Planta (1990)180:324-332

PI~..~[~ 9 Springer-Verlag 1990

A 1,4-/ -D-glucan-synthase system from Dictyostelium discoideum R.L. Blanton* and D.H. Northcote Department of Biochemistry,University of Cambridge, Tennis Court Road, Cambridge CB2 1QW, UK

Abstract. Particulate membrane preparations have been isolated from culminating Dictyostelium discoideum cells. The preparations incorporated glucose from uridine 5'diphosphate-glucose into a glucose polymer or polymers. These have been shown to be homopolymers of fl-linked glucose. A high percentage (78% by methylation analysis) of the linkages formed are 1,4-linkages and a lower percentage (12%) are 1,3-linkages. The glucan-synthase complex present in the particulate membrane preparation has an apparent Km of 0.28 mM and a Vm,x of 1.59 nmo1. min-1. (rag protein)-1. The enzyme system is dependent upon Mg 2 + and cellobiose for maximal activity, but is inhibited by millimolar levels of Ca 2+. Particulate membrane preparations were made from cells at various times during a synchronous developmental time course and demonstrated that the glucan-synthase activity appeared at the tight-aggregate stage of development. Key words: Cellulose synthesis - Dictyostelium synthase - Stalk formation (Dictyostelium)

Glucan

Introduction An understanding of the control and regulation of cellulose synthesis is fundamental to understanding plant development because cellulose is the primary structural component of the plant cell wall (Preston 1974). It, is becoming increasingly evident that the cell wall has a critical role in determining the rate and pattern of plant * Present address and address for correspondence: Department of

Biological Sciences, Texas Tech University, P.O. Box 4149, Lubbock, TX 79409-3131, USA Abbreviations: EDTA=ethylenediaminetetraacetic acid; EGTA = ethylene glycol-bis(fl-aminoethylether)-N,N,N',N'-tetraacetic acid; Hepes=4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid; UDP = uridine 5'-diphosphate; UDPGIc = UDP-glucose

development, in mediating plant responses to environmental stimuli and stress, and in housing a variety of enzymes critical to plant function (Haigler 1985; see Cassab and Varner 1988; July and Zaerr 1987). However, there is a lack of a reliable in-vitro assay system for cellulose synthase from higher plants. Membrane preparations from higher-plant cells incorporate large amounts of glucose from uridine 5'-diphosphate glucose (UDPGlc) into a glucose polymer, but this invariably turns out to be predominantly/%l,3-1inked rather than /%l,4-1inked. Various hypotheses have been advanced to explain this phenomenon: perhaps the callose and cellulose-synthase systems are the same and cell disruption activates callose synthesis preferentially (Jacob and Northcote 1985; Delmer et al. 1985; Delmer 1987); or perhaps a regulatory protein as part of the cellulosesynthase complex is rapidly degraded upon cell disruption (Delmer 1988). The presence of cellulose in the life cycle of the cellular slime mold Dictyostelium discoideum is first detected during late aggregation (Sussman and Sussman 1969; Sussman 1972). Vegetative and aggregating amoebae do not synthesize cellulose, nor are the amoebae capable of forming microcysts, which in other cellular slime mold species have walls containing cellulose (Raper 1984). Only when aggregates become consolidated and begin to develop an organizing tip is cellulose made. Cellulose microfibrils are found in the slime sheath that surrounds consolidated and tipped aggregates, pseudoplasmodia, and sorocarps (Raper and Fennell 1952; Hohl and Jehli 1973; Freeze and Loomis 1977), in the stalk tube and stalk cell walls (Raper and Fennell 1952; Mfihlethaler 1956; Gezelius and R~mby 1957; George and Hohl 1969; Freeze and Loomis 1978), and in the spore walls (Mfihlethaler 1956; Hemmes et al. 1972). Because D. discoideum exhibits induced cellulose synthesis and is easily manipulated experimentally, it is a good model organism for the study of eukaryotic cellulose synthesis (Delmer 1983). Reports in the literature have indicated that incorporation of glucose from UDPGlc into an alkali-insolu-

