- Email: [email protected]
Galactosyl transfer reactions catalysed by pneumococcal α-galactosidase
ARCHIVES
OF
BIOCHEMISTRY
Galactosyl
AND
BIOPHYSICS
Transfer
301-313
108,
Reactions
(1964)
Catalysed
by
Pneumococcal
c+Galactosidase’ YU-TEH From
the Department
LI
&!I. R. SHETLAR
AND
of Biochemistry, Oklahoma
University of Oklahoma City, Oklahoma
Received
School of Medicine,
June 18, 1964
It was found that the a-galactosidase isolated from culture broths of Diplococcus pneumoniae synthesized both reducing and nonreducing oligosaccharides through transferring reactions. For synthesizing reducing oligosaccharides, the enzyme preparation transfers a-n-galactosyl groups from suitable oligosaccharides to various acceptors forming linkages which are predominately or-(1 + 6), but other a-linkages are also formed. Incubation of melibiose with pneumococcal a-galactosidase produced 2 nonreducing and 7 reducing oligosaccharides as detected by paper chromatography. One of the nonreducing oligosaccharides contained only galactose; the other contained both galactose and glucose. Among the reducing oligosaccharides, 4 were identified as galactobiose, galactotriose, manninotriose, and verbascotetraose. Three others were tentatively identified as isomers of galactotriose, manninotriose, and verbascotetraose. When raffinose was incubated with this enzyme, galactobiose, galactotriose, stachyose, verbascose, and 2 sucrose-containing oligosaccharides were formed. These results were confirmed by using sugars labeled with Cd. When melibiose was incubated with the enzyme in the presence of n-glucose-l-Cl4, rapid incorporation of radioactivity into melibiose was found. Similarly, radioactive raffinose was formed from enzymic digests of raffinose in the presence of sucrose-Un-galactose, sucrose, o-mannose, n-fructose, n-glucosamine, D04. D-Glucose galactosamine, ‘2-deoxy-n-galactose, maltose, lactose, cellobiose, D- and L-arabinose, D- and L-xylose, n-lyxose, and n-ribose were able to function as acceptors for the WDgalactosyl group. INTRODUCTION
Oligosaccharides containing one or more a-n-galactosyl groups in their structures are widely distributed in the plant kingdom. These n-galactosyl groups are commonly found in nature joined to sugars such as n-glucose, n-galactose, sucrose, to certain polysaccharides, and to a few nonsugars such as glycerol and inositol (1). In spite of this wide distribution of a-galactosides, a-galactosidases are relatively unexplored. ar-Galactosidase activity has been reported to be present in yeast, filamentous fungi, plant seeds, and the digestive tract of some 1 This investigation was supported from the National Institute of Health
by a grant (CA 03081).
animals (2-4). Bailey has reported the presence of a-galactosidase activity in a rumen strain of Streptococcus bovis and in a rumen ciliate, Epidinium ecaudatum (5, 6). Howhave ever, none of these preparations been studied in the purified state. Furthermore, the galactosyl transfer reactions catalyzed by these enzymes have not been studied in detail. In a previous communication (7), we reported the isolation and purification of an a-galactosidase from the culture broth of Diplococcus pneumoniae. This enzyme was also found to catalyze transfer as well as hydrolytic reactions, forming oligosaccharides with a-(1+6)galactosidic linkages. Further investigation, 301
302
LI AND
as reported below, has revealed that the pneumococcal a-galactosidase preparation synthesizes both reducing and nonreducing oligosaccharides by transfer reactions. The enzyme preparation also transfers a-ngalactosyl groups to suitable acceptors forming linkages other than the a-(1+6) linkage. MATERIALS
AND
METHODS
Chemicals. Melibiose, raffinose, 2-deoxy-nand 3-O-methyl-n-glucopyranose were glucose, purchased from California Corporation for Biochemical Research, Los Angeles, California. Stachyose, D- and L-fucose, and o-sorbitol were from Sigma Chemical Company, St. Louis, Missouri. n-Glucose-l-phosphate, n-galactose-l-phosphate, dulcitol, and 2-deoxy-o-galactose were the products of Nutritional Biochemical Corporation, Cleveland, Ohio. Planteose was kindly provided by Dr. Dexter French, Department of Biochemistry and Biophysics, Iowa State University, Ames, Iowa. Galactobiose2 was a gift of Dr. Roy L. Whistler, Department of Biochemistry, Purdue University, Lafayette, Indiana. Verbascose was kindly made available by Dr. R. W. Bailey, Plant Chemistry Division, Department of Scientific and Industrial Research, New Zealand. Manninotriose was prepared by partial acid hydrolysis of stachyose as described by French (1). o-Glucosel-04 (5.3 mc per mmole), n-galactose-l-Cl4 (4.8 mc per mmole), sucrose-Cl4 (3.1 mc per mmole, uniformly labeled), n-glucosamine-l-Cl4 (8.3 mc per mmole), and n-galactosamine-l-Cl4 (2.1 mc per mmole) were products of New England Nuclear Corporation, Boston, Massachusetts. n-Mannosel-Cl4 (3.3 mc per mmole), n-fructose-C14 (4.2 mc per mmole, uniformly labeled), lactose-l-C4 (1.5 mc per mmole), and maltose-l-Cl4 (0.1 mc per mmole) were also obtained from California Corporation for Biochemical Research, Los Angeles, California. Preparation of a-galactosidase. The enzyme preparation used in this study was first purified in large quantities from culture broths of a strain of D. pneumoniae (type VI) isolated from patient material (7). The procedure, utilizing (NH3 $04 fractionation, heat treatment, and absorption on red blood cells, has been described previously (8). This partially purified enzyme was further purified by filtering it through a Sephadex G-200 column. The Sephadex was prepared by suspending 5 gm of Sephadex G-200 in about 500 ml of imidazole buffer pH 6.2, r/2 = 0.15 left at 4°C overnight. The gel suspension was then transferred 2 Galactobiose in this manuscript 60-a-n-galactopyranosyl-n-galactose.
refers
to
SHETLAR to a chromatographic tube (2 X 30 cm). A 25.cm high column was obtained by sedimentation; 2 ml of the pneumococcal a-galactosidase prepared as described above (contained 50 mg protein with specific activity of 90 units of a-galactosidase per milligram protein) was applied on the top and allowed to filter (the unit of enzyme is defined in the previous paper) (8). The column was then eluted at 2 ml per hour with imidazole buffer, pH 6.2, p/2 = 0.15. Two-ml fractions were collected, and the protein of each fraction was determined by the method of Lowry et al. (9). The cr-galactosidase activity of these fractions was determined by using o-nitrophenyl-a-n-galactopyranoside as a subst,rate as described previously (8). It was found that or-galactosidase activity emerged in the first protein peak. The active fractions were pooled, being concentrated by ultrafiltration through Visking cellophane tubing under 40 lb pressure, and were used as the enzyme source in the subsequent experiments. By this procedure, the specific activity increased 3 times, and the yield of enzyme activity was about BOoYe.cu-Galactosidase prepared by this procedure was found to be free from neuraminidase, @-galactosidase, p-glucosaminidase, @-fructosidase, 01- and @glucosidase, and phosphatase. When the purified a-galactosidase was subjected to analytical ultracentrifugation, two sedimentation peaks were found, indicating that the preparation was not homogeneous. Work is in progress to prepare larger amounts of the enzyme in order to allow further purification. Assay for galactosyl transfer reaction. Galactosyl transfer reaction of pneumococcal a-galactosidase was examined by the following procedure. Substrate and enzyme were dissolved in imidazole buffer pH 6.2, p/2 = 0.15, as previously described (8). Enzyme solution (0.25 ml contains 150 units) was incubated with 0.25 ml of a solution containing 0.5 M substrate or, in some instances, 0.5 M acceptor. The incubation was maintained at 40°C. At various times the enzyme was inactivated by heating in a boiling water bath for 5 minutes. After removal of precipitated protein by centrifugation, 5 or 10 ~1 of the reaction mixture was applied to Whatman No. 1 paper (50 cm in length), and the products were resolved by descending paper chromatography using the solvent system, n-butanol-pyridine-O.l N HCl (5:3:2 by vol.). Better resolution of the newly formed oligosaccharides was obtained by using a multiple-development technique, with three successive developments. Analytical methods. Ammonical AgN03, benaidine trichloroacetic acid, alkaline triphenyl tetrazolium chloride, and diphenylamine-anilinephosphate (10) were used to locate reducing
GALACTOSYL
TRANSFER
REACTIONS
303
sugars. The 2.aminobiphenyl reagent of Gordon et al. (11) and the periodate-permanganatebeneidine reagent of Wo!from and Miller (12) were used to detect both reducing and nonreducing oligosaccharides. The chromatogram was heated at 110” for 30 minutes after staining with a-aminobiphenyl. Fructose-containing oligosaccharides were detected by spraying the chromatogram with naphthoresorcinol
(10).
The
monosaccharide
composition of the oligosaccharides was quantitatively
determined
by the methods
Girgis (13), after hydrolysis
of Khadem
and
of oligosaccharides
with 2th N HCI for 4 hours. The radioactivity of the sugars was detected on the chromatograms by a Forro windowless gas-flow counter probe. Radio-
autograms were prepared by placing chromatograms in contact with Kodak No-Screen X-ray film for 3-7 weeks. RESULTS
OLIGOSACCHARIDES PRODUCED IN MELIBIOSE DIGESTS Figure 1 is a chromatogram showing production of 8 new areas staining with 2aminobiphenyl when melibiose was incubated with pneumococcal a-galactosidase. These areas were designated as compounds A, B, C, D, E, F, G, and H according to their mobilities. Of these 8 areas, A, B, C,
D, E, and F were also readily detected by ammonical AgN03 and benzidine reagents, alkaline triphenyltetrazolium chloride and diphenylamine-aniline-phosphate reagents, suggesting that these are reducing compounds. Compounds G and H fail to react with the above reagents for reducing sugar, but react slowly with 2-aminobiphenyl and periodate-permanganate-benzidine reagents, indicating that these may be nonreducing sugars. In order to prove that these new spots were actually
formed
by galactose
transfer
reaction and not from the degradation products of glucose, galactose, or enzyme itself, 150 units of enzyme were incubated with 0.5 ml of 0.25 M n-glucose, n-galactose, or mixtures of D-galactose and n-glucose, respectively, under conditions similar to those used for melibiose, for 1, 2, and 4 hours. After the incubation, 10 ~1 of the mixture was applied to Whatman No. 1 paper and resolved with the solvent system described above, for the detection of any
FIG. 1. Paper chromatogram of products in digests of melibiose with pneumococcal a-galactosidase. 8, Standard; Gl, n-glucose; Ga, n-galactose; M, melibiose; Ma, manninotriose. 0.25 ml solution of melibiose (0.5 M) was incubated with 0.25 ml enzyme solution containing 150 units of a-galactosidase. The paper chromatogram was stained with 2-aminobiphenyl reagent and heated at 110°C for 30 minutes.
