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Cytochemical localization of N-acetyl-β-d -glucosaminidase in hyphae of Mucor racemosus
EXPERIMENTAL MYCOLOGY 3, 164-173 (1979)
Cytochemical Localization of N-Acetyl-~-D-glucosaminidase in Hyphae of Mucor racemosus HARVEY C. HOCH,* GERD HXNSSLER,t AND HANS-JOACHIM REISENER ~
Department of Plant Pathology, New York State Agricultural Experiment Station, Cornell University, Geneva, New York 14456, and t Institute fiir Physikalische Biologie, Technische Hochschule Aachen, Bundesrepublik Deutschland Accepted for Publication January 25, 1979; revised April 10, 1979 HOCH, H. C., H.&NSSLER, G., ANn REISENER, H-J. 1979. Cytochemical localization of N-aeetyl-~-D-glucosaminidase in hyphae of Mucor racemosus. Experimental Mycology 3, 164-173. Light microscopic localization of N-acetyl-fl-D-glucosaminidase activity by the hydrolysis of a-naphthyl N-aeetyl-fl-D-glucosaminide and by the subsequent coupling of the released a-naphthol with Fast Red B and p-nitrobenzene diazonium tetrafluoroborate was obtained in Mucor racemosus. Enzyme activity appeared as discrete spherical sites, from < 0.3 to 1 /ml in diameter, in the primary hyphae. In the secondary and tertiary branch hyphae of the older portions of the M. racemosus colony the sites were often as large as 3 ~m in diameter. The sites of enzyme activity occurred sparsely and were randomly distributed in the hyphal tips. They were most numerous in older portions of these hyphae. Hydolysis of the p-nitrophenyl N-acetyl-fl-D-glucosaminide was obtained with cell-free extracts of M. racemous, Rhizoctonia solani, Cortic!um sp., and Phycomyces nitens; however, eytochemieal localizations of enzyme activity were observed only in M. racemosus. Four cytochemical substrates and 11 coupling agents were investigated. Only a-naphthyl N-acetyl-p-D-glucosaminide was satisfactory as a substrate. Fast Red B and p-nitrobenzene diazonium tetrafluoroborate were the only coupling reagents that yielded stable discrete reaction products. INDEX DESCRIPTORS: N-acetyl-B-D-glucosaminidase; Mueor racemosus; cytochemistry.
Chitinolytie enzymes are involved in several aspects of development and degradation of filamentous fungi. They are associated with: hyphal tip elongation and hyphal branch formation (Gooday, 1978); maintenance of dikaryotie conditions through dissolution of dolipore septa and fusion of hyphal walls (Casselton, 1978; Gull, 1978; Iten and Matile, 1970; Reyes et al., 1977); degradation of chitinous debris; autolysis; and penetration of chitinous walls and exoskeletons of various hosts (Hoch and Fuller, 1977; Unestam, 1966). To be able to visualize the sites of ehitinase and chitobiase (EC 3.2.1.14), and
N-acetyl-fl-D-glucosaminidase (EC 3.2.1.30) activities would enhance our ability to study in more detail some of the above events. However, activities of these enzymes have not been cytochemically localized in fungi with any degree of reliability or resolution that would lend meaningful and accurate information about their sites of activity. N-Acetyl-fl-D-glucosaminidase activities have been localized by both light and electron microscopy in mammalian cells with several synthetic naphthol-linked substrates (Pearse, 1972; Pugh, 1973), and
164 0147-5975/79/020164-10502.00/0 Copyright © 1979 by AcademicPress, Inc. All rights of reproductionin any form reserved.
N-ACETYL-/3-D-GLUCOSAMINIDASE IN MUCOR also with a substituted indoxyl substrate (Pugh, 1973). In this paper we report on the cytochemieal localization of N-acetyl-/3-D-glucosaminidase in hyphae of Mucor racemosus using the substrate a-naphthyl N-acetyl-/3D-glucosaminide. We also present our results with three other fungi and with various other substrates and capture reagents.
165
ium: buffer:sand). The resulting triturate was centrifuged at 2500g and the supernatant collected. To 0.2 ml of the crude intracellular enzyme extract or culture broth (extracellular enzyme); 0,8 ml of 0.1 M citrate buffer, pH 5.5, containing 28/z~ pnitrophenyl N-aeetyl-/~-D-glucosaminidewas added, shaken, and allowed to incubate at 37°C for 30 rain. Next, 2.0 ml 0.5 M NaOH was added to stop the reaction and to develop the color of the liberated p-nitroMATERIALS AND METHODS phenol which was determined spectrophoThe fungi used in this investigation tometrically. In addition to p-nitrophenyl were: Corticium sp. from Dr. M. Boosalis, N-acetyl-/9-I~-glucosaminide, the four cytoUniversity of Nebraska, Lincoln; Mucor chemical substrates were also tested with racemosus Fres. f. sphaerosporus ( Hagem ) the enzyme preparations. They were used Schipper ( + s t r a i n ) , isolate CBS 115.08 at the same concentration as for cytochemfrom Centraalbureau voor Schimmelcul- istry but were postcoupled with Fast Dark tures, Baarn, Netherlands; Phycomyces Blue R (Sigma Chemical Co.) or p-nitronitens (Kunze) van Tiegh. & Le Monn., benzene diazonium tetrafluoroborate (East( + strain), isolate H 241 from Dr. R. T. man Kodak Co., Rochester, New York). In Hanlin, University of Georgia, Athens; and addition to the above treatment, purified Rhizoctonia solani Kiihn from Dr. D. P. N-acetyl-/3-I)-glueosaminidases from AsperMaxwell, University of Wisconsin, Madigillus niger and Jack beans (Sigma Chemson. All fungi were maintained on potatoical Co.) and from beef kidney (Boehringerdextrose agar at ca. 21°C. For determina- Mannheim GmbH, Bioehemica, Mannheim) tions of intracellular and extracellular Nwere used to test the substrates and subacetyl-/3-D-glucosaminidase activity, the sequent color reactions. Heat-inactivated fungi were grown in 125-mi Erlenmeyer controls were employed. flasks containing 25 ml of either sodium Since the primary purpose of this study polypectate medium ( Maxwell and was eytoehemistry, not biochemistry, of Lumsden, 1970) or 1% colloidal chitin N- acetyl-/3- D- glueosaminidase, quantative (Lingappa and Lockwood, 1962) plus values for enzyme activities for the various essential salts. They were grown at ca. fungi, based on milligrams protein, were 21°C in still culture for 8 days. For cytonot determined. Thus, optimum pH, chemistry they were grown on chitin (1%)presence or absence of enzyme activities, agar (2%)-coated microscope slides supand substrate specificity were determined ported on V-shaped glass rods placed on speetrophotometrieally for the colored rewater-moistened filter paper in petri plates. action products and were based on the wet Intracellular and extracellular enzyme weight of the myeelia. These data were activities were determined using p-nitrophenyi N-acetyl-/3-D-glucosaminide (Sigma used as a basis for the eytoehemieal inChemical Co., St. Louis, Mo.). Mycelia of vestigations. For eytochemistry, the fungi were fixed the four fungi were collected on a Buchner in situ on the slides for 10 min with 1% funnel, rinsed with 4°C 0.02 ~t citrate formaldehyde containing 10% methanol in buffer, pH 5.5, and ground with a cold mortar and pestle with washed quartz sand 0.1 ~ citrate (Na +) buffer, pH 6.5. The and citrate buffer ( 1 : 2: 4; w: v: w; rnycel- slides were then rinsed for 5 rain with 0.1
ttOCH, HANSSLER, AND REISENER
166
TABLE 1 Relative Hydrolysis of Synthetic Substrates by Intracellular N-Acetyl-fi-Dglucosaminidases from Various Fungi~ Enzyme source
Purified, commercial Rhizoctonia solani Corticium sp. Mucor racernosus Phycomyces nitens
Control°
N-Acetyl-~-D-glyeosaminidesubstrate p-Nitrophenyl
a-Naphthyl
q- + + +
÷ q- q- +
q_ ( q_ ) b
.
