Porcine A Blood Group-specific IV-Acetylgalactosaminyltransferase

Porcine A blood group-specific N-acetylgalactosaminyl-transferase required either Mn2+, Cd2+, or Zn2+ for activity and 2'-O-alpha-fucosylgalactosides as acceptor substrates. The presence of detergent stabilizes the enzyme but is not essential for catalysis. To obtain information about the kinetic mechanism of the transferase reaction, initial rate parameters have been determined using 2'-fucosyllactose or A--mucin as acceptors, and Mn2+ or Cd2+ as cosubstrates. 2'-Fucosyllactose is a competitive inhibitor with respect to A--mucin and a noncompetitive inhibitor with respect to UDP-N-acetylgalactosamine. UDP inhibits noncompetively with respect to acceptor; thus UDP-N-acetylgalactosamine or acceptor can bind to the transferase via an equilibrium random pathway. The transferase converts human O blood type erythrocytes of A blood types. After exhaustive glycosylation, 3 X 10(6) N-acetylgalactosaminyl residues were incorporated per cell. Gel electrophoretic analysis of the labeled erythrocyte membranes indicates that glycoproteins with apparents molecular weights from 30,000 to 100,000 have been glycosylated; glycolipids account for only 15% of the labeled material, although pure H-glycolipid is a good acceptor. The transferase, with its strict acceptor specificity, can thus be used as a tool to study the biosynthesis and function of glycolipids and glycoproteins.


Porcine
A blood group-specific N-acetylgalactosaminyltransferase required either Mn*+, Cd'+, or Zn" for activity and 2'-0-cu-fucosylgalactosides as acceptor substrates. The presence of detergent stabilizes the enzyme but is not essential for catalysis.
To obtain information about the kinetic mechanism of the transferase reaction, initial rate parameters have been determined using 2'-fucosyllactose or Amucin as acceptors, and MnZ+ or Cd'+ as cosubstrates. 2'-Fucosyllactose is a competitive inhibitor with respect to Amucin and a noncompetitive inhibitor with respect to UDP-IV-acetylgalactosamine. UDP inhibits noncompetitively with respect to acceptor; thus UDP-N-acetylgalactosamine or acceptor can bind to the transferase via an equilibrium random pathway.
The transferase converts human 0 blood type erythrocytes of A blood type. After exhaustive glycosylation, 3 x 10" N-acetylgalactosaminyl residues were incorporated per cell. Gel electrophoretic analysis of the labeled erythrocyte mem-branes indicates that glycoproteins with apparent molecular weights from 30,000 to 100,000 have been glycosylated; glycolipids account for only 15% of the labeled material, although pure H-glycolipid is a good acceptor. The transferase, with its strict acceptor specificity, can thus be used as a tool to study the biosynthesis and function of glycolipids and glycoproteins.
The enzymatic properties of the transferase are reported here, including kinetic studies that point to similarities between the mechanism of the transferase and that of galactosyltransferase (EC 2.4.1.38) from bovine milk (2). In addition, the pure transferase has been found to efficiently convert human 0 blood type erythrocytes to type A.
Hydrolysis in 1 M acetic acid for 1% h at 100" yielded only fucose, lactose, and some unreacted oligosaccharide, detected by paper chromatography as above. It was resistant to Escherichia coli p-galactosidase unless simultaneously treated with beef kidney cY-L-fucosidase and was thus distinguished from 3-fucosyllactose, another oligosaccharide reported to occur in human milk (6).

Assay of UDP-GalNAc:Gal
Transferme" Methods 1 and 2 have been described in the preceding paper (1).

Use of Glycolipids as Acceptors
Reaction mixtures were as given in Method 1, except that asialo-PSM A-was replaced by glycolipids, which were first dissolved in chloroform:methanol (2:1, v/v), mixed with the detergent, and dried at room temperature under a stream of Nz before adding the other components. In the experiments described here, transfer of GalNAc was determined by Method 1 since the labeled glycolipids appeared in the void volume of Sephadex G-50. Similar results were obtained with Method 3 or electrophoresis on Whatman No. 3MM paper in 1% tetraborate.

