BIOCHIMICA ET BIOPHYSICA ACTA 405

BBA 25381

THE ACID-SOLUBLE NUCLEOTIDES OF MILK

III. OCCURRENCE OF UDP-N-ACETYLLACTOSAMINE AND

UDP-D-XYLOSE IN PIG'S MILK AND COLOSTRUM

AKIRA KOBATA AND SUZUOKI-ZIR0 Biological Research Laboratories, Research and Development Division, Takeda Chemical Industries Ltd., Osaka (Japan)

(Received January 29th, 1965)

SUMMARY

The acid-soluble nucleotides of pig's milk and colostrum were analyzed by ion-exchange chromatography.

Both milk samples had almost the same nucleotide pattern. The major nucleo- tide constituents were UDP-sugars and GDP-mannose. UDP-N-acetyllactosamine and UDP-D-xylose were detected in both milk samples. Evidence demonstrating the small contamination of UDP-N-acetylgalactosamine <--galactoside in pig's milk UDP-N-acetyllactosamine is presented.

INTRODUCTION

Ever since the discovery of the unique nucleotide composition of sheep's milk 1-~, several investigators have reported on the nucleotide constituents of cow's milk 4-6, goat's milk ~,9, and human milkLS,8. These reports show that the nucleotide pattern of milk is, contrary to those of mammalian tissues, species-specific and quite different from that of the mammary gland. Moreover, milk contains many unique nucleotides : e.g. sheep's milk and cow's colostrum were shown to contain GDP-fucosel-3,5; goat's

colostrum, UDP-N-acetylglucosamine (4 +-~ I) galactopyranosyl ~-sialic acid and

UDP-N-acetylglucosamine (6 ~ I) galactopyranosyl ~- sialic acidg; and human milk,

UDP-N-acetyllactosamine and UDP-N-acetyllactosamine (2 ~-~--I) L-fucopyrano- sideS,lo.

We have been interested in the species specificities in the nucleotide patterns of milk. The present report gives results of the analyses of the acid-soluble nucleotides of pig's milk and colostrum.

MATERIALS AND METHODS

Unless otherwise noted, all compounds, reagents and methods used in these studies were the same as those described in the preceding paper 5.

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406 A. KOBATA, S. ZIRO

Milk samples All milk samples were frozen by dry ice immediately after being collected from

mammary glands and stored below - -4 °° . Experimental samples were obtained from cows of Yorkshire and Berkshire strains: colostra, 1-2 days after parturition; milk 19-62 days after parturition.

Nucleotides UDP-N-acetyllactosamine was prepared from human milk according to the

procedure previously described 8.

Carbohydrates D-Xylose, L-xylose, D-arabinose, D-lyxose and D-ribose were purchased from

Nutritional Biochemical Corp. N-Acetyl-D-galactosamine from Seikagaku Kogyo Co. Ltd., Tokyo. D-Galactose, D-glucose, N-acetyl-D-glucosamine, D-glucosamine and D-galactosamine from Wako Pure Chemical Co. Ltd., Osaka. N-Acetylglucosaminol was synthesized from N-acetylglucosamine according to the method of CRIMMIN 11.

Lactobacillus cell suspension Lactobacillus pentosus strain 124-2 was kindly supplied by Dr. J. O. LAMPEX.

The cells were grown in the D-xylose medium 12 at 37 ° for 48 h, transferred to fresh medium and grown for another 48 h. The cells were harvested by centrifugation, washed twice with ice-cold 1% KC1, and suspended in water (IOO mg wet weight per ml). The suspension was used immediately for an experiment.

Paper chromatography and paper electrophoresis Descending chromatography was carried out on acid-washed Toyo Roshi filter

paper No. 5IA. Solvents used were as follows: (A) n-butanol-e thanol-water (4: i : i , v/v); (B) 80% aq. phenol-conc, ammonia (2oo:1, v/v); (C) upper layer of ethyl acetate-acetic acid-water (3: 1:3, v/v); (D) isopropanol-water (4: I, v/v); (E) 95 % e thanol - I M ammonium acetate (pH 7.5) (75:3 °, v/v); (F) upper layer of ethyl ace ta te-pyr id ine-water (2 : I :2, v/v) ; (G) n-butanol-acetic acid-water (12:3:5, v/v). With Solvents B and C, the ascending technique was employed. On a preparative scale, the sample was applied as a band to the paper strip. The compounds were then eluted from the chromatogram with water.