areas which would react with silver nitrate, benzidine, and 2-aminobiphenyl reagents. No new spots were detected in such incubation mixtures. These results also suggest that the pneumococcal cr-galactosidase is not, able to catalyze the synthesis of oligosaccharides directly from monosaccharides. When the digestion of melibiose was continued for up to 24 hours, all of the oligosaccharides produced in the earlier stage of
304
LI AN11 SHETLAR
the incubation disappeared. Glucose and galactose were the only two sugars that could be detected on the paper chromatogram (8). These results indicated that the new spots which appeared in the chromatogram as shown in Fig. 1 were oligosaccharides resulting from glycosyl transfer reaction. The monosaccharide composition of these oligosaccharide areas is presented in Table I. If one considers the acceptors for galactose in the incubation mixture, melibiose and water are the only two acceptors available in the first stages. When water acts as an acceptor, the product will be the ordinary hydrolysis. If melibiose acts as an acceptor, the result will be the formation of an oligosaccharide containing 2 moles of galactose and 1 mole of glucose. This oligosaccharide may, in turn, serve as an acceptor to form new oligosaccharides. If the enzyme transfers the galactose moiety to form cY-(l-+6)-galactosidic linkages, manninotriose may be formed from melibiose, verbascotetraose from manninotriose, galactobiose from galactose, and melibiose from glucose. In support of this postulation, compounds C and F of the melibiose digests had mobilities on paper chromatograms of manninotriose and galactobiose, respectively (Figs. 1 and 3). Compound C was found in the largest quantities at the earliest stage in incubation and contained galactose and TABLE
I
MONOSACCHARIDE COMPOSITION OF NEW OLIGOSACCHARIDES PRODUCED IN THE ENZYMIC DIGEST OF MELIBIOSE Monosaccharide composition
Compounds
Rglucoso
A B C D E F 0 H
0.036 0.068 0.122 0.162 0.234 0.315 0.473 0.631
“t”$
a Not determined.
+ + + + + + + +
Glucose
+ + + + +
Molar ratio, Galactose:Glucose
3.2:1 2.9:1 2.4:1 1.8:1
-(I
Glucose termination Goloctose termination
3CStochyose)
\ I I (Verboscose
I 0
I I I I 2 3 Goloctose units per terminal sugar
FIG. 2. Paper chromatographic mobilities of some of the oligosaccharides produced in the melibiose and raffinose digests. Logarithms of R giucose of various oligosaccharides were plotted against the homolog number (expressed as number of galactose per terminal sugar). (-A-A-) Oligosaccharides produced in melibiose digests with glucose as terminal; (-O-O-) oligosaccharides produced in the raffinose digest with sucrose as terminal; (-X-X-) oligosaccharides contained only galactose.
glucose in a ratio of approximately 2: 1 (Table I). This compound was therefore concluded to be manninotriose synthesized from the melibiose by acceptance of 1 mole of galactose. Compound A contained galactose and glucose in ratio of approximately 3: 1; this suggests that this oligosaccharide was formed from manninotriose (compound C) by acceptance of one mole of galactose. By plotting the logarithm of the Rglueose of glucose, melibiose, compound C (manninotriose) and compound A, against the molecular size according to the method of French et al. (14, 15), a straight line was obtained (Fig. 2). This indicates that these 4 sugars belong to homologous series. In this case the series has a glucose residue at the reducing end, and increases in size by
GALACTOSYL
TRANSFER
progressive addition of galactosyl residues linked ~(1-6). Compound A is the tetraose in this series and is therefore concluded to be verbascotetraose, which would be formed from manninotriose (compound C) by acceptance of 1 mole of galactose. This compound was found to have the R, ruoose of authentic verbascotetraose on paper chromatograms. As shown in Table I, the monosaccharide composition of compounds E and B were similar to manninotriose and verbascotetraose, respectively, which may indicate that these two oligosaccharides are isomers of manninotriose and verbascotetraose, respectively. When compound E was partially hydrolyzed with 0.2 N HCl at 106” for 2 hours and then subjected to paper chromatography, two new spots were found with mobilities in the disaccharide range. One of these spots had the mobility of melibiose; the other moved slightly faster than melibiose and contained only galactose. This disaccharide reacted with 2,3,5triphenyl tetrazolium chloride as described by Avigad et al. (16), indicating that this disaccharide was not 2-O-a-n-galactopyranosyl-n-galactopyranose. It was therefore tentatively concluded that compound E is a trisaccharide in which one galactose is attached to the galactose moiety of melibiose through an a-(1+3) or (r-(1-+4) linkage. By similar reasoning, compound B might be a tetrasaccharide in which one galactose is attached to manninotriose through an a-(1-+3) or a-(1+4) linkage. Compound H contained both glucose and galactose. Since this compound was nonreducing by the tests described above and had the fastest mobility of all the oligosaccharides formed, it was tentatively concluded that this compound is a nonreducing disaccharide formed from glucose by the acceptance of 1 mole of galactose during the galactose transfer reaction. The synthesis of nonreducing oligosaccharide by a transfer reaction has not been previously observed. Generally, transfers of glycosyl units do not occur to the anomeric carbon. Further work is in progress to clarify this point. Compounds D, I’, and G contained only
REACTIONS
305
galactose. Among these three compounds, compounds F and G had chromatographic mobilities in the disaccharide region, and the mobility of compound D was in the trisaccharide region. Compound F was found to have the same chromatographic mobility as authentic galactobiose (Fig. 3), which would be formed from galactose by acceptance of 1 mole of galactose transferred from melibiose. Compound G was nonreducing; this compound was concluded
FIG. 3. Enzymic digestion of melibiose in the presence of D-galactose. 0.25 ml solution of melibiose (0.5 M) and n-galactose (0.5 J4) was incubated with 0.25 ml of enzyme solution containing 150 units of a-galactosidase. S, Standard; Gl, D-glucose; Ga, D-galactose; M(ordinate), melibiose; Gb, galactobiose; Ma, manninotriose. M(absissa), melibiose was incubated with a-galactosidase. M + Ga, melibiose was digested with cu-galactosidasein the presence of D-galactose.
306
LI AND SHETLAli
to be a nonreducing disaccharide which was also synthesized from galactose by acceptance of 1 mole of galactose during the galactose transfer reactions. To study further the formation of compounds G, F, and D, 0.25 ml of a solution of melibiose (0.5 M) and galactose (0.5 M) was incubated with 0.25 ml of or-galactosidase (150 units), and the rate of the formation of compounds G, F and D was compared with the formation of these compounds in melibiose digests without addition of galactose. If these compounds are synthesized by transferring galactose directly to galactose, the formation of compounds G, F, and D should be increased when a large amount of galactose was added to the incubation mixture to serve as an acceptor. The result of this experiment is presented in Fig. 3. Compounds D, F, and G were synthesized in larger amounts when melibiose was digested in the presence of galactose. The increase in synthesis of compound F was most prominent. The formation of the other oligosaccharides was retarded when melibiose was digested with galactose, indicating that galactose competes with other acceptors (i.e., melibiose, manninotriose, etc.) for acceptance of galactose. It was therefore concluded that compounds F, G, and D are formed from galactose through acceptance of 1 or 2 moles of galactose, respectively, during the transferring reaction. When the logarithm of the Rgiucose of galactose, compound G (galactobiose) and compound D are plotted against its molecular size, a straight-line relationship is not found. This indicates that compound D is not the trisaccharide of the homologous series which includes galactobiose. When compound D was subjected to partial acid hydrolysis with 0.2 N HCI at 100” for 2 hours and chromatographed on paper as described above, two disaccharide spots appeared together with that of galactose. One of these two disaccharides had a chromatographic mobility identical to galactobiose, while the other spot moved slightly faster than melibiose. This faster moving disaccharide reacted with 2,3,5-triphenyl tetrazolium chloride, indicating that this
disaccharide was not 2-O-cr-n-galactopyranosy]-n-galactopyranose, as in the case of the faster moving disaccharide produced by partial acid hydrolysis of compound E. It was therefore concluded that compound D is a trisaccharide in which one galactose is attached to galactobiose through an (Y-(1 -+3) or a-(1+4) linkage. Since galactobiose was synthesized rapidly and in such quantity, the acceptance of 1 mole of galactose by galactobiose to synthesize galactotriose (0 - a -D - galp - (l-+6) - 0 - (Y -D - galp (I-6)n-galp) would be expected. It was found that, when the logarithm of Rgiucose of galactose, galactobiose, and compound C were plotted against its homolog number, a straight line relationship was obtained (Fig. 2). Compound C was found by analysis to contain both galactose and glucose and had the paper chromatographic mobility of manninotriose. However, the molar ratio of galactose to glucose was higher than 2:1(2.4:1), as indicated in Table I. This high galactose to glucose ratio might imply contamination of manninotriose with galactotriose in compound C. ENZYMIC DIGESTION OF MELIBIOSE IN THE PRESENCE OF .' D-GALACTOSE-l-C'4
In order to test this hypothesis of the interrelationship of galactose, compounds F, G, D, and galactotriose, an isotope labeling experiment was utilized. If compounds F, G, and D were synthesized from galactose or galactobiose as described above, these three compounds should be labeled when melibiose is incubated with cr-galactosidase in the presence of n-galactose-1-CY4. Furthermore, if galactotriose was produced which overlapped the manninotriose area (compound C), the manninotriose area should be labeled. The result of this experiment is presented in part of Fig. 4. When 0.25 ml of solution of melibiose (0.5 M) and n-galactose-l-Cl4 (1 PC) was incubated with 0.25 ml of enzyme containing 150 units cr-galactosidase for 2 hours and then analyzed by paper chromatography, the radioautogram as shown in Fig. 4 resulted. Compounds F and G were heavily labeled. There was also a definite labeling of
GALACTOSYL
TRANSFER
REACTIONS
307
compound D. As predicted by plotting logarithm of RRiucoss of galactose, galactobiose, and galactotriose according to their homolog number as described before, a significant labeling spot (compound B’) was located at the lower tip of the manninotriose spot. This spot was tentatively identified as galactotriose synthesized by transfer of galactose to galactobiose. ENZYMIC DIGESTION OF MELIBIOSE IN THE PRESENCE OF D-GLucOSE-~-C'~
In order to investigate the reversibility of enzyme action catalyzed by galactosyl transfer reaction, 0.25 ml of solution of melibiose (0.5 M) and n-glucose-l-Cl4 (1 PC) was incubated with 0.25 ml of enzyme containing 150 units of cu-galactosidase at 40’ for 2 hours. Chromatograms and radioautograms were prepared as described above. Examination of the radioautogram (Fig. 5) revealed that the melibiose was heavily labeled, indicating that glucose was capable of accepting galactose from melibiose to form melibiose. Significant labeling of compounds C and E and some labeling of compounds A, B, and H was found. Some labeling of compound G was also detected in this experiment. This could be due to the contamination of the glucosel-C4 used by galactose-1-C4. The n-glucosel-Cl4 used in this experiment was later found to contain approximately 5% of radioactive galactose and 10% of an unknown radioactive substance with a mobility faster than glucose. FIG. 4. A paper chromatogram (A) and a radioautograph (B) of eneymic digest of melibiose and raffinose in the presence of n-galactose-l-C14. S, Standard; Gl, u-glucose; Ga, n-galactose; Su, sucrose; M, melibiose; Gb, galactobiose; R, raffinose; Ma, manninotriose; St, stachyose; M + Ga*, 0.25 ml solution of melibiose (0.5 M) and n-galactose-l-C4 (1 PC) was incubated with 0.25 ml enzyme solution containing 150 units a-galactosidase; R + Ga*, 0.25 ml solution of raffinose (0.5 M) and n-galactose-l-C14 (1 ~c) was incubated with 0.25 ml of enzyme solution containing 150 units of a-galactosidase. Incubation was maintained at 40°C for 2 hours.