Hemagglutination Inhibition
These tests were carried out in phosphate-buffered saline (0.073 M NaCl, 0.018 M KH,PO,, 0.057 M Na,HPO,, pH 7.2). Appropriately diluted antisera (100 ~1) were added to serial dilutions (100 ~1; dilution factor = l/2) of the mucin to be tested. After allowing the mixtures to stand for 45 min at room temperature, 100 ~1 of a 1% suspension of the erythrocytes were added. The degree of agglutination was read after allowing the erythrocytes to settle for 2 h at room temperature.

Glycosylation of Erythrocytes
The incubation mixture (100 pl) contained 13 pmol of NaCl, 1 pmol of imidazole-HCl, pH 7.0, 1 pmol of MnCl,, 0.1 mg of bovine serum albumin, 10 nmol of UDP-[1J4ClGalNAc (4000 cpmlnmol), 4 milliunits of UDP-GalNAc:Gal transferase (in control tubes inactivated 4 min at loo"), and erythrocytes (blood type 0 or A) as a 50% suspension. After incubation at 37" for 1 h, the cells were washed three times with 5 ml 0.15 M NaCl and used for hemagglutination tests. To determine the extent of ["ClGalNAc transfer, cells were counted using an internal standard to correct for quenching.
To determine the blood type of the erythrocytes a 1% suspension of the erythrocytes to be tested (100 ~1) and 100 ~1 of 0.15 M NaCl were added to serial dilutions (100 ~1; dilution factor = i/2) of antiserum. The degree of agglutination was read after allowing the erythrocytes to settle for 2 h at room temperature. One volume of packed erythrocytes was mixed rapidly with 40 volumes of cold 10 m&f TrislHCl, pH 7.5, membranes were collected by centrifugation in a Sorvall HB-4 rotor for 30 min at 11,000 rpm, resuspended in 20 volumes of Tris buffer, centrifuged again, and then resuspended in 1 volume of Tris buffer. These membranes, or the erythrocytes prior to disruption, were incubated with the transferase as outlined above, except that the incubation mixture contained 6 nmol of UDP-GalNAc (88,000 cpm/nmol), 5 milliunits of transferase, and erythrocytes as a 67% suspension (or an equivalent amount of erythrocyte membranes).

Gel Electrophoresis of Labeled Membranes
Membranes were dissociated in sodium dodecyl sulfate-urea-mercaptoethanol and subjected to polyacrylamide gel electrophoresis as described earlier (1). To determine the distribution of radioactivity, gels were frozen after electrophoresis and cut into 2-mm slices. Each slice was incubated in a plastic vial for 24 h at 37" and then 24 h at room temperature in 0.5 ml of Protosol (New England Nuclear):toluene:water (9:lO:l). To each vial, 3.75 ml of scintillator fluid (4 g of I,bdiphenyloxazole, 0.1 g of 1,4-bis[2-(Bphenyloxazolyl)lbenzene, and 6 g of benzoic acid in 1 liter of toluene) were then added for counting.

Determination of @ Parameters
The generalized rate equation for three-substrate reactions, as proposed by Dalziel (8), was reduced either to the form of Equation 1, e=cJ,+ -@L + @A VO UDP-GalNAc Acceptor UDP-GalNAc. Acceptor for initial rate measurements performed at a saturating Mn*+ concentration, or to the form of Equation 2, $=ao+ %I @" @ --+ UDP-GalNAc + Metap+ UG-GalNAc MetaP+ (2) 0 for initial rate measurements performed at a saturating acceptor concentration.
These equations were used to calculate @ parameters from initial rate data with the help of primary and secondary double reciprocal plots and regression analysis as outlined earlier (2).