R.L. Blanton and D.H. Northcote: Dictyostelium 1,4-fl-D-glucan synthase ble p r o d u c t b y m e m b r a n e p r e p a r a t i o n s o f D. discoideum ( W a r d a n d W r i g h t 1965; L o o m i s a n d T h o m a s 1976) a n d Polysphondylium pallidum ( P h i l i p p i a n d P a r i s h 1981) o c curred, but none of these reports included the essential p r o d u c t a n a l y s i s w h i c h w a s n e c e s s a r y to d e m o n s t r a t e t h a t a fl-l,4-1inked p o l y m e r w a s f o r m e d . T h i s h a s b e e n d o n e in t h e p r e s e n t i n v e s t i g a t i o n .

Material and methods Chemicals and radiochemicals. Culture media components were either from Difco (Detroit, Mich., USA) or Oxoid (Basingstoke, Hampshire, UK). Uridine 5'-diphosphate-D-glucose and guanidine 5'-diphosphate (GDP)-D-glucose were from Sigma (Poole, Dorset, UK), GDP-f-[-U-14C]-D-glucose and 6-[-U-a4C]-D-glucose were from Amersham International (Amersham, Bucks., UK), and UDPdi-[U-14C]-D-glucose was from Amersham International or New England Nuclear (Wilmington, Del., USA). Tinopal LPW (Fluorescent Brightening Agent 28) was a gift from Ciba-Geigy, Inc. (Greensboro, N.C., USA). fl-(1-3)Glucanase purified from Rhizopus and cellulase purified from Streptomyces were gifts from E.T. Reese (U.S. Army Laboratories, Natick, Mass., USA). Proteinase K was from Sigma. All other chemicals were of analytical grade or of the highest purity available. Organism and culture. Dictyostelium discoideum Raper strain AX-2 (kindly provided by Dr. R.R. Kay; Laboratory for Molecular Biology, MRC, Cambridge, UK) stocks were maintained at 8~ on SM agar (Sussman 1966) with Klebsiella aerogenes as a food source. These stocks were renewed monthly from spores stored at 4 ~ on silica gel. New axenic stocks were prepared monthly from the plate cultures. Amoebae were grown in HL-5 medium (Watts and Ashworth 1970) at 22 ~ C with shaking at 130~150 rpm. Synchronous development. Cells in late exponential (5.106-8 9 cells-m1-1) growth were harvested by centrifugation, washed twice with KK2 buffer (16.5 mM KH2PO4, 3.9 mM K2HPO4, 2 mM MgSO4), resuspended in KK2 and plated to a final density of 2.4.106 cells.cm -2 in Petri dishes with 1.5% K K 2 agar. After spreading and drying the plates were kept in a moist box at 22 ~ C with dim overhead illumination. 10 6

Crude-membrane preparation. Developing sorocarps (early culmination, usually 14-16 h following plating) from 24-32 plates were collected with a glass scraper and deposited in 10-15 ml NaHepes [4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid, Na salt] disruption buffer [20 mM NaHepes; 40 mM sodium pyrophosphate; 0.57 mM PMSF (phenylmethylsulfonyl fluoride); 2 mM E D T A (ethylenediaminetetraacetic acid); 1 mM E G T A (ethylene glycolbis(fl-aminoethyl ether)-N,N,N',N'-tetraacetic acid); 5 mM 1,10phenanthroline; 2 m M N-carbobenzoxy-L-phenylalanine; 0.5% ethanol; 0.4 mM dithiothreitol, pH 7.0; modified from GoodloeHolland and Luna 1987] in a 30-ml Corex centrifuge tube. The cells were disrupted with five 10-s bursts (with 10-s pauses) at top power (for a microtip) of a Dawe Soniprobe Type 7532A, Heat Systems-Ultrasonics Model W-225R (Kinematica, Lucerne, Switzerland), or a Tekmar Model TM-300 sonicator (Tekmar Co., Cincinnati, Ohio, USA). The lysate was filtered through a 45-~tm mesh Nitex screen and centrifuged in a Sorvall SS34 rotor (Sorvall Instruments, Stevenage, Herts., UK) for 5 min at 2000 rpm. The supernatant was transferred to a 50-ml polyallomer centrifuge tube, disruption buffer added to the half-filled point, and the tube centrifuged in an SS34 rotor at 18000 rpm for 15 min. The supernatant was discarded and the pellet resuspended in disruption buffer with gentle Dounce homogenization and the tube centrifuged as before. The supernatant was discarded, the pellet resuspended in NaHepes