.
5-Bromoindoxyl
Naphthol ASBI-
Naphthol ASCL-
q- q- q- q-
q- q- q- q-
q- q- + +
.
+ (+)
.
.
.
+ + + (-) q- ( - )
+ + + .
.
+ + + .
- (-)
.
.
.
.
. --
--
.
.
a q_, -t- -t-, q- q- q-, q- -t- -t- -b represent increasing enzyme activities; --indicates no enzyme activity. bValues in parentheses represent data for extraeellular enzyme activity. Heat-inactivated extract of M . racemosus. citrate (Na ÷) buffer, pH 6.5, followed by additional rinse for 10 min with 0.1 citrate (Na ÷) buffer, pH 5.5 The slide cultures were next flooded with the appropriate enzyme substrate-diazonium salt coupling solution. They were incubated at 21°C for 5 to 30 min, after which time a cover glass was gently positioned over the hyphae. The material was immediately examined using oil immersion 100× objectives and photographed. Various combinations of the following enzyme substrates and diazonium salts were tested: a-naphthyl N-aeetyl-fi-D-glueosaminide (Serva Feinbiochemica, Heidelberg ); napthol ASBI- and naphthol ASLC-N-acetyl-B-Dglucosaminide (Sigma Chemical Co. ); and 5-bromoindoxyl N-aeetyl-/?-D-glucosaminide (Aldrich Chemical Co., Milwaukee, Wis.); Fast Red B; Fast Red ITR; Fast Red Violet LB; Fast Blue B; Fast Blue tlR; Fast Blue BBN; Fast Dark Blue R; Fast Violet B; Fast Garnet GBC; hexazotized pararosaniline (all above from Sigma Chemical Co.); potassium ferricyanide (for the 5-bromoindoxyl derivative); and p-nitrobenzene diazonium tetrafluoroborate. All substrates were first dissolved in ethylene glycol monomethyl ether (15 mg/0.4 ml) to which was added 10 ml of 0.1 ~ citrate buffer,
pH 5.5. The diazonium salts were added to this mixture at various rates, but generally at 0.05 ml (50 mg dissolved in 10 ml distilled water) per milliliter of substrate solution. The solution was filtered after 5 min and used immediately. Cytochemical controls consisted of (i) heat-inactivated enzyme (100°C for 15 min following fixation), (ii) incubation in solutions free of the synthetic enzyme substrate, and (iii) incubation in complete substrate-diazonium salt media containing 10 t'~ N-acetylglueosaminonolactone, a specific competitive inhibitor of N-aeetyl-fl-D-glucosaminidase activity. The laetone was prepared according to procedure (a) as reported by Findlay e t al. ( 1 9 5 8 ) except that the starting substrate was glueosaminic acid (Sigma Chemical Co. ) rather than glucosamine. RESULTS Optimum pH values for intracellular Nacetyl-fl-D-glueosaminidase activities as determined with p-nitrophenyl N-acetyl-flD-glucosaminide were 5.4 for both C o r t i c i u m sp. and R. s o I a n i , and 4.8 and 5.0 for M . r a c e m o s u s and P. n i t e n s , respectively. Optimum pH values were not determined for extraeellular enzyme activities.
N-ACETYL-#-~-GLUCOSAMINIDASE IN MUCOR
167
FIGs. 1 and 2. Mucor racemosus colony on colloidal chitin agar. The colloidal chitin substrate became transparent when hydrolyzed by chitinolytic enzymes from M. racemosus (Fig. 1). The cleared zone (darker area) was first noticeable 0.5 to 1 cm from the advancirrg colony margin (dashed line). The same area as in Fig. 1, but developed for N-acetyl-~-D-glucosaminidase activity with a-naphthyl N-aeetyl-/3-D-glucosarninideand p-nitrobenzene diazonium tetrafluoroborate, is shown in Fig. 2. Based on mycelial wet weight, M. racemosus had considerably more intraeellular N-
period. N-AcetyI-fl-D-glueosaminidase activity was apparent in the hyphae of M. acetyl-fi-D-glucosaminidase activity than raceraosus and not in the medium ( Fig. 2 ). did the other fungi (Table 1). Extracellular Colloidal chitin was not hydrolyzed by enzyme activity was found only in broths tl. solani or P. niten~ within 120 h, the from R. solani and C o r t i c i u m sp. cultures. longest growth period observed. The cytoplasm in the first 100 ~m of the Intracellular N-acetyl-fl-D-glucosaminidase preparations fi'om all fungi tested hydro- hyphal tip of M. r a c e m o s u s is dense with lyzed the p-nitrophenol-substituted sub- numerous organelles generally <1 ~m in strate; however, only M. r a c e m o s u s prep- diameter (Fig. 3). The density of the cytoarations hydrolyzed the ~-napthol- and 5- plasm in this region makes it difficult to bromoindoxyl-substituted substrates. The distinguish the various organelles. Large naphthol ASBI- and naphthol ASLC-N- vacuoles constitute most of the cellular aeetyl-fl-D-glucosaminide substrates were contents in older portions of the fungal not hydrolyzed by any of the intracellular colony (Fig. 4). These vacuoles are first enzyme preparations. All substrates were seen 90 to 120 ~am from the hyphal apex hydrolyzed by the three commercially puri- (Figs. 5 and 6). fied enzymes. Cytoehemical localization of N-acetyl-BColloidal chitin, in an agar medium, was D-glueosaminidase activity was achieved completely hydrolyzed by M. r a c e m o s u s only in M . r a c e m o s u s (Figs. 5-12). Hy(Figs. 1 and 2) and C o r t i c i u m sp. The drolysis of the cytochemical substrates was chitin agar medium was cleared, indicative not observed in the other test organisms. of hydrolysis, following 48 h growth (Fig. Only two of the cytochemieal substrates, 1). Chitin in the first 0.5 to 1 em of the ,-naphthyl N-acetyl-/3-D-glucosaminide 'and advancing colony margins was not cleared, 5-bromoindoxyl N-acetyl-fl-D-glucosaminide, but was cleared within the next 24-h were hydrolyzed to yield colored reaction
168
HOCH, HNNSSLEtt, AND tlEISENEtt
N-ACETYL-/3-D-GLUCOSAMINIDASE IN products when reacted with the appropriate capture reagent. The data summarized in Table 2 indicate that only two of the diazonium salts proved satisfactory as coupling reagents for the ~-napthol moiety released from the glueosamide substrate. The 5-bromoindoxyl derivative was not a satisfactory substrate in these tests since the colored reaction product was always diffuse. Potassium ferricyanide, the recommended oxidizing reagent for the 5-bromoindoxyl moiety (Pughl 1973), did not work in these studies (except with the commercially purified enzymes). Red to reddish-brown reaction products of ~-napthol and p-nitrobenzene diazonium tetrafluoroborate or Fast Red B salt were observed as discrete particles in M . r a c e m o s u s . The particles, presumably membrane-bound vesicles, varied from <0'.3 to 1 /~m in diameter. They were distributed sparsely throughout the cytoplasm in the first 200 to 400 ~m of the hyphal tip (Figs. 5, 6, and 11). A very few (one to five) sites of activity could often be detected in the apical 50 ~m. The distribution and number of sites of activity illustrated in Fig. 1i were representative for most hyphal tips, while that in Fig. 5 was considered exceptionally high. At a distanee of 300 to 500 ~m from the hyphal apex the concentration of the sites of activity gradually increased to the concentration depicted in Figs. 7 to 9 and 12. The sites of enzyme activity were distinctly separate from the large vacuoles shown in Fig. 4 (Figs. 6, 8, and 9) and for the most part were distributed peripherally around the
