Isolation of GDP-L -galactose from the albumen gland of Helix pomatia

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Isolation of GDP-L-galactose from the albumen gland of Helix pomatia Tile secretory material of the albumen gland portion of the reproductive system of pulmonate snails contains a galactose polymer, galactogen, which accumulates in large quantities and is subsequently deposited with the eggs I. Galactogen is composed of 86 % D-galactose and 14 % L-galactose 2. Previous work has shown that UDPD-galactose is the precursor of the D-galactose residues of this polysaccharide a. We have now undertaken to examine the origin of tile L-galactose residues. The present communication describes the isolation of GDP-L-galactose from albumen glands. This sugar nucleotide has previously been found by Su AND HASSlD4 in the red alga, Porphyra perforata, where it was postulated to be an intermediate in the synthesi~ of the L-galactose residues of galactan. Helix pomatia was collected from a field in Jackson, Michigan, a colony of this strictly European species having been introduced into Jackson about 194o. One hundred albumen glands (I5o g wet wt.) obtained from freshly killed snails, were frozen in liquid nitrogen, transferred to IO vol. of 8o % ethanol and homogenized in a high-speed blendor. The homogenate was boiled for IO min, filtered through ce!ite, defatted with 5 vol. of chloroform-methanol (2:1, v/v) and concentrated to 5o ml under reduced pressure. The extract, containing 75 ° absorbance units (measured at 260 m/~), was placed on a column of Dowex-I formate resin (8 % cross-linked, 2o0-4oo mesh, 20 cm ~ 1.8 cm). Elution was carried out with the ammonium formate system described b y HURLBERT5 with a mixer volume of 5oo ml. An ultraviolet absorbing peak which occurred between 5oo ml and 69 ° ml after changing the reservoir liquid to 1.6 M ammonium formate was adsorbed on to acid-washed Norit A and eluted with ioo ml of 5 ° %, ethanol containing o.i ml of concentrated NH4OH. The eluate was concentrated, applied in a 7-inch strip to the origin of W h a t m a n 3 MM paper, and purified by chromatography in e t h a n o l - I M ammonium acetate, p H 3.8, (75:3o, v/v) for 2o h. Since GDP-galactose and GDP-mannose are inseparable under these conditions 4, the area with the chromatographic mobility of GDP-mannose was eluted from the paper. It had an ultraviolet spectrum identical to that of guanosine. An aliquot, subjected to high-w)ltage electrophoresis in o.05 M citrate buffer (pH 4-5) (8o volts/cm, 3o rain) migrated as a single spot, again with the mobility of GDPmannose. As shown in Fig. 1, paper chromatography of the remainder of the material in isobutyric a c i d - I M NH4OH (lO:6, v/v) for 52 h resulted in the separation from GDP-mannose of an ultraviolet absorbing spot (Fraction I) which ultimately proved to contain GDP-L-galactose. This area was eluted from the paper, yielding o.21 ~mole of nucleotide. Its ultiaviolet absorption spectrum was identical to that of guanosine at p H 7 and p H I. Chemical analysis showed that the nucleotide contained, per ~mole of guanosine; 1.96/~moles of phosphate 6, and o.97/,mole of reducing sugar 7 liberated b y hydrolysis at pH 2 and calculated as galactose. In order to identify the reducing sugars present, aliquots of Fraction I were brought to p H 2 by the addition of HCI, hydrolyzed 15 min at IOO° and then deionized by passage through a small column (i cm :~ o.5 cm) of Amberlite MB-3. The sugars were analyzed by paper chromatography, paper electrophoresis, and gas-liquid chromatography as shown in Fig. 2. On the basis of these three criteria, the preBiochim. Biophys. dcta, i 2 t (t966) I 9 2 - I 9 5

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dominant sugar component was shown to be galactose. Sugars with the chromatographic mobility of mannose and glucose were also present in trace amounts. Characterization of the galactose component of the sugar nucleotide as the L-isomer was first indicated by its unreactivity in the n-galactose oxidase test (D-galactose:oxygen oxidoreductase, EC 1.1.3.9) (Table I). Positive evidence that the sugar was L-galactose was obtained by its conversion to a keto-sugar in the presence

0 :o ¢) Z

Fig. 1. C h r o m a t o g r a p h i c s e p a r a t i o n of G D P - L - g a l a c t o s e (Fraction I) f r o m G D P - m a n n o s e . G D P D-[14Clmannose (2. IO-3/~mole, 15oo c o u n t s / m i n ) w a s a d d e d as m a r k e r , a n d r a d i o a c t i v i t y located w i t h a strip scanner. T h e c h r o m a t o g r a m was developed in isobutyric acid - i M N H 4 O H (IO : 6, v/v) for 5 2 h a n d n u c l e o t i d e s located b y u l t r a v i o l e t light absorption.