Requirements
for Enzymatic Activity of UDP-GalNAc:Gal Transferase Donor-The results given in Table I show that UDP-GalNAc is the only glycose donor used efficiently by the enzyme. UDP-GlcNAc and UDP-Gal had poor but measurable donor activity; the other nucleotide sugars tested were inactive.
Acceptor -The acceptor specificity of the enzyme is given in Table II. In the absence of added acceptor, no radioactivity was detected using the gel filtration assay (Method 1); when the ion exchange assay (Method 3) was used, however, a low level of enzyme-catalyzed conversion of UDP-GalNAc into a neutral compound (presumably GalNAc) was discerned. All good acceptors were 2'-0-fucosyl+galactosides and either oligosaccharides (e.g. 2'-fucosyllactose), glycoproteins (PSM A-), or glycolipids (H-glycolipid).
Substances with blood group A or Leb activity were poor acceptors, although they contain the Fucol + 2Gal grouping.
Enzyme -The transferase was inactivated at 60" with a halftime of about 3 min, and at 65" with a half-time of less than a min (Fig. 1). The activity for hydrolysis of UDP-GalNAc was inactivated in parallel with loss of transferase activity, suggesting that the transferase rather than a small amount of contaminating enzyme hydrolyzes UDP-GalNAc. In standard assays (Method 11, GalNAc transfer to asialo-PSM A-was proportional to the amount of enzyme present in the assay mixture in the range of 0 to 50 microunits (not shown).

Incubation
Time -The amount of GalNAc incorporated into asialo-PSM A-increased linearly with time for at least 4 h ( Fig. 2). In this experiment, as well as in all kinetic experiments to follow, conditions were chosen that consumed less than 10% of the UDP-GalNAc, since after more than 10% Assays were carried out by Method 1 or 3 as indicated, except that the following donors were substituted for UDP-GalNAc: 5 nmol of UDP- 6,900 cpm/nmol; 0.1 pmol of UDP-lU-14ClGal, 200 cpmlnmol; 5 nmol of GDP-lUJ4ClFuc, 11,900 cpmlnmol; 5 nmol of CMP- [4,5,6,7,8,9J4ClNeuAc,7,200  Assays were carried out by Method 1 or 3, except that in place of the standard acceptors other substances were added as stated in the table. To permit detection of low levels of activity, incubation time and amount of enzyme added were increased where appropriate. The rates are given relative to asialo-PSM A-.

Accept&
Quantity Transferase Relative activitv rate The following substances were also tested and found to have less than 1% of the acceptor activity of asialo-PSM A-: completely deglycosylated ovine submaxillary mucin and PSM; a,-acid glycoprotein; antifreeze glycoprotein 8; lacto-N-neotetraosyl ceramide; Lea-glycolipid, L-fucose alone or together with lactose; L-arabinose; n-glucose; maltose.
transfer, rates decreased significantly due to product inhibition by UDP. Prolonged incubation of the transferase under standard assay conditions, however, in the absence of substrates, led to a slow decrease in enzyme activity with a halftime of about 4 days (data not shown). Thus the enzyme could be expected to support a reaction for a considerable length of time if product inhibition is circumvented.
Buffer-As seen in Fig. 3, transferase activity was largely unaffected by the type and pH of the buffer employed between pH 5 and 8. Transferase activity decreased below pH 5, and appeared to increase above pH 7.5. In Tris buffer, however, oxidation and precipitation of Mn2+ occurred, and use of Mes buffer at pH 6 was therefore considered more suitable for standard assays.
Metals-No transferase activity was observed when Mn2+ was omitted from the standard assay mixture, but several other cations, notably Cd2+ and Zn2+, supported enzymic activity (Table III). Among salts not listed, LiCl, NaCl, and the hydrochlorides of trimethylamine, spermine, and spermidine could not substitute for MnCl,. When two cations were used in the same incubation mixture (Table IV), inhibition of transferase activity resulted in most cases. Among the cations tested, Cu2+ was the most inhibitory.
Only Cd2+ and Zn*+, used at low concentration, helped to increase transferase activity over the level seen with nonsaturating Mn2+ alone.
Temperature -The rate of GalNAc transfer was determined as a function of temperature using saturating substrates. The results are shown in Fig. 4 in the form of an Arrhenius plot. A linear relationship between log V,/e and the reciprocal of the absolute temperature was obtained between 0" and 42". From the slope of the line, the energy of activation for the ratelimiting step of the reaction was calculated to be 21.6 kcal/mol.