MUCOB
169
hyphal axis. The eentral portion of the hyphal axis was comprised mostly of larger vacuoles and thus most of the remaining cytoplasm, including the sites of enzyme localization, were excluded from this central zone. This feature was particularly noticeable when the plane of focus was moved from just below the hyphal surface (Fig. 8) to a longitudinally median plane of focus (Fig. 9). The distribution and size of sites of enzyme activity were different in the secondary and tertiary branch hyphae of older portions of the M . r a c e m o s u s colony than in the larger primary hyphae. These sites varied from <0.3 to >3 tzm in diameter and often were delimited by the diameter of these hyphae (Fig. 10). When diffusion of the reaction product occurred, these were the sites at which diffusion was first detected. No sites of enzyme localization could be detected in hyphae incubated in solutions minus the substrate (Figs. 13 and 14), in heat-treated hyphae incubated in complete media, or in hyphae incubated in complete media containing N-acetylglueosaminonolaetone. The small dark particles in Fig. 14 are not cytoehemical sites of enzyme localization, but instead are vesicles exhibiting a natural refractive index different from the ground cytoplasm. No differences were detected between the sites of enzyme activities localized with p-nitrobenzene diazonium tetrafluoroborate and Fast Red B (Figs. 5-9 vs 11 and 12). The red reaction product of Fast Red B, however, began to diffuse after 60 min,
Fits. 3 and 4. Nomarski interference-contrast light mierographs of formaldehyde-fixed hyphae of Mucor raceraosus. Figure 3, of the hyphal tip region, illustrates the dense nature of the cytoplasm containing small vesicles and very few large vacuoles. In older regions of the layphae, large vacuoles constitute most of the hyphal contents (Fig. 4). The magnification of these and all subsequent mierographs is X4200. Fins. 5-9. Bright field light micrographs of Mucor racemosus incubated in a medium containing a-naphthyl N-aeetyl-~-o-glucosaminide and p-nitrobenzene diazonium tetrafluoroborate to demonstrate N-acetyl-/~-D-glueosaminidase activity. Sites :of enzyme activity localization appear as discrete spherical deposits, from <0.3 ~zm to 1 #m in diameter. Distribution of these sites in the first 50 mn is sparse (Figs. 5 and 6). Dashed lines in Figs. 5 and 6 represent the same site in this hypha. Localization sites are concentrated 300 to 500 /tin and farther back from the hyphal tip (Fig. 7). Two planes of focus through the same hypha 1 cm from the byphal tip (Figs. 8 and 9).
170
HOCH, H/tiNSSLER, AND ttEISENER
N-ACETYL.fl-D-GLUCOSAMINIDASE IN MUCOR
171
DISCUSS,ION whereas the reaction product formed with p-nitrobenzene diazonium tetrafluoroborate The N-acetyl-fl-D-glueosaminidases proremained stable for more than 3 h. duced by the four fungi are apparently There were no detectable differences in substrate specific. While all fungi tested the sites of enzyme activity localization in hydrolyzed p-nitrophenyl N-acetyl-fl-D-gluhyphae of M. racemosus grown on chitin cosaminide, only M. racemosus could hyagar vs sodium polypeetate agar. drolyze any of the other synthetic subThe background color in the agar substrates. Since the commercially purified enstrate of both diazonium salts at the concenzyme preparations hydrolyzed all five subtration used in the incubation media was strates, it was unlikely that inhibition, due negligible. In the hyphae, a very slight to the diazonium salt, contributed to the yellow background color was noted but inability of the fungal enzyme preparations did not interfere with the detection of the to hydrolyze the substrates. Enzyme aceytochemieal reaction product color. Treatment of the hyphae influenced the tivators must be contemplated as one reason that the cytochemieal substrates time of the cytochemieal reaction product were not hydrolyzed by the fungal prepformation as well as product diffusion. Unfixed hyphae were compared with formal- arations; however, evidence from other chitinolytic systems indicates activators dehyde- and glutaraldehyde-fixed hyphae. Hyphae fixed with glutaraldehyde (1% in not required for these enzymes (Jeuniaux, 0.1 ~ citrate buffer, pH 6.5, followed by a 1963). N-Acetyl-fl-D-glucosaminidase has recitrate buffer, pH 5.5, rinse) developed a portedly been localized cytoehemieally in red-colored reaction product with both M. hiemalis Wehmer, Aspergillus niger van diazonium salts very rapidly (within 1 rain Tieghem, Saccharomyces cerevisiae Meyen, at 91°C). The color development was enand Can dida aIbieans ( Itobin ) Berkhout tirely diffuse, with particulate sites of using 5-bromoindoxyl N-acetyl-fi-e-glucoslocalization developing after 10 rain. These sites, however, also become diffuse. Un- aminide as the substrate (Pugh and Cawfixed hyphae also rapidly developed a red son, 1977). Unfortunately, the evidence reaction product, generally with particulate presented for the localization of this ensites of localization. These sites likewise zyme by these authors was not adequately rapidly diffused, within 10 rain. The clear- illustrated or discussed; thus little informaest sites of enzyme localization developed tion could be gleaned from their report. in hyphae fixed with formaldehyde, as They indicated that the enzyme content was "greatest at the growiag tips" in exemplified in this paper. FIG. 10. Secondary and tertiary branch hyphae in the old portion of a Mucor racemosus colony. The sites of N-aeetyl-fl-D-glucosaminidaseactivity resulting from hydrolysis of c~naphthyl N-acetyl-C~-D-glueosaminide and subsequent capture of a-naphthol with p-nitrobenzene diazonium tetrafluoroborateare often 3 ~ln in diameter and delimited by the diameter of the hyphae. FIGs. 11 and 12. Hyphae of Mucor racemosus incubated in a medium containing a-naphthyl N-aeetyl-fl-D-glueosaminide and Fast Red B. Very few sites of enzyme activity localization are seen in the hyphal tip (Fig. 11). In ,older regions of the hypbae, the sites are more numerous and larger (Fig. 12). FIGS. 13 and 14. Hyphae of Mucor racemosus incubated in a medium containing p-nitrobenzene diazonium tetrafluoroboratebut not the enzyme substrate. No sites of enzyme activity developed. Fig. 13. Hyphal tip region. Fig. 14. Hyphal region comparable to Figs. 4, 7 to 9, and 11.
