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The use of Sephadex G-25 for the isolation of nucleotide sugar derivatives from Micrococcus lysodeikticus
378
PRELIMINARY NOTES
such as ovalbumin te,t*, orosomucoid Is and fetuin ~9. The molar ratio of neutral sugars to hexosamine is, however, considerably higher in the plant glycopeptide than that commonly found in the animal glycoproteins. We wish to thank Dr. E. MEYER of Central Soya, Chicago, for a generous gift of soybean meal. Thanks are due to Mrs. H. LATTER for skillful technical assistance. This work was supported by grant FG-Is-x2o from the United States Department of Agriculture.
Department of Biophysics. The Weizmann Institute of Science, Rehovoth (Israel)
H A L I N A L1S NATHAN SHARON E P H R A I M KATCHALSKI
t F. R. BETTELHEIM-JEVONS, Advan. Protein Chem., t3 0 9 5 8 ) 35. tt R. W . JEANLOZ, Advan. Eneymol., z 5 (t963) 433. 3 R. G. SPIRO. New EnglandJ. Med., 269 (1963) 566. 616. 4 I. E. L1F,NER, J. Nutrition. 49 (t953) 527. 5 A. TISELIUS, S. HJERTEN AND t~. LEVIN, Arch. Biochem. Biophys.. 65 (1950) 13 z. 6 1. E. LmNER, Arch. Biochem. Biophys., 54 0 9 5 5 ) 23. S. XVADA, M. J. PALL^NSCH AND 1. E. LIENER, J. Biol. Chem., 233 (I958) 3 9 5 s j . E. EASTOE, Nature. I73 (1954) 54o. 0 M. D u u o l s , K. A. GILLES. J. K. HAMILTON, P. A. REBERS AND F. SMITH, .4hal. Chem., 28 (1956) 35 o. 10 A . HALLI{'N, .4cta Chem. Scand., ]4 0 9 6 0 ) 2249. II S. GARDELL, Acta Chem. Stand., 5 (I951) 195. l~ p. j . STOFFYN AUD R. W. JEANLOZ, Arch. Biochem. Biophys., 52 (I954) 373. I3 S. O H K I N U R A AND T. SHINOHARA, Nature, zo2 (1964) 593. 14 D. H. SPACKMAN, W. H. STEIN AND S. MOORE, ,4hal. Chem., 3 ° (1958) ti9o. is 1. WERNER AND L. ODIN, Acta Soc. Meal. Upsaliensis, 57 0 9 5 2 ) 230le R. H. NUENKE AND L. W. CUNNINGHAM, J. Biol. Chem., 236 (I961) 2452. IT A. P. FLETCHER, G. S. MARKS, R. D. MARSHALL A N D A. NEUBERGER, Biochem. J., 87 (IQ63) 265. Is S. KAMIYAMA AND K. SCHMID, Biochim. Biophys. Acta, 58 0962) 8o. 19 R. G. SPlRO, J. Biol. Chem., 237 (I962) 382.
