LLOYD
Isolation, Characterization, and Partial Structure of Peptido Galactomannans from the Yeast Form of Cladosporium werneckii" Kenneth 0. Lloydt
ABSTRACT : Extraction
of yeast cells of Cladosporium werneckii with phosphate buffer (pH 7) at 100' and fractionation of the product with hexadecyltrimethylammonium bromide in borate buffer yielded a galactomannan (14 % D-galactose and 78 Z D-mannose) having covalently bound phosphate (3 Z) and peptide (1 1 %). The polymer has a high molecular weight (150,000-200,000) and contains both a- and /3-linked sugar residues. Methylation analysis and identification of the resulting O-methyl sugars by gas-liquid chromatography showed that the mannose is mainly 1+2 linked with smaller amounts of 1+6- and 1-3-substituted residues being present. There are very few branch points in the structure (mainly 2,6-linked mannose). About 1 2 x of the mannose and all of the galactose residues are present at the nonreducing ends of chains. The galactose is found in both D-pyranosyl and D-furanosyl forms. Fractionation of the peptidopolysaccharide on a DEAE-Sephadex column gave a series of fractions (I-VII) varying in their galactose, mannan, peptide, and phosphate contents. The fractions eluted with dilute salt were, in general, lower in phosphate and higher in galac-
I
n addition to insoluble, skeletal polysaccharides, such as chitin and @-glucans,the cell walls of yeasts and fungi contain water-soluble polysaccharides, usually mannans (for recent reviews see, Bartnicki-Garcia, 1968, Phaff, 1963, and Nickerson, 1963). Depending on the conditions used for extraction, these mannans contain varying amounts of covalently bound phosphate and protein (Falcone and Nickerson, 1956; Eddy, 1958; Korn and Northcote, 1960; Barker et al., 1963; Kessler and Nickerson, 1959; Sentandreu and Northcote, 1968). The phosphate is diesterified when the complexes are isolated under mild conditions (Slodki, 1962; Stewart and Ballou, 1968; McLellan and Lampen, 1968). Although the structure of the soluble polysaccharides of many fungi is understood in some detail (for a review, see Gorin and Spencer, 1968) there is as yet no satisfactory picture of the role which phosphate and protein play in the overall structure of these cell wall complexes. There is some evidence that the mannan of Saccharomyces cerecisiae constitutes the outermost layer of the cell (Mundkur, 1960) although this view has recently been criticized by Bacon et al. (1969). Their presence
* From the Department of Biochemistry and Dermatology, College of Physicians and Surgeons, Columbia University, New York, New York, 10032. Received April 23, 1970. Supported by a grant (AI-08478) from the U. S . Public Health Service. t Research Career Development Awardee K4-AI-3823, U. S . Public Health Service.
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tose than were the later fractions. The fractionation mainly depends on the phosphate content but since digestion with Pronase yielded a product giving only one major peak on the DEAE-Sephadex column it is evident that the peptide component also plays a role in the chromatographic behavior. Methylation analysis of fractions I, 11, and IV gave the same O-methyl sugars as the original except that the proportions of the various sugars varied. The results indicate that the peptido galactomannan preparation consists of a family of polysaccharides having variable amounts of galactose and phosphate substituted on a common mannan structure. The peptide component is rich in serine and threonine (42% of the amino acids) and also in aspartic and glutamic acids. Cysteine, methionine, and aromatic amino acids are absent or present in very small amounts. Threonine is enriched in the product after digestion with Pronase. The amino acid composition is compared with the composition of similar products from Saccharomyces cereaisiae and Trichophyton mentagrophytes.
on the surface would be consistent with the observations that the soluble mannans are the surface antigens of S . cereuisiae (Sakaguchi et al., 1967; Suzuki et al., 1968), Candida (Hasenclever and Mitchell, 1964; Suzuki and Sunayama, 1968; Mitchell and Hasenclever, 1970), and Trichophyton (Grappel et al., 1967, 1968a,b, 1969; Barker et al., 1962). Cladosporium werneckii, the causative agent of tinea nigra palmaris, is a dimorphic fungus. This paper describes the isolation and characterization of a peptido galactomannan from the yeast form of the fungus. It is hoped that a detailed anaiysis of its structure will lead to an understanding of the nature of this peptido-phosphopolysaccharide complex and its role in the architecture of the cell wall. A preliminary account of this work has appeared (Lloyd, 1970). Materials Methyl 2,3,4-tri-O-methyl-a-~-mannoside, methyl 2,3-di0-methyl-cm-mannoside, 3,4,6-tri-O-methyl-~-mannose, and 3,4-di-O-methyl-~-mannose were kindly provided by Dr. C. E. Ballou. Methyl 2,3,6-tri-O-methyl-~-mannoside, 2,4-di0-methyl-D-mannose, and a mixture of all the di-O-methylD-mannoses were gifts from Dr. P. A. J. Gorin. 2,3,4,6- and 2,3,5,6-tetra-O-methyl-~-galactoses were prepared by methylating the corresponding glycosides (obtained by the method of Austin et al., 1963). A sample of 2,3,4,6-tetra-O-methylD-galactose was also obtained from Dr. E. A. Kabat.
