diverse biosynthetic deficiencies. 'Slime' cells fail to make chitin, yet they seem to contain the components for chitin biosynthesis [6,8,10,11] and also secrete the material necessary for cell- wall synthesis [15-19]. The properties and distri- bution of chitin synthetase are similar in both the wild-type and the 'slime' variant [7,8,10]. How- ever, results shown here on the stability of chitin synthetase preparations suggest the existence of one or more factors involved in the regulation of chitin synthetase that could be functionally al- tered a n d / o r mislocated in 'slime' cells; such factors would thus be responsible for the distinct enzyme stabilities observed. The existence of sol- uble a n d / o r membrane-bound factor(s) involved in chitin synthetase lability may account for the results presented here. In the case of 'sl ime' preparations, removal of such soluble factor(s) by gel filtration led to chitin synthetase stabilization

(i.e. the enzyme became stabilized in CFEbg and Slbg preparations); whereas this factor (or fac- tors) appeared to be exclusively membrane-bound in wild-type cells, as enzyme stabilization oc- curred only when the bulk of membranous struc- tures was separated from chitosomes by high- speed centrifugation. In any case, stabilization of the enzyme in the $54 preparat ion from the 'slime' variant also suggests that this factor could be partly membrane-bound in these cells.

It is not known whether this marked stability is related to the activation in vivo of chitin syn- thetase which, in turn, may account for the fail- ure of the 'slime' cells to make chitin. There is evidence for the existence of differences in the lipid content, lipid composition, or both, between chitosomes from 'slime' and wild-type cells [10]. Lipids appear to play a significant role in both the activity and the 'in vivo' activation of chitin synthetase [20-23] and, therefore, are involved in the stability of the enzyme.

A C K N O W L E D G M E N T S

This work was carried out in part while G.G. and J.P.M. were research visitors at the Depart- ment of Plant Pathology, University of California, Riverside, CA. We are indebted to Prof. S. Bart- nicki-Garcla for the facilities. This work was sup- ported in part by a grant (GM-33513) from the N.I.H., U.S.A. to S. Bartnicki-Garda. J.P.M. was a recipient of post-doctoral fellowships from the Tratado de Amistad y Cooperacidn entre Espafia y los Estados Unidos de America, and from the F.I.S.S.S. from the Ministerio de Sanidad y Se- guridad Social (Spain).

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