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Department of Chemistry, Rhodes College, 2000 North Parkway, Memphis, Tennessee 38112
Terry W. Hill 1
Department of Biology, Rhodes College, 2000 North Parkway, Memphis, Tennessee 38112
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ABSTRACT |
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Models of wall loosening in fungi and other walled eukaryotes require the action of proteins able to reduce the degree of linkage between components of the wall. In the oomycete Achlya ambisexualis, such a role has been proposed for a suite of endoglucanases that are secreted during branching and during the measurable wall softening associated with osmotic stress. We report here the isolation and characterization of one of these isoenzymes. The enzyme has a molecular weight of 32 kDa, a pH optimum of 6.75, a pI of 4.5, and a temperature optimum of 35 C. It is partially inhibited by sulfhydryl-binding reagents and completely inhibited by the tryptophan-binding reagent NBS. The enzyme has an endohydrolytic mode of action with substrate specificity towards glucans that contain ß-(1,4) linkages, either alone (carboxymethyl cellulose) or as mixed linkage (1,4–1,3)-ß-glucans (e.g., Avena glucan). It does not, however, degrade amorphous insoluble (phosphoric acid swollen) cellulose. Most significantly, the enzyme can also hydrolyze linkages in an Achlya cell wall fraction previously shown to consist of a mixed-linkage (1,4–1,3)-ß-glucan. This property is consistent with the long-standing hypothesis that the branching-related endoglucanases of oomycetes play a role in cell wall loosening.
Key words: cell walls, hyphal growth, oomycetes, proteases, wall loosening
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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, Harold 1990), a semi-rigid extracellular matrix composed of cross-linked polysaccharides and proteins (Sentandreu et al 1994). The mature wall also helps to contain the margin of the cell in the face of outwardly directed cytoplasmic forces, and it is an essential component of the mechanism by which a hypha brings invasive force to bear upon its environment (Money 2001, Money and Hill 1997).
At the same time, the wall is itself a product of the protoplast that it surrounds, and it is subject to precise modifications that reflect developmental programs of the cell (Harold 1995). Among these regulated wall properties is plasticity, the irreversible component of compliance (Cosgrove 1998). Sufficient plasticity, appropriately distributed within the fabric of the wall, is required in order for the cell to increase in size or develop in shape. Different growth patterns like spore swelling and hyphal tip extension depend, respectively, upon whether wall plasticity is globally or locally distributed (Wessels 1990). Experimental displacement of the determinants of apical wall plasticity can also alter the direction of a hypha's growth (Bracker et al 1997).
Debate continues about the mechanisms that regulate plasticity at an already-expanding site like a hyphal tip (Johnson et al 1996, Wessels 1990, Bartnicki-Garcia 1999). However, it is agreed that the introduction of de novo plasticity into a mature and noncompliant wall ("wall loosening"), as in the formation of branches or germ tubes, must involve agents that reduce the degree of linkage within or between the load-bearing components of the wall (Gow 1995, Cosgrove 1999). One category of agents that, in theory at least, could have such an effect consists of hydrolytic enzymes acting upon cell wall polymers (Brummell et al 1994, Fry 1995), and evidence exists to tie several such enzymes to wall remodeling events in higher plants (reviewed by Cosgrove 1999), as well as in fungi and oomycetes (reviewed by Gow 1995). Although the role of hydrolases in wall-loosening in higher plants is thought to be secondary to that of the nonhydrolytic expansin proteins (Cosgrove 2001), no counterpart for expansins has yet been discovered outside the plant kingdom, leaving hydrolases as the most likely wall-loosening agents in fungi and oomycetes.
Among oomycetes, the most-studied candidates for wall-loosening enzymes are the secreted endo-(1,4)-ß-glucanases (EC 3.2.1.4.) of Achlya ambisexualis (Thomas and Mullins 1967). The secretion of at least five electrophoretically distinct isoenzymes has been correlated with both local and isotropic wall-loosening events in this organism (Hill 1996, Money and Hill 1997). However, the basic biochemical properties of these enzymes, including the important question of their capacity to degrade polymers of a type actually found in Achlya cell walls, have not yet been investigated.
