Biosynthesis of Heparin SOLUBILIZATION, PARTIAL SEPARATION, AND PURIFICATION OF URIDINE DIPHOSPHATE-GALACTOSE: ACCEPTOR GALACTOSYLTRANSFERASES FROM MOUSE MASTOCYTOMA*

The biosynthesis of the neutral trisaccharide, 3-O-p-o-galactosyl-4-0-/?-D-galactosyl-n-xylose of the heparin-protein linkage region, microsomal the incorporation of galactose Partial acid hydrolysis of 14C-galactose-labeled endogenous acceptor yielded several fragments with the characteristics of neutral oligosaccharides isolated from the heparin-protein linkage region and, in addition, a compound with the chromatographic properties of N-acetyllactosamine.

The stepwise synthesis of the neutral trisaccharide was investigated with separate assays for the two galactosyl transferase reactions, with exogenous acceptors such as D-xylose (product, 4- Whereas only 4% of the UDP-galactose-D-xylose galactosyltransferase was brought into solution by treatment of the enzyme with detergent at neutral pH, about one-third of the transferase activity related to the synthesis of the second galactose moiety was recovered in the supernatant under similar conditions, indicating that the two reactions are catalyzed by two different enzymes. Cell-free systems from mouse mastocytoma and embryonic chick cartilage have been utilized in studies on the biosynthesis of the linkage regions of heparin (2) and chondroitin sulfate (3), respectively, and the transfer of xylose from UDP-xylose to an endogenous protein acceptor was reported. In addition, the synthesis of the 2 gala&se molecules of the linkage region has been investigated with a particulate enzyme preparation from embryonic chick cartilage (3,4). The use of exogenous fragments from the linkage region, such as n-xylose or Gal-CXyl as low molecular weight galactose acceptors, permitted the study of each transfer step separately. Substrate competition experiments indicated that the two galactosyl transfer reactions were catalyzed by two distinct catalytic centra, or enzymes (4).
A third galactosyltransferase which catalyzed the transfer of galactose to N-acetylglucosamine was also detected in the microsome particles.
This enzyme was solubilized by the same procedure as that used for the two transferases mentioned above but was discriminated from the latter by its stability toward heat and by fractionation with ethanol.
The present communication describes the transfer of galactose from UDP-galactose to endogenous as well as exogenous linkage region fragments in a cell-free system from mouse mastocytoma. Evidence is presented which indicates that the galactosyltransferase reactions are catalyzed by two different enzymes which may be partially separated by a selective solubilization procedure. MATERIALS AND Other reference compounds mentioned in the text were prepared as described (4).
A heparin-producing FMS mast cell tumor (5,6) was maintained in the solid state in (A/Sri x Leaden)Fl mice by subcutaneous and intramuscular transplantation in the hind legs every 10 to 14 days.

Analytical
Methods-Calorimetric procedures for the determination of pentose, hexose, and protein have been described (7)(8)(9). Prior to protein determination, the samples were dialyzed against distilled water or, alternatively, the protein material was precipitated with cold 10% trichloracetic acid and taken up in 1 M KOH. Oxidation of oligosaccharides with lead tetraacetate was carried out as described (4).
Munktell 314 papers were run for 20 hours, whereas the Whatman No. 3MM papers were irrigated for about 40 hours. Paper electrophoresis was carried out on a flat plate in Buffers D, 0.08 M pyridine-0.046 M acetic acid, pH 5.3 (80 volts per cm; 30 min), or E, 0.05 M borate buffer, pH 9.2 (25 volts per cm; 90 min). Guide strips were stained with aniline hydrogen phthalate (10). Paper chromatograms or electrophoretograms were analyzed for radioactivity with a Packard model 7201 strip scanner and the products were quantitatively determined by a Beckman liquid scintillation spectrometer. Digestion with /%Galactosidase-14C-Galactose-labeled oligosaccharides (1000 to 3000 cpm) were mixed with 0.1 M potassium phosphate buffer, pH 7.3, containing 1% albumin and enzyme (10 pg) in a total volume of 0.1 ml. Incubations were carried out at 30" for 15 hours and the products were analyzed by paper chromatography (Solvent B) or by paper electrophoresis (Buffer E) .