PEPTIDO GALACTOMANNANS
Methods Analytical Methods. Carbohydrate was determined by the phenol-sulfuric acid method (Dubois et al., 1956). Protein was determined by a micromodification of the Lowry-Folin method (Mage and Dray, 1965) and nitrogen by the ninhydrin method of Schiffman et al. (1964). Phosphate was estimated by the method described by Ames (1966). Component sugars of polysaccharides were determined after hydrolysis in trifluoroacetic acid (Alberheim et a/., 1967) as their hexitol acetates (Sawardeker et al., 1965). The sample (ca. 300 pg) was hydrolyzed in 300 pl of 2 N trifluoroacetic acid for 3.0 hr at 100' (with 100 pg of allitol as an internal standard). The reaction was carried out in a hydrolysis tube (Kontes Glass Co.); with the aid of an adapter to fit a Rinco evaporator all the subsequent steps could be conveniently carried out in the same tube. After evaporation to dryness in cacuo over PIOr and NaOH, the samples were reduced with sodium borohydride (0.5 mg) in 0.2 ml of 0.5 N NHIOH as described by Alberheim et al. (1967). After acidification and removal of borate with methanol, the samples were acetylated with 0.2 ml of acetic anhydride-pyridine ( 1 O : l ) at 100" for 1 hr. About 2 ml of water was added to the cooled solution and the acetates were extracted with chloroform. The chloroform solution was washed with water and evaporated to dryness. The residue was dissolved in 50 p1 of chloroform. Gas chromatography of the hexitol acetates was carried out on 6 ft X in. stainless steel columns containing 3 % ECNSS-M' at 185" by elution with NP at a rate of 30 ml/min (cf. Sawardeker et al., 1965; Lloyd and Kabat, 1967). Areas of the peaks were calculated using a Disc integrator and the ratio of each to the area of the internal standard was determined. By comparison with a standard mixture of sugars, the amount of each sugar in the sample could be calculated. Methylation of Polysaccharides. Methylation was carried out by the dimethylsulfinyl anion and methyl iodide method of Hakomori (1964) as described by Sandford and Conrad (1966). The sample was further methylated using methyl iodide and silver oxide in dimethylformamide (Kuhn et al., 1955). The following micromodification was used. Dry polysaccharide (3-12 mg) was dissolved in 5 ml of dry dimethyl sulfoxide by heating to 60" in a 25-ml erlenmeyer flask fitted with a serum stopper. The flask was then flushed with nitrogen using a hyDodermic needle and 1.0 ml of a solution of dimethyls;ifinyl sodium in dimethyl sulfoxide (Sandford and Conrad, 1966) was added with a syringe. After stirring vigorously with a magnetic stirrer for 5 hr, the solution was cooled and 0.4 ml of methyl iodide was added. Stirring was continued for 1.5 hr and the solution was then poured into ice-water. The solution was extracted three times with chloroform and the chloroform solution was washed three times with water and evaporated to dryness' The was remethy*ated with 0'25 m1 Of Oxide in O ' j 0 Of dimethyl iodide and 500 mg methy lformamide. Identification of o-Methyl sugars. The Polysaccharide was hydrolyzed in 1 ml of 80% formic acid at loo" and then for 15 hr in 0.5 N HZS04 at 100' (Garegg and L i d berg, 1960). The solution was neutralized with BaC03, and the 1 Abbreviations used are: ECNSS-M, a copolymer of ethylene glycol succinate polyester and a nitrile silicone polymer; TMSi, trimethylsilyl; Man, D-mannose; Gal, D-galactose.
precipitate was centrifuged and washed with ethanol. The combined solutions were evaporated to dryness. For identification of the 0-methyl sugars by gas chromatography, the sample was divided into two portions: (i) one was converted into the methyl glycosidesby heating in 0.75 ml of 2.5% methanolic HC1 at 100" for 2-3 hr. After neutralization with Ag2C03, the solution was allowed to evaporate to dryness at room temperature and atmospheric pressure. A portion of methyl glycosides was converted into the trimethylsilyl derivatives (Sweeley et af., 1963; cf: Lee and Ballou, 1965); (ii) the other portion was reduced to its hexitol derivatives with 2 mg of NaBH4in 0.5 ml of 0.5 N N H 4 0 H and then acetylated with 1 ml of acetic anhydride-pyridine as described above. The methyl glycosides and their TMSi derivatives were examined by gas chromatography on a 6 ft X ' / E in. column of 2 neopentylglycol succinate (Lee and Ballou, 1965; Bhattacharjee and Gorin, 1969) at 150" for the glycosides and at 130" for their TMSi derivatives. The 0-tetra-, tri-, and diin. methylhexitol acetates were chromatographed on 6 ft X columns of 3 % ECNSS-M at 160'; by raising the temperature to 185", the mono-0-methylhexitol acetates and mannitol acetate could be identified (Bjorndal et al., 1967). The identification of the peaks was confirmed by comparison to standard reference compounds. For quantitative determination of the O-methyl sugars the response factor relative to the corresponding 2,3,4,6-tetra-O-methyImannose derivative was determined using pure reference compounds. The identification of 0-methylmannoses by gas chromatography of their methyl glycosides and their 0-trimethylsilyl ethers has been described by Ballou and coworkers (Lee and Ballou, 1965; Stewart and Ballou, 1968) and by Bhattacharjee and Gorin (1969). In this study, gas chromatography of the O-methylhexitol acetates was also used, mainly for two reasons: (a) 2,3,4,6-tetra-O-methylmannitol diacetate could be separated from 2,3,5,6- and 2,3,4,6-tetra0-methylgalactitol diacetates (and a partial separation of the latter could be obtained), and (b) it enabled the degree of methylation of the polysaccharide to be checked by confirming the absence of mono-0-methylmannoses and mannose in the hydrolysate. Using a combination of all three methods the methyl ethers resulting from methylation of the galactomannan could be identified and quantitated. Experimental Section and Results Culture of Organism. C . werneckii was isolated from a patient with tinea nigra on the palm of the hand (isolate 625). It was converted into and maintained in the yeast phase by smearing relatively large inocula throughout the entire surface of Sabouraud-dextrose agar slants. Large-scale culture was carried out by inoculating 400 ml of dialyzed Sabouraud-dextrose broth in 1-1, culture flasks and shaking the flasks in a rotary shaker at 150 cycles/min at 28-30" for 7 days. This tirne period yielded the maxjmu, number of cells; the cultures were examined m~croscopica~~y and were found to be almost entirely (,95z) in the yeast form. The cells were harvested by centrifuging at 9ooo rpm in a Sorvall RB-2 centrifuge, washed withwater, and dried with acetone. The yield of acetone-dried powder was 3.8 gjl. Extraction of Cells and Isolation of Peptido Galactomannan (Scheme I). Acetone powder (100 g) was extracted with 1000
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LLOYD
GAL, TABLE I:
Analyses of Samples from Cetavlon Fractionation.