This report describes the isolation and basic biochemical characterization of one of the major secreted glucanase isoenzymes of A. ambisexualis. The choice of this specific enzyme was dictated principally by the fact that it is the only one among the five for which activity (seen in activity-stained electrophoretic gels) could be matched with an observable silver-stained polypeptide band (Hill 1996), which suggested that this particular isoenzyme is the one most likely to yield sequenceable amounts of pure protein. Another reason for initial focus on this isoenzyme is that it can be generated from a membrane-bound precursor by selective proteolysis (Hill et al 2002), suggesting a mechanism of developmental regulation of its activity.
Evidence will be presented that this enzyme has an endohydrolytic mode of action and a strong substrate specificity for noncrystalline glucans containing ß-(1,4) linkages. It will also be shown that the enzyme can independently hydrolyze components of a mixed-linkage glucan fraction of the Achlya cell wall, previously identified as containing ß-(1,4) linkages. These properties are consistent with hypotheses that attribute a morphogenetic role to this and related oomycete glucanases.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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. In order to maximize the production of endoglucanases, cultures were grown under osmotic stress (Money and Hill 1997) in a defined liquid medium (Hill 1996) modified to contain 400 mM sorbitol. Cultures (400 mL, inoculated with 1000 spore cysts/mL) were incubated at 25 C with reciprocal shaking (100 rpm, 3.5 cm excursion) for seven days, and the glucanase-containing medium was clarified by vacuum filtration through GF-A glass fiber filters (Whatman Intl. Ltd., Maidstone, England) and stored at -80 C.
Enzyme purification –
The culture filtrate (normally about 6 L) was concentrated at room temperature in an Amicon TCF-10 thin-channel ultrafiltration system (Amicon Inc., Beverly, Massachusetts) using about 0.4 MPa of nitrogen gas pressure and a PM-10 ultrafiltration membrane. The concentrated volume was about 50 mL, with a protein concentration between 0.5 and 1.0 mg per mL, using the method of Bradford (1976) with bovine serum albumin as a standard. Proteins were precipitated by dissolving solid ammonium sulfate at 5 C to bring the concentration to 90% saturation, followed by centrifugation at 10 000 x g for 20 min at 5 C.
The precipitate was resuspended in 1 mM HCl-histidine buffer, pH 6.3 ("column buffer"), containing 5% glycerol, and then applied to a Sephadex G-150 gel exclusion chromatography column (1.5 cm x 95 cm). Fractions of 2 mL were eluted at a 7-mL/h flow rate, and samples were assayed for endoglucanase activity viscometrically using carboxymethyl cellulose (CMC) as a substrate (Hill 1996). The viscometric assay was used throughout the isolation procedure.
The pooled E-III activity peak (Hill 1996) from the gel exclusion column was loaded in the 18-mL focusing chamber of a Rotofor® preparative isoelectric focusing apparatus (BioRad Laboratories, Hercules, California) and separated according to the manufacturer's recommendations using an Ampholyte mixture consisting of 90% "4/6" and 10% "3/10" Ampholytes. The peak fractions of endoglucanase activity from isoelectric focusing were desalted using a 1.5 cm x 10 cm column of Bio-Gel P-6DG (BioRad Laboratories, Hercules, California) at a flow rate of 38 mL/h. The resulting eluent was applied directly to an ion exchange column (1.5 cm x 8 cm) of QAE Sephadex A-25 resin. Retained proteins were eluted with a 100-mL linear gradient of 0 to 45 mM NaCl in column buffer at a flow rate of 18 mL/h. Salt concentration was monitored with a conductivity electrode.
Enzyme characterization –
SDS-PAGE was performed in 1-mm thick 10% w/v gels according to the method of Laemmli (1970). Silver staining followed Merril et al (1981). Endoglucanases in gels were visualized by activity staining after renaturation in situ, as described in Hill (1996). Analytical isoelectric focusing was performed in 0.5-cm x 10-cm tube gels according to BioRad Technical Bulletin 1030 (1975). Gel cylinders were cut into 0.5-cm slices and eluted overnight in 2 mL of 10 mM KCl, after which pH and endoglucanase activity (viscometric) were determined for each eluate.
The pH optimum of the isolated enzyme was determined viscometrically using isoionic (50 mM) buffers in order to avoid changes in viscometric flow rate due to differences in buffer ionic strength. The buffers and their pH ranges were: HCl-glutamic acid, pH 4–5; HCl-histidine, pH 5–6; NaOH-PIPES, pH 6–7.5; HCl-Tris, pH 7.5–9; NaOH-glycine, pH 9–10. The reaction temperature optimum was determined by incubating the enzyme in the reaction mixture used for viscometric assays for 30 min at the temperatures shown in Fig. 7, followed by boiling for 10 min to stop the reaction. Samples were then brought to 30 C, and the differences in the viscometric flow rates of parallel treated and control samples (without enzyme) for each temperature were used to calculate enzyme activity. Temperature stability of the enzyme was determined viscometrically after incubation of replicate enzyme samples in column buffer at the temperatures and for the times indicated in Fig. 8.
