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The cell wall of Helminthosporium sativum
ARCHIVES
OF
BIOCHEMISTRY
The
AND
Cell
BIOPHYSICS
Wall
134,
Of
285-289
Helminthosporiom
D. A. APPLEGARTH The
University
of
British
Columbia,
Received
28,
1969;
sativum’
AND G. BOZOIAN
Department
March
(1969)
of Paediatrics, accepted
Vancouver
August
9, British
Columbia
11, 1969
The chemical composition of thecell wall of Helminthosporium sativum is described. The wall contains equal amounts of glucosamine and galactosamine. The galactosamine polysaccharide can be separated from the glucosamine by extraction of the wall with hot alkali. The glucosamine polysaccharide, which is insoluble both in cold concentrated acid and hot alkali is digested by a commercial chitinase preparation. The results suggest t,hat two hexosamine polymers are present in the cell wall of this microorganism.
Air-borne molds have been implicated in the development of mold-sensitive asthma in some individuals (1,2). Examination of the components of cell walls of some common air-borne fungi is one of the prerequisites for more detailed work on the allergenicity of these fungi. The present communication describes the chemical analysis of the hyphal wall of Helminthosporium sativum, a commonly occurring member of the air-borne mold population of the Pacific North Western area of the United States and Canada.
taminat,ion when viewed by electron microscopy. Digestion of the cell walls with 0.170 trypsin solution at 37” did not modify the percentage of protein found in the wall nor did it modify the appearance of the wall under the electron microscope. By these criteria the wall preparation was felt to be relatively free of cytoplasmic contaminat.ion. Electron photomicrographs of the cell-wall preparation used for analyses have already been published (5).
CHEMICAL ANALYSES Most of the analytical techniques used were identical to methods used previously (4). Only t,he salient differences will be discussed below. Solvent systems used for paper chromatography of neutral monosaccharides and amino sugars were as follows. Ratios quoted are volume/volume. Solvent System A = n-butanol-acetic acid-water........ .(12:3:5) Solvent System B = n-butanol-pyridine-water. @:4:3) Solvent System C = ethyl acetatepyridine-water. (12:5:4) Solvent System D = n-butanol-etha(4:l:l) nol-water,
METHODS
PREPARATION OF CELL WALLS Helminthosporium sativum was obtained as a commercial culture (Hollister-Stier Laboratories, 1075 Division Street, Spokane, Washington). It was grown with continuous stirring in 1 liter Fernbach flasks containing 250 ml of a fully synthetic medium (3) previously recommended for the growth of molds used in antigen testing in patients with mold-sensitive asthma (3). The cultures were allowed to grow for 7-12 days and the mycelial pads harvested in a Buchner funnel. Cell walls were prepared according to previously described methods (4, 5). The cell-wall preparations showed no evidence of microsomal con-
NEUTRAL MONOSACCHARIDES After hydrolysis and assay by the previously described methods the percentage of galactose was confirmed with a galactose-oxidase method (6). Glucose was confirmed by the glucose-oxidase method (6) modified as described (4). Since, in both cases, all of the sugar liberated by the hy-
1 The work was made possible by the award of a Medical Research Council of Canada Term Grant. 285
286
APPLEGARTH TABLE
PERCENTAGE
BOZOIAN
I
COMPOSITION OF Helminthosporium
TABLE
OF THE CELL sativum
Ash Glucose Galactose Mannose Phosphate (as HzP03) Glucosamine Galactosamine Kjeldahl nitrogen Protein (by calculation) Readily extractable lipid Bound lipid
WALL
5.2 37.0 2.0 2.0 1.8 8.6 8.3 3.8 18.0 2.0 9.4
drolysis reacted with the specific enzymes, we may conclude that both glucose and galactose were of the n-configuration. The configuration of the mannose was not checked. Results are presented in Table I. AMINO
AND
SUGARS