Paper electrophoresis was carried out on the same paper (54 cm long) using the following buffer systems : (E-I) o.I M acetate buffer (pH 4.2), 58 V/cm, 20-40 rain; (E-2) 0.o5 M phosphate buffer (pH 7.4), 37 V/cm. 30-50 rain.

Quantitative analysis of galactose, glucosamine and galactosamine by gas chromatography A Hitachi Model KGL-2A gas chromatographic instrument equipped with a

hydrogen flame detector was used for this study. A U-shaped stainless tube (IOO × o.4 cm inner diameter) packed with I I %

(w/w) neopentylglycol adipate polyester (Applied Science Laboratories) coated on 8O-lOO mesh Chromosorb W (Applied Science Laboratories) was used. Columns were developed with helium at a flow rate of 95 ml/min at a temperature of 193 °. N-Acetyl- lactosamines (1.8-2.o #moles) isolated paper chromatographically (Solvent A) from the acid hydrolysate of UDP-N-acetyllactosamines were hydrolyzed in 0.4 ml of

Biochim. Biophys. Acta, lO7 (1965) 4o5-413

NUCLEOTIDES OF PIG'S MILK 407

I N HC1 by heating at 95 ° for I h. The solutions were evaporated to dryness under reduced pressure at room temperature; water was added and they were again eva- porated to dryness. This procedure was repeated three times. The residue was dis- solved by adding i ml of ice-cooled water. 0.2 ml of 5 % aq. acetic anhydride 14 and 0.2 ml of saturated sodium bicarbonate were added and the solution was kept at room temperature for 25 rain to acetylate free hexosamines. After being heated in a boiling- water bath for 3 rain, the reaction mixture was passed through a small (0.5 x 2 cm) Dowex-5o (H +) column. The column was washed with three portions of water. Eluates and washings were combined and lyophilized. The residues were dissolved in 0.2 ml of dry pyridine and trimethylsilanized with 20/~1 of trimethylchlorosilane and 20 #1 of hexamethyldisilazane 1~. Pyridine was removed from the reaction mixture according to the procedures of YAMAKAWA et al35, and the residue was dissolved in 20/~1 of hexane. 2-4/~1 of the solution were delivered to the gas chromatograph. N-Acetyl- glucosaminol was used as an internal standard.

RESULTS

Ultraviolet absorption curves for the acid-soluble fraction of colostrum and milk after charcoal treatment were determined. These curves in Fig. I show that uridine nucleotides are the main constituent in both milk and colostrum.

The amount of 26o-m/z absorbing substances and cationic and neutral fractions were essentially the same in both samples (Table I). Moreover we found no qualitative differences in nucleotide pattern for colostrum and milk (Table I). A typical chromato-

E i i i ~ i 1

! ( " ' \ ,

Y IY \,..t. 0'3~- ~,

2 3 0 2 5 0 270 2 9 0 Wave leng th (rnlJ)

~ Colos t r 'um

. . . . . Mi lk

Fig. i. Ultraviolet absorption spectra of perchloric acid extracts from pig's milk and colostrum.

Biochim. Biophys. Acta, lO 7 (1965) 4o5-413

408

T A B L E I

N U C L E O T I D E C O N S T I T U E N T S IN P I G ' S MILK AND COLOSTRUM

A. KOBATA, S. ZIRO

Peak No. Constituent Content in btmoles/Ioo ml

Colostrum Ivlilk

II 5 ' -AMP i .7 I I I 3 ' ,5 '-cyclic AM P 0. 3 IV 5 ' -GMP 6.3 VI 5 ' -UMP 45.6 VI I U D P - N - a c e t y l l a c t o s a m i n e (UY1) 7.2 V I I I G D P - m a n n o s e 34.3 I X U D P - N - a c e t y l h e x o s a m i n e 61.2 X U D P - h e x o s e 57.2 XI UDP-D-xylose (UY~) 7-7 X I I G D P 6.8 X I I I U D P + UDP -g l ucu ron i c acid 14.o

Tota l a b s o r b a n c y un i t s a t 26o m/z per IOO ml of mi lk sample

Cationic and neu t r a l f rac t ions ( absorbancy un i t s a t 260 nap per IOO ml of mi lk sample)

2797 2712

28o 268

4.0 I .O

5.5 31.5

5-5 22.5 77.5 44.0

4.0 21.5 15.O

2.0

E28omp / E2eomp 2"01 . ,

1.0

0

. . . . . . . . . . . . . " " .

' ° . " . ° . . . . . . . . . . . . . . . . . . . . . . . , . . . . . . . ° °

Vl Vlll IX X Im,- Ik-

Xlll XII .