172
HOCH, H}iNSSLER, AND ItEISENER
TABLE 2 Performance of Various Capture Reagents for 5-Bromoindoxyl and a-Naphthol Moieties Hydrolyzcd from N-Acetyl-B-D-glucosaminidein situ in Mucor racemosus Enzyme substrate
Capture reagents
5-BromoindoxylN-aeetylB-D-glucosaminide Fast Red B Fast Red Violet LB Fast Blue B p-Nitrobenzene diazonium tetrafluoroborate Hexatized pararosiline Fast Garnet GBC Fast Red ITR Fast Violet B Fast Blue RR Fast Blue BBN K + Ferricyanide
Not particulate, diffuse Not particulate, diffuse Some particulate, but diffuse Not particulate, very diffuse Not particulate, very diffuse
~-Naphthy[ N-acetyl-C~-Dglucosaminise Particulate, excellent Not particulate, diffuse Not particulate, very diffuse Particulate, excellent Not particulate, very diffuse Some particulate, but diffuse Not particulate, diffuse Particulate, but diffuse Particulate, but diffuse Particulate, but diffuse
No reaction
M u c o r and AspergiUus. Based on our results with M. racemosus, N-acetyl-fl-vglueosaminidase activity was greatest in older portions of the hyphae, at least 300 to 500 ~m from the tip. Such distribution of enzyme activity also corresponded with the earliest indication of clearing of the colloidal chitin afar. Distribution of N-acetyl-fi-v-glueosaminidase sites of activity in M. r a c e m o s u s was similar to that observed for acid phosphatase activity detected with 5-benzoyI-2naphthyl phosphate (Na +) and coupled with p-nitrobenzene diazonium tetrafluoroborate (H~inssler and Hoch, unpublished). Beaded sites of enzyme activity reported for acid phosphatase in some fungi (H~inssler et al., t975) were not seen for N-aeetylfl-D-glueosaminidase in M. racemosus. ~-Naphthyl N- acetyl- fi- D- glucosaminide as a substrate fox"its corresponding enzyme has been used in various mammalian systems (Pugh, 1973). Its use, however, has been abandoned in favor of the naphthol ASBI- and ASLC- substituted substrates because its final reaction product was considered too diffuse (Pugh, 1973; Pugh and Walker, 1961). The liberated a-naphthol
was coupled with Fast Blue B, Fast Bed TI/, Fast Red RC, and Fast Garnet GBC. Fast Garnet GBC yielded the most reliable colored reaction product but it was not particulate; eor~sequently, a histological rather than a cytological localization was achieved (Pugh and Walker, 1961). Free ~-naphthol was found to couple most efficiently and to provide nondiffusable reaction products with several diazonium salts including Fast Blue B, Fast Garnet GBC, Fast Red ITR, and Fast Violet B (Pearse, 1972). Fast Red B was not considered satisfactory. Contrary to these observations, we found Fast Red B quite satisfactory and the other salts not acceptable (Table 2). p-Nitrobenzene diazonium tetrafluoroborate had not been evaluated with a-naphthol previously. The differences noted between our results and those of other workers concerning the most satisfactory diazonium salt may be due to several factors, e.g., fixation procedure, cell system involved, and concentration of the reagents, pH, and buffer of the incubation system. Therefore, the best set of conditions and reagents must be determined for each system.
N-ACETYL-/3-D-GLUCOSAMINIDASE IN MUCOR
173
containing vacuoles in hyphal tip cells of ScIerotium rolfsii. 1. Bacteriol. 124: 997-1006. Hocri, H. C., AND FtrLLE~, M. S. 1977. Mycoparasitic relationships. I. Morphological features of interactions between Pythium acanthicum and several fungal hosts. Arch. Microbiol. II1: 207224. IT~r, W., AND MATn~, P. 1970,. Role of chitinase and other lysosomal enzymes of Coprinus lagopus and the autolysis of fruiting bodies. J. Gen Microbiol. 61: 301-309. JE~Nt~ux, C. 1963. Chitine et chitinolyse, un ACKNOWLEDGMENTS ehapitre de la biologic moldculaire. Masson, This investigation was made possible through Paris. travel fellowships from the College of Agriculture LI~'CAPPA, Y., ann LOCKWOOD, J. L. 1962. Chitin and Life Sciences of Cornell University and the media for selective isolation and culture of American Phytopathological Society, and through actinomycetes. Phytopathology 52: 317-323. support form Hatch Project No. NYG 25482 to MAXWELL, D. P., ANI) LUMSDEN, R. D. 1970. H. C. Hoch. Support is also acknowledged from Oxalic acid production by ScIerotinia scIeroDeutsche Forschungsgemeinschaft to C. H~nssler. tiorum in infected bean and in culture. Phytopathology 60: 1395-1398. itEFERENCES PEAaSE, A. G. E. 1972. Histochemistry, Theoretical and Applied, 3rd ed., Vol. 2. Churchill LivingCASSELTOX, L. A. 1978. Dikaryon formation in stone, Edinburgh. higher Basidiomycetes. In The Filamentous Fungi. Developmental Mycology (J. E. Smith P~:o~, D. 1973. Glyeosidases. N-Acetyl-~3-glucosanainidase. In Electron Microscopy of Enzymes. and D. R. Berry, Eds.), Vol. 3, pp. 275-297. Principles and Methods (M. A. Hayat, Ed.), Edward Arnold, London. Vol. 1, pp. 104-115. Van Nostrand Reinhold, FINDLAY, J., LEvvY, G. A., AND MARStt, C. A. New York. 1958. Inhibition of glycosidases by aldonolactones of correspnoding configuration. 2. Inhibi- PUCH, D., AND CAWSON, R. A. 1977. The cytotors of fl-N-aeetylglucosaminidase. Biochem. ]. chemical localization of acid hydrolases in four common fungi. Cell. Mol. Biol. 22: 125-132. 69: 467-476. GOODAY, G. W. 1978. The enzylnology of hyphal Pua~I, D., ANn W*LKEn, P. G, 1961. The localizagrowth. In The Filamentous Fungi. Develoption of N-acetyl-/3-glucosaminidase in tissues. mental Mycology (J. E. Smith and D. R. Berry, ]. Histochem. Cytochem. 9: 24"2-- 250. Eds.), Vol. 3, pp. 51-77. Edward Arnold, REYES, F., LAHOZ, R. L., AND CO~NACO, P. 1977. London. Autolysis of Neurospora crassa in different GULL, K. 1978. Form and function of septa in culture conditions and release of ~-N-aeetylfilamentous fungi. In The Filamentous Fungi. glucosaminidase and chitinase. Trans. Brit. Developmental Mycology (J. E. Smith and Mycol. Soc. 68: 357-361. D. R. Berry, Eds.), Vol. 3, pp. 78-93. Edward UNESTAM, T. 1966. Chitinolytie, cellulolytic, and Arnold, London. pectinolytic activity in vitro of some parasitic H~_NSSLER, C., MAXWELL, D. P., AND MAXWELL, and saprophytic Oomycetes. Physiol. Plant. 19: M. D. 1975. Demonstration of acid phosphatase15-30.
The procedure reported herein provides t h e i n f o r m a t i o n n e c e s s a r y for t h e l o c a l i z a t i o n of t h e h y d r o l y t i c e n z y m e , N - a c e t y l - p D - g l u c o s a m i n i d a s e , in c e r t a i n fungi. T h e localizations were small and discrete, with s h a r p outlines, T h e i n v o l v e m e n t of this e n z y m e in v a r i o u s c e l l u l a r f u n c t i o n s of f u n g i (e.g., a n a s t o m o s i s , autolysis, a n d g r o w t h ) c a n n o w b e s t u d i e d in m o r e detail.