The hydrolysis conditions used for the analysis and identification of the amino sugars present in the wall were as before (4). The identity of the amino sugars was established as follows. The amino sugars liberated by acid hydrolysis of the cell walls were chromatographed on Whatman 3 MM paper developed in solvent system A. Areas corresponding to the amino sugars were eluted with water and the resulting compounds cochromatographed with authentic samples of glucosamine, galactosamine, and mannosamine in solvent systems A, B, and C. In all solvent systems only glucosamine and galactosamine could be detected. The amino sugars were oxidized with ninhydrin (7) and the resulting pentoses cochromatographed with standard solutions of arabinose, xylose, lyxose, and ribose in solvent system D for periods of up to 48 hr. When the chromatograms were developed with solvent system D for 24 hr, dried, and developed for another 24 hr all four pentoses could be separated. Only arabinose and lyxose could be detected in the products from the ninhydrin oxidation of the cell-wall amino sugars. Finally the amino sugar mixture from the cell wall was N-acetylated (8) and the resulting products cochromatographed with authentic samples of N-acetyl glucosamine, N-acetyl galactosamine, and N-acetyl mannosamine in solvent systems A and B. In solvent system A, N-acetyl glucosamine and N-acetyl galactosamine did not separate but the mixture of these sugars separated easily from N-acetyl mannosamine. In solvent system B all three sugars were separable. Only N-acetyl glucosamine and N-acetyl galactosamine were de-
AMINO
II
ACID COMPOSITION Helminthosporium
Amino Acid
OF WALLS sativum
pMole/mg
Lysine Histidine Arginine Cysteic Aspartic Threonine Serine Glutamic Proline Glycine Alanine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine
0.05 0.0075 0.0224 0.015 0.107 0.0775 0.0975 0.1215 0.0975 0.095 0.1225 0.0825 0.0075 0.045 0.08 0.0325 0.0375
wall
OF
70 of Total
4.6 0.7 2.0 1.4 9.9 7.1 9.0 10.3 9.0 8.9 11.2 7.6 0.7 4.1 7.5 3.0 2.0
tected, and no N-acetyl mannosamine was present. The above data show that the only amino sugars present in the cell wall of Helminthosporium sativum were glucosamine and galactosamine. The quantitative amount of each of these amino sugars was established in two separate ways. After prior purification of amino sugars by elution from Dowex-50 (H+) form with 0.3 N HCl the calorimetric technique of Ludoweig and Benaman (9) was used. This method was found to be the most successful and certainly the easiest method to determine the percentages of glucosamine and galactosamine. Identical answers were obtained using the Beckman Spinco amino acid analyzer after a hydrolysis time of 16 hr at 100” in 6 N HCl. AMINO
ACIDS
Samples of cell wall were hydrolyzed in 6 N hydrochloric acid at 100” for 16, 40, and 72 hr. The amino acids were analyzed with a Beckman 120C Spinco amino acid analyzer. The authors are grateful to Dr. Gordon Dixon and Miss Dorothy Kaufman for these analyses. The values obtained are quoted in Table II and are the maximum figures obtained for each amino acid at the three hydrolysis times. In practice there was no more than 10% variation between the values obtained at each hydrolysis time for any amino acid. LIPIDS Readily extracted lipids and bound lipids were assayed according to the procedures of BartnickiGarcia and Nickerson (10).
THE
CELL
WALL
OF HELMINTHOSPORIUM
Estimation of Protein Content An estimate of the amount of protein in the wall was obtained using the Kjeldahl gen value and subtracting the contribution nitrogen content by the amino sugars. PURINES
AND
present nitroto this
PYRIMIDINES
Ultraviolet-absorption spectra of 4 N HCl hydrolyzates of the cell walls indicated the presence of traces of purine and pyrimidine bases (approximately 0.4%). This is of the same order of magnitude BS that found by Bartnicki-Garcia and Nickerson (10) in cell walls of Mucor rouxii. A molar extinction coefficient of 34,800 for an equimolar mixture of adenine, guanine, cytosine, and uracil (total molecular weight 509) was used for the calculation. The ultraviolet-absorption spectrum of the acid hydrolyzate of the cell walls was identical to that from walls of Mucor rouzii reported by Bartnicki-Garcia and Nickerson. WORK ON THE POLYMERS OF
hrmo THE
SUGAR WALL