IV

0 2 0 40 6 0 8 0 100 120 140 160 180

t t t t t IN-FA 4N-F'A 4N-FA'¢'O.2M-AF 4N-FA+O.4M-AF" 4N-F'A+O.BM-AF

Fig. 2. Ion -exchange c h r o m a t o g r a m of t he acid-soluble f rac t ion of p ig ' s milk. E x t r a c t equ iva len t to 20 ml of mi lk was sepa ra ted on a 2o0-400 m e s h Dowex-I X8 (formate) c o l u m n (4o × o.5 cm). Abbrev ia t ions : FA, formic acid; AF, a m m o n i u m formate .

Biochim. Biophys. Acta, lO 7 (1965) 4o5-413

NUCLEOTIDES OF PIG'S MILK 409

gram for the nucleotides of pig's milk is shown in Fig. 2. No differences could be seen between Berkshire and Yorkshire milk.

Nucleotides for each peak were identified as shown in Table I by the analytical procedure described in the preceding paper 5.

TABLE II P A P E R C H R O M A T O G R A P H I C M O B I L I T I E S O F S U G A R F R O M U Y 1

RD_galactose values

Solvent A C F G

N-Acetyllactosamine o.33 o.95 i .o 7 o.42 6-O-fl-D-Galactopyranosyl-N-acetylglucosamine o.21 o.88 o.97 o.22 Sugar from UY 1 0.33 0-95 1-o7 0.42

Identification o/ UDP-N-acetyllactosamine (UYI) The eluting position (Peak VII) of this fraction resembled to that of UDP-N-

acetyllactosamine found in human milk and colostrum. After being purified by re- chromatography (sodium formate linear gradient system) and paper chromatography with Solvent E, the main nucleotide of this fraction showed the ultraviolet spectrum of nridine nucleotide both in acidic (o.i N HC1) and in alkaline (o.i N KOH) solutions, and contained 1.93 moles of total phosphate and 0.97 mole of acid-labile phosphate per mole of uridine. After hydrolysis in o.oi N HC1 for 20 min at 95-98°, i mole of reducing substance was released, and two ultravi0!et light-absorbing spots corre- sponding to UDP (major) and UMP (minor) were de~ected by paper electrophoresis (E-I and E-2). Hydrolysis of the UDP in o.oi N HC1 for 3 h at 95-980 liberated equimolar amounts of UMP and inorganic phosphate. The UMP was completely hydrolyzed into uridine and inorganic phosphate by the action of bull semen 5'-nucleo- tidase (ECyI.3.5) , but was not attacked by Bacillus subtilis 3'-nucleotidase (EC 3.1.3.6).

The reducing substance was studied by paper chromatography with four solvent systems, and only one spot corresponding to N-acetyllactosamine was detected with the periodate-benzidine reagent (Table II). This spot was negative to MORGAN- ELSOY reagent 16 and reacted faintly with alkaline AgNOa reagent.

About 0. 9 #mole of this saccharide was recovered by elution from paper chroma- tograms. When heated in i N HC1 at ioo ° for 80 rain, approximately equimolar amounts of galactose and hexosamine were liberated. This galaetose was shown to have the D-configuration by the utilization experiment with Saccharomyces fragilis strain IFO o541 (ref. 17). The sugar mixture was subjected to ninhydrin degradation TM, and examined by paper chromatography with Solvent A. The chromatogram (Fig. 3) shows galactose and arabinose together with a small amount of lyxose.

Separation of sugar components of UDP-N-acetyllactosamine isolated from pig's milk by gas chromatography

N-Acetyllactosamine (1.86/,moles) isolated from pig's milk UDP-N-acetyl- lactosamine and 1.95 #moles isolated from human milk UDP-N-acetyllactosamine were hydrolyzed with acid, and N-acetylated. These sugar mixtures were trimethyl-

Biochim. Biophys. Acta, lO 7 (1965) 4o5-413

410 A. KOBATA, S. ZIR()

silanized and analyzed by gas chromatography. The gas chromatogram (Fig. 4) shows that the sugar moiety of human milk UDP-N-acetyllactosamine was composed of equimolar amounts of galactose and glucosamine, whereas pig's milk nucleotide was

L~

@

At(

GI¢"

• - ~ Ga l

0

O r i g i n I I I I :

Fig. 3. Paper chromatogram of the ninhydrin degradation products• IOO m/~moles of UY 1 were hydrolyzed with I N HC1 (IOO °, I h), degraded with 2 % ninhydrin and 4 % pyridine mixture and examined by paper chromatography in Solvent A. Abbreviations: Glc, glucose; Lyx, lyxose; Gal, galactose; Ara, arabinose.