Preparation
of Galactosyltransjerases from Mouse Mastocytoma -All procedures were carried out at O-4". Tumor-bearing mice were stunned by a blow and killed by cervical dislocation. The tumors, weighing 0.5 to 1.0 g, were removed and dissected free from adherent tissue. After homogenization in a Virtis 45 homogenizer with 2 volumes of a buffer containing Tris-acetate (50 MM, pH 7.4), KC1 (70 mu), and EDTA (1 mu), the crude material was subjected to centrifugation as depicted in Fig. 1. The microsomal fraction, sedimenting at 1 x 105 x g, was utilized in the initial experiments with exogenous substrates and for studies with the endogenous acceptors.
Solubilization of GalactosyltransferasesThe microsomal fraction was suspended in the buffer mentioned above and Tween 20 was added (final concentration, 2%). The solution was then brought to pH 10.4 to 10.6 by addition of concentrated ammonium hydroxide and quickly readjusted to pH 7.4 by addition of glacial acetic acid (cj. Reference 11). After centrifugation at 1.59 x lo6 x g for 2 hours, the top 10 ml from each tube (total volume, 13 ml) were withdrawn and aliquots were tested for enzymatic activity as described below.
Transfer of Gala&se to Endogenous Acceptor-The microsomal fraction derived from four tumors (0.5 ml; 8 mg of protein) was incubated with UDP-galactoseJ4C (1 PCi; 240 PCi per pmole) at 37" for 3 hours. The reaction was stopped by the addition of 1 volume of cold 10% trichloracetic acid and a neutral sugar fraction was isolated after partial acid hydrolysis of the washed precipitate essentially as described (4), with omission of the papain treatment.
Transfer of Gala&se to Exogenous Acceptors-Transfer of galactose to low molecular weight substrates such as xylose or Gall-Xyl was studied by incubating the acceptor (0.5 to 5 pmoles) at 37" for 0.5 to 6 hours with UDP-galactose-14C, enzyme fraction (0.01 to 0.75 mg of protein in 0.05 ml of the Tris-acetate buffer mentioned above), and MnClz (10 to 20 MM), in a total volume of 0.06 ml (for exact experimental conditions, see the legends to figures and tables). The kinetic parameters were determined with solubilized enzyme preparations which were obtained by treatment of the microsomal fraction with detergent and alkali as described above.
The reactions were stopped by heating the tubes in a boiling water bath for 2 to 3 min and the mixtures were spotted on paper strips (4 x 40 cm). After removal of anionic derivatives by electrophoresis (Buffer D), the papers were dried and cut at a distance of about 2 cm from the site of application, toward the anode. A wick of filter paper was attached with a sewing machine, and the neutral sugar fraction, which had migrated slightly toward the cathode, was subjected to chromatography in Solvent A. The radioactivity on the chromatograms was located with a strip scanner. Control incubations without and ZZZ, Gal-3-Gal-4-Xyl.
added acceptor were included in all experiments and indicated whether further purification from interfering, labeled substances was necessary. In such cases, the latter materials were adequately separated from the product by subjecting the eluted fraction to paper electrophoresis in Buffer E.

RESULTS
Transfer of Galactose to Endogenous Acceptor-Incubation of the particulate enzyme with UDP-galactose-14C resulted in incorporation of 3.35 X lo5 cpm (230/, of added radioactivity) into trichloracetic acid-precipitable material. Following acid hydrolysis (1~ HCI; 100" for 3 hours) and passage of the neutralized solution through columns (2 X 4 cm) of Dowex 1 and Dowex 50, all of the radioactivity in the eluate migrated as did galactose on paper chromatography (Solvent B) or paper electrophoresis (Buffer E). About one-third of the radioactivity was recovered as a neutral sugar fraction following partial acid hydrolysis (pH 1.5; 100" for 5 hours) of the 14C-galactose-labeled trichloracetic acid precipitate and passage through ion exchange columns. Paper chromatography (Solvent A) of this fraction gave the distribution of radioactivity shown in Fig. 2. The sections indicated were eluted and rechromatographed or further purified by paper electrophoresis in Buffer E. Section A yielded a fraction with the chromatographic properties (Solvents A and C) of JV-acetyllactosamine (33 150 cpm; cf. Reference 12), Section B contained a fraction which migrated similar to Gal-4-Xyl (5280 cpm; Solvent A and Buffer E), and Sections C and D showed the presence of materials with mobilities similar to those of Gal-3-Gal (307 cpm) and Gal-3-Gall-Xyl (300 cpm), respectively (paper chromatography, Solvent B and paper electrophoresis, Buffer D). Digestion of each fraction with /3-galactosidase released all of the radioactivity as free galactose (paper chromatography, Solvent B). Treatment of Fraction C with lead tetraacetate resulted in the formation of a compound with the electrophoretic mobility of Gal-2-Lyx.