Two chemical tests and one enzymic test, for presence of chitin were performed. Ten milligrams of wall was extracted with 1 N acetic acid (5 ml) at 100” for 20 min. The acetic acid extract was removed by centrifugation and tested for the presence of chitosan by the addition of Gram’s iodine solution. No color developed, so presumably chitosan is absent from these cell walls. The acid-extracted residue was heated with 24% potassium hydroxide at 160” for 6 hr, then acidified with concentrated sulfuric acid (about 0.5 ml.) and Gram’s iodine solution added dropwise. No red-violet color was seen, whereas in a sample of Peniciullium pat&urn (11) used as a control the red-violet color of chitosan was easily visible. In summary, the preceding reactions did not indicate the presence of chitin. The cell wall (100 mg) was extracted with concentrated hydrochloric acid (10 ml) at 4” for 10 min. The mixture was filtered through glass wool into cold 50yo alcohol (50 ml). Neither a precipitate nor an opalescence was visible. The hydrochloric acid-treated wall was washed well with water and examined by phase-contrast microscopy. The morphology of the wall appeared to be completely unaffected by this acid treatment. A control experiment performed on cell walls of Penicillium patulum (11) yielded a visible precipitate of chitin. In the case of Penicillium patulum the extracted wall residue was rendered so amorphous by the treatment with cold hydrochloric acid that it could not be washed free of glass wool to be examined microscopically. By the two methods de-
the
SATIWM
scribed no chitin was detected in the wall of Helminthosporium sativum. The wall preparation (15 mg) was digested with chitinase (6) in 0.02 M acetate buffer (15 ml) pH 5.5, at 25” for 96 hr. After this treatment the residual cell wall was hydrolyzed with 4 N hydrochloric acid at 100” for 24 hr and the percentage of glucosamine and galactosamine assayed. This material contained 4.9% glucosamine and 9.4% galactosamine. This value for galactosamine was not significantly different from the value found before treatment with chitinase. However, some 40-50yo of the glucosamine in the wall had been removed, so presumably some 40-5Oojo of the glucosamine polymer in the wall was solubilized by chitinase. Because of the discrepancy in the results of simple chemical tests for chitin and the susceptibility of the wall to chitinase digestion, a further sample of wall (10 mg) was stirred with a chitinase preparation at 25” for 72 hr. The resulting suspension was centrifuged and the supernatant fraction assayed in a modified Morgan-Elson reaction (12) using standards of N-acetyl glucosamine prepared in the same buffer. The equivalent of 0.25 mg N-acetyl hexosamine (measured as N-acetyl glucosamine) was released from the lo-mg sample of cell wall. An aliquot of the supernatant fluid from the chitinase digestion was desalted with a mixture of Dowex-50 x 2 (H+ form) and Duolite-A 4 (OHForm), then deproteinized by shaking with several aliquots of a mixture of chloroform-t-amyl alcohol (5:l). The resulting aqueous solution was made 4 molar with respect to hydrochloric acid and the acid solution heated at 100” for 24 hr. After drying down in the usual manner the mixture was chromatographed in solvent system C. Only glucosamine was visible. These results confirmed that the cell wall of Helminthosporium sativum was digested by a chitinase preparation to yield a product which on hydrolysis liberates glucosamine as it,s only amino sugar. The galactosamine polymer in the wall was found to be capable of dissolution in hot 10% sodium hydroxide. A sample of the cell wall (100 mg) was extracted with 1 N potassium hydroxide (10.0 ml) at 6” for 2 hr. The wall suspension was recovered by filtration, washed with water, and resuspended in 10% sodium hydroxide. This suspension was heated at 100” for 30 min, centrifuged, and the supernatant fraction decanted into a fivefold excess of ethanol containing 5% (vol/vol) acetic acid. The ethanolic suspension was stored at 4” for 16 hr and the solid precipitate collected, dried over phosphorus pentoxide in vacua, then triturated with water. The aqueous supernatant portion was collected and dried over phosphorus pentoxide. The residual (water-insoluble) solid
288
APPLEGARTH
AND
BOZOIAN