Analysis
TIME OF HYDROLYSIS (hrs)
1: Release of sugars from peptido galactomannan by acid hydrolysis. Solid lines: 1 N trifluoroacetic acid at 100'. Broken lines: 0.01 N trifluoroacetic acid at 100".
PronaseTreated FracFraction D tion B
Fraction A
Fraction B
Fraction C
(%I
(%)
(%)
(%)
(%)
1.8 11.3 9.6 3.2 78 14 0 92
1.6 10.0 8.9 0.4 44 7 47 98
1.2 7.5 8.1 3.6 8 0.2 77 86
0.8 5.0 2.3 5.0 81 13 0 94
6.1
6.2
4.0
6.5
3.2 N Nasproteina 20.0 Folinprotein 10.3 Po4 5.7 69 Mannose Galactose 12 Glucose 7 Total 88 carbohydrate Man:Galratio 6.1
FIGURE
ml of 0.05 M potassium phosphate buffer (pH 7.0) by stirring at 100" for 2 hr (cf. Peat et al. (1961) who used 0.019 M citrate pH 7 at 140"). After centrifuging at 9000 rpm for 30 min the residue was reextracted using 1000 ml and then 750 ml of buffer. To the combined extracts was added sodium acetate (50 g) and ethanol (3 volumes). The precipitate was centrifuged, dissolved in 120 ml of water, and the solution was dialyzed extensively against distilled water. The solution was freeze-dried to give 3.7 g of a light brown powder. Extract (3.6 g) was then dissolved in water (100 ml) and hexadecyltrimethylammonium bromide (Cetavlon) (3.6 g) in water (50 ml) was added. After standing at room temperature overnight, the precipitate was centrifuged and washed with water (50 ml). FRACTION A. The precipitate was dissolved in 1 M NaCl (15 ml) by shaking overnight at room temperature. The re-
SCHEME I extract (3.6g)
A + Cetavlon
J.
precipitate (40mg)
solution
fraction A
precipitate (1.86g)
borate (pH8.8)
solution
lfraction]
lborate (pH 9.5)
1
solution
precipitate (320 mg) fraction C
I
ethanol
precipitate (470mg) fraction D
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a
N value X 6.25.
sulting solution was clarified by centrifugation and ethanol (3 volumes) was added. The resulting precipitate was isolated by centrifugation and washed with 2 acetic acid in ethanol. The residue was dissolved in water (6 ml), dialyzed exhaustively, and reisolated by freeze-drying; yield 40 mg. FRACTION B (PEPTIDO GALACTOMANNAN). To the combined solution and washing (200 ml) was added 1 % boric acid (100 ml). The solution was stirred and the pH was adjusted to 8.8 by the careful addition of 2 N NaOH. After standing overnight at room temperature, the precipitate was centrifuged and washed with 0.5% sodium borate (pH 8.8). The residue was dissolved in 2 % acetic acid (50 ml) and sodium acetate (1 g) and ethanol (3 volumes) were added to the solution. The resulting precipitate was removed by centrifugation and washed with 2 % acetic acid in ethanol and then ethanol. The product was dried over P205in uacuo; yield 1.86 g. FRACTION C. The pH of the supernatant was adjusted to pH 9.5 with 2 N NaOH and the product was isolated as for fraction B; yield 320 mg. FRACTION D. Ethanol (3 volumes) was added to the supernatant. The resulting precipitate was centrifuged and washed with 2 % acetic acid in ethanol and then ethanol; yield 396 mgThe isolation procedure is summarized in Scheme I. Analytical data on the fractions are given in Table I. Properties of the Peptido Galactomannan (Fraction B). The product was an off-white powder, easily soluble in water and having an [a],,-24" ( c 0.21). It was entirely excluded from a Sephadex G-100 column. Chromatography on a column of Sephadex G-200 gave a single broad peak (a portion of which was in the excluded volume). These results indicate a molecular weight in the 150,000-200,000 range. Zdentifcation of Sugars and Amino Acids. The component sugars of fraction B were identical with mannose and galactose by gas chromatography as their hexitol acetates and as their 0-trimethylsilyl derivatives (Sweeley et al., 1963) and by thin-layer chromatography on silica gel using the method of Huber et al. (1968). A sample was hydrolyzed in 2 N
PEPTIDO GALACTOMANNANS
5
700 600
_ _ _ _ _ _---_-
w 500 3
z
a Z
40G
300
= 200 IO0
0
TUBE NUMBER
2: Chromatography of peptido galactomannan (fraction B) on DEAE-Sephadex column (60 X 2.9 cm). A two-step gradient from 1 M NaCl in 0.005 M sodium phosphate buffer (pH 7.1,2500ml) to 0.02 M NaCl in 0.005 M sodium phosphate buffer (pH 7.1, 1750 ml) was used, Fractions of 6.2 ml were collected. (0)Mannose and ( 0 )protein.