• C

o 4 s ~'2 ~& '

Time (min) 20 2 4 28 32

c o

o

0 32

F

E G

4 8 12 16 2 0 24 28 T ime (rnin)

Fig. 4. Gas chromatograms of the acid hydrolysates of N-acetyllactosamines. I, sample from human milk UDP-N-acetyllactosamine; II, sample from UY x. A, B, C, galactose; D, N-acetyl- glucosaminol (authentic); E, N-acetylgalactosamine; F, G, N-acetylglucosamine.

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NUCLEOTIDES OF PIG'S MILK

T A B L E I I I

P A P E R C H R O M A T O G R A P H I C M O B I L I T I ] ~ S O F T H E S U G A R F R O M U Y . 2

RD.xylose values

Solvent A B F D

o-Arab inose o.9 ° I. z5 D-Xylose I.OO I.OO I.OO I.OO D-Lyxose I.O6 I .o6 D-Ribose I. I 2 1.33 Sugar f rom UY~ I.OO 0.99 I .oo I.OO

4zz

Xyl

GIc

0

-O • 0 o- - - ~ i g I I i i i , O t ~

D-xylose k-xwtose sugor D-xylose k-xylose sugar enzyme Authentic from from blonk Authentic

+ enzyme untreotecl

Fig. 5. Decompos i t ion of t he xylose isolated f rom UYz by Lactobacillus pentosus. Paper ch roma to - g r a m in Solvent A is shown. " + e n z y m e " : each suga r (o . i /2mole in Ioo/ t l ) was i ncuba t ed wi th 5o/xl of Lac tobac i l lus cell suspens ion and 50/zl of 0.2 M p h o s p h a t e buffer (pH 7.4) a t 37 °. Af te r 90 min , 2oo #1 of e thano l were added, and the m i x t u r e was hea t ed to boiling. Af te r cent r i fugat ion , t he s u p e r n a t a n t solut ion was evapo ra t ed to d ryness by aera t ion and t h e n appl ied to paper . The spo t s ind ica ted by arrows are due to endogenous mater ia l s in the bacter ia l ceils. Abbrev ia t ions : Xy], xy lose , Glc, glucose.

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412 A. KOBATA, S. ZIRO

composed of equimolar amounts of galactose and glucosamine plus galactosamine. By planimetry, it was found that the sugar moiety of pig's milk nucleotide contained approx, o.o 9 mole galactosamine and o.8 mole glucosamine per mole galactose.

Identification of UDP-D-xylose (UY2) By rechromatography (sodium formate linear gradient system), almost all

the 26o-m# absorbing substance was eluted as a single peak, the absorption spectrum of which was similar to that of uridine in o.I N HC1 and in o.I N KOH. The molar ratio of uridine, total phosphate and acid-labile phosphate was 1.oo:2.o1:o.96. On paper electrophoresis (E-I and E-2 system), the substance migrated as one spot a little faster than UDP-N-acetylglucosamine. After hydrolysis in o.oi N HC1 for 2o min at 95-98°, UDP was liberated with release of 0.9o mole reducing power. The UDP was found to be uridine 5'-pyrophosphate by the same examination as used for UDP-N-acetyllactosamine. This reducing substance was negative to MORGAN--ELSON reagent but was positive to alkaline AgNO3, periodate-benzidine reagent and p-anisi- dine reagent. With the last reagent, the reducing sugar gave a red color indicating that it was a pentose. By an orcinol test 19, it was shown that the nucleotide contained I mole pentose other than the ribose moiety of nucleotide per mole uridine. Finally the pentose was identified as xylose by paper chromatography using four different solvent systems (Table I I I ) . This xylose was shown to have the D-configuration by using Lactobacillus pentosus (Fig. 5; see also the experimental section). These results indicate that the nucleotide is UDP-D-xylose.

DISCUSSION

The evidence of high species specificity in nucleotide constituents of milk has already been stressed in the preceding paper 5. The results of this study add further evidence of the specificity. Pig's milk has the same nucleotide pat tern as pig's colostrum, and in this respect is similar to human milk. But its pat tern is more like that of cow's colostrum, sheep's milk and mare 's milk, the main constituents of which are UDP-sugars.

DENAMUR et al. 2° reported that pig's milk is rich in GDP-fucose, but in our experiments GDP-fucose was not detected. We cannot explain this discrepancy.