OLIGOSACCHARIDES PRODUCED IN RAFFINOSE DIGESTS
Studies were made of the products produced by the enzymic digestion of raffinose. At least 5 new oligosaccharides in the raffinose digests were detected by paper chromatography (Fig. 6). These oligosaccharrides were named as compounds 1, 2, 3, 4, and 5 according to their mobilities. Compound 3 was formed most rapidly in large quantities and had an Rciuoose corresponding to that of authentic stachyose. It was tentatively concluded that this compound is stachyose, formed from raffinose by the
308
LI AND
SHETLAR
FIG. 6. Paper chromatogram of products in digests of raffinose with pneumococcal cu-galxtosidase. S, Standard; Gl, n-glucose; Ga, D-galactose; Su, sucrose; M, melibiose; R, raffinose; St, stachyose. 0.25 ml solution of raffinose (0.5 M) was incubated with 0.25 ml enzyme solution containing 150 units of a-galactosidase.
FIG. 5. A paper ehromatogram (A) and a radioautograph (B) of enzymic digest of melibiose in the presence of D-glucose-l-U4 and enzymic digest of raffinose in the presence of n-glucose and D-glucose-l-W. S, Standard; Gl, n-glucose; Ga, n-galactose; M, melibiose; Gb, galactosbiose; R, raffinose; Ma, manninotriose; St, stachyose. M + Gl*, 0.25 ml solution of melibiose (0.5 M) and n-glucose-l-Cl4 (1 ~c) was incubated with 0.25 ml enzyme solution containing 150 units a-galactosidase; R + Gl + Gl*, 0.25 ml of solution of raffinose (0.5 M), n-glucose (0.5 M) and n-glucose-l-C’4 (1 PC) was incubated with 0.25 ml of enzyme solution containing 150 units of a-galactosidase. Incubation was maintained at 40°C for 2 hours.
acceptance of 1 molecule of galactose. This conclusion was strengthened by plotting the logarithms of R, luoose of sucrose, raffinose, compound 3, and compound 1 against the homolog numbers (Fig. 2). A straight line was obtained suggesting that these 3 oligosaccharides belong to the homologous series with sucrose as the terminal sugar and increasing in number by the progressive addition of galactose. It was therefore concluded that compound 1 is verbascose, formed from compound 3 (stachyose) by the acceptance of 1 molecule of galactose. Compound 1 was later found to have the R Rlucose of authentic verbascose. When compound 4 was hydrolyzed with 0.1 N
CALACTOSYL
TRANSFER
HCl for 5 minutes at loo”, fructose and a spot with mobility identical to compound E of the melibiose digests were found. Quantitative analysis of the monosaccharide composition of this spot revealed that it contained galactose and glucose in a ratio of 2: 1. This result strongly suggested that compound 4 was also synthesized from raffinose by the acceptance of galactose; however, the galactose was joined to the galactose moiety of raffinose with a linkage other than a-(1+6) as was the case in the synthesis of compounds D and E. Compound 2 contained galactose, glucose, and fructose, and had a mobility slightly higher than compound 1. This compound was tentiatively assumed to be an isomer of compound 1 which was synthesized from stachyose by acceptance of 1 molecule of galactose. This observation was further supported by the following experiment. When the raflinose digest was hydrolyzed with 0.1 N HCl at 100” for 15 minutes, to preferentially hydrolyze the fructosyl residues of the various oligosaccharides, it was found that the pattern of the residual as obtained by paper oligosaccharides, were essentially similar chromatography, to that of the melibiose digest. This would suggest that compounds 1, 2, 3, and 4 (Fig. 6) of the raffinose digest were converted into compounds A, B, C, and E (Fig. 1) by dilute acid hydrolysis. Melibiose and galactobiose were also detected after hydrolysis with the dilute acid. Further proof of the presence of galactobiose in the raffinose digest will be discussed later. Compound 5 contained only galactose. This compound failed to react with reagents for reducing sugar, indicating that this compound was nonreducing. This compound had the chromatographic mobility of a disaccharide. It was therefore concluded that compound 5 is a nonreducing disaccharide which was formed from galactose. Compound 5 was identical to compound G in the melibiose digest (Fig. 4). Upon careful examination of the paper chromatogram as shown in Fig. 6, raffinose digests were found to contain a spot with a mobility corresponding to that of melibiose. This spot was first
REACTIONS
309
considered to be due to the contamination of the enzyme with ,&fructosidase which would split off fructose from raffinose to produce melibiose. However, it was found that the cu-galactosidase preparation used in this study was not able to hydrolyze sucrose. When the chromatogram was stained with naphthoresorcinol for fructose, this spot also gave a positive test, indicating that this spot contained fructose. This compound contained equimolar amounts of fructose, glucose, and galactose. This suggests that this compound may be an isomer of raffinose which was synthesized from sucrose by acceptance of 1 molecule of galactose. The possibility that this compound might be a planteose was ruled out because planteose had the same chromatographic mobility as raflinose in the solvent systems used. ENZYMIC DIGESTION OF RAFFINOSE THE PRESENCE OF IFGLUCOSE
IN
As described above, cr-galactosidase apparently transfers a-galactose from melibiose to glucose-l-Cl4 to form radioactive melibiose. A similar study with raffinose was made by incubation of 0.25 ml solution of raffinose (0.5 M) and n-glucose (0.5 M) with 0.25 ml of enzyme containing 150 units of a-galactosidase. If melibiose were synthesized, one would also anticipate the production of oligosaccharides corresponding to compounds C, E, and H as described above. The result of this experiment is presented in Fig. 7. After 2 hours of incubation, 4 additional chromatographic areas were produced from this digest as compared to the original raffinose digests; these areas were designated as 3’, 4’, 4!‘, and 5’. These 4 additional areas failed to react with naphthoresorcinol reagent. suggesting that they did not contain sucrose. All of the compounds produced were reducing except compounds 5 and 5’. Compound 4” was produced in very large quantities after a short period of incubation. This compound was found to have the Rgiucoseof authentic melibiose. It was therefore concluded that compound 4” is melibiose formed from the added glucose by acceptance of 1 molecule
310
LI AND
FIG. 7. Paper chromatogram of enzymic digest of raffinose in the presence of n-glucose. 0.25 ml solution of raffinose (0.5 M) and n-glucose (0.5 M) was incubated with 0.25 ml enzyme solution containing 150 units of ol-galactosidase. S, Standard; Gl, n-glucose; Ga, n-galactose; Su, sucrose; M, melibiose; R, raffinose; St, stachyose.
of galactose from r&nose. Compound 3’ contained both galactose and glucose, and had the Rglueoseof manninotriose (Fig. 5). Compound 4’ had the mobility of compound E of the melibiose digest. Compound 5’ was nonreducing and contained both glucose and galactose. This compound corresponded to compound H of the melibiose digest. ENZYMIC DIGESTION OF RAFFINOSE IN THE PRESENCE OF D-GALaCTOSE-l-C'4
As it was not possible to detect the formation of galactobiose or galactotriose in the raffinose digest due to overlapping of the spots, an effort was made to demonstrate
SHETLAR
the presence of these two compounds in the raffinose digest by use of n-galactose-lC14.When 0.25 ml of the solution of raffinose (0.5 M) and n-galactose-l-Cl4 (1 PC) was incubated with 0.25 ml of enzyme con taining 150 units of a-galactosidase at 40” for 2 hours, compounds 5, 4, 3, and raffinose spots were labeled as shown in Fig. 4. The labeling of the raffinose area did not mean that raffinose itself was actually labeled, as galactobiose overaps raffinose in this area. The raffinose digests were subjected to preparative chromatography; the raffinose areas were eluted and subjected to hydrolysis with 0.1 N HCl at 100” for 15 minutes to preferentially hydrolyze the fructosyl residues present in rafhnose. The hydrolyzate was then rechromatographed. The result indicated that the hydrolyzate con tained fructose, melibiose, and an area corresponding either to galactobiose or raffinose. However, this spot did not react with naphthoresorcinol and was radioactive, suggesting that this compound was not raffinose but galactobiose. Radioautograms indicated that compounds 3 and 4 were also radioactive. By comparison of the R,,,,,,, produced in the melibiose digests (Fig. 4), it was noted that compound 3 of the raffinose digest corresponds to compound B’ (galactotriose) and compound 4 to compound D. As shown in Fig. 4, the radioautogram of melibiose and rafhnose digests agree in regard to compounds 3 (B’) and 4 (D), although the original chromatogram from the melibiose and raffinose digest have overlapping in these areas. ENZYMIC DIGESTION OF RAFFINOSE IN THE PRESENCE OF D-GLUCOSE AND D-GLuCOSE-~-C'~
To indicate further that compounds 3’, 4’, 4!‘, and 5’ of the raffinose digest in the presence of glucose were actually formed from glucose, 0.25 ml of a solution of raffinose (0.5 M), n-glucose (0.5 L%?),and D-&Icase-l-C4 (1 PC) was incubated with 0.25 ml of enzyme solution containing 150 units of cr-galactosidase. After incubation at 40” for 2 hours, the distribution of radioactivity in the oligosaccharides formed was studied as previously described. As indi-
GALACTOSYL
TRANSFER
cated in Fig. 5, compound 4” (melibiose) was most heavily labeled; compounds 3’, 4’, and 5’ were also labeled. ENZYMIC DIGESTION OF RAFFINOSE IN THE PRESENCE OF SUCROSE-U-Cl4
In order to determine whether sucrose is able to accept galactose to form raffinose, 0.25 ml of a solution of raffinose (0.5 M) and sucrose-U-Cl4 (1 PC) was incubated with 0.25 ml of enzyme solution containing 150 units of a-galactosidase. After incubation at 40” for 30 minutes, the enzymic reaction was stopped and analyzed for the distribution of radioactivity in the various oligosaccharides. As indicated in Fig. 8, after 30 minutes of incubation, raffinose was heavily
FIG. 8. A paper chromatogram (A) and a radioautograph (B) of enzymic digest of raffinose in the presence of sucrose-U-C’4. S, Standard; Ga, u-galactose; M, melibiose; R, raffinose; St, stachyose. 0.25 ml solution of raffinose (0.5 M) and sucrose-Ii-CP (1 PC) was incubated at 40°C for 30 minutes.