Detergent and Nonspecific
Protein -The presence of Triton X-100 and bovine serum albumin did not appear to be essential for catalysis. A S-fold enhancement of the reaction rate was observed upon addition of Triton X-100 to the incubation mixture (Table V). It is likely that these agents primarily served to stabilize the enzyme or to keep it from aggregating.
As seen in Fig. 5, the enzyme rapidly lost activity when diluted into buffers lacking detergent and protein, either at 0" or 37". Addition of Mn2+ and acceptor gave only partial protection, whereas Triton X-100 and bovine serum albumin completely protected the enzyme against inactivation.

Characterization of Reaction Products
Quantitation of Acceptor Sites -Since the oligosaccharide side chains of pig submaxillary mucin are heterogeneous in structure, acceptor sites available for glycosylation by the transferase were quantitated enzymatically.
As shown in Fig.  6, when a large excess of transferase was used to glycosylate a small amount of asialo-PSM A-, the reaction stopped completely after 60 min. This must have been due to exhaustion of acceptor sites, since addition of more asialo-PSM A-immediately resulted in further incorporation of GalNAc. In a separate experiment (not shown) it was found that the transferase was unable to catalyze synthesis of UDP-GalNAc from reaction products in detectable amounts, and hence that the reaction equilibrium is much in favor of glycosylation.
It was calculated from the data that the particular preparation of asialo-PSM A-tested contained 0.45 pmol of acceptor sites/mg (dry weight). The same material contained 0.46 pmol of fucose/mg as estimated by the Dische-Shettles method (91, in accord with the earlier suggestion that only fucosylated galactose residues serve as acceptors for the transferase.
Conversion of PSM A-into PSM A+-To show that the isolated transferase is indeed the blood group A-specific enzyme, the immunological properties of the reaction products were examined.
Blood group A-negative PSM (not treated with neuraminidase) was exhaustively glycosylated with GalNAc in the same way as described for the previous experiment. In a control tube, the transferase was heat-inactivated before addition of the other components.