325

buffer (20 mM NaHepes, pH 7.0), and centrifuged at 15000 rpm for 10 min. The pellet was washed a second time. Following the second wash the pellet was resuspended in 300-750 gl of the appropriate buffer.

Glucan-synthase assays. Standard assay conditions were: 10 mM NaHepes; pH 7.0; 10 mM cellobiose; 5 mM MgCI:; and 0.5 mM uridine diphosphate [U-14C]-D-glucose (288.6 MBq. mmol-1). For most experiments the final reaction volume was 10 Ixl, with 5 ~tl membrane preparation (20-50 ~tg protein) in 20 mM NaHepes, 20 mM cellobiose, and 10 mM MgC12 being added to 5 ~tl 1.0 mM UDPGlc. Reaction times and variations in these conditions are noted in the figure captions. The reaction was stopped by adding 75 ~tl H20 and placing the reaction tube in a boiling-water bath for 5 min. The tube contents were spotted onto 2.5-cm circles of Whatman No. 1 filter paper, the papers dried in a stream of warm air, the filters placed in a plastic scintillation mini-vial and washed with six 1-min changes of H 2 0 (1 ml) with rotation in a vertical plane followed by six l-rain changes of 3:2 (v/v) chloroform:methanol (1 ml) with rotation (Jacob and Northcote 1985). The papers were then either dried in vacuo or in air, scintillation fluid added, and the radioactivity determined in a liquid scintillation counter. Collection and washing of the in-vitro product. For the chemical characterization of the in-vitro product, the reaction volume was 100 ~tl, 0.1 mM substrate 832.5 MB2-mmol-1) was used, and the reaction was run for 90 rain. The reaction was stopped by incubation for 2 rain in a boiling-water bath and the product was stored at - 2 0 ~ C. The thawed product was washed and separated from unreacted substrate by adding 1 mg carrier cellulose (except where noted otherwise) and 95% ethanol to make a final concentration of 70% ethanol. The tube was centrifuged at top speed in an Eppendoff microcentrifuge (Anderman & Co., Kingston-upon-Thames, Surrey, UK) for 2 min. The pellet was washed five times in 70% ethanol. The washed product was dried under N 2 and resuspended in 100 I-tl distilled water. Preparation of stalks. Non-radioactive stalks were collected from 24-h-old synchronous cultures, placed in distilled water, and stirred. The stalks were collected on 100-~tm Nitex mesh, washed vigorously with water while on the filter, and resuspended in fresh distilled water. This process was repeated until spores were no longer found in the filtrate. The stalks were lyophilized and lipids extracted by boiling the stalks with refluxing in 2:1 (w/v) chloroform:methanol (two changes) until the stalks were colorless. Radioactive stalks were prepared by spreading AX-2 amoebae on Millipore (Harrow, Mddx., UK) filters as described by Sussman (1966). At 16 h (mid-culminants) the filters were placed on 10 ~tl [U-14C]-D-glucose (111 M B q . m m o l - t). Moistened filter pads were placed on the dish lid to maintain humidity. After 2 h the filters were placed on a second 10-gl drop of [U-14C]-D-glucose until the fruiting bodies were fully formed. The stalks were then processed as described above.