linkages. The fact that no galactosamine could be found in acid hydrolyzates of the material solubilized by chitinase provides no evidence for areas of a mixed polymer in I 4 1 which there are glucosamine to galactosExtract (4.0,~~) Residue precipitated with Extracted with 10% NoOH amine links separated from one another by 5% aceticocid 100’ C, 30 minutes areas containing the chitinase-susceptible in EtOH I P-1-4 N-acetyl glucosaminyl linkages. In summary, the evidence presented indicates that two separate hexosamine polymers are Water soluble Wo,er nnroluble Residue (52.4mg) Extract ,4.2mg) Extmct(l I .4mg, Hydrolysis yields present in this cell wall. Hydrolysis yields Hydrolysis yields Glucoromlne 19.096 It will be noted that the iodine reaction Goloctoromine Galoctoramine Goloctoramine 1.0% used to prove the presence of chitin in fungi was negative. The other commonly used FIG. 1. Extraction of cell walls by alkali. method of proving the presence of chitin in a cell wall, that of extracting the chitin into was similarly dried. The wall residue, the watersoluble extract, and the water-insoluble extract cold concentrated hydrochloric acid, prewere all weighed, hydrolyzed in 4 N HCl at 100’ cipitating it by neutralization, or by addifor 24 hr and assayed for total hexosamine and tion of alcohol, and then checking the X-ray galactosamine. The results of these assays are diffraction pattern of the product (11) was shown diagrammatically in Fig. 1. The initial cold not available to us because we were unable potassium hydroxide extract was precipitated to extract the glucosamine polymer from the with absolute ethyl alcohol containing 5% (vol/ wall by using cold concentrated hydrovol) acetic acid but no attempt was made to idenchloric acid. The X-ray diffraction of the tify the components of this extract. Previous work whole wall would probably by meaningless (4) indicated that this extract would contain because of possible interference by the neutral glycans. galactosamine polymer. Until the discrepRESULTS AND DISCUSSION ancy between cbitinase digestion and both The cell wall of Helminthosporium sa- the calorimetric and extraction tests for the t&urn resembles cell walls of fungi pre- presenceof chitin can be further clarified the viously studied (4, 13) in being composed identification of chitin in fungi solely by methmainly of a glucose polysaccharide, poly- calorimetric or by acid-extractibility meric hexosamine, and some protein. The ods should be interpreted with caution. It has been obvious in the past that the most interesting finding is that both glucosamine and galactosamine are found in this calorimetric test for chitin was not so reliacell wall. Galactosamine has been reported ble as methods relying on X-ray powder previously in various fungi as a component diagrams of cold, concentrated acid extracts of whole mycelia (14) and of cell walls (13). of cell walls, but we appear now to have cast Glucosamine is a common component of some doubts on the validity of the acidestimation methods. Presumably a positive many fungal cell walls as the polymer chitin (13). From the fact that only a galactos- test will be meaningful but a negative test amine polymer can be extracted from the should be checked by an examination of acid hydrolyzates of cell walls to prove the abwalls by hot alkali and from the quantitative recovery of glucosamine from the re- sence of glucosamine in the wall. sidual cell wall after this extraction (see REFERENCES Fig. 1.) it is probable that the two amino 1. SOLOMON, W. R. Aero allergens in “A Manual sugars are present in separate molecules. of Clinical Allergy” (J. M. Sheldon, R. G. Furthermore, experiments presented here Lovell, and K. P. Mathews, eds.). Saunders, provide no evidence for the presence of any Philadelphia (1967). glucosamine to galactosamine links. The 2. PRINCE, H. E., ET AL. Ann. Allergy 19, 259 susceptibility of the glucosamine in the wall (1961). to digestion by chitinase suggeststhe exist3. S~HAFFER, N., MOLOMUT, N., AND CENTRE, ence of several p-1-4 N-acetyl glucosaminyl J. G., Ann. Allergy 17,380-384 (1959). CELL
WALL
(IOOmg)
Extraction with I N KOH 4’C. 2 hours under Nitrogen
I
1
1
THE
CELL
WALL
OF
HELMINTHOSPORIUM
4. APPLEGARTH, D. A., Arch. Biochem. Biophys. la0, 471-478 (1967). 5. APPLEGARTH, D. A., BOZOIAN, G., AND ZINTEL, B., Can. J. Microbial. 16, 312 (1969). 6. Worthington Biochemical Corporation, Freehold, New Jersey. 7. STOFFYN, I?. J., AND JEANLOZ, R. W., Arch. Biochem. Biophys. 62, 373 (1954). 8. CRUMPTON, M. J., Biochem. J. 73, 479 (1959). 9. LUDOWEIG, J., AND BENMBMAN, J. D., Anal. Biochem. 19, 80-88 (1967).
SATIVUM
289
10. BARTNICKI-GARCIA, S., AND NICKERSON, W. J., Biochim. Biophys. Acta68,102-119 (1962). 11. APPLEGARTH, D. A., AND BOZOIAN, G., J. Bacterial. 94, 1787 (1967). 12. REISSIG, J. L., STROMINGER, J. L., AND LELOIR, L. F., J. Biol. Chem. 217, 959-966 (1955). 13. BARTNICK-GARCIA, S., Ann. Rev. Microbial. 22, 87 (1968). 14. DISTLER, J. J., AND ROSEMAN, S., J. Biol. Chem. 236, 25-38 (1960).