No transfer of [W]GalNAc
to PSM A-was detected with heat-inactivated enzyme, whereas the active transferase incorporated 0.35 pmol of GalNAclmg of PSM A-, a value close to the 0.45 pmol of GalNAc/mg of asialo-PSM A-found in the previous experiment when allowing for a 20% weight loss after neuraminidase treatment.
Therefore most potential acceptor sites were glycosylated despite the presence of sialic acid.
The reaction products were also tested for their ability to inhibit. hemagglutination of blood type A human erythrocytes by blood type A antiserum (Table VI). The reaction product obtained with inactivated transferase had almost negligible inhibitory power, but that obtained with active transferase inhibited at a very low concentration, albeit not quite as well as authentic PSM A+. The inhibition was specific, since the reaction products did not inhibit agglutination of B/M erythrocytes by blood type B-and M-antisera, even at the highest concentrations used. These results suggest that at least part of  Acceptors -H-glycolipid from canine intestine could be completely glycosylated by the transferase; all of the reaction product was found in the organic phase after extraction with chloroform:methanol (2:1, v/v) and migrated as one spot with the same mobility as A-glycolipid upon thin layer 25 ~1 were withdrawn, mixed with 5 ~1 of 0.5 M EDTA, and GalNAc transfer was determined as described in Method 1. FIG. 3 (right). UDP-GalNAc:Gal transferase activity as a function of pH. Assay conditions are as given in Method 1, except that incubation mixtures contained 10 nmol of UDP-[l-WlGalNAc, 740 cpmlnmol, lacked bovine serum albumin, and were incubated for 30 min. Furthermore, 5 pmol of the following buffers were substituted for the standard buffer; acetic acid-Na+ (M), Mes-Na+ (A), imidazole-HCl CO), Tris/HCl (0). The abscissa represents pH of these buffers, measured at 22", and at a concentration of 0.1 M, rather than actual pH in the assay mixture. FIG. 4. Effect of temperature on the UDP-GalNAc:Gal transferase reaction. Assays were performed by Method 1, with the following modification., The temperature of incubation was varied between 0" and 65". Reactions were started by the addition of an appropriate amount of enzyme (between 12 and 1200 microunits) and incubated for a convenient time (between 4 and 200 min) to obtain incorporation between 400 and 2000 cpm at each temperature.
As a check on linearity, one assay mixture at each temperature contained 4 times less enzyme than the other and was incubated 4 times longer.  were calculated to have about 10" acceptor sites per cell. When tested for hemagglutination by A-antiserum, 0-erythrocytes previously incubated with inactivated transferase did not agglutinate.
However, 0-erythrocytes previously incubated with active transferase agglutinated with the same antiserum titer as authentic A-erythrocytes, indicating that their blood group specificity had indeed been changed from 0 to A.  Hemagglutination inhibition by products of transferase reaction Products of the transferase reaction were prepared by Method 1, using in one tube 12 milliunits of active transferase, and in the other the same amount of transferase that had been inactivated for 3 min at 100". Both incubation mixtures contained 50 yg of PSM A-in place of the asialo-PSM A-, and 50 nmol of UDP-[1-'4C]GalNAc (4000 cpm/nmol). Bovine serum albumin was omitted. After gel filtration, the products were tested for their inability to inhibit hemagglutination of A-, or B/M-erythrocytes by anti A-, B-, or M-serum, as described under "Methods." For comparison, authentic PSM A-and PSM A+ were also tested.

Inhibitor
Nanograms of inhibitor re-System quired to completely inhibit hemagglutination  In a separate experiment, membranes prepared from labeled erythrocytes were extracted with 1-butanol (10) in a procedure designed to separate glycoproteins and glycolipids, and the following distribution of "'C-labeled material was obtained: combined aqueous phases, 83%; insoluble material, 12%; combined butanol phases, 5%.

Kinetic Studies
Metal Dependence of GalNAc Transfer -The dependence of GalNAc transfer on the Mn'+ concentration is shown in Fig. 8. At pH 6, the standard pH for most of the experiments described here, 20 mM Mn"+ was sufficient to saturate the reaction, whereas 50 mM Mn'+ was required at pH 7.5. In the range of 0. Dependence of GalNAc transfer on Cd"+ concentration. As in Fig. 8 except that Cd*+ was substituted for Mn". and l/Mn*+ was obtained, and apparent Michaelis constants of 0.3 mM at pH 6 and 0.4 mM at pH 7.5 were calculated.
However, toward higher Mn"+ concentrations, the lines were deflected downward (substrate activation), suggesting that the Mn*+ might serve more than one purpose in catalysis. Substituting Cd*+ for Mn'+, a strikingly different cation dependence was observed (Fig. 9). Above 4 mM Cd*+, strong inhibition occurred at pH 6, but not at pH 7.5. The data obtained below 4 mM Ccl'+ were also strongly affected by pH as seen in the double reciprocal plot. The calculated apparent Michaelis constants were 0.1 mM at pH 6 and 1.2 mM at pH 7.5.
Determination of Initial Rate Parameters -Initial rates of GalNAc transfer were determined at pH 6 and saturating Mn'+ using six concentrations of UDP-GalNAc ranging from 5 to 125 pM and six concentrations of asialo-PSM A-ranging from 0.4 to 7.5 mglml (180 to 3375 PM with respect to acceptor sites; see Fig. 6). Primary and secondary double reciprocal plots of the data are shown in Figs. 11 and 12, respectively, of the supplementary material, which immediately follows the text and references.g A similar experiment was performed using the alternative substrate 2'-fucosyllactose at concentrations ranging from 30 to 750 PM, and the resulting primary and secondary plots are given in Figs. 13 and 14 of the supplementary material.
From these plots, initial rate parameters (see "Methods") which are listed in Table VIII were obtained. Initial rates of GalNAc transfer were also determined at  . Initial rate parameters obtained from these data are listed in Table IX. Inhibition