Protein determination. Protein was determined by the method of Markwell et al. (1978) with bovine serum albumin as standard.

Fluorescence microscopy. Stalks, sheaths, and spores were stained with 0.5 mM Tinopal LPW and observed with a violet filter set (405 nm excitation filter, 455 nm dichroic mirror with a 455 barrier filter, and a 475 nm supplemental barrier filter) on an Olympus BHS-T microscope (Olympus Optical Co., London, UK). Photomicrographs were taken on Kodak T-Max 400 film (Kodak, Hemel Hempsted, Herts., UK). Shadow-casting and electron microscopy. Stalks prepared as described above were rehydrated in distilled water and boiled for 30 min. The stalks were collected on a 100-1xm-mesh Nitex screen, washed with water, placed in 2% sodium dodecyl sulfate, and

326

R.L. Blanton and D.H. Northcote: Dictyostelium 1,4-fl-D-glucan synthase

boiled for 30 min. Following collection and extensive washing on the Nitex screen, the stalks were placed in 1 M NaOH, boiled for 30 rain, collected and washed and then treated with 1 M HC1 under the same conditions. The stalks were then dried onto freshly cleaved mica and shadowed with platinum-carbon at a 45 ~ angle. The replica was stabilized with deposition of carbon, floated off the mica on water, and cleaned by flotation on 2.5% Na-dichromate in 50% sulfuric acid for 30 min. The replicas were washed by three 5-min changes on distilled water, collected on carbon-stabilized Formvarcoated grids, and examined at 75 kV in an Hitachi (Tokyo, Japan) HU-11E electron microscope.

Fixation and electron microscopy. Membrane preparations were fixed for electron microscopy using the glutaraldehyde/tannic acid/ potassium ferrocyanide/osmium tetroxide procedure described by Goodloe-Holland and Luna (1987). Following the post-fixation step the membranes were embedded in 1.5% agar. Ultrathin sections were collected on carbon-stabilized Formvar-eoated grids and examined as described above. Enzyme-catalyzed hydrolysis. Radioactive stalks and in-vitro product were incubated in the following enzymes: (i) salivary amylase (prepared as described by Olaitan and Northcote 1962), 20 mM NaHepes, 25 ~ C, 4 h; (ii) 0.2 mg.m1-1 endo-l,3-fl-glucanase, 5 0 m M Na-acetate, 3 m M NAN3, pH5.5, 37~ 24h; (iii) 0.2 mg.m1-1 cellulase, 5 0 m M Na-acetate, 3 mM NaN 3, pH 5.5, 37 ~ C, 24 h; and (iv) 0.2 mg. m l - x proteinase K in 20 mM NaHepes, 37 ~ C, 24 h. The reactions were terminated by incubating the tubes at 100 ~ C for 10 min. After 20 mg carrier cellulose was added the tubes were allowed to stand 1-2 h, the contents deposited by vacuum filtration onto Whatman (Maidstone, Kent, UK) GF/C filters, the filters washed three times with 66% ethanol and once with methanol, the filters dried, scintillation fluid added, and the radioactivity retained on the filters determined. Radioactive stalks and washed in-vitro product were also digested as described and the products analyzed by paper chromatography. The digest without added carrier cellulose was deionized with the H + form of Amberlite IR-120 (BDH, Dagenham, UK), dried in a rotary evaporator, dissolved in water, and chromatographed in solvent A for 16-18 h or solvent B for 48 h. Glucose was run as a standard in solvent A and glucose, laminaribiose, maltose, and cellobiose were run as standards in solvent B.