REACTIONS
311
labeled, suggesting that the reaction was reversible due to the transfer of galactose to sucrose to form raffinose. At this stage, compounds 3, 4, and the compound with the R, lueoae identical to that of melibiose were also labeled, indicating that all of these compounds contained sucrose. ACCEPTOR SPECIFICITY OF PNEUMOCOCCAL CY-GALACTOSIDASE
Acceptor specificity of various monosaccharides, disaccharides, and hydroxyl amino acids was tested by incubating 0.25 ml of substrate (0.5 M melibiose or raffinose) and acceptor (0.5 M) with 0.25 ml of enzyme solution containing 150 units of 01galactosidase. Incubation was carried out at 40” for 2 hours; the new oligosaccharides produced were analyzed by paper chromatography as described above. When Dglucosamine, n-galactosamine, n-fructose, n-mannose, maltose, and lactose were tested for acceptor specificity, 1 PC of Dglucosamine-I-CY4, n-galactosamine-1-CY4, Dfructose-U-CY4, n~altose-l-C14, and lactosel-CY4, respectively, were incubated together with the acceptor. The incorporation of radioactivity in the new oligosaccharides produced was also checked by using a Forro windowless gas-flow counter probe. When glucosamine, galactosamine, and hydroxyamino acids were used as acceptors, the chromatograms were also sprayed with ninhydrin to locate new amino compounds. u-Mannose, n-fructose, n-glucosamine, Dgalactosamine, 2-deoxy-n-galactose, n-ribose, D- and L-arabinose, D- and L-xylose, n-lyxose, lactose, maltose, and cellobiose were found to be able to accept galactose. When n-mannose and cellobiose were used as acceptors, two new oligosaccharides were produced. The rest of the compounds produced only one new oligosaccharide; the structure of these new oligosaccharides was not determined. 2-Deoxy-n-glucose, 3 - 0 - methyl - D - glucose, D - galactose l-phosphate, n-galactose-6-phosphate, Dglucose-l-phosphate, n-glucose-6-phosphate, D- and L-fucose, u-glucuronic acid, Dgalacturonic acid, sorbitol, dulcitol, Dmannitol, inositol, nn-serine, nn-threonine, and L-hydroxyproline were not able to
312
LI AND
accept galactose under the condition scribed.
de-
DISCUSSION
Pneumococcal oc-galactosidase incubated with melibiose results in eight new spots staining with 2-aminobiphenyl as detected by paper chromatography. Discovery of more spots than those previously reported (8) is apparently due to the improvement made in chromatographic techniques. In the present study, better separation of various oligosaccharides produced in the enzymic digests was achieved by employing multiple development of the chromatogram. In addition, heating the chromatogram for 30 minutes rather than 5 minutes after staining with the 2-aminobiphenyl reagent leads to increased sensitivity. This modification is particularly effective in demonstrating nonreducing disaccharides. Although manninotriose, galactobiose, and galactotriose have been reported to be synthesized in the melibiose digests through galactosyl transfer reaction catalyzed by yeast, emulsin or streptococcal oc-galactosidase (1, 3, 5, 17, 18), pneumococcal a-galactosidase seems to have much greater transferring capacity than ol-galactosidases of other origins. Pneumococcal ol-galactosidase used in this study transfers a-n-galactosyl group to primary and secondary carbinol sites, as well as to anomeric sites of n-galactose and n-glucose. The production of nonreducing as well as reducing oligosaccharides in the melibiose and raffinose digests is of particular interest. Although the enzyme transfers the n-galactosyl group from an oligosaccharide to n-glucose, to n-galactose, and to sucrose to form linkages other than CZ-(l-+6), formation of the a-(1-+6) linkage appears to be favored, since compounds with this linkage occur more abundantly. The preferential transfer of the galactosyl residue to the primary alcohol group of the sugar molecule is similar to the galactosyl transfer reaction catalyzed by ,&galactosidase (19, 20). Although these two enzymes have different specificities toward the anomeric configuration of the galactosides, they are similar in their pattern of galactosyl transfer.
SHETLAR
The synthesis of oligosaccharide often proceeds through phosphorylation mechanisms by which phosphorylated sugars function as intermediates. The possibility that the galactosyl transfer reaction catalyzed by pneumococcal a-galactosidase is a type of phosphorolysis is unlikely for the following reasons: (1) the reaction proceeded in phosphate-free medium, (2) there was no evidence of the presence of phosphorylated intermediates in the melibiose and raffinose digests, and (3) the enzyme did not synthesize oligosaccharides from n-galactose-l-phosphate and suitable acceptors (i.e., n-g1ucose or n-galactose). Wallenfels et al. (3) have shown that in addition to hydrolytic and transferring activity, p-galactosidase is able to catalyze the synthesis of glycosides from free sugars and alcohols. However, no synthesis of oligosaccharides occurred from free sugars by pneumococcal cr-galactosidase. Glycosyl transfer reaction may be described as an anomeric substitution reaction. In some instances, the reaction brings about an inversion of the glycosidic linkage as in the case of maltose phosphorylase (21) and cellobiose phosphorylase (22). The enzyme preparation used in this study is free from p-galactosidase. The fact that the oligosaccharides produced in the earlier stages of incubation disappeared after prolonged incubation indicates that these oligosaccharides belong to the a-n-galactosidic series. This leads to the conclusion that galactose transfer reactions catalyzed by this enzyme preparation do not cause the inversion of the galactosidic linkage. When melibiose was incubated with the enzyme in the presence of n-glucose-1-C4, rapid incorporation of radioactivity into melibiose was found. Similarly, radioactive raffinose was obtained from enzymic digests of raffinose in the presence of sucrose-UCn. By incubation of raffinose in the presence of n-glucose to serve as an acceptor, melibiose was synthesized. A similar labeling pattern has been found in a galactosyl transfer reaction catalyzed by yeast ,& galactosidase using allolactose (B-O-P-Dgalactopryranosyl-n-glucose) as a substrate (23).
GALACTOSYL
TRANSFER
From the experimental results it can be concluded that n-glucose, n-galactose, sucrose, melibiose, and raffinose are potent acceptors of the a-n-galactosyl group. Although n-mannose, n-fructose, n-glucosamine, n-galactosamine, maltose, cellobiose, lactose, and all of the pentoses tested may also serve as acceptors, they are less reactive than the above-mentioned sugars. Since phosphorylation, reduction or oxidation of either the 1 or 6 position of n-glucose or n-galactose destroyed the function to serve as an acceptor, it may indicate that both the anomeric carbon and the Cc of n-glucose and n-galactose are important for accepting a galactosyl group. It is of interest to note that 2-deoxy-n-galactose, but not 2-deoxy-D-glucose, can serve as an acceptor. Methylation of C1 of n-glucose also resulted in destruction of the ability to serve as an acceptor. REFERENCES Chem. 9, 1. FRENCH, D., Advan. Carbohydrate 149 (1954). 2. PIGMAN, W. W., Advan. Enzymol. 4, 41 (1944). 3. WALLENFELS, K., AND MALHOTRA, 0. P., Advan. Carbohydrate Chem., 16, 239 (1961). 4. BANKS, W. M., Science 141, 1191 (1963). 5. BAILEY, R. W., Biochem. J. 86, 509 (1963). 6. BAILEY, R. W., AND HOWARD, B. H., Biochem. J. 87, 146 (1963). 7. LI, Y. T., AND SHETLAR, M. R., Proc. Sot. Exptl. Biol. Med. 106, 398 (1961).
REACTIONS
313
8. LI, Y. T., LI, S. C., AND SHETLAR, M. R., Arch. Biochem. Biophys. 108, 436 (1963). 9. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., AND RANDALL, R. J., J. Biol. Chem. 193, 265 (1951). 10. BLOCK, R. J., DURRUM, E. L., AND ZWEIG, G., “A Manual of Paper Chromatography and Paper Electrophoresis,” 2nd edition, pp. 178-188. Academic Press, New York, 1958. 11. GORDON, H. T., THORNBURG, W., AND WERUM, L. N., Anal. Chem. 28, 849 (1956). 12. WOLFROM, M. L., AND MILLER, J. B., Anal. Chem. 28, 1037 (1956). 13. KHADEM, H. E., AND GIRGIS, W., Anal. Chem. 33, 645 (1961). 14. FRENCH, D., AND KNAPP, 1~. W., J. BioZ. Chem. 187, 463 (1950). 15. FRENCH, D., AND WILD, G. M., J. Am. Chem. Sot. 76, 2612 (1953). 16. AVIGAD, G., ZELIKSON, R., AND HESTRIN, S., Biochem. J. 80, 57 (1961). 17. COURTOIS, J. E., Proc. Intern. Congr. Biochem., 4th, Vienna, 1958 Vol. I, p. 140 (1959). Macmillan (Pergamon), New York. 18. DEDONDER, R. A., Ann. Rev. Biochem. 30, 347 (1961). 19. WALLENFELS, K., BERNT, E., AND LIMBERG, G., Ann. Chem. 679, 113 (1953). 20. PAZUR, J. H., J. Biol. Chem. 208, 439 (1954). 21. FITTING, C., AND DOUDOROFF, M., J. Biol Chem. 199, 153 (1952). 22. AYERS, W. A., J. Biol. Chem. 234, 2819 (1959). 23. PAZUR, J. H., MARSH, J. M., AND TIPTON, C. L., J. Sm. Chem. Sot. 80, 1433 (1958).