Experiments-
To obtain information about the order of addition of substrates, 2'-fucosyllactose was used as an inhibitor of GalNAc transfer to asialo-PSM A-. Although 2'fucosyllactose is itself an acceptor, it acted as an inhibitor in this experiment, because its reaction product was not detected by the assay method used (only the reaction product of PSM was counted after its separation from the reaction product of 2'-fucosyllactose and from UDP-GalNAc on Sephadex G-50). It can be seen in Fig. 1OA that 2'-fucosyllactose is indeed a competitive inhibitor with respect to asialo-PSM A-. Fig. 1OB shows the effect of 2'-fucosyllactose on GalNAc transfer to asialo-PSM A-using UDP-GalNAc as the varied substrate. A typical noncompetitive pattern is observed. In a similar experiment, UDP was found to be a competitive inhibitor with respect to UDP-GalNAc (Fig. lOD), and a noncompetitive inhibitor with respect to asialo-PSM A- (Fig.  1OC). These results indicate addition of UDP-GalNAc and acceptor to the enzyme in random order.

DISCUSSION
The enzyme described here is a blood group A-specific UDP-GalNAc:Gal transferase as evidenced by the following facts. It occurs only in submaxillary glands of blood group A-positive animals. Potential acceptors must carry the grouping Gal(Z+ 1aFuc) to be effective substrates. The same acceptor specificity has been established for the partially purified blood group Aspecific transferases from human submaxillary glands (ll), human milk (12) by the human milk enzyme has been demonstrated to be Gal (3 t 1aGalNAc) (12); other A-specific transferases form in all likelihood the same linkage, giving rise to the blood group A determinant.
The transferase is capable of converting blood group Anegative into A-positive structures.
As demonstrated by hemagglutination inhibition assays, the transferase caused Anegative mucin to acquire the immunochemical characteristics of A-positive mucin. The fact that authentic A-positive mucin exhibited stronger A activity than the transferase reaction product may be due to the different origin and treatment of the two samples, or incomplete glycosylation, or simply experimental error. The slight A activity of the starting material might be caused by the presence of some contaminating Apositive mucin, or by cross-reaction of A antiserum with GalNAc residues bound to the polypeptide core. The porcine transferase also converts human 0-erythrocytes into cells that are indistinguishable from A-erythrocytes, as judged by agglutination with A-antiserum. In this respect, the enzyme is similar to A-specific transferases from human gastric mucosa (14) and human serum.4 After exhaustive glycosylation by the transferase, 3 x 10" GalNAc molecules are linked to each cell. This is in fair agreement with the known number (about 10") of immunoreactive A-type sites per human erythrocyte (15,16). The transferase glycosylates a variety of components of the erythrocyte membrane.
No attempt has been made to identify the labeled components, but it appears that the label is predominantly associated with glycoproteins, in contrast to the view (17, 18) that glycoproteins carry only a minor portion of the blood group A activity of human erythrocytes. In a recent report (19), glycoproteins isolated from human 0-erythrocytes were glycosylated by a partially purified A-specific transferase from human milk (12); the gel electrophoretic pattern was similar to the one obtained here. Isolated glycolipids are good substrates for the transferase (Table II), and glycolipids of the isolated erythrocyte membrane are glycosylated more extensively than those of intact erythrocytes, suggesting that the latter may not be easily accessible to the enzyme.
The N-acetylgalactosaminyltransferase reaction involves three substrates (cation, UDP-GalNAc, and acceptor). It has been shown that many three-substrate mechanisms can be distinguished on the basis of complete initial rate studies (8). Therefore, the kinetic studies presented here were undertaken, and the following conclusions drawn.