OF
BIOCHEMISTRY
The
AND
Cell
BIOPHYSICS
Wall
134,
Of
285-289
Helminthosporiom
D. A. APPLEGARTH The
University
of
British
Columbia,
Received
28,
1969;
sativum’
AND G. BOZOIAN
Department
March
(1969)
of Paediatrics, accepted
Vancouver
August
9, British
Columbia
11, 1969
The chemical composition of thecell wall of Helminthosporium sativum is described. The wall contains equal amounts of glucosamine and galactosamine. The galactosamine polysaccharide can be separated from the glucosamine by extraction of the wall with hot alkali. The glucosamine polysaccharide, which is insoluble both in cold concentrated acid and hot alkali is digested by a commercial chitinase preparation. The results suggest t,hat two hexosamine polymers are present in the cell wall of this microorganism.
Air-borne molds have been implicated in the development of mold-sensitive asthma in some individuals (1,2). Examination of the components of cell walls of some common air-borne fungi is one of the prerequisites for more detailed work on the allergenicity of these fungi. The present communication describes the chemical analysis of the hyphal wall of Helminthosporium sativum, a commonly occurring member of the air-borne mold population of the Pacific North Western area of the United States and Canada.
taminat,ion when viewed by electron microscopy. Digestion of the cell walls with 0.170 trypsin solution at 37” did not modify the percentage of protein found in the wall nor did it modify the appearance of the wall under the electron microscope. By these criteria the wall preparation was felt to be relatively free of cytoplasmic contaminat.ion. Electron photomicrographs of the cell-wall preparation used for analyses have already been published (5).
CHEMICAL ANALYSES Most of the analytical techniques used were identical to methods used previously (4). Only t,he salient differences will be discussed below. Solvent systems used for paper chromatography of neutral monosaccharides and amino sugars were as follows. Ratios quoted are volume/volume. Solvent System A = n-butanol-acetic acid-water........ .(12:3:5) Solvent System B = n-butanol-pyridine-water. @:4:3) Solvent System C = ethyl acetatepyridine-water. (12:5:4) Solvent System D = n-butanol-etha(4:l:l) nol-water,
METHODS
PREPARATION OF CELL WALLS Helminthosporium sativum was obtained as a commercial culture (Hollister-Stier Laboratories, 1075 Division Street, Spokane, Washington). It was grown with continuous stirring in 1 liter Fernbach flasks containing 250 ml of a fully synthetic medium (3) previously recommended for the growth of molds used in antigen testing in patients with mold-sensitive asthma (3). The cultures were allowed to grow for 7-12 days and the mycelial pads harvested in a Buchner funnel. Cell walls were prepared according to previously described methods (4, 5). The cell-wall preparations showed no evidence of microsomal con-
NEUTRAL MONOSACCHARIDES After hydrolysis and assay by the previously described methods the percentage of galactose was confirmed with a galactose-oxidase method (6). Glucose was confirmed by the glucose-oxidase method (6) modified as described (4). Since, in both cases, all of the sugar liberated by the hy-
1 The work was made possible by the award of a Medical Research Council of Canada Term Grant. 285
286
APPLEGARTH TABLE
PERCENTAGE
BOZOIAN
I
COMPOSITION OF Helminthosporium
TABLE
OF THE CELL sativum
Ash Glucose Galactose Mannose Phosphate (as HzP03) Glucosamine Galactosamine Kjeldahl nitrogen Protein (by calculation) Readily extractable lipid Bound lipid
WALL
5.2 37.0 2.0 2.0 1.8 8.6 8.3 3.8 18.0 2.0 9.4
drolysis reacted with the specific enzymes, we may conclude that both glucose and galactose were of the n-configuration. The configuration of the mannose was not checked. Results are presented in Table I. AMINO
AND
SUGARS