Since GalS-Gal is converted into the latter disaccharide under similar conditions (13), this result, in conjunction with the chromatographic mobility, suggests the identity of Fraction C with the disaccharide, Gal-3-Gal.
Although a detailed structural study of Fractions B to D was not undertaken in the present investigation, the properties observed were in good accord with the characteristics of neutral oligosaccharides previously isolated from the protein-polysaccharide linkage regions of chondroitin sulfate and heparin (13,14). The results lend support to the conclusion that a significant portion of the galactose incorporated into the trichloracetic acidprecipitable material in the particulate mouse mastocytoma preparation is transferred to precursors of the heparin-protein linkage region.
The presence in the partial acid hydrolysate of a fragment with the properties of N-acetyllactosamine indicated that other

Paper electrophoresis
(Buffer E) of (upper tracing) the fraction migrating similar to Gal-3-Gal-4-Xyl on paper chromatography in Solvent A (cf. Fig. 4) and (lower tracing) the analogous fraction from the control experiment without added acceptor. The standards on the guide strip are (I) galactose and (II) Gal-3-Gal-4-Xyl.
types of galactosyl transfer reactions also occurred in the enzyme preparation.
This observation is further discussed below. Transfer of Galuctose to Xylose and Gal-$Xy&Figs.
3a and 4a show the distributions of radioactivity obtained on paper chromatography after incubating the particulate enzyme with UDP-galactose-14C and xylose or Gal-4-Xyl.
Since some interference with endogenous products was observed, the fractions migrating similarly to Gal-4-Xyl or Gal-3-Gal-4-Xyl were eluted with water and subjected to paper electrophoresis in Buffer E. Such a purification step is illustrated in Fig. 5, which shows the complete separation from Gal-3-Gal-4-Xyl of the interfering substances. Further characterization of the products was obtained as follows.
Treatment of the presumed Gal-4-Xyl with ,&galactosidase or with 1 M HCl at 100" for 3 hours yielded galactose as the only product (paper chromatography, Solvent B and paper electrophoresis, Buffer E). On partial acid hydrolysis (HCl, pH 1.5; 100" for 5 hours) only two compounds, corresponding in migration to undegraded Gall-Xyl and to galactose, were observed (paper chromatography, Solvent A). The product of incubation migrated as did authentic Gal-4-Xyl in chromatographic and electrophoretic systems which separate the position isomers of galactosylxylose (Solvents A and C; Buffer E; cf. Reference 13). When applied to a column (2 X 140 cm) of Sephadex G-25, eluted with 10% ethanol, the radioactivity appeared at the effluent volume of Gal-4-Xyl. Since the addition of xylose to the reaction mixture was a prerequisite for product formation, it is concluded that the radioactive substance was identical with Gal-4-Xyl. The product obtained with Gal-4-Xyl and UDP-galactose-14C possessed the chromatographic (Solvent A) and electrophoretic (Buffer E; Fig. 5) properties of Gal-3-Gal-4-Xyl. Digestion FIG. 6. Radioscan of the chromatogram obtained after subjecting the presumed Gal-3-Gal-4-Xyl (12,000 cpm) to partial acid hydrolysis as described in the text.