Cytochemical Localization of N-Acetyl-~-D-glucosaminidase in Hyphae of Mucor racemosus HARVEY C. HOCH,* GERD HXNSSLER,t AND HANS-JOACHIM REISENER ~
Department of Plant Pathology, New York State Agricultural Experiment Station, Cornell University, Geneva, New York 14456, and t Institute fiir Physikalische Biologie, Technische Hochschule Aachen, Bundesrepublik Deutschland Accepted for Publication January 25, 1979; revised April 10, 1979 HOCH, H. C., H.&NSSLER, G., ANn REISENER, H-J. 1979. Cytochemical localization of N-aeetyl-~-D-glucosaminidase in hyphae of Mucor racemosus. Experimental Mycology 3, 164-173. Light microscopic localization of N-acetyl-fl-D-glucosaminidase activity by the hydrolysis of a-naphthyl N-aeetyl-fl-D-glucosaminide and by the subsequent coupling of the released a-naphthol with Fast Red B and p-nitrobenzene diazonium tetrafluoroborate was obtained in Mucor racemosus. Enzyme activity appeared as discrete spherical sites, from < 0.3 to 1 /ml in diameter, in the primary hyphae. In the secondary and tertiary branch hyphae of the older portions of the M. racemosus colony the sites were often as large as 3 ~m in diameter. The sites of enzyme activity occurred sparsely and were randomly distributed in the hyphal tips. They were most numerous in older portions of these hyphae. Hydolysis of the p-nitrophenyl N-acetyl-fl-D-glucosaminide was obtained with cell-free extracts of M. racemous, Rhizoctonia solani, Cortic!um sp., and Phycomyces nitens; however, eytochemieal localizations of enzyme activity were observed only in M. racemosus. Four cytochemical substrates and 11 coupling agents were investigated. Only a-naphthyl N-acetyl-p-D-glucosaminide was satisfactory as a substrate. Fast Red B and p-nitrobenzene diazonium tetrafluoroborate were the only coupling reagents that yielded stable discrete reaction products. INDEX DESCRIPTORS: N-acetyl-B-D-glucosaminidase; Mueor racemosus; cytochemistry.
Chitinolytie enzymes are involved in several aspects of development and degradation of filamentous fungi. They are associated with: hyphal tip elongation and hyphal branch formation (Gooday, 1978); maintenance of dikaryotie conditions through dissolution of dolipore septa and fusion of hyphal walls (Casselton, 1978; Gull, 1978; Iten and Matile, 1970; Reyes et al., 1977); degradation of chitinous debris; autolysis; and penetration of chitinous walls and exoskeletons of various hosts (Hoch and Fuller, 1977; Unestam, 1966). To be able to visualize the sites of ehitinase and chitobiase (EC 3.2.1.14), and
N-acetyl-fl-D-glucosaminidase (EC 3.2.1.30) activities would enhance our ability to study in more detail some of the above events. However, activities of these enzymes have not been cytochemically localized in fungi with any degree of reliability or resolution that would lend meaningful and accurate information about their sites of activity. N-Acetyl-fl-D-glucosaminidase activities have been localized by both light and electron microscopy in mammalian cells with several synthetic naphthol-linked substrates (Pearse, 1972; Pugh, 1973), and
164 0147-5975/79/020164-10502.00/0 Copyright © 1979 by AcademicPress, Inc. All rights of reproductionin any form reserved.
N-ACETYL-/3-D-GLUCOSAMINIDASE IN MUCOR also with a substituted indoxyl substrate (Pugh, 1973). In this paper we report on the cytochemieal localization of N-acetyl-/3-D-glucosaminidase in hyphae of Mucor racemosus using the substrate a-naphthyl N-acetyl-/3D-glucosaminide. We also present our results with three other fungi and with various other substrates and capture reagents.
165
ium: buffer:sand). The resulting triturate was centrifuged at 2500g and the supernatant collected. To 0.2 ml of the crude intracellular enzyme extract or culture broth (extracellular enzyme); 0,8 ml of 0.1 M citrate buffer, pH 5.5, containing 28/z~ pnitrophenyl N-aeetyl-/~-D-glucosaminidewas added, shaken, and allowed to incubate at 37°C for 30 rain. Next, 2.0 ml 0.5 M NaOH was added to stop the reaction and to develop the color of the liberated p-nitroMATERIALS AND METHODS phenol which was determined spectrophoThe fungi used in this investigation tometrically. In addition to p-nitrophenyl were: Corticium sp. from Dr. M. Boosalis, N-acetyl-/9-I~-glucosaminide, the four cytoUniversity of Nebraska, Lincoln; Mucor chemical substrates were also tested with racemosus Fres. f. sphaerosporus ( Hagem ) the enzyme preparations. They were used Schipper ( + s t r a i n ) , isolate CBS 115.08 at the same concentration as for cytochemfrom Centraalbureau voor Schimmelcul- istry but were postcoupled with Fast Dark tures, Baarn, Netherlands; Phycomyces Blue R (Sigma Chemical Co.) or p-nitronitens (Kunze) van Tiegh. & Le Monn., benzene diazonium tetrafluoroborate (East( + strain), isolate H 241 from Dr. R. T. man Kodak Co., Rochester, New York). In Hanlin, University of Georgia, Athens; and addition to the above treatment, purified Rhizoctonia solani Kiihn from Dr. D. P. N-acetyl-/3-I)-glueosaminidases from AsperMaxwell, University of Wisconsin, Madigillus niger and Jack beans (Sigma Chemson. All fungi were maintained on potatoical Co.) and from beef kidney (Boehringerdextrose agar at ca. 21°C. For determina- Mannheim GmbH, Bioehemica, Mannheim) tions of intracellular and extracellular Nwere used to test the substrates and subacetyl-/3-D-glucosaminidase activity, the sequent color reactions. Heat-inactivated fungi were grown in 125-mi Erlenmeyer controls were employed. flasks containing 25 ml of either sodium Since the primary purpose of this study polypectate medium ( Maxwell and was eytoehemistry, not biochemistry, of Lumsden, 1970) or 1% colloidal chitin N- acetyl-/3- D- glueosaminidase, quantative (Lingappa and Lockwood, 1962) plus values for enzyme activities for the various essential salts. They were grown at ca. fungi, based on milligrams protein, were 21°C in still culture for 8 days. For cytonot determined. Thus, optimum pH, chemistry they were grown on chitin (1%)presence or absence of enzyme activities, agar (2%)-coated microscope slides supand substrate specificity were determined ported on V-shaped glass rods placed on speetrophotometrieally for the colored rewater-moistened filter paper in petri plates. action products and were based on the wet Intracellular and extracellular enzyme weight of the myeelia. These data were activities were determined using p-nitrophenyi N-acetyl-/3-D-glucosaminide (Sigma used as a basis for the eytoehemieal inChemical Co., St. Louis, Mo.). Mycelia of vestigations. For eytochemistry, the fungi were fixed the four fungi were collected on a Buchner in situ on the slides for 10 min with 1% funnel, rinsed with 4°C 0.02 ~t citrate formaldehyde containing 10% methanol in buffer, pH 5.5, and ground with a cold mortar and pestle with washed quartz sand 0.1 ~ citrate (Na +) buffer, pH 6.5. The and citrate buffer ( 1 : 2: 4; w: v: w; rnycel- slides were then rinsed for 5 rain with 0.1
ttOCH, HANSSLER, AND REISENER
166
TABLE 1 Relative Hydrolysis of Synthetic Substrates by Intracellular N-Acetyl-fi-Dglucosaminidases from Various Fungi~ Enzyme source
Purified, commercial Rhizoctonia solani Corticium sp. Mucor racernosus Phycomyces nitens
Control°
N-Acetyl-~-D-glyeosaminidesubstrate p-Nitrophenyl
a-Naphthyl
q- + + +
÷ q- q- +
q_ ( q_ ) b
.