The hydrolysis conditions used for the analysis and identification of the amino sugars present in the wall were as before (4). The identity of the amino sugars was established as follows. The amino sugars liberated by acid hydrolysis of the cell walls were chromatographed on Whatman 3 MM paper developed in solvent system A. Areas corresponding to the amino sugars were eluted with water and the resulting compounds cochromatographed with authentic samples of glucosamine, galactosamine, and mannosamine in solvent systems A, B, and C. In all solvent systems only glucosamine and galactosamine could be detected. The amino sugars were oxidized with ninhydrin (7) and the resulting pentoses cochromatographed with standard solutions of arabinose, xylose, lyxose, and ribose in solvent system D for periods of up to 48 hr. When the chromatograms were developed with solvent system D for 24 hr, dried, and developed for another 24 hr all four pentoses could be separated. Only arabinose and lyxose could be detected in the products from the ninhydrin oxidation of the cell-wall amino sugars. Finally the amino sugar mixture from the cell wall was N-acetylated (8) and the resulting products cochromatographed with authentic samples of N-acetyl glucosamine, N-acetyl galactosamine, and N-acetyl mannosamine in solvent systems A and B. In solvent system A, N-acetyl glucosamine and N-acetyl galactosamine did not separate but the mixture of these sugars separated easily from N-acetyl mannosamine. In solvent system B all three sugars were separable. Only N-acetyl glucosamine and N-acetyl galactosamine were de-
AMINO
II
ACID COMPOSITION Helminthosporium
Amino Acid
OF WALLS sativum
pMole/mg
Lysine Histidine Arginine Cysteic Aspartic Threonine Serine Glutamic Proline Glycine Alanine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine
0.05 0.0075 0.0224 0.015 0.107 0.0775 0.0975 0.1215 0.0975 0.095 0.1225 0.0825 0.0075 0.045 0.08 0.0325 0.0375
wall
OF
70 of Total
4.6 0.7 2.0 1.4 9.9 7.1 9.0 10.3 9.0 8.9 11.2 7.6 0.7 4.1 7.5 3.0 2.0
tected, and no N-acetyl mannosamine was present. The above data show that the only amino sugars present in the cell wall of Helminthosporium sativum were glucosamine and galactosamine. The quantitative amount of each of these amino sugars was established in two separate ways. After prior purification of amino sugars by elution from Dowex-50 (H+) form with 0.3 N HCl the calorimetric technique of Ludoweig and Benaman (9) was used. This method was found to be the most successful and certainly the easiest method to determine the percentages of glucosamine and galactosamine. Identical answers were obtained using the Beckman Spinco amino acid analyzer after a hydrolysis time of 16 hr at 100” in 6 N HCl. AMINO
ACIDS
Samples of cell wall were hydrolyzed in 6 N hydrochloric acid at 100” for 16, 40, and 72 hr. The amino acids were analyzed with a Beckman 120C Spinco amino acid analyzer. The authors are grateful to Dr. Gordon Dixon and Miss Dorothy Kaufman for these analyses. The values obtained are quoted in Table II and are the maximum figures obtained for each amino acid at the three hydrolysis times. In practice there was no more than 10% variation between the values obtained at each hydrolysis time for any amino acid. LIPIDS Readily extracted lipids and bound lipids were assayed according to the procedures of BartnickiGarcia and Nickerson (10).
THE
CELL
WALL
OF HELMINTHOSPORIUM
Estimation of Protein Content An estimate of the amount of protein in the wall was obtained using the Kjeldahl gen value and subtracting the contribution nitrogen content by the amino sugars. PURINES
AND
present nitroto this
PYRIMIDINES
Ultraviolet-absorption spectra of 4 N HCl hydrolyzates of the cell walls indicated the presence of traces of purine and pyrimidine bases (approximately 0.4%). This is of the same order of magnitude BS that found by Bartnicki-Garcia and Nickerson (10) in cell walls of Mucor rouxii. A molar extinction coefficient of 34,800 for an equimolar mixture of adenine, guanine, cytosine, and uracil (total molecular weight 509) was used for the calculation. The ultraviolet-absorption spectrum of the acid hydrolyzate of the cell walls was identical to that from walls of Mucor rouzii reported by Bartnicki-Garcia and Nickerson. WORK ON THE POLYMERS OF
hrmo THE
SUGAR WALL