Received July I6th, I964 Biochim. Biophv,..4eta. 83 (1964) 37 ° 378
I'N 8 I 0 0 4
The use of Sephadex 6-25 for the isolation of nucleotide sugar derivatives from Micrococcus lysodeikticus Since the original isolation of uridine nucleotide sugar peptide derivatives from
Staphylococcus aureus t, similar nucleotides have been shown to be present in a variety of other microorganisms 2. These compounds have usually been isolated from cell extracts by the use of ion exchange chromatography, followed by adsorption on, and elution from, charcoal. In the course of work aimed at the isolation of nucleotide sugar peptides from Micrococcus lysodeikticus, we found that these compounds can be separated from other nucleotide derivatives and low molecular weight metabolites by the use of Sephadex G-25. This separation is apparently based on the fact that Sephadex contains a small amount of carboxyl groups, which enable it to act as an anion exchanger in solutions of low ionic strength, in addition to acting as a molecular sieve s , 4. In a typical experiment IO g of dried M. lysodeikticus cells (Miles Laboratories, Biochim. Biophys. Acta, 83 (1964) 3 7 8 38o
PRELIMINARY NOTES
379
Elkhart, Ind.) were extracted with 50 ml of trichloroacetic acid (xo %, w/v) for 70 min in the cold, and then centrifuged. The trichloroacetic acid was removed by extraction with ether, and a small amount of a glucose-containing polymer (probably identical with the glucose-N-acetyl aminomannuronic acid polymer isolated from M . lysode//~/cus by P~EKINS*), was removed by precipitation with 2.5 vol. of ethanolic potassium acetate (o.5 %, w/v). The clear supernatant was then concentrated under vacuum at room temperature to 6 ml. The sample, which contained 22 pmoles of N-acetylhexosamine (estimated* after hydrolysis in o.ox N HCI for x5 rain at xoo°, using N-acetylglucosamine as standard), was applied to a column of Sephadex G-25 (2.6 cm × 8o cm) which had been equilibrated with o.ox N acetic acid. Elution was carried out with o.ox N acetic acid. Under these conditions the acidic nucleotides are excluded from the Sephadex. A graph of the elution pattern (Fig. x) shows that three peaks of ultraviolet-absorbing material are obtained. Peak I and Peak II contain essentially all of the amino sugar nucleotides present in the extract, while Peak III contains the remainder of the nucleotides in the extract. I
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240
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oo
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4--
0
40
i : i : J 40
80
=
i
120
J
160
200 240 m! EbUT(O
280
3~0
360
400
0
Fig. I. Elution pattern obtained when an extract of xo g of dry wt. of M. lysodeikti6-us in a volume of 6 ml is applied to a column of Sephadex G-25, and elution is carried out with o.oi M acetic acid. The solid line represents the ultraviolet &bsorption at 26o mp. The dashed line represents the N-acetyl-amino sugar content (expressed in Klett units/o. I ml, using the Klett-Summerson photo-colorimeter, with Filter No. 54) after hydrolysis in o.oz N HCI (see text for details).
Paper chromatography of Peak I using the ethanol-i M ammonium acetate (pH 7.5) (7.5: 3, v/v) system and isobutyric acid-conc, ammonia-water (66: x :33, v/v) system yielded two major components with Rp values identical to those of authentic samples of UDP-N-acetylmuramyl-alanyl-glutamyl-lysine and UDP-N-acetylmuramyl-alanyl-glutamyl-lysyl-alanyl-alanine, prepared from S. aureus inhibited by oxamycin7 and penicillin s, respectively. The ultraviolet spectrum, both in acid (o.ox N HCI) and alkali (o.ox N NaOH) was typical of uridine. Analysis of acid hydrolysates (6 N HCI, xxo°, 8 h) of each of the two components from Peak I on the Spinco model amino acid analyser revealed the presence of lysine, glutamic acid and alanine in the ratio of x:x:x. 4 in the first compound and x:x:3 Biochim. Biophys. Acta, 83 (x964) 378-380
380
PRELIMINARY NOTES
in the second. In addition to these three amino acids, the first component contained o.z mole threonine and the second o.z mole of glycine per mole of glutamic acid. The ratio of uridine to phosphate to glutarnie acid in both compounds was x : z . z : I . Paper chromatography of Peak II in ethanol-x M ammonium acetate (pH 7-5) (7.5:3, v/v) and isobutyric acid-conc, a m m o n i a - w a t e r (66: x :33, v/v) showed that it contained one major component. This component had an Re that differed from those of UDP-N-acetylglucosamine or UDP-N-acetylmuramic acid. Following hydrolysis in o.ox N HC1 at xoo ° for xo min, and estimation of the N-acetylhexosamine content of the hydrolysate b y the Morgan-Elson reaction 6, a low color yield was obtained relative to the phosphate and uridine content. Upon paper chromatography of this acid hydrolysate in n-butanol-acetic a c i d - w a t e r (25:6:25, v/v) three reducing spots were detected with the silver nitrate reagentL The major one had a mobility" relative to N-acetylglucosamine RAa = 0.85, and the other two spots had Raa ~ 0.45 and x.z respectively. No N-acetylglucosamine or N-acetylmuramic acid could be detected on the paper chromatogram. The hydrolysed nucleotide derivative gave a positive carbazole reaction ~°. The ratio of the uronic acid to uridine was o.I 7 : r, using glucuronic acid as standard. After hydrolysis in 6 N HCI at I I o ° for 8 h, no amino acids could be detected on paper with ninhvdrin. Further work on identification of this compound is in progress. We wish to thank Prof. E. KATCHALSKIfor his continued interest and suggestions. This work was supported in part by Grant AI-o3528 from the National Institutes of Health, U.S. Public Health Service. One of the authors (S. R.) is a special Fellow of the National Heart Institute, National Institutes of Health, U.S. Public Health Service.