OF
BIOCHEMISTRY
Galactosyl
AND
BIOPHYSICS
Transfer
301-313
108,
Reactions
(1964)
Catalysed
by
Pneumococcal
c+Galactosidase’ YU-TEH From
the Department
LI
&!I. R. SHETLAR
AND
of Biochemistry, Oklahoma
University of Oklahoma City, Oklahoma
Received
School of Medicine,
June 18, 1964
It was found that the a-galactosidase isolated from culture broths of Diplococcus pneumoniae synthesized both reducing and nonreducing oligosaccharides through transferring reactions. For synthesizing reducing oligosaccharides, the enzyme preparation transfers a-n-galactosyl groups from suitable oligosaccharides to various acceptors forming linkages which are predominately or-(1 + 6), but other a-linkages are also formed. Incubation of melibiose with pneumococcal a-galactosidase produced 2 nonreducing and 7 reducing oligosaccharides as detected by paper chromatography. One of the nonreducing oligosaccharides contained only galactose; the other contained both galactose and glucose. Among the reducing oligosaccharides, 4 were identified as galactobiose, galactotriose, manninotriose, and verbascotetraose. Three others were tentatively identified as isomers of galactotriose, manninotriose, and verbascotetraose. When raffinose was incubated with this enzyme, galactobiose, galactotriose, stachyose, verbascose, and 2 sucrose-containing oligosaccharides were formed. These results were confirmed by using sugars labeled with Cd. When melibiose was incubated with the enzyme in the presence of n-glucose-l-Cl4, rapid incorporation of radioactivity into melibiose was found. Similarly, radioactive raffinose was formed from enzymic digests of raffinose in the presence of sucrose-Un-galactose, sucrose, o-mannose, n-fructose, n-glucosamine, D04. D-Glucose galactosamine, ‘2-deoxy-n-galactose, maltose, lactose, cellobiose, D- and L-arabinose, D- and L-xylose, n-lyxose, and n-ribose were able to function as acceptors for the WDgalactosyl group. INTRODUCTION
Oligosaccharides containing one or more a-n-galactosyl groups in their structures are widely distributed in the plant kingdom. These n-galactosyl groups are commonly found in nature joined to sugars such as n-glucose, n-galactose, sucrose, to certain polysaccharides, and to a few nonsugars such as glycerol and inositol (1). In spite of this wide distribution of a-galactosides, a-galactosidases are relatively unexplored. ar-Galactosidase activity has been reported to be present in yeast, filamentous fungi, plant seeds, and the digestive tract of some 1 This investigation was supported from the National Institute of Health
by a grant (CA 03081).
animals (2-4). Bailey has reported the presence of a-galactosidase activity in a rumen strain of Streptococcus bovis and in a rumen ciliate, Epidinium ecaudatum (5, 6). Howhave ever, none of these preparations been studied in the purified state. Furthermore, the galactosyl transfer reactions catalyzed by these enzymes have not been studied in detail. In a previous communication (7), we reported the isolation and purification of an a-galactosidase from the culture broth of Diplococcus pneumoniae. This enzyme was also found to catalyze transfer as well as hydrolytic reactions, forming oligosaccharides with a-(1+6)galactosidic linkages. Further investigation, 301
302
LI AND
as reported below, has revealed that the pneumococcal a-galactosidase preparation synthesizes both reducing and nonreducing oligosaccharides by transfer reactions. The enzyme preparation also transfers a-ngalactosyl groups to suitable acceptors forming linkages other than the a-(1+6) linkage. MATERIALS
AND
METHODS
Chemicals. Melibiose, raffinose, 2-deoxy-nand 3-O-methyl-n-glucopyranose were glucose, purchased from California Corporation for Biochemical Research, Los Angeles, California. Stachyose, D- and L-fucose, and o-sorbitol were from Sigma Chemical Company, St. Louis, Missouri. n-Glucose-l-phosphate, n-galactose-l-phosphate, dulcitol, and 2-deoxy-o-galactose were the products of Nutritional Biochemical Corporation, Cleveland, Ohio. Planteose was kindly provided by Dr. Dexter French, Department of Biochemistry and Biophysics, Iowa State University, Ames, Iowa. Galactobiose2 was a gift of Dr. Roy L. Whistler, Department of Biochemistry, Purdue University, Lafayette, Indiana. Verbascose was kindly made available by Dr. R. W. Bailey, Plant Chemistry Division, Department of Scientific and Industrial Research, New Zealand. Manninotriose was prepared by partial acid hydrolysis of stachyose as described by French (1). o-Glucosel-04 (5.3 mc per mmole), n-galactose-l-Cl4 (4.8 mc per mmole), sucrose-Cl4 (3.1 mc per mmole, uniformly labeled), n-glucosamine-l-Cl4 (8.3 mc per mmole), and n-galactosamine-l-Cl4 (2.1 mc per mmole) were products of New England Nuclear Corporation, Boston, Massachusetts. n-Mannosel-Cl4 (3.3 mc per mmole), n-fructose-C14 (4.2 mc per mmole, uniformly labeled), lactose-l-C4 (1.5 mc per mmole), and maltose-l-Cl4 (0.1 mc per mmole) were also obtained from California Corporation for Biochemical Research, Los Angeles, California. Preparation of a-galactosidase. The enzyme preparation used in this study was first purified in large quantities from culture broths of a strain of D. pneumoniae (type VI) isolated from patient material (7). The procedure, utilizing (NH3 $04 fractionation, heat treatment, and absorption on red blood cells, has been described previously (8). This partially purified enzyme was further purified by filtering it through a Sephadex G-200 column. The Sephadex was prepared by suspending 5 gm of Sephadex G-200 in about 500 ml of imidazole buffer pH 6.2, r/2 = 0.15 left at 4°C overnight. The gel suspension was then transferred 2 Galactobiose in this manuscript 60-a-n-galactopyranosyl-n-galactose.
refers
to
SHETLAR to a chromatographic tube (2 X 30 cm). A 25.cm high column was obtained by sedimentation; 2 ml of the pneumococcal a-galactosidase prepared as described above (contained 50 mg protein with specific activity of 90 units of a-galactosidase per milligram protein) was applied on the top and allowed to filter (the unit of enzyme is defined in the previous paper) (8). The column was then eluted at 2 ml per hour with imidazole buffer, pH 6.2, p/2 = 0.15. Two-ml fractions were collected, and the protein of each fraction was determined by the method of Lowry et al. (9). The cr-galactosidase activity of these fractions was determined by using o-nitrophenyl-a-n-galactopyranoside as a subst,rate as described previously (8). It was found that or-galactosidase activity emerged in the first protein peak. The active fractions were pooled, being concentrated by ultrafiltration through Visking cellophane tubing under 40 lb pressure, and were used as the enzyme source in the subsequent experiments. By this procedure, the specific activity increased 3 times, and the yield of enzyme activity was about BOoYe.cu-Galactosidase prepared by this procedure was found to be free from neuraminidase, @-galactosidase, p-glucosaminidase, @-fructosidase, 01- and @glucosidase, and phosphatase. When the purified a-galactosidase was subjected to analytical ultracentrifugation, two sedimentation peaks were found, indicating that the preparation was not homogeneous. Work is in progress to prepare larger amounts of the enzyme in order to allow further purification. Assay for galactosyl transfer reaction. Galactosyl transfer reaction of pneumococcal a-galactosidase was examined by the following procedure. Substrate and enzyme were dissolved in imidazole buffer pH 6.2, p/2 = 0.15, as previously described (8). Enzyme solution (0.25 ml contains 150 units) was incubated with 0.25 ml of a solution containing 0.5 M substrate or, in some instances, 0.5 M acceptor. The incubation was maintained at 40°C. At various times the enzyme was inactivated by heating in a boiling water bath for 5 minutes. After removal of precipitated protein by centrifugation, 5 or 10 ~1 of the reaction mixture was applied to Whatman No. 1 paper (50 cm in length), and the products were resolved by descending paper chromatography using the solvent system, n-butanol-pyridine-O.l N HCl (5:3:2 by vol.). Better resolution of the newly formed oligosaccharides was obtained by using a multiple-development technique, with three successive developments. Analytical methods. Ammonical AgN03, benaidine trichloroacetic acid, alkaline triphenyl tetrazolium chloride, and diphenylamine-anilinephosphate (10) were used to locate reducing
GALACTOSYL
TRANSFER
REACTIONS
303
sugars. The 2.aminobiphenyl reagent of Gordon et al. (11) and the periodate-permanganatebeneidine reagent of Wo!from and Miller (12) were used to detect both reducing and nonreducing oligosaccharides. The chromatogram was heated at 110” for 30 minutes after staining with a-aminobiphenyl. Fructose-containing oligosaccharides were detected by spraying the chromatogram with naphthoresorcinol
(10).
The
monosaccharide
composition of the oligosaccharides was quantitatively
determined
by the methods
Girgis (13), after hydrolysis
of Khadem
and
of oligosaccharides
with 2th N HCI for 4 hours. The radioactivity of the sugars was detected on the chromatograms by a Forro windowless gas-flow counter probe. Radio-
autograms were prepared by placing chromatograms in contact with Kodak No-Screen X-ray film for 3-7 weeks. RESULTS
OLIGOSACCHARIDES PRODUCED IN MELIBIOSE DIGESTS Figure 1 is a chromatogram showing production of 8 new areas staining with 2aminobiphenyl when melibiose was incubated with pneumococcal a-galactosidase. These areas were designated as compounds A, B, C, D, E, F, G, and H according to their mobilities. Of these 8 areas, A, B, C,
D, E, and F were also readily detected by ammonical AgN03 and benzidine reagents, alkaline triphenyltetrazolium chloride and diphenylamine-aniline-phosphate reagents, suggesting that these are reducing compounds. Compounds G and H fail to react with the above reagents for reducing sugar, but react slowly with 2-aminobiphenyl and periodate-permanganate-benzidine reagents, indicating that these may be nonreducing sugars. In order to prove that these new spots were actually
formed
by galactose
transfer
reaction and not from the degradation products of glucose, galactose, or enzyme itself, 150 units of enzyme were incubated with 0.5 ml of 0.25 M n-glucose, n-galactose, or mixtures of D-galactose and n-glucose, respectively, under conditions similar to those used for melibiose, for 1, 2, and 4 hours. After the incubation, 10 ~1 of the mixture was applied to Whatman No. 1 paper and resolved with the solvent system described above, for the detection of any
FIG. 1. Paper chromatogram of products in digests of melibiose with pneumococcal a-galactosidase. 8, Standard; Gl, n-glucose; Ga, n-galactose; M, melibiose; Ma, manninotriose. 0.25 ml solution of melibiose (0.5 M) was incubated with 0.25 ml enzyme solution containing 150 units of a-galactosidase. The paper chromatogram was stained with 2-aminobiphenyl reagent and heated at 110°C for 30 minutes.