The standards indicated are: I, galactose; ZZ, Gal-3-Gal; and III, Gal-3-Gall-Xyl. with j%galactosidase released all of the radioactivity as galactose (Solvent B and Buffer E). Further characterization of the radioactive product was obtained by partial acid hydrolysis under the conditions described above for Gall-Xyl. As expected for a compound with the structure, Gal-3-Gall-Xyl, labeled in the nonreducing, terminal position, three hydrolytic fragments were observed on paper chromatography in Solvent A (Fig. 6): free galactose (9370 cpm), Gal-a-Gal (705 cpm), and Gal-3-Gal-4-Xyl (620 cpm).
Treatment of a sample of the presumed Gal-3-Gal with lead tetraacetate resulted in the production of a compound which migrated similarly to Gal-2-Lyx, well separated from the unoxidized compound, which had moved to the position of Gal-3-Gal (paper electrophoresis, Buffer E). These observations clearly indicate the formation of a p-1 -+ 3 linkage between the two galactose moieties and the presence, in the mouse mastocytoma preparation, of a UDP-galactose-Gal-4-Xyl galactosyltransferase.
Transfer of Galactose to N-Acetylglucosamine-Since the partial acid hydrolysate of the l4C-galactose-labeled endogenous acceptors had shown the presence of a fragment with the mobility of N-acetyllactosamine, free N-acetylglucosamine was also tested as acceptor for galactose.
The product formed showed migration characteristics identical with those of the endogenous fragment and yielded galactose on treatment with P-galactosidase.
Of the three exogenous substrates tested in the present study, N-acetylglucosamine was the most active galactose acceptor (Table  I) No appreciable loss of activity was observed after keeping the microsomal fraction at pH 9.8 for 18 hours at 4". The treatment of the particulate enzyme with Tween under alkaline conditions resulted in the solubilization of considerable amounts of galactosyltransferase activity from the particles, as seen by analysis of the supernatant fraction following centrifugation at 1.59 x lo6 x g for 2 hours.
With xylose and Gall-Xyl as substrates, analysis of the various enzyme fractions (Fig. 1) revealed that the 25 to 50% ammonium sulfate fraction contained 20 to 25% of the total activity of the crude homogenate, whereas the specific activities of the two reactions had increased 6-to S-fold, respectively.
Interestingly, the ratios of the two products formed varied for the solubilized and particulate enzyme fractions, indicating a preferential release from the microsomes of the UDP-galactose-Gal-4-Xyl galactosyltransferase. Selective Solub&ation of UDP-gala&se-Gal+Xyl Galactosyltransferase-The findings described above prompted more de- It therefore seems likely that the observed pH profile is related to enzyme activity rather than enzyme stability.) Aliquots were mixed with (a) xylose (2 pmoles tailed studies on the solubilization procedure in attempts to separate the two galactosyltransferases under investigation. In the absence of added detergent, no appreciable enzyme activity was found in the supernatant following centrifugation of the alkali-treated particles. However, a considerable portion of the UDP-galactose-Gal-4-Xyl galactosyltransferase activity was brought, into solution by treatment with Tween 20 at, pH 7.2. This is illustrated in Fig. 7, which shows the release of the transferase activities into the supernatant fluid as a function of pH. By contrast, more than 80% of the UDP-galactose-n-xylose galactosyltransferase activity was recovered in the particulate fraction in the pH range 7.2 to 9.2, only 4% being solubilized by the detergent at pH 7.2.
The selective solubilization of the UDP-galactose-Gal-4-Xyl galactosyltransferase by treatment with Tween at neutral pH provides strong evidence that the two galactosyl transfer reactions described above, which may represent the synthesis of the galactose moieties in the heparin-protein linkage region, are catalyzed by two different enzymes.
A similar conclusion, on the basis of substrate competition experiments, was reached in studies on the biosynthesis of the chondroitin sulfate-protein linkage region (4).
Kinetic Studies-The two solubilized enzymes, obtained after treatment of the microsomal fraction at pH 10.6 in the presence of Tween 20 and centrifugation at, 1.59 x 105 x g, were further characterized as follows. Product formation was linear with time for 3 hours but continued at a slightly reduced rate for at least 5 hours.
Product formation was also proportional to the concentration of protein within the range tested (0.25 to 2.5 mg per ml).