Pig's milk contains UDP-N-acetyllactosamine which was initially found in human milk. But the experimental results show that this nucleotide fraction of pig's milk is contaminated with about IO % of galactosamine-containing nucleotide. All efforts to isolate this minor component from UDP-N-acetyllactosamine were in vain, indicating that the minor component has a similar structure to UDP-N-acetyllactos- amine, probably UDP-N-acetylgalactosamine (4 <-- i) galactose. The negative reaction of the disaccharide moiety to the MORGAN--ELSON assay supports this configuration. The occurrence of UDP-D-xylose in pig's milk is the first finding that the nucleotide also exists in the animal kingdom. Cow's colostrum and goat 's milk also contain this nucleotide ~1. This nucleotide was isolated from mung bean seedlings by GINSBURG et al. 2s, and its biosynthetic routes were thoroughly studied by FEINGOLD et al. 24. On the other-hand, even the occurrence of D-xylose itself in mammals had long been doubted. But recently, LINDAHL AND RODt~N 2a reported that mucopolysaccharides such as heparin and chondroitin sulfate exist naturally in coniugate form with protein

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NUCLEOTIDES OF PIG'S MILK 4 1 3

t h r o u g h t h e x y l o s e - s e r i n e l i n k a g e . T h i s e v i d e n c e s u g g e s t s t h a t U D P - D - x y l o s e m a y

p l a y a n i m p o r t a n t ro l e i n t h e b i o g e n e s i s o f m u c o p o l y s a c c h a r i d e s .

ACKNOWLEDGEMENTS

The authors are indebted to Dr. K. KANAZAWA and Dr. E. 0HMURA for their encouragement. The authors also wish to express their thanks to Dr. D. NIWA of the National Institute of Animal Industry M.A.F. for the collection of pig's milk and colostrum, to Dr. S. IGARASHI for 3'-nucleotidase, to Mr. I. SUHARA for 5'-nucleotidase, and to Mr. T. IIDA and Mr. T. ASAI for technical assistance.

R E F E R E N C E S

I R. DENAMUR, G. FAUCONNEAU AND G. GUNTZ, Compt. Rend., 246 (1958) 492. 2 R. DENAMUR, G. FAUCONNEAU AND G. GUNTZ, Compt. Rend., 246 (1958) 652. 3 R. DENAMUR, G. FAUCONNEAU AND G. GUNTZ, Compt. Rend., 246 (1958) 2820. 4 A. DEUTSCI-I AND S. MATTSSON, Alnarp Report, No. 63 (196o) 2. 5 A. KOBATA, SUZUOKI-Z. AND M. KIDA, J. Biochem. Tokyo, 51 (1962) 277. 6 T. JOHKE AND T. GOTO, J. Dairy Sei., 45 (1962) 735- 7 A. DEUTSCH AND R. NILSSON, Z. Physiol. Chem., 321 (196o) 246. 8 A. KOBATA, J. Biochem. Tokyo, 53 (1963) 167- 9 G. W'. JOURDIAN, F. SHIMIZU AND S. ROSEMAN, Federation Proc., 20 (1961) 161.

io A. KOBATA, J. Bioehem. Tokyo, in t he press. I I W. R. C. CRIMMIN, J. Chem. Sou., (1957) 2838. 12 J. o . LAMPEN, J. Biol. Chem., 204 (1953) 999. 13 C. C. SWEELEY, R. BENTLEY, M. MAKITA AND W. W. WELLS, J. Am. Chem. Soc., 85 (1963) 2497. 14 S. ROSEMAN AND J. LUDOWlEG, J. Am. Chem. Sou., 76 (1954) 3Ol. 15 T. YAMAKAWA AND N. UETA, Japan. J. Exptl. Med., 34 (1964) 37. 16 M. R. J. SALTON, Biochim. Biophys. Aura, 34 (1959) 3 °8. 17 S. SuzuKI, J. Biol. Chem., 237 (1962) 1393. 18 J. L. STREMINGER AND M. S. SMITH, J. Biol. Chem., 234 (1959) 1828. 19 W. MEJBAUM, Z. Physiol. Chem., 258 (1939) 117- 20 R. DENAMUR, G. FAUCONNEAU AND J. GUNTZ, Ann. Biol. Animale Biochim. Biophys., I

(1961) 74- 2I A. KOBATA AND 1Y[. TSUOA, unpub l i shed da ta . 22 U. LINDAHL AND L. RODI~N, Biochem. Biophys. Res. Commun., 17 (1964) 254. 23 V. GINSBURG, P. K. STUMPF AND W. Z. HASSID, J. Biol. Chem., 223 (1956) 977. 24 D. S. FEINGOLD, E. F. NEUFELD AND W. Z. HASSID, J. Biol. Chem., 235 (196o) 91o.

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