L-qOloCtose

\ ilO Retention

20

30

I

time (min)

Fig. 2. A. H i g h - v o l t a g e p a p e r electrophoresis (80 V per cm) in 0.05 M s o d i u m t e t r a b o r a t e (pH 9.2) for 30 min. T h e s u g a r s were visualized w i t h silver n i t r a t e 11. (a) glucose, (b) galactose, (c) fucose, (d) m a n n o s e , (e) r h a m n o s e ; stds, s t a n d a r d sugars. B. P a p e r c h r o m a t o g r a p h y in b u t a n o l - p y r i d i n e w a t e r (6 : 4 : 3, b y vol.) for 2o h. Sugars were visualized w i t h silver n i t r a t e 11. (a) r h a m n o s e , (b) fucose, (c) m a n n o s e , (d) glucose a n d (e) galactose. C. G a s - l i q u i d c h r o m a t o g r a p h y . T h e t r i m e t h y l s i l y l d e r i v a t i v e s of 5-1o # g of s u g a r s a m p l e were p r e p a r e d according to t h e m e t h o d of SWEELEY et al. TM. T h e i n s t r u m e n t used was t h e F a n d M Model 16o9, w i t h a h y d r o g e n flame ionization detector. T h e column, 6 ft. b y o.25 in. o u t e r diameter., p a c k e d w i t h 3 % SE-52 silicone p o l y m e r as t h e liquid phase, was o p e r a t e d a t 14 °0 . N i t r o g e n was used as t h e carrier gas. R e a s o n s for t h e incomplete equilibration of t h e c¢ a n d fl a n o m e r s of F r a c t i o n I h y d r o l y s a t e are u n k n o w n . However, it m a y be d u e to t h e t h o r o u g h deionization of t h e sample. T h e a u t h o r s are i n d e b t e d to Dr. J. PLIMMER for g u i d a n c e in this analysis. Biochim: Biophys. Acta. 121 (1966) 1 9 2 - I 9 5

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of L-fucose i s o m e r a s e ( D - a r a b i n o s e k e t o l - i s o m e r a s e , E C 5.3.1.3). T h i s e n z y m e h a d b e e n s h o w n b y GREEN AND COHEN s t o b e specific f o r s u g a r s w i t h t h e L-fucose c o n f i g u r a t i o n a t C-2, C-3 a n d C-4, s u c h as L-fucose, L - g a l a c t o s e , D - a r a b i n o s e , a n d D-altrose. I t p r o v e d t o b e i n a c t i v e w i t h D - g a l a c t o s e , D- a n d L - m a n n o s e , a n d D-glucose, w h e n 0.05 ° / , m o l e of t h e s e s u g a r s w a s i n c u b a t e d u n d e r t h e c o n d i t i o n s d e s c r i b e d in T a b l e I. TABLE I IDENTIFICATION

OF

L-GALACTOSE

IN

FRACTION

Substrate added

i~mole substrate converted by (t*mole)

D-galactose oxidase*

L-f~tcoSe isomerase* *

o-Galactose

O.OLO o.o5o

O.OLO o.o5o

o.ooo o.ooo

L-Galactose

o.o io 0.o50

o.ooo o.ooo

o.oo7 0.035

Fraction I hydrolysate* * *

o.o16

o.ooo §

0.008

* For the assay of D-galactose by galactose oxidase ~, the "galactostat" reagents (Worthington Biochemical Corp.) were used. Suggested volumes were reduced so that the absorbance obtained from the oxidation of O.OLO/~nlole of D-galactose was o. 135. * * The enzyme was prepared according to the method of GREEN AND COHEN8 from Escherichia coli Bal~ (a gift from Dr. S. S. COHEN) grown in a medium containing 0.8 mg per ml of D-glucose and 0.2 mg per ml of D-arabinose as the sole carbon source. The enzyme extract was treated with solid ammonium sulfate (4.36 g per IO ml extract). The precipitate was redissolved in distilled water, dialyzed against water for 2o h, and finally centrifuged at 40000 rev./min for I h in a Spinco Model L ultracentrifuge. The reaction mixture contained in 0.075 ml: substrate as indicated, 0. 5/mlole of sodium tetraborate (pH 8.0) and 0.06 ml of Spinco supernatant fluid. Control incubations lacking enzyme or substrate were set up concurrently. The tubes were incubated for 5 h at 37 °. At the end of this time the complete reaction mixtures were tested for the presence of ketose TM. The assay was scaled down five-fold so that O.OLO/,mole of tagatose, used as a standard, had an absorbance of 0.270 at its maxinlum, 560 m/,. *** Hydrolysis at pH 2, as described in text. § The oxidation of O.OLOtmaole of D-galactose was not inhibited by the addition of o.o16 lmlole of Fraction I hydrolysate. A s c a n b e s e e n in T a b l e I, o n e - h a l f of t h e t o t a l r e d u c i n g s u g a r of F r a c t i o n I w a s c o n v e r t e d t o a k e t o s e , p r e s u m a b l y t a g a t o s e ; if t h i s v a l u e is c o r r e c t e d for t h e i n c o m p l e t e (7o%) c o n v e r s i o n of a u t h e n t i c L - g a l a c t o s e , o n e c a n c a l c u l a t e t h a t Lg a l a c t o s e c o m p r i s e d 75 % of tile s u g a r m o i e t y of t h e n u c l e o t i d e . T h u s , o.16 t~mole of G D P - L - g a l a c t o s e h a s b e e n i s o l a t e d f r o m 15o g of H. pomatia a l b u m e n g l a n d s . T h e b i o s y n t h e s i s a n d s u b s e q u e n t r e a c t i o n s of t h i s c o m p o u n d are u n d e r i n v e s t i g a t i o n . O n e of t h e a u t h o r s ( E . M . G . ) is a P o s t d o c t o r a l F e l l o w of t h e U n i t e d S t a t e s Public H e a l t h Service.