Paper chromatography. Descending paper chromatography was done with 460-mm-long Whatman No. 1 paper developed with one of two solvent systems: Solvent A, ethyl acetate:pyridine:water (8:2:1, by vol.) or Solvent B, propanol: ethyl acetate :water (7:1:2, by vol.). Sugar markers and products from procedures with nonradioactive stalks were detected with the alkaline silver-nitrate reagent (Trevelyan et al. 1950). Radioactive products were detected by cutting the chromatogram into 40.10 mm z strips, which were placed in glass vials, and scintillation fluid added (Dalessandro et al. 1986).

Results

In-vivo-product analysis. The D. discoideum stalk bound the r-linked glucan-specific (Wood 1980) fluorochrome Tinopal LPW (Fig. 1). Shadowed preparations revealed the microfibrillar nature of the stalk (Fig. 2). Total acid hydrolysis of nonradioactive stalks analyzed by gas-liquid chromatography yielded 92.6% glucose, 5.7% mannose, and 1.7% galactose. Acetolysis gave only cellobiose. Alpha-amylase did not release any mono- or disaccharides from the stalks. The //-1,3 glucanase was reported by Jacob and Northcote (1985) to have no fl-l,4-glucanase activity; however, we did find the enzyme has a very small amount of fl-l,4-glucanase activity on powdered cellulose and trace amounts of glucose were detected from radioactive stalks incubated in this enzyme. Jacob and Northcote (1985) reported, and we have confirmed, that the //-1,4 glucanase has both fl-1,4- and fl-l,3-glucanase activities, as tested against microcrystalline cellulose and fl-l,6-free pachyman. Ra-

Total acid hydrolysis and acetolysis. Total acid hydrolysis of radioactive and non-radioactive stalks and washed radioactive in-vitro product were performed as described by Dalessandro et al. (1986). The hydrolyzed and acetolyzed products of the radioactive stalks and in-vitro product and the acetolyzed products of the non-radioactive stalks were detected by paper chromatography as described below, with 16-18 h in Solvent A being used for the hydrolyzed materials and 48 h in Solvent B for the acetolyzed materials. Products of the hydrolyzed non-radioactive stalks were detected by gasliquid chromatography, under the conditions described by Dalessandro et al. (1986). Periodate oxidation. Periodate oxidations were performed (for 5 and 9 d) on the washed radioactive product as described by Fry (1988), except that the oxidized and reduced product was subjected to total acid hydrolysis as described above. Samples were desalted by dialysis against distilled water using 1000-MW cut-off Spectra/ Pot dialysis tubing (Spectrum Medical Industries, Los Angeles, Cal., USA). The neutralized samples were chromatographed in solvent B for 18 h. This system gave good separation of glucose, erythritol, and glycerol. Methylation analysis. Methylation of radioactive in-vitro product and stalks was as described by Dalessandro et al. (1986), except that following each dialysis the contents of the dialysis bag were freeze-dried.

Fig. 1. Stalk ofDictyostelium discoideum stained with 0.5 mM Tinopal LPW. The stalk tube and stalk cell wails fluoresce brightly. • 340; bar = 50 ~tm Fig. 2. Shadowed preparation of the outer surface of the stalk tube of D. discoideum. Note the fibrillar nature of the stalk, x 95000; bar = 200 nm

R.L. Blanton and D.H. Northcote: Dictyostelium t,4-fl-D-glucan synthase

327

Table l. Relative solubility of the radioactive in-vitro product resulting from the incubation of crude membrane preparation from Dictyostellium disoideum ceils with UDP-[U-14C]-D-glucose. The material was incubated in the appropriate solvent. After 20 mg carrier cellulose was added the reaction tube contents were filtered onto glass-fiber filters, washed three times with water, once with methanol, the filters dried, scintillation fluid added, and the retained radioactivity determined using a liquid scintillation counter Treatment