areas which would react with silver nitrate, benzidine, and 2-aminobiphenyl reagents. No new spots were detected in such incubation mixtures. These results also suggest that the pneumococcal cr-galactosidase is not, able to catalyze the synthesis of oligosaccharides directly from monosaccharides. When the digestion of melibiose was continued for up to 24 hours, all of the oligosaccharides produced in the earlier stage of
304
LI AN11 SHETLAR
the incubation disappeared. Glucose and galactose were the only two sugars that could be detected on the paper chromatogram (8). These results indicated that the new spots which appeared in the chromatogram as shown in Fig. 1 were oligosaccharides resulting from glycosyl transfer reaction. The monosaccharide composition of these oligosaccharide areas is presented in Table I. If one considers the acceptors for galactose in the incubation mixture, melibiose and water are the only two acceptors available in the first stages. When water acts as an acceptor, the product will be the ordinary hydrolysis. If melibiose acts as an acceptor, the result will be the formation of an oligosaccharide containing 2 moles of galactose and 1 mole of glucose. This oligosaccharide may, in turn, serve as an acceptor to form new oligosaccharides. If the enzyme transfers the galactose moiety to form cY-(l-+6)-galactosidic linkages, manninotriose may be formed from melibiose, verbascotetraose from manninotriose, galactobiose from galactose, and melibiose from glucose. In support of this postulation, compounds C and F of the melibiose digests had mobilities on paper chromatograms of manninotriose and galactobiose, respectively (Figs. 1 and 3). Compound C was found in the largest quantities at the earliest stage in incubation and contained galactose and TABLE
I
MONOSACCHARIDE COMPOSITION OF NEW OLIGOSACCHARIDES PRODUCED IN THE ENZYMIC DIGEST OF MELIBIOSE Monosaccharide composition
Compounds
Rglucoso
A B C D E F 0 H
0.036 0.068 0.122 0.162 0.234 0.315 0.473 0.631
“t”$
a Not determined.
+ + + + + + + +
Glucose
+ + + + +
Molar ratio, Galactose:Glucose
3.2:1 2.9:1 2.4:1 1.8:1
-(I
Glucose termination Goloctose termination
3CStochyose)
\ I I (Verboscose
I 0
I I I I 2 3 Goloctose units per terminal sugar
FIG. 2. Paper chromatographic mobilities of some of the oligosaccharides produced in the melibiose and raffinose digests. Logarithms of R giucose of various oligosaccharides were plotted against the homolog number (expressed as number of galactose per terminal sugar). (-A-A-) Oligosaccharides produced in melibiose digests with glucose as terminal; (-O-O-) oligosaccharides produced in the raffinose digest with sucrose as terminal; (-X-X-) oligosaccharides contained only galactose.
glucose in a ratio of approximately 2: 1 (Table I). This compound was therefore concluded to be manninotriose synthesized from the melibiose by acceptance of 1 mole of galactose. Compound A contained galactose and glucose in ratio of approximately 3: 1; this suggests that this oligosaccharide was formed from manninotriose (compound C) by acceptance of one mole of galactose. By plotting the logarithm of the Rglueose of glucose, melibiose, compound C (manninotriose) and compound A, against the molecular size according to the method of French et al. (14, 15), a straight line was obtained (Fig. 2). This indicates that these 4 sugars belong to homologous series. In this case the series has a glucose residue at the reducing end, and increases in size by
GALACTOSYL
TRANSFER
progressive addition of galactosyl residues linked ~(1-6). Compound A is the tetraose in this series and is therefore concluded to be verbascotetraose, which would be formed from manninotriose (compound C) by acceptance of 1 mole of galactose. This compound was found to have the R, ruoose of authentic verbascotetraose on paper chromatograms. As shown in Table I, the monosaccharide composition of compounds E and B were similar to manninotriose and verbascotetraose, respectively, which may indicate that these two oligosaccharides are isomers of manninotriose and verbascotetraose, respectively. When compound E was partially hydrolyzed with 0.2 N HCl at 106” for 2 hours and then subjected to paper chromatography, two new spots were found with mobilities in the disaccharide range. One of these spots had the mobility of melibiose; the other moved slightly faster than melibiose and contained only galactose. This disaccharide reacted with 2,3,5triphenyl tetrazolium chloride as described by Avigad et al. (16), indicating that this disaccharide was not 2-O-a-n-galactopyranosyl-n-galactopyranose. It was therefore tentatively concluded that compound E is a trisaccharide in which one galactose is attached to the galactose moiety of melibiose through an a-(1+3) or (r-(1-+4) linkage. By similar reasoning, compound B might be a tetrasaccharide in which one galactose is attached to manninotriose through an a-(1-+3) or a-(1+4) linkage. Compound H contained both glucose and galactose. Since this compound was nonreducing by the tests described above and had the fastest mobility of all the oligosaccharides formed, it was tentatively concluded that this compound is a nonreducing disaccharide formed from glucose by the acceptance of 1 mole of galactose during the galactose transfer reaction. The synthesis of nonreducing oligosaccharide by a transfer reaction has not been previously observed. Generally, transfers of glycosyl units do not occur to the anomeric carbon. Further work is in progress to clarify this point. Compounds D, I’, and G contained only
REACTIONS
305
galactose. Among these three compounds, compounds F and G had chromatographic mobilities in the disaccharide region, and the mobility of compound D was in the trisaccharide region. Compound F was found to have the same chromatographic mobility as authentic galactobiose (Fig. 3), which would be formed from galactose by acceptance of 1 mole of galactose transferred from melibiose. Compound G was nonreducing; this compound was concluded
FIG. 3. Enzymic digestion of melibiose in the presence of D-galactose. 0.25 ml solution of melibiose (0.5 M) and n-galactose (0.5 J4) was incubated with 0.25 ml of enzyme solution containing 150 units of a-galactosidase. S, Standard; Gl, D-glucose; Ga, D-galactose; M(ordinate), melibiose; Gb, galactobiose; Ma, manninotriose. M(absissa), melibiose was incubated with a-galactosidase. M + Ga, melibiose was digested with cu-galactosidasein the presence of D-galactose.
306
LI AND SHETLAli
to be a nonreducing disaccharide which was also synthesized from galactose by acceptance of 1 mole of galactose during the galactose transfer reactions. To study further the formation of compounds G, F, and D, 0.25 ml of a solution of melibiose (0.5 M) and galactose (0.5 M) was incubated with 0.25 ml of or-galactosidase (150 units), and the rate of the formation of compounds G, F and D was compared with the formation of these compounds in melibiose digests without addition of galactose. If these compounds are synthesized by transferring galactose directly to galactose, the formation of compounds G, F, and D should be increased when a large amount of galactose was added to the incubation mixture to serve as an acceptor. The result of this experiment is presented in Fig. 3. Compounds D, F, and G were synthesized in larger amounts when melibiose was digested in the presence of galactose. The increase in synthesis of compound F was most prominent. The formation of the other oligosaccharides was retarded when melibiose was digested with galactose, indicating that galactose competes with other acceptors (i.e., melibiose, manninotriose, etc.) for acceptance of galactose. It was therefore concluded that compounds F, G, and D are formed from galactose through acceptance of 1 or 2 moles of galactose, respectively, during the transferring reaction. When the logarithm of the Rgiucose of galactose, compound G (galactobiose) and compound D are plotted against its molecular size, a straight-line relationship is not found. This indicates that compound D is not the trisaccharide of the homologous series which includes galactobiose. When compound D was subjected to partial acid hydrolysis with 0.2 N HCI at 100” for 2 hours and chromatographed on paper as described above, two disaccharide spots appeared together with that of galactose. One of these two disaccharides had a chromatographic mobility identical to galactobiose, while the other spot moved slightly faster than melibiose. This faster moving disaccharide reacted with 2,3,5-triphenyl tetrazolium chloride, indicating that this
disaccharide was not 2-O-cr-n-galactopyranosy]-n-galactopyranose, as in the case of the faster moving disaccharide produced by partial acid hydrolysis of compound E. It was therefore concluded that compound D is a trisaccharide in which one galactose is attached to galactobiose through an (Y-(1 -+3) or a-(1+4) linkage. Since galactobiose was synthesized rapidly and in such quantity, the acceptance of 1 mole of galactose by galactobiose to synthesize galactotriose (0 - a -D - galp - (l-+6) - 0 - (Y -D - galp (I-6)n-galp) would be expected. It was found that, when the logarithm of Rgiucose of galactose, galactobiose, and compound C were plotted against its homolog number, a straight line relationship was obtained (Fig. 2). Compound C was found by analysis to contain both galactose and glucose and had the paper chromatographic mobility of manninotriose. However, the molar ratio of galactose to glucose was higher than 2:1(2.4:1), as indicated in Table I. This high galactose to glucose ratio might imply contamination of manninotriose with galactotriose in compound C. ENZYMIC DIGESTION OF MELIBIOSE IN THE PRESENCE OF .' D-GALACTOSE-l-C'4
In order to test this hypothesis of the interrelationship of galactose, compounds F, G, D, and galactotriose, an isotope labeling experiment was utilized. If compounds F, G, and D were synthesized from galactose or galactobiose as described above, these three compounds should be labeled when melibiose is incubated with cr-galactosidase in the presence of n-galactose-1-CY4. Furthermore, if galactotriose was produced which overlapped the manninotriose area (compound C), the manninotriose area should be labeled. The result of this experiment is presented in part of Fig. 4. When 0.25 ml of solution of melibiose (0.5 M) and n-galactose-l-Cl4 (1 PC) was incubated with 0.25 ml of enzyme containing 150 units cr-galactosidase for 2 hours and then analyzed by paper chromatography, the radioautogram as shown in Fig. 4 resulted. Compounds F and G were heavily labeled. There was also a definite labeling of
GALACTOSYL
TRANSFER
REACTIONS
307
compound D. As predicted by plotting logarithm of RRiucoss of galactose, galactobiose, and galactotriose according to their homolog number as described before, a significant labeling spot (compound B’) was located at the lower tip of the manninotriose spot. This spot was tentatively identified as galactotriose synthesized by transfer of galactose to galactobiose. ENZYMIC DIGESTION OF MELIBIOSE IN THE PRESENCE OF D-GLucOSE-~-C'~
In order to investigate the reversibility of enzyme action catalyzed by galactosyl transfer reaction, 0.25 ml of solution of melibiose (0.5 M) and n-glucose-l-Cl4 (1 PC) was incubated with 0.25 ml of enzyme containing 150 units of cu-galactosidase at 40’ for 2 hours. Chromatograms and radioautograms were prepared as described above. Examination of the radioautogram (Fig. 5) revealed that the melibiose was heavily labeled, indicating that glucose was capable of accepting galactose from melibiose to form melibiose. Significant labeling of compounds C and E and some labeling of compounds A, B, and H was found. Some labeling of compound G was also detected in this experiment. This could be due to the contamination of the glucosel-C4 used by galactose-1-C4. The n-glucosel-Cl4 used in this experiment was later found to contain approximately 5% of radioactive galactose and 10% of an unknown radioactive substance with a mobility faster than glucose. FIG. 4. A paper chromatogram (A) and a radioautograph (B) of eneymic digest of melibiose and raffinose in the presence of n-galactose-l-C14. S, Standard; Gl, u-glucose; Ga, n-galactose; Su, sucrose; M, melibiose; Gb, galactobiose; R, raffinose; Ma, manninotriose; St, stachyose; M + Ga*, 0.25 ml solution of melibiose (0.5 M) and n-galactose-l-C4 (1 PC) was incubated with 0.25 ml enzyme solution containing 150 units a-galactosidase; R + Ga*, 0.25 ml solution of raffinose (0.5 M) and n-galactose-l-C14 (1 ~c) was incubated with 0.25 ml of enzyme solution containing 150 units of a-galactosidase. Incubation was maintained at 40°C for 2 hours.