The enzymes were active over a wide pH range with maximal activity:atypHi7.2 to 7.5 (Fig. 8). The transfer of gala&se to xylose as a function of the nucleotide concentration gave an approximate K, value of 0.3 x 10T4 M for UDP-galactose ( Fig. 9). At fixed nucleotide concentration, half-maximal velocity occurred at an acceptor concentration of 1.8 X 10m2 M (Fig. 10). Similarly, the corresponding K, values for the UDP-galactose-Gal4-Xyl galactosyltransferase were 2.5 X 10m4 M and 2.1 X 10-z M for UDP-galactose and Gal-4-Xyl, respectively. Addition of a divalent cation, notably manganese, was required for product formation. Under the conditions tested, Co2+ could only partially replace Mn* (about 10%; Fig. 11). The particulate enzymes, which had not been subjected to the treatment with detergent and alkali, were less specific as to metal requirements.
Both Mn* and Co* gave comparable yields and the presence of Mg2+ or Ca* also stimulated the enzymes significantly.
No product formation was detected with Cu2+. Other properties of the particulate enzyme system which were tested, notably pH dependence and the formation of product as a function of time, were similar to those observed for the solubilized-galactosyltransferases.  total activity of the UDP-galactose-GlcNAc galactosyltransferase was recovered in this fraction.
A minor portion of the UDPgalactose-Gal-4-Xyl transferase activity was also present in the alcohol supernatant fraction, adding further support to the contention that the two galactosyl transfer reactions which complete the synthesis of the neutral trisaccharide, Gal-S-Gal4-Xyl, are catalyzed by two different enzymes.
It is of note that the activities of these two enzymes were significantly impaired by the treatment with ethanol under the conditions described in Table  II.
For each reaction, only 20 to 30% of the total enzymatic activity was recovered.
In contrast, essentially all of the UDP-gala&se-GlcNAc galactosyltransferase activity was regained in the supernatant and pellet fractions. Further discrimination between the three transferases was obtained in heat inactivation studies. Table III shows the amounts of product formed from xylose, Gal-PXyl, and N-acetylglucosamine, respectively, when incubated with UDP-galactose and aliquots of the 1.59 x 105 x g supernatant fraction which had been previously treated for 1 or 5 min at 50" or 55". Whereas product formation from xylose had practically vanished after heating the preparation at 55" for 5 min, the acceptor activity with N-acetylglucosamine had decreased but slightly. By comparison, the UDP-galactose-Gal-4-Xyl galactosyltransferase showed intermediate characteristics when tested for its lability toward heat.
These results clearly suggest that the three galactosyl transfer reactions detected in the mouse mastocytoma preparation are mediated by different enzymes which may be discriminated from each other by fractionation as well as by inactivation procedures.
The interpretation of the results obtained with the endogenous acceptors present in the tumor homogenate is hampered by the lack of knowledge of their structures. Possibly, the UDPgalactose-GlcNAc galactosyl transfer reaction may reflect the synthesis, under physiological conditions, of a similar linkage from UDP-galactose and endogenously bound N-acetylglucosamine residues.
It is of note that the transfer of galactose to endogenous material was substantially impeded by the presence of N-acetylglucosamine in the reaction mixture.4 This result favors but does not conclusively establish a kinship between an enzyme catalyzing the transfer of galactose to a residue of Nacetylglucosamine present in the endogenous acceptor, and the UDP-galactose-GlcNAc galactosyltransferase.
Since almost half of the galactose was transferred from UDP-galactose to free Nacetylglucosamine under the conditions used, a similar inhibitory effect might have been caused by depletion of the nucleotide pool. The separation of the UDP-galactose-GlcNAc galactosyltransferase from the other two transferases under study is in agreement with the results obtained with a particulate enzyme system from embryonic chick cartilage which catalyzes the same reactions (12). In contrast, however, the latter enzyme was extremely labile toward heat, whereas the solubilized enzyme from the mast cell tumor exhibited a marked stability when tested under similar conditions.
A more comprehensive characterization of the mouse mastocytoma enzyme was not undertaken in the present study and it is not known at present whether this discrepancy may indicate different physiological roles for the two UDP-galactose-GlcNAc galactosyltransferases investigated.