Department of Biophysics, The Weizmann Institute of Science, Rehovoth (Israel)
SPENCER ROSENTHAL NATHAN SHARON
t j . T. PARK, J. Biol. Chem., I94 (1952) 877, 885, 897. • J. L. STROMt•GRR, in I. C. GUNSALUS AND a . Y. S'fANIER, The Bacteria, krol. 3, A c a d e m i c Press, N e w Y o r k , I962, p. 413 . a B. GELOTTg, J. Chromatog., 3 ([96o) 33 o. 4 A. N. GLAZER AND D. WELLNER, Nature, I94 (1962) 862. 6 H. R. P s n x l r ; s , Biochem. J., 86 (x963) 475. • J. L. RslssxG, J. L. SrROmSOER ASt~ L. F. LRLOm, J. Biol. Chem., 217 (1956) 959. 7 j . L. STROMI~CGRR, R. H. TllRENN AND S. S. SCOTT, J. Am. Chem. Soc., 8t (I959) 3803 . s j . L. S'rROMINGER, J. Biol. Chem., 224 (x957) 509. i N. SrIARON AND R. W. JEAm.OZ, J. Biol. Chem., 235 (I96o) L S0 Z. DlSCHE, J. Biol. Chem., I67 (1947) 189.
Received July 24th, z964 Biochim. Biophys. Acla, 83 (t964) 3 7 8 - 3 8 0
PRELIMINARY NOTES
such as ovalbumin te,t*, orosomucoid Is and fetuin ~9. The molar ratio of neutral sugars to hexosamine is, however, considerably higher in the plant glycopeptide than that commonly found in the animal glycoproteins. We wish to thank Dr. E. MEYER of Central Soya, Chicago, for a generous gift of soybean meal. Thanks are due to Mrs. H. LATTER for skillful technical assistance. This work was supported by grant FG-Is-x2o from the United States Department of Agriculture.
Department of Biophysics. The Weizmann Institute of Science, Rehovoth (Israel)
H A L I N A L1S NATHAN SHARON E P H R A I M KATCHALSKI
t F. R. BETTELHEIM-JEVONS, Advan. Protein Chem., t3 0 9 5 8 ) 35. tt R. W . JEANLOZ, Advan. Eneymol., z 5 (t963) 433. 3 R. G. SPIRO. New EnglandJ. Med., 269 (1963) 566. 616. 4 I. E. L1F,NER, J. Nutrition. 49 (t953) 527. 5 A. TISELIUS, S. HJERTEN AND t~. LEVIN, Arch. Biochem. Biophys.. 65 (1950) 13 z. 6 1. E. LmNER, Arch. Biochem. Biophys., 54 0 9 5 5 ) 23. S. XVADA, M. J. PALL^NSCH AND 1. E. LIENER, J. Biol. Chem., 233 (I958) 3 9 5 s j . E. EASTOE, Nature. I73 (1954) 54o. 0 M. D u u o l s , K. A. GILLES. J. K. HAMILTON, P. A. REBERS AND F. SMITH, .4hal. Chem., 28 (1956) 35 o. 10 A . HALLI{'N, .4cta Chem. Scand., ]4 0 9 6 0 ) 2249. II S. GARDELL, Acta Chem. Stand., 5 (I951) 195. l~ p. j . STOFFYN AUD R. W. JEANLOZ, Arch. Biochem. Biophys., 52 (I954) 373. I3 S. O H K I N U R A AND T. SHINOHARA, Nature, zo2 (1964) 593. 14 D. H. SPACKMAN, W. H. STEIN AND S. MOORE, ,4hal. Chem., 3 ° (1958) ti9o. is 1. WERNER AND L. ODIN, Acta Soc. Meal. Upsaliensis, 57 0 9 5 2 ) 230le R. H. NUENKE AND L. W. CUNNINGHAM, J. Biol. Chem., 236 (I961) 2452. IT A. P. FLETCHER, G. S. MARKS, R. D. MARSHALL A N D A. NEUBERGER, Biochem. J., 87 (IQ63) 265. Is S. KAMIYAMA AND K. SCHMID, Biochim. Biophys. Acta, 58 0962) 8o. 19 R. G. SPlRO, J. Biol. Chem., 237 (I962) 382.