H20 ,

24% 24% 24% 24% Fig. 3. A representative sample of the glucan-synthesizing crude membrane preparation from early-culminating cells of D. discoideum (14-16 h after plating). • 21300; bar= 1 I~m dioactive and non-radioactive stalks when incubated with fl-l,4 glucanase yielded glucose and cellobiose. When the radioactive and non-radioactive stalks were subjected to methylation and hydrolysis only 2,3,6tri-O-methyl-D-glucose was found. N o 2,3,4,6-tetra-Omethyl-D-glucose was detected. The absence of di-Omethylated sugars indicated that the methylation procedure used probably gave a complete methylation of the polymer. M e m b r a n e preparation. The crude m e m b r a n e preparation was seen in the electron microscope to be a mixture of closed vesicles of diverse dimensions (Fig. 3). Some vesicles contained cytoplasmic remnants, but no whole cells were ever observed in the sectioned material or in light-microscope preparations (not shown). In-vitro-product analysis. Complete acid hydrolysis of the radioactive products from incubation of m e m b r a n e s with UDP-[U-14C]-D-glucose gave only [14C-]glucose. Acetolysis of the in-vitro product released predominantly cellobiose, but also a trace a m o u n t of laminaribiose. The synthesized product was incubated with alpha-amylase, fl-l,3 glucanase, fl-l,4 glucanase, and proteinase K and the extent of the hydrolysis monitored either by the detection of hydrolysis products or by the loss of the product when the material was washed on glass-fiber filters. Proteinase K and alpha-amylase were found to have no effect upon the product. When incubated with fl-l,4 glucanase nearly all the product (93%) was lost from the filter and glucose and cellobiose were detected in the hydrolysate. When incubated with the fl-l,3 glucanase, some product (8%) was lost from the filters and small amounts of glucose but no laminaribiose, were detected in the hydrolysate. The material was extensively removed from the glassfiber filters by solubility in 24% K O H both at r o o m temperature and at 100 ~ C (Table 1). However, when the reducing end of the polymer was protected by including 0.5 M N a B H 4 in the alkali the solubility was much re-

% of maximum retained activity

21~ C, 30 min KOH, 21~ C, 2 h KOH, 100~ C, 20 min KOH+0.5 M NaBH4, 21~ C, 2 h KOH+0.5 M NaBH4, 100~ C, 2 h

100% 14 13 68 65

Table 2. Periodate oxidation of the radioactive in-vitro product resulting from the incubation of crude membrane preparation from D. discoideum with UDP-[U-t4C]-D-glucose. Values are the percentage of the total recovered radioactivity associated with the glucose, erythritol, or glycerol, corrected for number of carbons in each molecule. Total recovered activity: 5 d, 4224cpm; 9 d, 784 cpm Oxidation time (d)

Glucose Erythritol Glycerol

5

9

23 74 4

21 72 7

Table 3. Methylation analysis of the radioactive in-vitro product resulting from the incubation of crude membrane preparation from D. discoideum with UDP-[U-laC]-o-glucose. Total recovered activity: 626 cpm % of total radioactivity recovered 2,3,6-Tri-O-methyl-D-glucose 2,4,6-Tri-O-methyl-D-glucose 2,3,4,6-Tetra-O-methyl-D-glucose

78 t2 10

duced. It appeared, therefore, that the solubility in the alkali was the consequence of degradation. The radioactive in-vitro product was subjected to periodate oxidation followed by complete hydrolysis. The results are summarized in Table 2. Erythritol was the major product detected, but a significant quantity of glucose was also detected. A low percentage of glycerol was found. The relatively constant proportions of erythritol and glucose between the 5- and 9-d oxidations indicated that the oxidation was probably complete and that glucose was formed as the result of the presence of 1-3-linkages. The radioactive in-vitro product after methylation and hydrolysis gave three derivatives (Table 3). 2,3,6-TriO-methyl-D-glucose was the predominant methylated sugar recovered, but 2,4,6-tri-O-methyl-D-glucose and 2,3,4,6-tetra-O-methyl-D-glucose were also detected.