.
5-Bromoindoxyl
Naphthol ASBI-
Naphthol ASCL-
q- q- q- q-
q- q- q- q-
q- q- + +
.
+ (+)
.
.
.
+ + + (-) q- ( - )
+ + + .
.
+ + + .
- (-)
.
.
.
.
. --
--
.
.
a q_, -t- -t-, q- q- q-, q- -t- -t- -b represent increasing enzyme activities; --indicates no enzyme activity. bValues in parentheses represent data for extraeellular enzyme activity. Heat-inactivated extract of M . racemosus. citrate (Na ÷) buffer, pH 6.5, followed by additional rinse for 10 min with 0.1 citrate (Na ÷) buffer, pH 5.5 The slide cultures were next flooded with the appropriate enzyme substrate-diazonium salt coupling solution. They were incubated at 21°C for 5 to 30 min, after which time a cover glass was gently positioned over the hyphae. The material was immediately examined using oil immersion 100× objectives and photographed. Various combinations of the following enzyme substrates and diazonium salts were tested: a-naphthyl N-aeetyl-fi-D-glueosaminide (Serva Feinbiochemica, Heidelberg ); napthol ASBI- and naphthol ASLC-N-acetyl-B-Dglucosaminide (Sigma Chemical Co. ); and 5-bromoindoxyl N-aeetyl-/?-D-glucosaminide (Aldrich Chemical Co., Milwaukee, Wis.); Fast Red B; Fast Red ITR; Fast Red Violet LB; Fast Blue B; Fast Blue tlR; Fast Blue BBN; Fast Dark Blue R; Fast Violet B; Fast Garnet GBC; hexazotized pararosaniline (all above from Sigma Chemical Co.); potassium ferricyanide (for the 5-bromoindoxyl derivative); and p-nitrobenzene diazonium tetrafluoroborate. All substrates were first dissolved in ethylene glycol monomethyl ether (15 mg/0.4 ml) to which was added 10 ml of 0.1 ~ citrate buffer,
pH 5.5. The diazonium salts were added to this mixture at various rates, but generally at 0.05 ml (50 mg dissolved in 10 ml distilled water) per milliliter of substrate solution. The solution was filtered after 5 min and used immediately. Cytochemical controls consisted of (i) heat-inactivated enzyme (100°C for 15 min following fixation), (ii) incubation in solutions free of the synthetic enzyme substrate, and (iii) incubation in complete substrate-diazonium salt media containing 10 t'~ N-acetylglueosaminonolactone, a specific competitive inhibitor of N-aeetyl-fl-D-glucosaminidase activity. The laetone was prepared according to procedure (a) as reported by Findlay e t al. ( 1 9 5 8 ) except that the starting substrate was glueosaminic acid (Sigma Chemical Co. ) rather than glucosamine. RESULTS Optimum pH values for intracellular Nacetyl-fl-D-glueosaminidase activities as determined with p-nitrophenyl N-acetyl-flD-glucosaminide were 5.4 for both C o r t i c i u m sp. and R. s o I a n i , and 4.8 and 5.0 for M . r a c e m o s u s and P. n i t e n s , respectively. Optimum pH values were not determined for extraeellular enzyme activities.
N-ACETYL-#-~-GLUCOSAMINIDASE IN MUCOR
167
FIGs. 1 and 2. Mucor racemosus colony on colloidal chitin agar. The colloidal chitin substrate became transparent when hydrolyzed by chitinolytic enzymes from M. racemosus (Fig. 1). The cleared zone (darker area) was first noticeable 0.5 to 1 cm from the advancirrg colony margin (dashed line). The same area as in Fig. 1, but developed for N-acetyl-~-D-glucosaminidase activity with a-naphthyl N-aeetyl-/3-D-glucosarninideand p-nitrobenzene diazonium tetrafluoroborate, is shown in Fig. 2. Based on mycelial wet weight, M. racemosus had considerably more intraeellular N-
period. N-AcetyI-fl-D-glueosaminidase activity was apparent in the hyphae of M. acetyl-fi-D-glucosaminidase activity than raceraosus and not in the medium ( Fig. 2 ). did the other fungi (Table 1). Extracellular Colloidal chitin was not hydrolyzed by enzyme activity was found only in broths tl. solani or P. niten~ within 120 h, the from R. solani and C o r t i c i u m sp. cultures. longest growth period observed. The cytoplasm in the first 100 ~m of the Intracellular N-acetyl-fl-D-glucosaminidase preparations fi'om all fungi tested hydro- hyphal tip of M. r a c e m o s u s is dense with lyzed the p-nitrophenol-substituted sub- numerous organelles generally <1 ~m in strate; however, only M. r a c e m o s u s prep- diameter (Fig. 3). The density of the cytoarations hydrolyzed the ~-napthol- and 5- plasm in this region makes it difficult to bromoindoxyl-substituted substrates. The distinguish the various organelles. Large naphthol ASBI- and naphthol ASLC-N- vacuoles constitute most of the cellular aeetyl-fl-D-glucosaminide substrates were contents in older portions of the fungal not hydrolyzed by any of the intracellular colony (Fig. 4). These vacuoles are first enzyme preparations. All substrates were seen 90 to 120 ~am from the hyphal apex hydrolyzed by the three commercially puri- (Figs. 5 and 6). fied enzymes. Cytoehemical localization of N-acetyl-BColloidal chitin, in an agar medium, was D-glueosaminidase activity was achieved completely hydrolyzed by M. r a c e m o s u s only in M . r a c e m o s u s (Figs. 5-12). Hy(Figs. 1 and 2) and C o r t i c i u m sp. The drolysis of the cytochemical substrates was chitin agar medium was cleared, indicative not observed in the other test organisms. of hydrolysis, following 48 h growth (Fig. Only two of the cytochemieal substrates, 1). Chitin in the first 0.5 to 1 em of the ,-naphthyl N-acetyl-/3-D-glucosaminide 'and advancing colony margins was not cleared, 5-bromoindoxyl N-acetyl-fl-D-glucosaminide, but was cleared within the next 24-h were hydrolyzed to yield colored reaction
168
HOCH, HNNSSLEtt, AND tlEISENEtt
N-ACETYL-/3-D-GLUCOSAMINIDASE IN products when reacted with the appropriate capture reagent. The data summarized in Table 2 indicate that only two of the diazonium salts proved satisfactory as coupling reagents for the ~-napthol moiety released from the glueosamide substrate. The 5-bromoindoxyl derivative was not a satisfactory substrate in these tests since the colored reaction product was always diffuse. Potassium ferricyanide, the recommended oxidizing reagent for the 5-bromoindoxyl moiety (Pughl 1973), did not work in these studies (except with the commercially purified enzymes). Red to reddish-brown reaction products of ~-napthol and p-nitrobenzene diazonium tetrafluoroborate or Fast Red B salt were observed as discrete particles in M . r a c e m o s u s . The particles, presumably membrane-bound vesicles, varied from <0'.3 to 1 /~m in diameter. They were distributed sparsely throughout the cytoplasm in the first 200 to 400 ~m of the hyphal tip (Figs. 5, 6, and 11). A very few (one to five) sites of activity could often be detected in the apical 50 ~m. The distribution and number of sites of activity illustrated in Fig. 1i were representative for most hyphal tips, while that in Fig. 5 was considered exceptionally high. At a distanee of 300 to 500 ~m from the hyphal apex the concentration of the sites of activity gradually increased to the concentration depicted in Figs. 7 to 9 and 12. The sites of enzyme activity were distinctly separate from the large vacuoles shown in Fig. 4 (Figs. 6, 8, and 9) and for the most part were distributed peripherally around the