Received July I6th, I964 Biochim. Biophv,..4eta. 83 (1964) 37 ° 378
I'N 8 I 0 0 4
The use of Sephadex 6-25 for the isolation of nucleotide sugar derivatives from Micrococcus lysodeikticus Since the original isolation of uridine nucleotide sugar peptide derivatives from
Staphylococcus aureus t, similar nucleotides have been shown to be present in a variety of other microorganisms 2. These compounds have usually been isolated from cell extracts by the use of ion exchange chromatography, followed by adsorption on, and elution from, charcoal. In the course of work aimed at the isolation of nucleotide sugar peptides from Micrococcus lysodeikticus, we found that these compounds can be separated from other nucleotide derivatives and low molecular weight metabolites by the use of Sephadex G-25. This separation is apparently based on the fact that Sephadex contains a small amount of carboxyl groups, which enable it to act as an anion exchanger in solutions of low ionic strength, in addition to acting as a molecular sieve s , 4. In a typical experiment IO g of dried M. lysodeikticus cells (Miles Laboratories, Biochim. Biophys. Acta, 83 (1964) 3 7 8 38o
PRELIMINARY NOTES
379
Elkhart, Ind.) were extracted with 50 ml of trichloroacetic acid (xo %, w/v) for 70 min in the cold, and then centrifuged. The trichloroacetic acid was removed by extraction with ether, and a small amount of a glucose-containing polymer (probably identical with the glucose-N-acetyl aminomannuronic acid polymer isolated from M . lysode//~/cus by P~EKINS*), was removed by precipitation with 2.5 vol. of ethanolic potassium acetate (o.5 %, w/v). The clear supernatant was then concentrated under vacuum at room temperature to 6 ml. The sample, which contained 22 pmoles of N-acetylhexosamine (estimated* after hydrolysis in o.ox N HCI for x5 rain at xoo°, using N-acetylglucosamine as standard), was applied to a column of Sephadex G-25 (2.6 cm × 8o cm) which had been equilibrated with o.ox N acetic acid. Elution was carried out with o.ox N acetic acid. Under these conditions the acidic nucleotides are excluded from the Sephadex. A graph of the elution pattern (Fig. x) shows that three peaks of ultraviolet-absorbing material are obtained. Peak I and Peak II contain essentially all of the amino sugar nucleotides present in the extract, while Peak III contains the remainder of the nucleotides in the extract. I
I
|
I
!
I
I
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I
I
I I ,t It ! it
240
[ ~ t
m
2oo
a0
¢) 1-
E 160
w
12:
oo
8
4--
0
40
i : i : J 40
80
=
i
120
J
160
200 240 m! EbUT(O
280
3~0
360
400
0
Fig. I. Elution pattern obtained when an extract of xo g of dry wt. of M. lysodeikti6-us in a volume of 6 ml is applied to a column of Sephadex G-25, and elution is carried out with o.oi M acetic acid. The solid line represents the ultraviolet &bsorption at 26o mp. The dashed line represents the N-acetyl-amino sugar content (expressed in Klett units/o. I ml, using the Klett-Summerson photo-colorimeter, with Filter No. 54) after hydrolysis in o.oz N HCI (see text for details).