328

R.L. Blanton and D.H. Northcote: Dictyostelium 1,4-fl-D-glucan synthase

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Fig. 4. Time course of glucan-synthase activity of the crude membrane preparation from early-culminating cells of D. discoideum (14-16 h after plating). The reaction mixtures contained: 10 or 100nmol UDPGIc, 0.155nmol UDP-[U-14C]-Glc, 500nmol MgC12, 1 mmol cellobiose, 1 mmol NaHepes, pH 7, and 50 ~tl crude membrane preparation (1.9 mg protein for 1 mmol UDPGIc, 0.38 mg protein for 0.1 mM UDPGlc) in a total volume of 100 pl. Ten identical tubes were prepared for each UDPGlc concentration. The reactions were kept at 22~ and tubes removed and boiled at the appropriate time

and the V m a x 1.59+0.028 n m o l . m i n - ~ . ( m g protein) -1, as determined with the aid of a nonlinear regression c o m puter p r o g r a m (Duggleby 1981) using the data presented in Fig. 6. The kinetic values m a y be of limited significance since we were using crude m e m b r a n e preparations and the values m a y relate to two transglueosylase activities. W h e n G D P - [ U - 1 4 C ] - D - g l u c o s e was supplied as the substrate, no i n c o r p o r a t i o n of radioactivity was observed.

Fig. 5. Effect of protein (enzyme) concentration on the synthesis of radioactive glucan by crude membrane preparations from cells of D. discoideum (14-16 h after plating). The reactions were run under standard conditions for 20 rain

Effects of cellobiose and divalent cations on enzyme activity. The effect of cellobiose on the enzyme activity is

Fig. 6. Effect of varying UDPGIc concentrations on the kinetics of the glucan-synthase system from crude membrane preparations of D. discoideum cells (14-16 h after plating). The reaction was otherwise run under standard conditions for 5 min. Time-course experiments with each substrate concentration were performed and demonstrated that in all cases the reaction was linear for at least 5 min Fig. 7. Effect of cellobiose concentration on the glucan-synthase activity of crude membrane preparations from D. discoideum cells (14-16 h after plating). The reaction was performed under standard conditions for 20 min

shown in Fig. 7, of m a g n e s i u m ions in Fig. 8, and of calcium ions in Fig. 9. Cellobiose and m a g n e s i u m ions were required for maximal activity and both reached saturation at a r o u n d 5 m M . N o enzyme activity was detected in the presence of 5 m M E D T A . Calcium ions inhibited the enzyme activity. W h e n incubated with 5 m M E G T A the enzyme activity was 84% of the m a x i m u m detected at 0 m M added Ca 2+. This indicates that m i c r o m o l a r concentrations of Ca 2 + were p r o b a b l y required for m a x i m u m activity. The lower activity in the presence of E G T A could have been caused by the inhibition of fl-l-3-synthase activity.

Effects of temperature and pH on enzyme activity. The

Kinetics of the glucan synthase. The time course of the in-vitro glucan synthesis is shown in Fig. 4. The reaction was linear for at least 30 min with either 0.1 or 1 m M substrate. The reaction was linear for up to 50 I~g protein per 10 gl reaction volume in a 20 min reaction (Fig. 5). The influence of substrate concentration on reaction rate is shown in Fig. 6. The a p p a r e n t Km is 0.28_+0.03 m M

effect of p H on enzyme activity is shown in Fig. 10. The o p t i m u m p H was a r o u n d p H 7.0. The temperature optim u m (data not shown) was determined to be 22 ~ C.

The induction of glucan-synthase activity. The developmental time course of the enzyme activity is shown in Fig. 11. E n z y m e activity was first detected at a very low level in aggregates that had stopped streaming and had become consolidated. The specific activity rapidly in-

R.L. Blantonand D.H. Northcote: Dictyostelium1,4-/%D-glucansynthase

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A 1,4-β-D-glucan-synthase system from Dictyostelium discoideum.

Particulate membrane preparations have been isolated from culminating Dictyostelium discoideum cells. The preparations incorporated glucose from uridi...
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