Two chemical tests and one enzymic test, for presence of chitin were performed. Ten milligrams of wall was extracted with 1 N acetic acid (5 ml) at 100” for 20 min. The acetic acid extract was removed by centrifugation and tested for the presence of chitosan by the addition of Gram’s iodine solution. No color developed, so presumably chitosan is absent from these cell walls. The acid-extracted residue was heated with 24% potassium hydroxide at 160” for 6 hr, then acidified with concentrated sulfuric acid (about 0.5 ml.) and Gram’s iodine solution added dropwise. No red-violet color was seen, whereas in a sample of Peniciullium pat&urn (11) used as a control the red-violet color of chitosan was easily visible. In summary, the preceding reactions did not indicate the presence of chitin. The cell wall (100 mg) was extracted with concentrated hydrochloric acid (10 ml) at 4” for 10 min. The mixture was filtered through glass wool into cold 50yo alcohol (50 ml). Neither a precipitate nor an opalescence was visible. The hydrochloric acid-treated wall was washed well with water and examined by phase-contrast microscopy. The morphology of the wall appeared to be completely unaffected by this acid treatment. A control experiment performed on cell walls of Penicillium patulum (11) yielded a visible precipitate of chitin. In the case of Penicillium patulum the extracted wall residue was rendered so amorphous by the treatment with cold hydrochloric acid that it could not be washed free of glass wool to be examined microscopically. By the two methods de-
the
SATIWM
scribed no chitin was detected in the wall of Helminthosporium sativum. The wall preparation (15 mg) was digested with chitinase (6) in 0.02 M acetate buffer (15 ml) pH 5.5, at 25” for 96 hr. After this treatment the residual cell wall was hydrolyzed with 4 N hydrochloric acid at 100” for 24 hr and the percentage of glucosamine and galactosamine assayed. This material contained 4.9% glucosamine and 9.4% galactosamine. This value for galactosamine was not significantly different from the value found before treatment with chitinase. However, some 40-50yo of the glucosamine in the wall had been removed, so presumably some 40-5Oojo of the glucosamine polymer in the wall was solubilized by chitinase. Because of the discrepancy in the results of simple chemical tests for chitin and the susceptibility of the wall to chitinase digestion, a further sample of wall (10 mg) was stirred with a chitinase preparation at 25” for 72 hr. The resulting suspension was centrifuged and the supernatant fraction assayed in a modified Morgan-Elson reaction (12) using standards of N-acetyl glucosamine prepared in the same buffer. The equivalent of 0.25 mg N-acetyl hexosamine (measured as N-acetyl glucosamine) was released from the lo-mg sample of cell wall. An aliquot of the supernatant fluid from the chitinase digestion was desalted with a mixture of Dowex-50 x 2 (H+ form) and Duolite-A 4 (OHForm), then deproteinized by shaking with several aliquots of a mixture of chloroform-t-amyl alcohol (5:l). The resulting aqueous solution was made 4 molar with respect to hydrochloric acid and the acid solution heated at 100” for 24 hr. After drying down in the usual manner the mixture was chromatographed in solvent system C. Only glucosamine was visible. These results confirmed that the cell wall of Helminthosporium sativum was digested by a chitinase preparation to yield a product which on hydrolysis liberates glucosamine as it,s only amino sugar. The galactosamine polymer in the wall was found to be capable of dissolution in hot 10% sodium hydroxide. A sample of the cell wall (100 mg) was extracted with 1 N potassium hydroxide (10.0 ml) at 6” for 2 hr. The wall suspension was recovered by filtration, washed with water, and resuspended in 10% sodium hydroxide. This suspension was heated at 100” for 30 min, centrifuged, and the supernatant fraction decanted into a fivefold excess of ethanol containing 5% (vol/vol) acetic acid. The ethanolic suspension was stored at 4” for 16 hr and the solid precipitate collected, dried over phosphorus pentoxide in vacua, then triturated with water. The aqueous supernatant portion was collected and dried over phosphorus pentoxide. The residual (water-insoluble) solid
288
APPLEGARTH
AND
BOZOIAN
linkages. The fact that no galactosamine could be found in acid hydrolyzates of the material solubilized by chitinase provides no evidence for areas of a mixed polymer in I 4 1 which there are glucosamine to galactosExtract (4.0,~~) Residue precipitated with Extracted with 10% NoOH amine links separated from one another by 5% aceticocid 100’ C, 30 minutes areas containing the chitinase-susceptible in EtOH I P-1-4 N-acetyl glucosaminyl linkages. In summary, the evidence presented indicates that two separate hexosamine polymers are Water soluble Wo,er nnroluble Residue (52.4mg) Extract ,4.2mg) Extmct(l I .4mg, Hydrolysis yields present in this cell