Paper chromatography of Peak I using the ethanol-i M ammonium acetate (pH 7.5) (7.5: 3, v/v) system and isobutyric acid-conc, ammonia-water (66: x :33, v/v) system yielded two major components with Rp values identical to those of authentic samples of UDP-N-acetylmuramyl-alanyl-glutamyl-lysine and UDP-N-acetylmuramyl-alanyl-glutamyl-lysyl-alanyl-alanine, prepared from S. aureus inhibited by oxamycin7 and penicillin s, respectively. The ultraviolet spectrum, both in acid (o.ox N HCI) and alkali (o.ox N NaOH) was typical of uridine. Analysis of acid hydrolysates (6 N HCI, xxo°, 8 h) of each of the two components from Peak I on the Spinco model amino acid analyser revealed the presence of lysine, glutamic acid and alanine in the ratio of x:x:x. 4 in the first compound and x:x:3 Biochim. Biophys. Acta, 83 (x964) 378-380
380
PRELIMINARY NOTES
in the second. In addition to these three amino acids, the first component contained o.z mole threonine and the second o.z mole of glycine per mole of glutamic acid. The ratio of uridine to phosphate to glutarnie acid in both compounds was x : z . z : I . Paper chromatography of Peak II in ethanol-x M ammonium acetate (pH 7-5) (7.5:3, v/v) and isobutyric acid-conc, a m m o n i a - w a t e r (66: x :33, v/v) showed that it contained one major component. This component had an Re that differed from those of UDP-N-acetylglucosamine or UDP-N-acetylmuramic acid. Following hydrolysis in o.ox N HC1 at xoo ° for xo min, and estimation of the N-acetylhexosamine content of the hydrolysate b y the Morgan-Elson reaction 6, a low color yield was obtained relative to the phosphate and uridine content. Upon paper chromatography of this acid hydrolysate in n-butanol-acetic a c i d - w a t e r (25:6:25, v/v) three reducing spots were detected with the silver nitrate reagentL The major one had a mobility" relative to N-acetylglucosamine RAa = 0.85, and the other two spots had Raa ~ 0.45 and x.z respectively. No N-acetylglucosamine or N-acetylmuramic acid could be detected on the paper chromatogram. The hydrolysed nucleotide derivative gave a positive carbazole reaction ~°. The ratio of the uronic acid to uridine was o.I 7 : r, using glucuronic acid as standard. After hydrolysis in 6 N HCI at I I o ° for 8 h, no amino acids could be detected on paper with ninhvdrin. Further work on identification of this compound is in progress. We wish to thank Prof. E. KATCHALSKIfor his continued interest and suggestions. This work was supported in part by Grant AI-o3528 from the National Institutes of Health, U.S. Public Health Service. One of the authors (S. R.) is a special Fellow of the National Heart Institute, National Institutes of Health, U.S. Public Health Service.
Department of Biophysics, The Weizmann Institute of Science, Rehovoth (Israel)
SPENCER ROSENTHAL NATHAN SHARON
t j . T. PARK, J. Biol. Chem., I94 (1952) 877, 885, 897. • J. L. STROMt•GRR, in I. C. GUNSALUS AND a . Y. S'fANIER, The Bacteria, krol. 3, A c a d e m i c Press, N e w Y o r k , I962, p. 413 . a B. GELOTTg, J. Chromatog., 3 ([96o) 33 o. 4 A. N. GLAZER AND D. WELLNER, Nature, I94 (1962) 862. 6 H. R. P s n x l r ; s , Biochem. J., 86 (x963) 475. • J. L. RslssxG, J. L. SrROmSOER ASt~ L. F. LRLOm, J. Biol. Chem., 217 (1956) 959. 7 j . L. STROMI~CGRR, R. H. TllRENN AND S. S. SCOTT, J. Am. Chem. Soc., 8t (I959) 3803 . s j . L. S'rROMINGER, J. Biol. Chem., 224 (x957) 509. i N. SrIARON AND R. W. JEAm.OZ, J. Biol. Chem., 235 (I96o) L S0 Z. DlSCHE, J. Biol. Chem., I67 (1947) 189.
Received July 24th, z964 Biochim. Biophys. Acla, 83 (t964) 3 7 8 - 3 8 0