FIGURE
sulfuric acid for 2 hr and the component sugars were isolated by preparative paper chromatography in 1-butanol-acetic acid-water (3 :1 : 1, v/v) and purified by chromatography on a Bio-Gel P2 column, D-Mannose was identified as its phenylhydrazone (mp and mmp 198-200', [a], +28.5', c 0.22, pyridine) and D-galactose as its benzylphenylhydrazone (mp and mmp 157-158', [a],-14.7', c0.20, pyridine). Amino acids in the sample were estimated on an amino acid analyzer after hydrolysis in 6 N HCl at 110' for varying periods of time (Table 11). Only traces of glucosamine (ca. 1 %) were found, both in the amino acid analyzer and by gas chromatography of its N-acetyl-0-trimethylsilyl derivative after hydrolysis in 2 N HCl for 2 hr (Lloyd and Kabat, 1969). Acid Hydrolysis of Galactomannan. Portions of fraction B were hydrolyzed in 2 and 0.01 N trifluoroacetic acid (as described in the methods section) for varying lengths of time. The proportion of each sugar liberated was calculated; the results are shown in Figure 1. About 2 7 Z of the galactose was released by 0.01 N acid, showing that it was in the furanose form. The remainder was released only by 2 N acid; it was more readily liberated than the mannose indicating that it occurs mostly in terminal positions. Fractionation of Peptido Galactornannan (Fraction B ) on DEAE-Sepliadex Column. Chromatography of fraction B (380 mg) on a DEAE-Sephadex column (60 X 2.9 cm) by elution with a NaCl gradient from 0.02 to 0.6 M gave a complex pattern of fractions (Figure 2). Fractions I-VI1 were isolated and 11, 111, IV, and V were rechromatographed. Analysis of the fractions (Table 111) showed that they varied in their galactose, mannose, protein, and phosphate contents; the fractions eluted with dilute salt were, in general, lower in phosphate and higher in galactose than the later fractions. Digestion of Peptido Galactornannan (Fraction B ) with Pronase. Fraction B (54 mg) was dissolved in 1 ml of 0.01 M sodium bicarbonate buffer (pH 8.2) containing 0.01 M CaC12. Pronase (Calbiochem, 2.0 mg) was added in 2.0 ml of the same buffer. The mixture was incubated at 37' for 2 days (with a drop of toluene). The solution was heated at 100" for 5 min and the resulting precipitate was removed by centrifugation. The solution was chroma2 1 am grateful to Dr. A. Gold, Department of Biochemistry, for
tographed on a column (60 X 1.8 cm) of Sephadex G-25. The polysaccharide was separated from a second peak which contained only ninhydrin-reactive material (and salts). Chromatography of the polysaccharide fraction on a DEAESephadex column by elution with a NaCl gradient from 0.02 to 0.6 M gave one major peak (Figure 3). This component was reisolated by dialysis and freeze-drying; yield 18 mg. Analytical data are given in Table I. Methylation Analysis. Peptido galactomannan (fraction B) was methylated and hydrolyzed as described in the Methods
TABLE 11:
Amino Acid Analyses. of Peptido Galactomannans. Pronase-Digested Fraction B
Fraction B Amino Acid LYS His Arg ASP Thr Ser Glu Pro GlY Ala CYS Val Met Ile Leu TYr Phe Total amino acids (as of fraction)
mpmole/ mg
,ug/mg
m,umole/ mg
16.4 4.4 7.1 64.2 248.0 153.0 80.9 51 .O 64.5 120.0 0 44.7 Trace 30.8 37.0 Trace 19.9
2.4 0.7 1.2 8.5 29.6 16.1 11.9 5.9 4.8 10.7 0 5.2 Trace 4.0 4.8 Trace 3.3
10.8 4.7 Trace 11.6 118.0 51.2 29.6 11.6 11.2 45.7 0 23.6 Trace 4.2 3.9 Trace Trace
M/mg 1.6 0.8 Trace 1.5 14.0 5.4 4.3 1.3 0.8 4.1 0 2.8 Trace 0.6 0.5
Trace Trace
11
3.8
a Averages of 24- and 48-hr hydrolysis values except for threonine and serine which were extrapolated to zero time.
these analyses.
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TABLE III:
Analyses and Yields of Peptido Galactomannan Fractions from DEAE-Sephadex Column.