wall. Hydrolysis yields Hydrolysis yields Glucoromlne 19.096 It will be noted that the iodine reaction Goloctoromine Galoctoramine Goloctoramine 1.0% used to prove the presence of chitin in fungi was negative. The other commonly used FIG. 1. Extraction of cell walls by alkali. method of proving the presence of chitin in a cell wall, that of extracting the chitin into was similarly dried. The wall residue, the watersoluble extract, and the water-insoluble extract cold concentrated hydrochloric acid, prewere all weighed, hydrolyzed in 4 N HCl at 100’ cipitating it by neutralization, or by addifor 24 hr and assayed for total hexosamine and tion of alcohol, and then checking the X-ray galactosamine. The results of these assays are diffraction pattern of the product (11) was shown diagrammatically in Fig. 1. The initial cold not available to us because we were unable potassium hydroxide extract was precipitated to extract the glucosamine polymer from the with absolute ethyl alcohol containing 5% (vol/ wall by using cold concentrated hydrovol) acetic acid but no attempt was made to idenchloric acid. The X-ray diffraction of the tify the components of this extract. Previous work whole wall would probably by meaningless (4) indicated that this extract would contain because of possible interference by the neutral glycans. galactosamine polymer. Until the discrepRESULTS AND DISCUSSION ancy between cbitinase digestion and both The cell wall of Helminthosporium sa- the calorimetric and extraction tests for the t&urn resembles cell walls of fungi pre- presenceof chitin can be further clarified the viously studied (4, 13) in being composed identification of chitin in fungi solely by methmainly of a glucose polysaccharide, poly- calorimetric or by acid-extractibility meric hexosamine, and some protein. The ods should be interpreted with caution. It has been obvious in the past that the most interesting finding is that both glucosamine and galactosamine are found in this calorimetric test for chitin was not so reliacell wall. Galactosamine has been reported ble as methods relying on X-ray powder previously in various fungi as a component diagrams of cold, concentrated acid extracts of whole mycelia (14) and of cell walls (13). of cell walls, but we appear now to have cast Glucosamine is a common component of some doubts on the validity of the acidestimation methods. Presumably a positive many fungal cell walls as the polymer chitin (13). From the fact that only a galactos- test will be meaningful but a negative test amine polymer can be extracted from the should be checked by an examination of acid hydrolyzates of cell walls to prove the abwalls by hot alkali and from the quantitative recovery of glucosamine from the re- sence of glucosamine in the wall. sidual cell wall after this extraction (see REFERENCES Fig. 1.) it is probable that the two amino 1. SOLOMON, W. R. Aero allergens in “A Manual sugars are present in separate molecules. of Clinical Allergy” (J. M. Sheldon, R. G. Furthermore, experiments presented here Lovell, and K. P. Mathews, eds.). Saunders, provide no evidence for the presence of any Philadelphia (1967). glucosamine to galactosamine links. The 2. PRINCE, H. E., ET AL. Ann. Allergy 19, 259 susceptibility of the glucosamine in the wall (1961). to digestion by chitinase suggeststhe exist3. S~HAFFER, N., MOLOMUT, N., AND CENTRE, ence of several p-1-4 N-acetyl glucosaminyl J. G., Ann. Allergy 17,380-384 (1959). CELL
WALL
(IOOmg)
Extraction with I N KOH 4’C. 2 hours under Nitrogen
I
1
1
THE
CELL
WALL
OF
HELMINTHOSPORIUM
4. APPLEGARTH, D. A., Arch. Biochem. Biophys. la0, 471-478 (1967). 5. APPLEGARTH, D. A., BOZOIAN, G., AND ZINTEL, B., Can. J. Microbial. 16, 312 (1969). 6. Worthington Biochemical Corporation, Freehold, New Jersey. 7. STOFFYN, I?. J., AND JEANLOZ, R. W., Arch. Biochem. Biophys. 62, 373 (1954). 8. CRUMPTON, M. J., Biochem. J. 73, 479 (1959). 9. LUDOWEIG, J., AND BENMBMAN, J. D., Anal. Biochem. 19, 80-88 (1967).
SATIVUM
289
10. BARTNICKI-GARCIA, S., AND NICKERSON, W. J., Biochim. Biophys. Acta68,102-119 (1962). 11. APPLEGARTH, D. A., AND BOZOIAN, G., J. Bacterial. 94, 1787 (1967). 12. REISSIG, J. L., STROMINGER, J. L., AND LELOIR, L. F., J. Biol. Chem. 217, 959-966 (1955). 13. BARTNICK-GARCIA, S., Ann. Rev. Microbial. 22, 87 (1968). 14. DISTLER, J. J., AND ROSEMAN, S., J. Biol. Chem. 236, 25-38 (1960).