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) using glucose as a standard. Activity against the ß-glucosidase substrate p-phenyl-ß-D-glucopyranoside (pNPG) was assayed in a total volume of 125 µL, containing 13-mM pNPG and 16-mM pH 6.75 PIPES buffer for 2 h at 35 C. The reaction was stopped by adding 200 µL of 200-mM Na2CO3, and absorbance was read at 410 nm. A molar extinction coefficient of 18 000 was employed.
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Reagents –
Achlya cell walls were isolated and fractionated into HCl-soluble (ß-[1,3–1,6]-glucan), NaOH-soluble (ß-[1,3–1,4]-glucan with occasional [1,6] side branches), and residual fractions (principally cellulose) by the methods of Reiskind and Mullins (1981a). In addition, we used wall fractions kindly provided by Drs. J. B. Reiskind and J. T. Mullins. Phosphoric acid-swollen cellulose (PASC) was prepared from cellulose powder type CF 11 (Whatman Intl. Ltd., Maidstone, England) by the method of Walseth (1952). Chitosan and pustulan were purchased from Calbiochem (San Diego, California). CMC (type 7-MF) was a gift of Aqualon Co. (Wilmington, Delaware). Ampholytes were from BioRad Laboratories (Hercules, California). Avena glucan was purchased from the Department of Food Science and Nutrition, University of Minnesota (St. Paul, Minnesota). All other reagents were from Sigma Chemical Company (St. Louis, Missouri).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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. At this culture age, activity of the target isoenzyme is found in peak E-III (Fig. 2A, lane 2). In preparative isoelectric focusing (Fig. 3), the pooled E-III activity resolved as a single broad peak, with highest activity in fractions having pH's close to 4.8. The only glucanase activity in the fractions between pH 4.5 and 5.0 was that of the target enzyme (Fig. 2A, lane 3). Activity from these desalted fractions bound to the ion exchange resin and was eluted at a gradient concentration of approximately 20 mM NaCl (Fig. 4). The final sample contained a single band in SDS-PAGE having an estimated molecular mass under reducing conditions (silver stain) of approximately 32 kDa and under nonreducing conditions (silver stain or activity stain) of approximately 25 kDa (Fig. 2A, lanes 4 and 5). A partial amino acid sequence of the 45 N-terminal residues was determined to be: DIXTG IAPSS YSTAV QTNSQ FQPAI DELKK HSVAT TYVXN NGNSI.
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. The isoelectric point of the purified endoglucanase was determined by analytical isoelectric focusing to be approximately 4.5 (Fig. 5). The reaction pH optimum is approximately 6.75 (Fig. 6) and the reaction temperature optimum is about 35 C (Fig. 7). The enzyme is reasonably stable during incubation at temperatures up to 25 C, while activity is rapidly lost at temperatures exceeding 45 C (Fig. 8). Upon extended storage at -20 C, activity declined with a half-life of approximately 75 d, even in the presence of 20% glycerol (data not shown). This decay could be avoided by storage at -80 C.
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), was hydrolyzed. Results using the wall fractions generated in this study were comparable to those using extracts provided by Reiskind and Mullins. The enzyme caused a rapid reduction in the viscosity of soluble substrates, while causing only a small increase in the number of reducing groups (Fig. 9). Under the conditions employed, the relative viscosity of CMC decreased by approximately 85%, while less than 1% of the estimated number of glucose equivalents in the substrate were released. The result for Avena glucan was similar.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). Glucanase activity is not released from cultures that are no longer growing (Hill and Mullins 1979).
Hill (1996) originally reported the molecular mass of the glucanase isolated in this study to be 24 kDa, based upon its mobility in electrophoretic activity stains (under nonreducing conditions) and gel filtration. As shown in lane 5 of Fig. 2A, however, the molecule migrates as a 32-kDa protein under reducing conditions, and this is taken as the polypeptide's true molecular mass. Such a discrepancy is often observed with proteins whose tertiary structures are very compact and are stabilized by one or more disulfide bonds (Owen et al 1980).