Nitrogen N as protein” Folin protein
PO4 Mannose Galactose Total carbohydrate Man :Gal ratio ID (de& Weight (mg) a
1.1 7.2 5.4 0.6 75 26 101 2.9 -22 18
2.0 12.7 10.5 1.3 81 17 98 4.7 -31 30
2.0 12.7 8.2 2.2 88 7 95 12.5 - 34 30
1. o 6.3 5.0 5.1 85 13 98 6.5 -23 115
1.5 3.9 2.9 6.1 76 12 88 6.3 -25 15
2.7 16.8 9.8 4.0 61 7 67 8.8 - 27 8
2.4 14.9 8.2 3.7 64 8 72 8.0 - 17 14
N value X 6.25.
section. The resulting 0-methyl ethers were identified by gas-liquid chromatography of three different derivatives. Representative gas chromatograms are shown in Figure 4; the presence of large amounts of 3,4,6-tri-O-methyl-~mannose and of only trace amounts of di-0-methyl-Dmannoses is clearly illustrated in Figure 4A,C. Quantitative data for the ratios of the sugars are given in Table IV. The proportion of tetra-0-methyl-D-galactose in the mixture was approximately the same as the proportion of galactose in the galactomannan thus confirming that all the galactose residues occupy terminal positions. Both 2,3,5,6- and 2,3,4,6tetra-0-methyl-D-galactose were identified but since they were not completely resolved (Figure 4D) only an approximate estimate of their proportion (3 :2) was obtained. Methylation analysis of galactomannans I, 11, and IV from the DEAE-Sephadex column showed that they contained the same linkages as the original galactomannan although in slightly different proportions (Table IV). The amount of tetra-0-methyl-D-galactose was proportional to the amount of galactose in that fraction showing that the extra galactose in fractions I and I1 all occurs at the nonreducing ends of chains.
H
400
n
1
0
50
100 I50 TUBE NUMBER
0
200
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Extraction of yeast cells with phosphate buffer (pH 7) at 100” yielded a mixture of soluble galactomannan and glycogen. Fractionation of the mixture by precipitation of the borate complex of the mannan by Cetavlon (Barker et al., 1957) avoided the use of high pH. The major product had 14% of D-galactose and 78% of D-mannose with covalently bound phosphate (3 %) and peptide (11 %) also being present. This peptido gala~tomannan~ had a molecular weight (estimated by gel filtration) in the 150,000-200,000 range. The optical rotation ([a],,-24”) indicated an approximately equal number of a- and P-linked residues in the polymer. The majority of mannans isolated from fungi have been a-linked polysaccharides (Gorin and Spencer, 1968) although recently Gorin et al. (1969a,b) have reported a number of mannans with both cy- and P-linked residues. The constant ratio of galactose to mannan in fractions A, B, and C suggested that a single galactomannan had been isolated. However, chromatography of the peptido galactomannan on a DEAE-Sephadex column by elution with increasing salt concentrations yielded a series of fractions (Figure 2) which varied in their phosphate and galactose contents. The fractions eluted with dilute salt were lower in phosphate and higher in galactose than were the later fractions (except fraction 111). The fractionation is due mainly to differences in phosphate content but digestion with Pronase to a yield a product giving only one major peak on DEAE-Sephadex indicates the differences in the peptide portion also play a role. Barker et al. (1967) showed that a galactomannan from Trichophyton mentagrophytes could be fractionated into two components on a DEAESephadex column; the first fraction was higher in galactose than the second (the phosphate content is not given). Simi-
250
FIGURE 3 : Chromatography of peptido galactomannan after digestion with Pronase on DEAE-Sephadex column (60 X 1.8 cm). A twostep gradient from 1 M NaCl in 0.005 M sodium phosphate buffer (pH 7.1, 600 ml) to 0.02 M NaCl in 0.005 M sodium phosphate buffer (pH 7.1,400 ml) was used. Fractions of 2.5 ml were collected. (0)Mannose and ( 0 )protein.
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a These materials have been designated “peptido polysaccharides” to contrast them with glycoproteins and glycopeptides. I n glycoproteins the protein moiety is the major constituent and the carbohydrate plays a minor role. The term “glycopeptide” has come to refer to low molecular weight fragments derived from glycoproteins and so it is suggested that peptido polysaccharide be used to refer to materials in which a polysaccharide is linked to relatively small amounts of peptide or protein.
PE PTID0 GA L A CT0MA N NA N S
TABLE IV:
Identification and Quantitation of 0-Methyl Sugars from Methylated Galactomannans by Gas-Liquid Chromatography.
0-Methyl Sugars
Re1 A.
2,3,4,6-Tetra-O-methylmannose 1 .oo 2,3,5,6-Tetra-O-methylgalactose 1.00 2,3,4,6-Tetra-0-methylgalactose 1.22 3,4,6-Tri-O-rnethylmannose 1.96 2,3,4-Tri-O-rnethylmannose 2.05 2,4,6-Tri-O-methylmannose 2.53 3,4-Di-O-methylmannose 3.50 Tetra-O-methyl galactoses ( %) in methylation products Galactose content (%) of galactomannan
Elution Bb
Times C.