MUCOB
169
hyphal axis. The eentral portion of the hyphal axis was comprised mostly of larger vacuoles and thus most of the remaining cytoplasm, including the sites of enzyme localization, were excluded from this central zone. This feature was particularly noticeable when the plane of focus was moved from just below the hyphal surface (Fig. 8) to a longitudinally median plane of focus (Fig. 9). The distribution and size of sites of enzyme activity were different in the secondary and tertiary branch hyphae of older portions of the M . r a c e m o s u s colony than in the larger primary hyphae. These sites varied from <0.3 to >3 tzm in diameter and often were delimited by the diameter of these hyphae (Fig. 10). When diffusion of the reaction product occurred, these were the sites at which diffusion was first detected. No sites of enzyme localization could be detected in hyphae incubated in solutions minus the substrate (Figs. 13 and 14), in heat-treated hyphae incubated in complete media, or in hyphae incubated in complete media containing N-acetylglueosaminonolaetone. The small dark particles in Fig. 14 are not cytoehemical sites of enzyme localization, but instead are vesicles exhibiting a natural refractive index different from the ground cytoplasm. No differences were detected between the sites of enzyme activities localized with p-nitrobenzene diazonium tetrafluoroborate and Fast Red B (Figs. 5-9 vs 11 and 12). The red reaction product of Fast Red B, however, began to diffuse after 60 min,
Fits. 3 and 4. Nomarski interference-contrast light mierographs of formaldehyde-fixed hyphae of Mucor raceraosus. Figure 3, of the hyphal tip region, illustrates the dense nature of the cytoplasm containing small vesicles and very few large vacuoles. In older regions of the layphae, large vacuoles constitute most of the hyphal contents (Fig. 4). The magnification of these and all subsequent mierographs is X4200. Fins. 5-9. Bright field light micrographs of Mucor racemosus incubated in a medium containing a-naphthyl N-aeetyl-~-o-glucosaminide and p-nitrobenzene diazonium tetrafluoroborate to demonstrate N-acetyl-/~-D-glueosaminidase activity. Sites :of enzyme activity localization appear as discrete spherical deposits, from <0.3 ~zm to 1 #m in diameter. Distribution of these sites in the first 50 mn is sparse (Figs. 5 and 6). Dashed lines in Figs. 5 and 6 represent the same site in this hypha. Localization sites are concentrated 300 to 500 /tin and farther back from the hyphal tip (Fig. 7). Two planes of focus through the same hypha 1 cm from the byphal tip (Figs. 8 and 9).
170
HOCH, H/tiNSSLER, AND ttEISENER
N-ACETYL.fl-D-GLUCOSAMINIDASE IN MUCOR
171
DISCUSS,ION whereas the reaction product formed with p-nitrobenzene diazonium tetrafluoroborate The N-acetyl-fl-D-glueosaminidases proremained stable for more than 3 h. duced by the four fungi are apparently There were no detectable differences in substrate specific. While all fungi tested the sites of enzyme activity localization in hydrolyzed p-nitrophenyl N-acetyl-fl-D-gluhyphae of M. racemosus grown on chitin cosaminide, only M. racemosus could hyagar vs sodium polypeetate agar. drolyze any of the other synthetic subThe background color in the agar substrates. Since the commercially purified enstrate of both diazonium salts at the concenzyme preparations hydrolyzed all five subtration used in the incubation media was strates, it was unlikely that inhibition, due negligible. In the hyphae, a very slight to the diazonium salt, contributed to the yellow background color was noted but inability of the fungal enzyme preparations did not interfere with the detection of the to hydrolyze the substrates. Enzyme aceytochemieal reaction product color. Treatment of the hyphae influenced the tivators must be contemplated as one reason that the cytochemieal substrates time of the cytochemieal reaction product were not hydrolyzed by the fungal prepformation as well as product diffusion. Unfixed hyphae were compared with formal- arations; however, evidence from other chitinolytic systems indicates activators dehyde- and glutaraldehyde-fixed hyphae. Hyphae fixed with glutaraldehyde (1% in not required for these enzymes (Jeuniaux, 0.1 ~ citrate buffer, pH 6.5, followed by a 1963). N-Acetyl-fl-D-glucosaminidase has recitrate buffer, pH 5.5, rinse) developed a portedly been localized cytoehemieally in red-colored reaction product with both M. hiemalis Wehmer, Aspergillus niger van diazonium salts very rapidly (within 1 rain Tieghem, Saccharomyces cerevisiae Meyen, at 91°C). The color development was enand Can dida aIbieans ( Itobin ) Berkhout tirely diffuse, with particulate sites of using 5-bromoindoxyl N-acetyl-fi-e-glucoslocalization developing after 10 rain. These sites, however, also become diffuse. Un- aminide as the substrate (Pugh and Cawfixed hyphae also rapidly developed a red son, 1977). Unfortunately, the evidence reaction product, generally with particulate presented for the localization of this ensites of localization. These sites likewise zyme by these authors was not adequately rapidly diffused, within 10 rain. The clear- illustrated or discussed; thus little informaest sites of enzyme localization developed tion could be gleaned from their report. in hyphae fixed with formaldehyde, as They indicated that the enzyme content was "greatest at the growiag tips" in exemplified in this paper. FIG. 10. Secondary and tertiary branch hyphae in the old portion of a Mucor racemosus colony. The sites of N-aeetyl-fl-D-glucosaminidaseactivity resulting from hydrolysis of c~naphthyl N-acetyl-C~-D-glueosaminide and subsequent capture of a-naphthol with p-nitrobenzene diazonium tetrafluoroborateare often 3 ~ln in diameter and delimited by the diameter of the hyphae. FIGs. 11 and 12. Hyphae of Mucor racemosus incubated in a medium containing a-naphthyl N-aeetyl-fl-D-glueosaminide and Fast Red B. Very few sites of enzyme activity localization are seen in the hyphal tip (Fig. 11). In ,older regions of the hypbae, the sites are more numerous and larger (Fig. 12). FIGS. 13 and 14. Hyphae of Mucor racemosus incubated in a medium containing p-nitrobenzene diazonium tetrafluoroboratebut not the enzyme substrate. No sites of enzyme activity developed. Fig. 13. Hyphal tip region. Fig. 14. Hyphal region comparable to Figs. 4, 7 to 9, and 11.