Although a considerable amount of N-terminal sequence information was obtained (43 of the first 45 residues), no significant sequence similarity was found to any already-characterized protein in the databases. This may reflect the unique phylogeny of the oomycetes, which are little studied and evolutionarily distinct from plants, animals, and fungi (Cavalier-Smith 1986). We are unaware of any other published sequence information for oomycete glucanases, against which to compare. Overall, the basic physical properties of the Achlya 32-kDa glucanase (molecular weight, pI, pH optimum, and temperature optimum) lie easily within the range of values shown by other glucanases from plants and microbes, including those thought to play morphogenetic roles. However these data do not suggest affinities to any specific physiological class of glucanases, since, with the possible exception of a tendency for plant abscission cellulases to have a highly alkaline pI (e.g., Sexton et al 1990), no consistent set of physical characters unites glucanases playing common metabolic roles. Physical characteristics reported for the Phytophthora endo-(1,4)-ß-glucanase (Bodenmann et al 1985) are: MW = 21 kDa, pHopt = 6.0, pI = 3.2, and Topt = undetermined. These data fail to indicate any obvious similarity between the two proteins.
The 32-kDa glucanase's pH optimum of 6.75 is in excellent agreement with the optimum observed for assimilative growth in aquatic oomycetes (Powell et al 1972). The narrowness of the curve suggests the possibility that activity in vivo could be regulated by local pH changes in the manner observed for auxin-induced wall loosening in higher plants (Rayle and Cleland 1992). However in Achlya only minimal differences in extracellular pH have been measured between growing and non-growing regions of a hypha (Gow et al 1984), and there is no evidence to suggest that wall loosening in oomycetes or fungi may be regulated in such a way.
The enzyme's endohydrolytic mode of action (i.e., more or less random cleavage at interior sites in the polymer, rather than release of disaccharides from the nonreducing end) is indicated by its considerably greater effect upon substrate viscosity than upon the release of reducing equivalents (Brummel et al 1994) and by its insensitivity to the inhibitors deoxynojirimycin and gluconolactone, which principally inhibit exoglucanases and ß-glucosidases, respectively, at the concentrations employed (Reese et al 1971). The complete inhibition of activity by NBS suggests the presence of at least one essential tryptophan residue, most likely located at the active site in a substrate-binding or catalytic role (Macarrón et al 1995). Although the effect of NBS has been reported for only a minority of plant and microbial glucanases, to our knowledge all that have been tested have proven to be highly sensitive to this reagent, possibly indicating a conserved mode of action among at least some types of endoglucanases.
The partial sensitivity to the inhibitors NEM, Hg2+, and HMBA suggests the existence of at least one important sulfhydryl group (Ozaki and Ito 1991), though not one essential to activity under the reaction conditions employed. The absence of inhibition by either EDTA or EGTA indicates that no divalent cations are required. Aside from the thiol-inhibitor Hg2+, the only other tested ions that had significant effects upon activity were Mn2+ and Ca2+, both of which were mildly inhibitory. Patterns of ion inhibition and of sensitivity to sulfhydryl-binding agents vary greatly among plant and microbial glucanases, and their significance, if any, is not understood.
Of more likely significance is the enzyme's strong substrate specificity towards soluble glucans containing ß-(1,4) linkages (Table II). The complete absence of activity against the insoluble homopolymer PASC is surprising since enzymes active against CMC normally also hydrolyze amorphous swollen cellulose (Goyal et al 1991). However, precedents for inactivity of CMCases against PASC do exist (e.g., Hatfield and Nevins 1986, Ozaki and Ito 1991). Activity against the KOH-soluble, but not the HCl-soluble, fraction of Achlya walls (Table III) is consistent with the presence of ß-(1,4) linkages in the former and their absence in the latter (Reiskind and Mullins 1981a). Lack of action against the residual wall fraction most likely reflects the insoluble crystalline nature of its cellulose component. The enzyme's failure to show measurable activity against whole wall fragments, despite their content of KOH-soluble glucan, suggests that these polymers are less accessible when associated with other wall components. It is possible then, that effective hydrolysis of this wall fraction in situ requires either the cooperation of still further kinds of enzymes (the 32 kDa glucanase is, after all, but one of several associated with wall softening in Achlya) or conditions not replicated in our experiments.