OLIGOSACCHARIDES PRODUCED IN RAFFINOSE DIGESTS
Studies were made of the products produced by the enzymic digestion of raffinose. At least 5 new oligosaccharides in the raffinose digests were detected by paper chromatography (Fig. 6). These oligosaccharrides were named as compounds 1, 2, 3, 4, and 5 according to their mobilities. Compound 3 was formed most rapidly in large quantities and had an Rciuoose corresponding to that of authentic stachyose. It was tentatively concluded that this compound is stachyose, formed from raffinose by the
308
LI AND
SHETLAR
FIG. 6. Paper chromatogram of products in digests of raffinose with pneumococcal cu-galxtosidase. S, Standard; Gl, n-glucose; Ga, D-galactose; Su, sucrose; M, melibiose; R, raffinose; St, stachyose. 0.25 ml solution of raffinose (0.5 M) was incubated with 0.25 ml enzyme solution containing 150 units of a-galactosidase.
FIG. 5. A paper ehromatogram (A) and a radioautograph (B) of enzymic digest of melibiose in the presence of D-glucose-l-U4 and enzymic digest of raffinose in the presence of n-glucose and D-glucose-l-W. S, Standard; Gl, n-glucose; Ga, n-galactose; M, melibiose; Gb, galactosbiose; R, raffinose; Ma, manninotriose; St, stachyose. M + Gl*, 0.25 ml solution of melibiose (0.5 M) and n-glucose-l-Cl4 (1 ~c) was incubated with 0.25 ml enzyme solution containing 150 units a-galactosidase; R + Gl + Gl*, 0.25 ml of solution of raffinose (0.5 M), n-glucose (0.5 M) and n-glucose-l-C’4 (1 PC) was incubated with 0.25 ml of enzyme solution containing 150 units of a-galactosidase. Incubation was maintained at 40°C for 2 hours.
acceptance of 1 molecule of galactose. This conclusion was strengthened by plotting the logarithms of R, luoose of sucrose, raffinose, compound 3, and compound 1 against the homolog numbers (Fig. 2). A straight line was obtained suggesting that these 3 oligosaccharides belong to the homologous series with sucrose as the terminal sugar and increasing in number by the progressive addition of galactose. It was therefore concluded that compound 1 is verbascose, formed from compound 3 (stachyose) by the acceptance of 1 molecule of galactose. Compound 1 was later found to have the R Rlucose of authentic verbascose. When compound 4 was hydrolyzed with 0.1 N
CALACTOSYL
TRANSFER
HCl for 5 minutes at loo”, fructose and a spot with mobility identical to compound E of the melibiose digests were found. Quantitative analysis of the monosaccharide composition of this spot revealed that it contained galactose and glucose in a ratio of 2: 1. This result strongly suggested that compound 4 was also synthesized from raffinose by the acceptance of galactose; however, the galactose was joined to the galactose moiety of raffinose with a linkage other than a-(1+6) as was the case in the synthesis of compounds D and E. Compound 2 contained galactose, glucose, and fructose, and had a mobility slightly higher than compound 1. This compound was tentiatively assumed to be an isomer of compound 1 which was synthesized from stachyose by acceptance of 1 molecule of galactose. This observation was further supported by the following experiment. When the raflinose digest was hydrolyzed with 0.1 N HCl at 100” for 15 minutes, to preferentially hydrolyze the fructosyl residues of the various oligosaccharides, it was found that the pattern of the residual as obtained by paper oligosaccharides, were essentially similar chromatography, to that of the melibiose digest. This would suggest that compounds 1, 2, 3, and 4 (Fig. 6) of the raffinose digest were converted into compounds A, B, C, and E (Fig. 1) by dilute acid hydrolysis. Melibiose and galactobiose were also detected after hydrolysis with the dilute acid. Further proof of the presence of galactobiose in the raffinose digest will be discussed later. Compound 5 contained only galactose. This compound failed to react with reagents for reducing sugar, indicating that this compound was nonreducing. This compound had the chromatographic mobility of a disaccharide. It was therefore concluded that compound 5 is a nonreducing disaccharide which was formed from galactose. Compound 5 was identical to compound G in the melibiose digest (Fig. 4). Upon careful examination of the paper chromatogram as shown in Fig. 6, raffinose digests were found to contain a spot with a mobility corresponding to that of melibiose. This spot was first
REACTIONS
309
considered to be due to the contamination of the enzyme with ,&fructosidase which would split off fructose from raffinose to produce melibiose. However, it was found that the cu-galactosidase preparation used in this study was not able to hydrolyze sucrose. When the chromatogram was stained with naphthoresorcinol for fructose, this spot also gave a positive test, indicating that this spot contained fructose. This compound contained equimolar amounts of fructose, glucose, and galactose. This suggests that this compound may be an isomer of raffinose which was synthesized from sucrose by acceptance of 1 molecule of galactose. The possibility that this compound might be a planteose was ruled out because planteose had the same chromatographic mobility as raflinose in the solvent systems used. ENZYMIC DIGESTION OF RAFFINOSE THE PRESENCE OF IFGLUCOSE
IN
As described above, cr-galactosidase apparently transfers a-galactose from melibiose to glucose-l-Cl4 to form radioactive melibiose. A similar study with raffinose was made by incubation of 0.25 ml solution of raffinose (0.5 M) and n-glucose (0.5 M) with 0.25 ml of enzyme containing 150 units of a-galactosidase. If melibiose were synthesized, one would also anticipate the production of oligosaccharides corresponding to compounds C, E, and H as described above. The result of this experiment is presented in Fig. 7. After 2 hours of incubation, 4 additional chromatographic areas were produced from this digest as compared to the original raffinose digests; these areas were designated as 3’, 4’, 4!‘, and 5’. These 4 additional areas failed to react with naphthoresorcinol reagent. suggesting that they did not contain sucrose. All of the compounds produced were reducing except compounds 5 and 5’. Compound 4” was produced in very large quantities after a short period of incubation. This compound was found to have the Rgiucoseof authentic melibiose. It was therefore concluded that compound 4” is melibiose formed from the added glucose by acceptance of 1 molecule
310
LI AND
FIG. 7. Paper chromatogram of enzymic digest of raffinose in the presence of n-glucose. 0.25 ml solution of raffinose (0.5 M) and n-glucose (0.5 M) was incubated with 0.25 ml enzyme solution containing 150 units of ol-galactosidase. S, Standard; Gl, n-glucose; Ga, n-galactose; Su, sucrose; M, melibiose; R, raffinose; St, stachyose.
of galactose from r&nose. Compound 3’ contained both galactose and glucose, and had the Rglueoseof manninotriose (Fig. 5). Compound 4’ had the mobility of compound E of the melibiose digest. Compound 5’ was nonreducing and contained both glucose and galactose. This compound corresponded to compound H of the melibiose digest. ENZYMIC DIGESTION OF RAFFINOSE IN THE PRESENCE OF D-GALaCTOSE-l-C'4
As it was not possible to detect the formation of galactobiose or galactotriose in the raffinose digest due to overlapping of the spots, an effort was made to demonstrate
SHETLAR
the presence of these two compounds in the raffinose digest by use of n-galactose-lC14.When 0.25 ml of the solution of raffinose (0.5 M) and n-galactose-l-Cl4 (1 PC) was incubated with 0.25 ml of enzyme con taining 150 units of a-galactosidase at 40” for 2 hours, compounds 5, 4, 3, and raffinose spots were labeled as shown in Fig. 4. The labeling of the raffinose area did not mean that raffinose itself was actually labeled, as galactobiose overaps raffinose in this area. The raffinose digests were subjected to preparative chromatography; the raffinose areas were eluted and subjected to hydrolysis with 0.1 N HCl at 100” for 15 minutes to preferentially hydrolyze the fructosyl residues present in rafhnose. The hydrolyzate was then rechromatographed. The result indicated that the hydrolyzate con tained fructose, melibiose, and an area corresponding either to galactobiose or raffinose. However, this spot did not react with naphthoresorcinol and was radioactive, suggesting that this compound was not raffinose but galactobiose. Radioautograms indicated that compounds 3 and 4 were also radioactive. By comparison of the R,,,,,,, produced in the melibiose digests (Fig. 4), it was noted that compound 3 of the raffinose digest corresponds to compound B’ (galactotriose) and compound 4 to compound D. As shown in Fig. 4, the radioautogram of melibiose and rafhnose digests agree in regard to compounds 3 (B’) and 4 (D), although the original chromatogram from the melibiose and raffinose digest have overlapping in these areas. ENZYMIC DIGESTION OF RAFFINOSE IN THE PRESENCE OF D-GLUCOSE AND D-GLuCOSE-~-C'~
To indicate further that compounds 3’, 4’, 4!‘, and 5’ of the raffinose digest in the presence of glucose were actually formed from glucose, 0.25 ml of a solution of raffinose (0.5 M), n-glucose (0.5 L%?),and D-&Icase-l-C4 (1 PC) was incubated with 0.25 ml of enzyme solution containing 150 units of cr-galactosidase. After incubation at 40” for 2 hours, the distribution of radioactivity in the oligosaccharides formed was studied as previously described. As indi-
GALACTOSYL
TRANSFER
cated in Fig. 5, compound 4” (melibiose) was most heavily labeled; compounds 3’, 4’, and 5’ were also labeled. ENZYMIC DIGESTION OF RAFFINOSE IN THE PRESENCE OF SUCROSE-U-Cl4
In order to determine whether sucrose is able to accept galactose to form raffinose, 0.25 ml of a solution of raffinose (0.5 M) and sucrose-U-Cl4 (1 PC) was incubated with 0.25 ml of enzyme solution containing 150 units of a-galactosidase. After incubation at 40” for 30 minutes, the enzymic reaction was stopped and analyzed for the distribution of radioactivity in the various oligosaccharides. As indicated in Fig. 8, after 30 minutes of incubation, raffinose was heavily
FIG. 8. A paper chromatogram (A) and a radioautograph (B) of enzymic digest of raffinose in the presence of sucrose-U-C’4. S, Standard; Ga, u-galactose; M, melibiose; R, raffinose; St, stachyose. 0.25 ml solution of raffinose (0.5 M) and sucrose-Ii-CP (1 PC) was incubated at 40°C for 30 minutes.