1.00 1 .oo 1.20, 1.27 C.47,0.52 0.69 0.82 0.34
1 .oo 1.15 1.25 1.97 2.45 2.00 5.30
Ratios (Wt) of 0-Me Sugars in:
IT
Original
I
1.00 1.08d
1.00 5.25d
1.00 2.01d
100 1.22d
5.95 0.52 1.28 0.14 11 14
8.70 0.88 1.42 0.56 29 26
6.35 0.24 1.34 0.45 19 17
6.00 0.55 0.75 0.13 13 13
IV
A: Methyl glycosides on neopentylglycol succinate column (150'). * B: TMSi derivatives of methyl glycosides on neopentylglycol succinate column (130O). c C : 0-Methylhexitol acetates on ECNSS-M column (160'). Ratio of 2,3,5,6-tetra-O-methyl: 2,3,4,6-tetra-O-methylgalactose was approximately 3 :2. 0
larly, Stewart and Ballou (1968) showed a complex pattern for Kloeckera brevis mannan by chromatography on DEAESephadex. They also showed an increase in phosphate (and decrease in peptide content) in going from low to high salt concentrations. The question arises as to whether this fractionation indicates a number of unrelated galactomannans or whether the components have a basic underlying polysaccharide structure which is substituted by varying amounts of other residues. That the latter is the case for the galactomannan from C. werneckii was shown by methylation analysis. Methylation analysis of the original galactomannan (fraction B) showed that the mannose is largely 1-2 linked with smaller amounts of 1-6and 143-substituted residues being preset (Table IV). There are very few branch points in the structure (mainly 2,6-linked mannose). The very low proportion of branch points in the polymer as compared to the number of terminal nonreducing end groups of mannose and galactose is remarkable (see Table IV for the ratio of di-O-methylmannose to tetra-0-methylhexoses). About 12 of the mannose residues are present at the nonreducing ends of chains. All the galactose residues are terminal which is the case in other fungal galactomannans also (Bishop et al., 1962, 1965, 1966; Barker et al., 1963; Gorin et al., 1969a,b). However this polysaccharide is unusual in that a portion of the galactose is in the D-furanosyl form while the remainder is in the D-pyranosyl configuration. This conclusion was supported by hydrolysis studies (Figure 1) although only about half of the galactose found to be in the furanosyl form by methylation analysis was released by 0.01 N acid at 100"; the reason for this is unclear. Methylation analysis of three of the fractions from the DEAE-Sephadex column showed that they had the same basic 1-2-linked mannose structure (with smaller amounts of 1+3 and 1+6 linkages). The tetra-O-methylgalactose content was higher in fractions I and I1 showing that the extra galactose was all terminal. Since the amount of 3,4-di0-methylmannose was also higher in these fractions it is possible that the galactose is substituted on to the C-6 position of 1-2-linked mannose. However this might not be the
m I
0
4
8
I ill
I2
16
PO
24 26 0 4 ELUTION TIME [ M I N U T E S )
6
12
16
21
FIGURE 4: Gas chromatograms of O-methyl sugars from methylated galactomannans. (A) Methyl glycosides from methylated galactoand mannan, fraction B: I, methyl 2,3,4,6-tetra-O-methylmannoside methyl 2,3,5,6-tetra-O-methylgalactoside; 11, methyl 2,3,4,6-tetra-Ornethylgalactoside; 111, methyl 3,4,6 and 2,3,4-tri-O-methylmannosides; IV, methyl 2,4,6-tri-O-methylmannoside; V, methyl 3,4-di0-methylmannoside. (B) Trimethylsilyl derivatives of methyl glycosides from methylated galactomannan, fraction B : I, methyl 3,4-di-O-methyl-2,6-di-O-trimethylsilylmannoside; 11, methyl 3,4,6tri-0-methyl-2-0-trimethylsilylmannoside; 111, methyl 2,3,4-tri-Omethyl-6-0-trimethylsilylmannoside; IV, methyl 2,4,6-tri-O-rnethyltrimethylmannoside; V, methyl 2,3,4,6-tetra-O-methylmannoside and 2,3,5,6-tetra-O-methylgalactoside. (C) O-Acetyl-O-methyl-hexitols from methylated galactomannan,fraction B: I, 1,5-di-O-acetyl2,3,4,6-tetra-O-methylmannitol and 1,4-di-O-acetyl-2,3,5,6-tetra-Omethylgalactitol; 11, 1,2,5-tri-O-acetyl-3,4,6-tri-O-methylmannitol and 1,3,5-tri-O-acetyl-2,4,6-tri-O-methylmannitol; 111, 1,5,6-tri-O-
acetyl-2,3,4-tri-O-methylmannitol; V, 1,2,5,6-tetra-O-acetyl-3,4-di0-methylmannitol. (D) 0-Acetyl-0-methylhexitols from methylated glactomannan I separated at 140" on ECNSS-M column: I, 1,5-di-O-acetyl-2,3,4,6-tetra-O-methylmannitol; 11, 1,4-di-O-acetyl2,3,5,6-tetra-O-methylgalactitol;111, 1,5-di-O-acetyl-2,3,4,6-tetra-
0-methylgalactitol.