The KOH-soluble mixed-linkage glucan of Achlya walls is thought to participate in cross-linking of the wall's crystalline cellulose microfibrils (Reiskind and Mullins 1981b), thus functioning in the same manner as the matrix components of the higher plant cell wall. Wall matrix components, rather than cellulose microfibrils, are assumed to be the sites of action of the plasticizing agents envisioned in models of wall loosening in higher plants (e.g., Carpita and Gibeaut 1993, Rose and Bennett 1999), both because the loose structure of matrix components should make them more accessible to enzyme action and because experimental observations have shown growth-related degradation of matrix molecules (reviewed in Cosgrove 1999). By its ability to hydrolyze linkages in the KOH-soluble matrix component of the Achlya cell wall, the 32-kDa endoglucanase described in this study seems well suited to participate in the wall-modifying role that has been proposed for the suite of glucanases secreted during branching and accommodation to osmotic stress.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Accepted for publication May 31, 2002.
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LITERATURE CITED |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Bartnicki-Garcia S., 1999 Glucans, walls, and morphogenesis: on the contributions of J. G. H. Wessels to the golden decades of fungal physiology and beyond. Fungal Genet Biol 27:119-127[Medline]
Bodenmann J, Heiniger U, Hohl HR., 1985 Extracellular enzymes of Phytophthora infestans: Endo-cellulase, ß-glucosidases, and 1,3-ß-glucanases. Can J Microbiol 31:75-82
Bracker CE, Murphy DJ, Lopez-Franco R., 1997 Laser microbeam manipulation of cell morphogenesis in growing fungal hyphae. SPIE 2983:67-80
Bradford MM., 1976 A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye-binding. Anal Biochem 72:248-254[Medline]
Brummell DA, Lashbrook CC, Bennett AB., 1994 Plant endo-1,4-ß-D-glucanases. Structure, properties, and physiological function. In: Himmel ME, Baker JO, Overend RP, eds. Enzymatic conversion of biomass for fuels production. Washington DC: American Chemical Society. p 100–129
Carpita NC, Gibeaut DM., 1993 Structural models of primary cell walls in flowering plants: consistency of molecular structure with the physical properties of the walls during growth. Plant J 3:1-30[Medline]
Cavalier-Smith T., 1986 The kingdom Chromista: origin and systematics. In: Round FE, Chapman DJ, eds. Progress in phycological research. Vol. 4. Bristol: Biopress Ltd. p 309–247
Cosgrove DJ., 1998 Cell wall loosening by expansins. Plant Physiol 118:333-339
———. 1999 Enzymes and other agents that enhance cell wall extensibility. Annu Rev Plant Physiol Plant Molec Biol 50:391-417[Medline]
———. 2001 Wall structure and wall loosening. A look backwards and forwards. Plant Physiol 125:131-134
Dygert S, Li LH, Florida D, Thoma JA., 1965 Determination of reducing sugar with improved precision. Anal Biochem 13:367-374[Medline]
Fry SC., 1995 Polysaccharide-modifying enzymes in the plant cell wall. Annu Rev Plant Physiol Plant Mol Biol 46:497-520
Gooday GW., 1995 Cell walls. In: Gow NAR, Gadd GM, eds. The growing fungus. London: Chapman & Hall. p 41–62
Gow NAR., 1995 Tip growth and polarity. In: Gow NAR, Gadd GM, eds. The growing fungus. London: Chapman & Hall. p 277–299
———, Kropf DL, Harold FM., 1984 Growing hyphae of Achlya bisexualis generate a longitudinal pH gradient in the surrounding medium. J Gen Microbiol 130:2967-2974[Medline]
Goyal A, Ghosh B, Eveleigh D., 1991 Characteristics of fungal cellulases. Bioresour Technol 36:37-50
Harold FM., 1990 To shape a cell: an inquiry into the causes of morphogenesis of microorganisms. Microbiol Rev 54:381-431
———. 1995 From morphogenes to morphogenesis. Microbiology 141:2765-2778[Medline]
Hatfield R, Nevins DJ., 1986 Purification and properties of an endoglucanase isolated from the cell walls of Zea mays seedlings. Carbohydr Res 148:265-278