REACTIONS
311
labeled, suggesting that the reaction was reversible due to the transfer of galactose to sucrose to form raffinose. At this stage, compounds 3, 4, and the compound with the R, lueoae identical to that of melibiose were also labeled, indicating that all of these compounds contained sucrose. ACCEPTOR SPECIFICITY OF PNEUMOCOCCAL CY-GALACTOSIDASE
Acceptor specificity of various monosaccharides, disaccharides, and hydroxyl amino acids was tested by incubating 0.25 ml of substrate (0.5 M melibiose or raffinose) and acceptor (0.5 M) with 0.25 ml of enzyme solution containing 150 units of 01galactosidase. Incubation was carried out at 40” for 2 hours; the new oligosaccharides produced were analyzed by paper chromatography as described above. When Dglucosamine, n-galactosamine, n-fructose, n-mannose, maltose, and lactose were tested for acceptor specificity, 1 PC of Dglucosamine-I-CY4, n-galactosamine-1-CY4, Dfructose-U-CY4, n~altose-l-C14, and lactosel-CY4, respectively, were incubated together with the acceptor. The incorporation of radioactivity in the new oligosaccharides produced was also checked by using a Forro windowless gas-flow counter probe. When glucosamine, galactosamine, and hydroxyamino acids were used as acceptors, the chromatograms were also sprayed with ninhydrin to locate new amino compounds. u-Mannose, n-fructose, n-glucosamine, Dgalactosamine, 2-deoxy-n-galactose, n-ribose, D- and L-arabinose, D- and L-xylose, n-lyxose, lactose, maltose, and cellobiose were found to be able to accept galactose. When n-mannose and cellobiose were used as acceptors, two new oligosaccharides were produced. The rest of the compounds produced only one new oligosaccharide; the structure of these new oligosaccharides was not determined. 2-Deoxy-n-glucose, 3 - 0 - methyl - D - glucose, D - galactose l-phosphate, n-galactose-6-phosphate, Dglucose-l-phosphate, n-glucose-6-phosphate, D- and L-fucose, u-glucuronic acid, Dgalacturonic acid, sorbitol, dulcitol, Dmannitol, inositol, nn-serine, nn-threonine, and L-hydroxyproline were not able to
312
LI AND
accept galactose under the condition scribed.
de-
DISCUSSION
Pneumococcal oc-galactosidase incubated with melibiose results in eight new spots staining with 2-aminobiphenyl as detected by paper chromatography. Discovery of more spots than those previously reported (8) is apparently due to the improvement made in chromatographic techniques. In the present study, better separation of various oligosaccharides produced in the enzymic digests was achieved by employing multiple development of the chromatogram. In addition, heating the chromatogram for 30 minutes rather than 5 minutes after staining with the 2-aminobiphenyl reagent leads to increased sensitivity. This modification is particularly effective in demonstrating nonreducing disaccharides. Although manninotriose, galactobiose, and galactotriose have been reported to be synthesized in the melibiose digests through galactosyl transfer reaction catalyzed by yeast, emulsin or streptococcal oc-galactosidase (1, 3, 5, 17, 18), pneumococcal a-galactosidase seems to have much greater transferring capacity than ol-galactosidases of other origins. Pneumococcal ol-galactosidase used in this study transfers a-n-galactosyl group to primary and secondary carbinol sites, as well as to anomeric sites of n-galactose and n-glucose. The production of nonreducing as well as reducing oligosaccharides in the melibiose and raffinose digests is of particular interest. Although the enzyme transfers the n-galactosyl group from an oligosaccharide to n-glucose, to n-galactose, and to sucrose to form linkages other than CZ-(l-+6), formation of the a-(1-+6) linkage appears to be favored, since compounds with this linkage occur more abundantly. The preferential transfer of the galactosyl residue to the primary alcohol group of the sugar molecule is similar to the galactosyl transfer reaction catalyzed by ,&galactosidase (19, 20). Although these two enzymes have different specificities toward the anomeric configuration of the galactosides, they are similar in their pattern of galactosyl transfer.
SHETLAR
The synthesis of oligosaccharide often proceeds through phosphorylation mechanisms by which phosphorylated sugars function as intermediates. The possibility that the galactosyl transfer reaction catalyzed by pneumococcal a-galactosidase is a type of phosphorolysis is unlikely for the following reasons: (1) the reaction proceeded in phosphate-free medium, (2) there was no evidence of the presence of phosphorylated intermediates in the melibiose and raffinose digests, and (3) the enzyme did not synthesize oligosaccharides from n-galactose-l-phosphate and suitable acceptors (i.e., n-g1ucose or n-galactose). Wallenfels et al. (3) have shown that in addition to hydrolytic and transferring activity, p-galactosidase is able to catalyze the synthesis of glycosides from free sugars and alcohols. However, no synthesis of oligosaccharides occurred from free sugars by pneumococcal cr-galactosidase. Glycosyl transfer reaction may be described as an anomeric substitution reaction. In some instances, the reaction brings about an inversion of the glycosidic linkage as in the case of maltose phosphorylase (21) and cellobiose phosphorylase (22). The enzyme preparation used in this study is free from p-galactosidase. The fact that the oligosaccharides produced in the earlier stages of incubation disappeared after prolonged incubation indicates that these oligosaccharides belong to the a-n-galactosidic series. This leads to the conclusion that galactose transfer reactions catalyzed by this enzyme preparation do not cause the inversion of the galactosidic linkage. When melibiose was incubated with the enzyme in the presence of n-glucose-1-C4, rapid incorporation of radioactivity into melibiose was found. Similarly, radioactive raffinose was obtained from enzymic digests of raffinose in the presence of sucrose-UCn. By incubation of raffinose in the presence of n-glucose to serve as an acceptor, melibiose was synthesized. A similar labeling pattern has been found in a galactosyl transfer reaction catalyzed by yeast ,& galactosidase using allolactose (B-O-P-Dgalactopryranosyl-n-glucose) as a substrate (23).
GALACTOSYL
TRANSFER
From the experimental results it can be concluded that n-glucose, n-galactose, sucrose, melibiose, and raffinose are potent acceptors of the a-n-galactosyl group. Although n-mannose, n-fructose, n-glucosamine, n-galactosamine, maltose, cellobiose, lactose, and all of the pentoses tested may also serve as acceptors, they are less reactive than the above-mentioned sugars. Since phosphorylation, reduction or oxidation of either the 1 or 6 position of n-glucose or n-galactose destroyed the function to serve as an acceptor, it may indicate that both the anomeric carbon and the Cc of n-glucose and n-galactose are important for accepting a galactosyl group. It is of interest to note that 2-deoxy-n-galactose, but not 2-deoxy-D-glucose, can serve as an acceptor. Methylation of C1 of n-glucose also resulted in destruction of the ability to serve as an acceptor. REFERENCES Chem. 9, 1. FRENCH, D., Advan. Carbohydrate 149 (1954). 2. PIGMAN, W. W., Advan. Enzymol. 4, 41 (1944). 3. WALLENFELS, K., AND MALHOTRA, 0. P., Advan. Carbohydrate Chem., 16, 239 (1961). 4. BANKS, W. M., Science 141, 1191 (1963). 5. BAILEY, R. W., Biochem. J. 86, 509 (1963). 6. BAILEY, R. W., AND HOWARD, B. H., Biochem. J. 87, 146 (1963). 7. LI, Y. T., AND SHETLAR, M. R., Proc. Sot. Exptl. Biol. Med. 106, 398 (1961).
REACTIONS
313
8. LI, Y. T., LI, S. C., AND SHETLAR, M. R., Arch. Biochem. Biophys. 108, 436 (1963). 9. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., AND RANDALL, R. J., J. Biol. Chem. 193, 265 (1951). 10. BLOCK, R. J., DURRUM, E. L., AND ZWEIG, G., “A Manual of Paper Chromatography and Paper Electrophoresis,” 2nd edition, pp. 178-188. Academic Press, New York, 1958. 11. GORDON, H. T., THORNBURG, W., AND WERUM, L. N., Anal. Chem. 28, 849 (1956). 12. WOLFROM, M. L., AND MILLER, J. B., Anal. Chem. 28, 1037 (1956). 13. KHADEM, H. E., AND GIRGIS, W., Anal. Chem. 33, 645 (1961). 14. FRENCH, D., AND KNAPP, 1~. W., J. BioZ. Chem. 187, 463 (1950). 15. FRENCH, D., AND WILD, G. M., J. Am. Chem. Sot. 76, 2612 (1953). 16. AVIGAD, G., ZELIKSON, R., AND HESTRIN, S., Biochem. J. 80, 57 (1961). 17. COURTOIS, J. E., Proc. Intern. Congr. Biochem., 4th, Vienna, 1958 Vol. I, p. 140 (1959). Macmillan (Pergamon), New York. 18. DEDONDER, R. A., Ann. Rev. Biochem. 30, 347 (1961). 19. WALLENFELS, K., BERNT, E., AND LIMBERG, G., Ann. Chem. 679, 113 (1953). 20. PAZUR, J. H., J. Biol. Chem. 208, 439 (1954). 21. FITTING, C., AND DOUDOROFF, M., J. Biol Chem. 199, 153 (1952). 22. AYERS, W. A., J. Biol. Chem. 234, 2819 (1959). 23. PAZUR, J. H., MARSH, J. M., AND TIPTON, C. L., J. Sm. Chem. Sot. 80, 1433 (1958).