172
HOCH, H}iNSSLER, AND ItEISENER
TABLE 2 Performance of Various Capture Reagents for 5-Bromoindoxyl and a-Naphthol Moieties Hydrolyzcd from N-Acetyl-B-D-glucosaminidein situ in Mucor racemosus Enzyme substrate
Capture reagents
5-BromoindoxylN-aeetylB-D-glucosaminide Fast Red B Fast Red Violet LB Fast Blue B p-Nitrobenzene diazonium tetrafluoroborate Hexatized pararosiline Fast Garnet GBC Fast Red ITR Fast Violet B Fast Blue RR Fast Blue BBN K + Ferricyanide
Not particulate, diffuse Not particulate, diffuse Some particulate, but diffuse Not particulate, very diffuse Not particulate, very diffuse
~-Naphthy[ N-acetyl-C~-Dglucosaminise Particulate, excellent Not particulate, diffuse Not particulate, very diffuse Particulate, excellent Not particulate, very diffuse Some particulate, but diffuse Not particulate, diffuse Particulate, but diffuse Particulate, but diffuse Particulate, but diffuse
No reaction
M u c o r and AspergiUus. Based on our results with M. racemosus, N-acetyl-fl-vglueosaminidase activity was greatest in older portions of the hyphae, at least 300 to 500 ~m from the tip. Such distribution of enzyme activity also corresponded with the earliest indication of clearing of the colloidal chitin afar. Distribution of N-acetyl-fi-v-glueosaminidase sites of activity in M. r a c e m o s u s was similar to that observed for acid phosphatase activity detected with 5-benzoyI-2naphthyl phosphate (Na +) and coupled with p-nitrobenzene diazonium tetrafluoroborate (H~inssler and Hoch, unpublished). Beaded sites of enzyme activity reported for acid phosphatase in some fungi (H~inssler et al., t975) were not seen for N-aeetylfl-D-glueosaminidase in M. racemosus. ~-Naphthyl N- acetyl- fi- D- glucosaminide as a substrate fox"its corresponding enzyme has been used in various mammalian systems (Pugh, 1973). Its use, however, has been abandoned in favor of the naphthol ASBI- and ASLC- substituted substrates because its final reaction product was considered too diffuse (Pugh, 1973; Pugh and Walker, 1961). The liberated a-naphthol
was coupled with Fast Blue B, Fast Bed TI/, Fast Red RC, and Fast Garnet GBC. Fast Garnet GBC yielded the most reliable colored reaction product but it was not particulate; eor~sequently, a histological rather than a cytological localization was achieved (Pugh and Walker, 1961). Free ~-naphthol was found to couple most efficiently and to provide nondiffusable reaction products with several diazonium salts including Fast Blue B, Fast Garnet GBC, Fast Red ITR, and Fast Violet B (Pearse, 1972). Fast Red B was not considered satisfactory. Contrary to these observations, we found Fast Red B quite satisfactory and the other salts not acceptable (Table 2). p-Nitrobenzene diazonium tetrafluoroborate had not been evaluated with a-naphthol previously. The differences noted between our results and those of other workers concerning the most satisfactory diazonium salt may be due to several factors, e.g., fixation procedure, cell system involved, and concentration of the reagents, pH, and buffer of the incubation system. Therefore, the best set of conditions and reagents must be determined for each system.
N-ACETYL-/3-D-GLUCOSAMINIDASE IN MUCOR
173
containing vacuoles in hyphal tip cells of ScIerotium rolfsii. 1. Bacteriol. 124: 997-1006. Hocri, H. C., AND FtrLLE~, M. S. 1977. Mycoparasitic relationships. I. Morphological features of interactions between Pythium acanthicum and several fungal hosts. Arch. Microbiol. II1: 207224. IT~r, W., AND MATn~, P. 1970,. Role of chitinase and other lysosomal enzymes of Coprinus lagopus and the autolysis of fruiting bodies. J. Gen Microbiol. 61: 301-309. JE~Nt~ux, C. 1963. Chitine et chitinolyse, un ACKNOWLEDGMENTS ehapitre de la biologic moldculaire. Masson, This investigation was made possible through Paris. travel fellowships from the College of Agriculture LI~'CAPPA, Y., ann LOCKWOOD, J. L. 1962. Chitin and Life Sciences of Cornell University and the media for selective isolation and culture of American Phytopathological Society, and through actinomycetes. Phytopathology 52: 317-323. support form Hatch Project No. NYG 25482 to MAXWELL, D. P., ANI) LUMSDEN, R. D. 1970. H. C. Hoch. Support is also acknowledged from Oxalic acid production by ScIerotinia scIeroDeutsche Forschungsgemeinschaft to C. H~nssler. tiorum in infected bean and in culture. Phytopathology 60: 1395-1398. itEFERENCES PEAaSE, A. G. E. 1972. Histochemistry, Theoretical and Applied, 3rd ed., Vol. 2. Churchill LivingCASSELTOX, L. A. 1978. Dikaryon formation in stone, Edinburgh. higher Basidiomycetes. In The Filamentous Fungi. Developmental Mycology (J. E. Smith P~:o~, D. 1973. Glyeosidases. N-Acetyl-~3-glucosanainidase. In Electron Microscopy of Enzymes. and D. R. Berry, Eds.), Vol. 3, pp. 275-297. Principles and Methods (M. A. Hayat, Ed.), Edward Arnold, London. Vol. 1, pp. 104-115. Van Nostrand Reinhold, FINDLAY, J., LEvvY, G. A., AND MARStt, C. A. New York. 1958. Inhibition of glycosidases by aldonolactones of correspnoding configuration. 2. Inhibi- PUCH, D., AND CAWSON, R. A. 1977. The cytotors of fl-N-aeetylglucosaminidase. Biochem. ]. chemical localization of acid hydrolases in four common fungi. Cell. Mol. Biol. 22: 125-132. 69: 467-476. GOODAY, G. W. 1978. The enzylnology of hyphal Pua~I, D., ANn W*LKEn, P. G, 1961. The localizagrowth. In The Filamentous Fungi. Develoption of N-acetyl-/3-glucosaminidase in tissues. mental Mycology (J. E. Smith and D. R. Berry, ]. Histochem. Cytochem. 9: 24"2-- 250. Eds.), Vol. 3, pp. 51-77. Edward Arnold, REYES, F., LAHOZ, R. L., AND CO~NACO, P. 1977. London. Autolysis of Neurospora crassa in different GULL, K. 1978. Form and function of septa in culture conditions and release of ~-N-aeetylfilamentous fungi. In The Filamentous Fungi. glucosaminidase and chitinase. Trans. Brit. Developmental Mycology (J. E. Smith and Mycol. Soc. 68: 357-361. D. R. Berry, Eds.), Vol. 3, pp. 78-93. Edward UNESTAM, T. 1966. Chitinolytie, cellulolytic, and Arnold, London. pectinolytic activity in vitro of some parasitic H~_NSSLER, C., MAXWELL, D. P., AND MAXWELL, and saprophytic Oomycetes. Physiol. Plant. 19: M. D. 1975. Demonstration of acid phosphatase15-30.
The procedure reported herein provides t h e i n f o r m a t i o n n e c e s s a r y for t h e l o c a l i z a t i o n of t h e h y d r o l y t i c e n z y m e , N - a c e t y l - p D - g l u c o s a m i n i d a s e , in c e r t a i n fungi. T h e localizations were small and discrete, with s h a r p outlines, T h e i n v o l v e m e n t of this e n z y m e in v a r i o u s c e l l u l a r f u n c t i o n s of f u n g i (e.g., a n a s t o m o s i s , autolysis, a n d g r o w t h ) c a n n o w b e s t u d i e d in m o r e detail.