Hill TW., 1996 Electrophoretic characterization of endo-(1,4)-ß-glucanases secreted during assimilative growth and antheridiol-induced branching in Achlya ambisexualis. Can J Microbiol 42:557-561
———, Loprete DM, Vu KN, Bayat Mokhtari SP, Hardin LV., 2002 Proteolytic activation of membrane-bound endo-(1,4)-ß-glucanase activity associated with cell wall softening in Achlya ambisexualis. Can J Microbiol 48:93-98[Medline]
———, Mullins JT., 1979 Hyphal tip growth in Achlya: enzymatic activities in mycelium and medium. Can J Bot 57:2145-2149
———, Pott MP., 1997 Regulation of extracellular proteases in Achlya ambisexualis. Can J Bot 75:440-444
Johnson BF, Calleja GB, Yoo BY., 1996 A new model for hyphal tip extension and its application to differential fungal morphogenesis. In: Chiu S-W, Moore D, eds. Patterns in fungal development. New York: Cambridge University Press. p 37–69
Laemmli UK., 1970 Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 227:680-685[Medline]
Macarrón R, Henrissat B, van Beeuman J, Dominguez JM, Claeyssens M., 1995 Identification of two tryptophan residues in endoglucanase III from Trichoderma reesei essential for cellulose binding and catalytic activity. In: Saddler N, Penner MH, eds. Enzymatic degradation of insoluble carbohydrates. Washington, DC: American Chemical Society. p 164–173
Merril CR, Goldman D, Sedman SA, Ebert MH., 1981 Ultrasensitive stain for proteins in polyacrylamide gels shows regional variation in cerebrospinal fluid proteins. Science (Washington, D.C.) 211:1437-1438
Money NP., 2001 Biomechanics of invasive hyphal growth. In: Howard RJ, Gow NAR, eds. The mycota VIII. Biology of the fungal cell. Berlin: Springer-Verlag. p 3–17
———, Hill TW., 1997 Correlation between endoglucanase secretion and cell wall strength in oomycete hyphae: implications for growth and morphogenesis. Mycologia 89:777-785
Owen MJ, Barber BH, Faulkes RA, Crumpton MJ., 1980 Albumin associated with purified pig lymphocyte plasma membrane. Biochem J 192:49-57[Medline]
Ozaki K, Ito S., 1991 Purification and properties of an acid endo-1,4-ß-glucanase from Bacillus sp. KSM-330. J Gen Microbiol 137:41-48[Medline]
Powell JR, Scott WW, Krieg NR., 1972 Physiological parameters of growth in Saprolegnia parasitica Coker. Mycopathol Mycol Appl 47:1-40[Medline]
Rayle DL, Cleland RE., 1992 The acid growth theory of auxin-induced cell elongation is alive and well. Plant Physiol 99:1271-1274
Reese ET, Parrish FW, Ettlinger M., 1971 Nojirimycin and D-glucono-1,5-lactone as inhibitors of carbohydrases. Carbohyd Res 18:381-388
Reiskind JB, Mullins JT., 1981a Molecular architecture of the hyphal wall of Achlya ambisexualis Raper. I. Chemical analyses. Can J Microbiol 27:1092-1099
———, ———. 1981b Molecular architecture of the hyphal wall of Achlya ambisexualis Raper. II. Ultrastructural analyses and a proposed model. Can J Microbiol 27:1100-1105
Rose JKC, Bennett AB., 1999 Cooperative disassembly of the cellulose-xyloglucan network of plant cell walls: parallels between cell expansion and fruit ripening. Trends Plant Sci 4:176-183[Medline]
Sentandreu R, Mormeneo S, Ruiz-Herrera J., 1994 Biogenesis of the fungal cell wall. In: Wessels JGH, Meinhardt F, eds. The mycota. I. Growth, differentiation, and sexuality. New York: Springer-Verlag. p 111–124
Sexton R, Del Campillo E, Duncan D, Lewis LN., 1990 The purification of an anther cellulase (ß(1:4)4-glucan hydrolase) from Lathyrus odoratus L. and its relationship to the similar enzyme found in abscission zones. Plant Sci 67:169-176
Thomas DdS, Mullins JT., 1967 Role of enzymatic wall-softening in plant morphogenesis: hormonal induction in Achlya. Science (Washington, D.C.) 156:84-85
Walseth CS., 1952 Occurrence of cellulase in enzyme preparations from microorganisms. TAPPI 35:228-233
Wessels JGH., 1990 Role of cell wall architecture in fungal tip growth generation. In: Heath IB, ed. Tip growth in plant and fungal cells. San Diego: Academic Press, Inc. p 1–29
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) and pH (
). Activity in fractions with pH's between 4.5 and 5.0 (delimited by dotted lines) was selected for further isolation