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TABLE
v: Comparison of Amino Acid Compositiona of
Peptido Polysaccharides from Various Fungi. C. werneckii
Pronase- S. cerevisaeb T. mentaDigested Glycogr0phytes.c Fraction B protein Peptide I d ( %> A2 (%) ( %>
Amino Acid
Fraction B (%)
LYS His Arg ASP Thr Ser Glu GlY
2.2 Trace Trace 7.7 27.0 14.6 10.8 5.4 4.4
4.2 Trace Trace 4.0 37.2 14.3 11.4 3.4 2.2
2.5 Trace Trace 9.9 28.5 14.6 9.9 7.8 4.8
2.5 2.7 2.0 5.5 25.8 15.4 7.5 16.7 6.9
Ala CYS Val Met Ile Leu TYr Phe
9.7 0 4.7 Trace 3.6 4.3 Trace 3.0
10.8 0 7.4 Trace 1.5 1.3 Trace Trace
9.0 0 6.1 0 2.1 2.2 1.3 1.3
5.8 0 5.4 0 3.1 2.3 0 1.8
Pro
Per cent of each amino acid in peptide. * Values recalculated from Sentandreu and Northcote (1968)’ c Values recalculated from Barker et al. (1967). d The other peptides from T. mentagrophytes have similar ratios of amino acids except that the proline content varies. a
case as it has been shown by precipitation with concanavalin A (K. 0. Lloyd, unpublished data, 1969) that galactomannans I and I1 also have greater amounts of terminal a-D-mannosyl residues. The covalent nature of the linkages between the peptide portion and the galactomannan moiety is suggested by the facts that: (a) the peptide is not removed during the Cetavlon fractionation, (b) it is not precipitated by 5 trichloroacetic acid (K. 0. Lloyd, unpublished data, 1969), (c) the Folin reactive material closely follows the carbohydrate peaks during fractionation on DEAE-Sephadex, and (d) the peptide component has a characteristic amino acid composition. Table I1 shows the amino acid composition of the peptido galactomannan. In its high serine and threonine content it resembles glycoproteins in which a large number of short carbohydrate chains are linked glycosidically to the OH groups of these residues in a peptide backbone. These glycoprotein are mainly animal in origin however a peptido galactomannan from T. mentagrophytes (Barker et al., 1967) and a peptido mannan from S . cerevisiae (Sentandreu and Northcote, 1968) have very similar compositions (Table V). In addition to their high serine and threonine contents they are characterized by having relatively high proportions of aspartic and glutamic acids and by the paucity or absence of aromatic amino acids and of cysteine and methionine.
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The importance of threonine in the structure of the peptido polysaccharide from C. werneckii is further emphasized by its enrichment (to 3 7 z of the peptide) in the product after digestion with Pronase (Table V). The microheterogeneity of peptido polysaccharides from yeasts and fungi demonstrated in this study and in the work of Barker et al. (1967) on T. mentagrophytes, of Stewart and Ballou (1968) on K. brevis, and of Preston et al. (1969) on Penicillium charlesii raises the question of the origin of this dispersity. It could be due to (a) the fact that the polysaccharides were isolated from nonsynchronous cultures containing cells in different stages of growth, (b) incomplete biosynthesis of some of the chains, and (c) degradation of some of the polysaccharide-protein complexes by the carbohydrases and proteinases which are produced in copious amounts by many fungi. Barker et al. (1967) found differences in heterogeneity between deep and surface cultures of T. mentagrophytes and emphasize the importance of the last possibility. Acknowledgments
I am grateful to Dr. M. Silva-Hutner for supplying the culture of C. werneckii and for her advice on mycology in general. I am also indebted to Minda A. Bitoon for expert technical help. References Alberheim, P., Nevins, D. J., English, P. D., and Karr, A. (1967), Carbohyd. Res. 5,340. Ames, B. N. (1966), Methods Enzymol. 8,115. Austin, P. W., Hardy, F. E., Buchanan, J. G., and Baddiley, J. (1963), J . Chem. SOC.,5350. Bacon, J. S. D., Farmer, V. C., Jones, D., and Taylor, I. F. (1969), Biochem. J. 114,557. Barker, S. A,, Basarab, O., and Cruickshank, C. N. D. (1967), Carbohyd. Res. 3,325. Barker, S. A., Cruickshank, C. N. D., and Holden, J. H. (1963), Biochim. Biophys. Acta 74,239. Barker, S. A., Cruickshank, C. N. D., Morris, J. H., and Wood, S . R. (1962), Immunology 5,627. Barker, S. A,, Stacey, M., and Zweifel, G. (1957), Chem. Ind. (London), 330. Bartnicki-Garcia, S . (1968), Annu. Rev. Microbiol. 22, 87. Bhattacharjee, S. S . , and Gorin, P. A. J. (1969), Can. J. Chem. 47,1207. Bishop, C. T., Blank, F., and Hranisavljevic-Jakovljevic, M. (1962), Can. J. Chem. 40,1816. Bishop, C. T., Perry, M. B., and Blank, F. (1966), Can. J. Chem. 44,2291. Bishop, C. T., Perry, M. B., Blank, F., and Cooper, F. P. (1965), Can. J . Chem. 43,30. Bjorndal, H., Lindberg, B., and Svensson, S. (1967), Acta Chem. Scand. 21,1801. Dubois, M., Gilles, K. A,, Hamilton, J. K., Rebers, P. A., and Smith, F. (1956), Anal. Chem. 28,350. Eddy, A. A. (1958), Proc. Roy. SOC.,Ser. B 149,425. Falcone, G., and Nickerson, W. J. (1956), Science 124, 272. Garegg, P. J., and Lindberg, B. (1960), Acta Chem. Scand. 14, 871.