National Institute of Arthritis and Metabolic Diseases, National Institutes of Health, Bethesda, Md. (U.S.A.)

ESTHER M. GOUDSMIT ELIZABETH F. NEUFELI)

I F. MAY, Z. Biol., 92 (1932) 325. 2 D. J. BELL AND E. BALDWIN, J. Chem. Soe., (1941) 125. 3 E. GOUDSMIT AND G. ASHWELL, Bioehem. Biophys. Res. Commun., 19 (1965) 417.

Biochim. Biophys. Acta, 121 (1966) 192-195

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4 J. C. Su AND W. Z. HASSID, Biochemistry, I (1962) 474. 5 R. H. HURLBERT, in S. P. COLOWICK AND •. O. KAPLAN, Methods in Enzymology, Vol. 3, Academic Press, New York, 1957, p. 796. 6 B. N. AMES AND D. T. DUBIN, j . Biol. Chem., 235 (196o) 769 . 7 J- T. PARK AND M. J. JOHNSON, J. Biol. Chem., 181 (1949) 149. 8 M. GREEN AND S. S. COHEN, J. Biol. Chem., 219 (1956) 557. 9 G. AVIGAD, D. AMARAL, C. ASENSIO AND B. L. HORECKER, j . Biol. Chem., 237 (1962) 2736. io Z. DlSCHE AND E. BORENFREUND, j . Biol. Chem., 192 (1951) 583 . i i E. F. L. J. ANET AND T. M. REYNOLDS, Nature, 172 (1953) 1188. 12 C. C. SVv'EELEY, R. BENTLEY, M. MAKITA AND W. W. WELLS, j . Am. Chem. Soc., 85 (1963) 2497.

Received January 26th, 1966 Biochim. Biophys. Aeta, 121 (1966) 192-195

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Oridine-diphosphoglucose-glucosyltransferase in human leukemic leukocytes Recently, increased amounts of uridine diphosphoglucose (UDPG) and uridine diphosphoacetylglucosamine in acute and chronic myelocytic leukemic leukocytes have been found 1. Since UDPG is the immediate precursor to glycogen, studies were initiated to investigate any possible correlation between glycogen and UDPG-glucosyltransferase (UDP glucose:~-I,4-glucan ~-4-glucosyltransferase, EC 2.4.1.11) activity in various human leukemic leukocytes. UDPG-glucosyltransferase assay. Leukocytes from approx. 30 ml of venous blood were separated as previously described 1. The method of LEL()IR AND GOLDEMBERG was used ~. The leukocytes were diluted with o.5-1.o ml of sucrose (0.25 M) containing EDTA (o.ooi M) and sonicated for I ½ min in a Raytheon (Model S-Io2A) sonic oscillator. The assay was performed within 4 h because of the rapid loss of activity in spite of storage at --80 °. The activity of the enzyme is expressed in /,moles of UDP formed per ml of homogenate per min. The specific activity is expressed: activity/mg protein. Protein concentration in the extract was measured by the method of LowRY3. The following were purchased from Sigma Chemical Co., St. Louis, Mo. : glycogen (from shellfish), glycine, glucose 6-phosphate, cysteine, UDPG, phospho(enol)pyruvic acid (tricyclohexylamine salt, crystalline), pyruvic kinase enzyme. Glycogen assay. The glycogen assay was modified from JOHNSON AND FUSARO~. Leukocytes were prepared and sonicated as described above. I.O ml of trichloroacetic acid (5 %) was added to 0. 5 ml of leukocyte extract and homogenized for 5 min; the precipitated protein was removed by centrifugation. To the supernatant, redistilled absolute ethanol was added to a 80 % concentration and allowed to stand at room temperature for 48 h. The precipitated glycogen was collected by centrifugation, redissolved in I.O ml distilled water, and thoroughly mixed on a Vortex mixer for 15 min. Dilutions of I/iO, 1/15, 1/2o were made. The standard glycogen solution contained 40/,g/ml. The remainder of the procedure was carried out exactly by the method of JOHNSON AND FUSARO. Diazyme was obtained from the Miles Chemical Co., N.J. The glucose oxidase and peroxidase were obtained from the Sigma Chemical Co., St. Louis, Mo. UDPG-glucosyltransferase and glycogen content were assayed in 6 normal individuals, 16 with chronic myelocytic leukemia, 8 with acute myeloblastic leukemia, Biochim. Biophys. Acta, 121 (1966) 195-197