The transferase reaction does not proceed by an enzymesubstituted (ping-pong) mechanism since the binary initial rate parameters, @)M,p and a,:,, are clearly present in the rate equation (Tables VIII and IX), whereas Dalziel has shown (8) that all enzyme-substituted mechanisms lack at least two of the three binary @ parameters.
Thus the transferase reaction involves a quaternary enzyme complex. Five types of quaternary complex mechanisms may be considered: random addition of all three substrates (a), partly random, partly compulsory addition where one of the substrates must bind first (b) , second (c) , or third (d) , and compulsory order of addition of all three substrates (e). All mechanisms of type b and most mechanisms of type e lack @)ML. or Qc, in their equation (8) and can therefore be eliminated.
The choice is narrowed further by inhibition studies. Compulsory addition of UDP-GalNAc followed by acceptor is not consistent with the finding that 2'-fucosyllactose is a competitive inhibitor with respect to PSM and a noncompetitive inhibitor with respect to UDP-GalNAc.
Likewise, compulsory addition of acceptor followed by UDP-GalNAc can be excluded because UDP, a competitive inhibitor with respect to UDP-GalNAc, is found to be a noncompetitive inhibitor with respect to acceptor. This eliminates all mechanisms of type b and e as well as two mechanisms of type d. At present, the remaining closely related mechanisms cannot be distinguished from one another. They all involve random order of addition of UDP-GalNAc and acceptor, but differ in the order of cation binding, which is either compulsory (in second or third place only), or random. The latter mechanism, which is perhaps the most likely, is shown in Scheme I. The left half of the scheme indicates that cation, donor, and acceptor bind to the enzyme via a random order equilibrium pathway to form a quaternary complex, which then enters the product phase in a rate-limiting step. Pathways involving alternative substrates are shown in the right half of the scheme.
For a random order equilibrium mechanism, all equilibrium constants for the dissociation of substrates from enzyme . substrate complexes can be calculated from initial rate parameters (8). Dissociation constants calculated from initial rate parameters determined at saturating Mn2+ or saturating   asialo-PSM A-are given in Tables X and XI, respectively. The values for K,,, K,,, and K,,, (=K,,,) were obtained from two different experiments and their close agreement serves to confirm that the model is internally consistent. Similarly the dissociation constants for 2'-fucosyllactose, K',:$ and K'143, were obtained either directly or from the inhibition experiment described above and agree within the limits of experimental error.
It may be noted that in the proposed mechanism, the dissociation constants involving the quaternary complex are identical with the classical Michaelis constants. Binding of cation and UDP-GalNAc to the enzyme is synergistic. For example, the presence of UDP-GalNAc in an enzyme complex facilitates the binding of Mn2+, since K,,, is an order of magnitude smaller than the corresponding K,,. Moreover, the presence of Mn2+ facilitates the binding of UDP-GalNAc. The synergistic effect is even stronger when Cd*+ is substituted for Mn*+. The kinetic mechanism of galactosyltransferase from bovine milk, recently investigated in this laboratory (2), is very similar to the mechanism proposed here for N-acetylgalactosaminyltransferase in that both enzymes bind donor and acceptor in random order. The two mechanisms differ, however, in the by guest on March 22, 2020 http://www.jbc.org/ Downloaded from order of cation binding. Galactosyltransferase must bind the cation first before donor and acceptor can bind. The order of cation binding of IV-acetylgalactosaminyltransferase has not been established, but the data reported here are inconsistent with a mechanism where the cation binds as a compulsory first substrate.
Acknowledgments-We wish to thank Dr. Bernard Kaufman and Dr. John Bell for helpful discussions concerning aspects of the work described here. We are grateful to Drs.