BONGKREKIC
ACID
INHIBITION OF ADENINE NUCLEOTIDE TRANSLOCATION
Gorin, P. A. J., and Spencer, J. F. T. (1968), Adoan. Carbohyd. Chem. 23,367. Gorin, P. A. J., Spencer, J. F. T., and Bhattacharjee, S. S. (1969a), Can. J. Chem. 47,1499. Gorin, P. A. J., Spencer, J. F. T., and Magus, R. J. (1969b), Can. J . Chem. 47,3569. Grappel, S. F., Blank, F., and Bishop, C . T. (1967), J . Bacteriol. 93,1001. Grappel, S . F., Blank, F., and Bishop, C . T. (1968a), J. Bacteriol. 95,1238. Grappel, S. F., Blank, F., and Bishop, C. T. (1968b), J . Bacteriol. 96,70. Grappel, S. F., Blank, F., and Bishop, C . T. (1969), J. Bacteriol. 97,23. Hakomori, S. (1964), J. Biochem. (Tokyo)55,205. Hasenclever, H. F., and Mitchell, W. 0. (1964), J. Zmmunol. 93,763. Huber, C . N., Scobell, H. D., Tai, H., and Fisher, E. E. (1968), Anal. Chem. 40, 207. Kessler, G., and Nickerson, W. J. (1959), J. Biol. Chem. 234, 2281. Korn, E. D., and Northcote, D. H. (1960), Biochem. J. 75,12. Kuhn, R., Tirschmann, H., and Low, I. (1955), Angew. Chem. 67,32. Lee, Y .C., and Ballou, C . E. (1965), Biochemistry 4,257. Lloyd, K. 0. (1970), Fed. Proc., Fed. Amer. SOC.Exp. Biol. 29,637. Lloyd, K. O., and Kabat, E. A. (1967), Carbohyd. Res. 4, 165. Lloyd, K. O., and Kabat, E. A. (1969), Carbohyd. Res. 9, 41.
Mage, R., andDray, S. (1965), J. Zmmunol. 95,525. McLellan, W. L., and Lampen, J. 0. (1968), J. Bacteriol. 95, 967. Mitchell, W. O., and Hasenclever, H. F. (1970), Infect. Immunity I , 61. Mundkur, B. (1960), Exp. CellRes. 20,28. Nickerson, W. J. (1963), Bacteriol. Rev. 27,305. Peat, S., Turvey, J. R., and Doyle, D. (1961), J . Chem. SOC., 3918. Phaff, H. J. (1963), Annu. Reo. Microbiol. 17,15. Preston, J. F., Lapis, E., and Gander, J. E. (1969), Arch. Biochem. Biophys. 134,324. Sakaguchi, O., Suzuki, S., Suzuki, M., and Sunayama, H. (1967), Jap. J. Microbiol. I l , 119. Sandford, P. A., and Conrad, H. E. (1966), Biochemistry 5, 1508. Sawardeker, J. S., Sloneker, J. H., and Jeanes, A. (1965), Anal. Chem. 37,1602. Schiffman, G., Kabat, E. A., and Thompson, W. (1964), Biochemistry 3, 113. Sentandreu, R., and Northcote, D. H. (1968), Biochem. J. 109,419. Slodki, M. E. (1962), Biochim. Biophys. Acta57,525. Stewart, T. S., and Ballou, C. E. (1968), Biochemistry 7,1855. Suzuki, S., and Sunayama, H. (1968), Jap. J. Microbiol. 12, 413. Suzuki, S., Sunayama, H., and Saito, T. (1968), Jap. J . Microbiol. 12, 19. Sweeley, C . C., Bentley, R., Makita, M., and Wells, W. W. (1963), J. Amer. Chem. SOC.85,2497.
Factors Affecting the Inhibition of Adenine Nucleotide Translocase by Bongkrekic Acid* Peter J. F. Henderson, Henry A. Lardy, and Eileen Dorschner
ABSTRACT: The translocation of adenine nucleotides and their structural analogs across the membranes of rat liver mitochondria is inhibited by bongkrekic acid; the extent of inhibition achieved depends on the ratio of bongkrekic acid concentration to mitochondrial protein concentration, the temperature, and period of time for which the mitochondria are exposed to the inhibitor. By contrast, inactivation
W
elling et al. (1960) first reported that the antibiotic bongkrekic acid is an inhibitor of oxidative phosphorylation in rat heart mitochondria. Subsequent experiments with * From the Institute for Enzyme Research, University of Wisconsin, Madison, Wisconsin 53706. Received May 22, 1970. Supported by grants from the National Science Foundation, the National Institutes of Health, and the Life Insurance Medical Research Fund. This is paper 16 in the series: Antibiotics as Tools for Metabolic Studies.
of the translocase enzyme by atractyloside is relatively independent of these parameters. The greater potency of bongkrekic acid, when compared to atractyloside on a concentration basis, is probably due to the relatively weak antagonism by adenosine diphosphate, whereas adenine nucleotides competitively diminish inhibition by atractyloside.
mitochondria from rat liver have shown that bongkrekic acid inactivates an adenine nucleotide translocase and that this effect is probably responsible for the inhibition of ADP phosphorylation, ATPase, and other adenine nucleotide requiring reactions (Henderson and Lardy, 1970a,b). This conclusion is confirmed in this communication by further observations on the exchange of adenine nucleotides across the mitochondrial membrane, a direct assay of the adenine nucleotide translocase activity (Klingenberg and Pfaff, 1968).
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