Journal of Immunogenetics (1978) 5,107-1 16.

STUDY OF T H E

I N S E R A A N D R E D CELL M E M B R A N E S O F H U M A N A S U B G R O U P S

a-N- A C E T Y L G A L A C TOS A M I N Y L T R A N S F E R A S E

J . P . C A R T R O N , J . BADET, C . MULETAND C H . S A L M O N

INSERM U76, Service d’Immunologie du Centre National de Transfusion Sanguine, Paris, France

(Received 13 May 1977; revision received 30 September 1977)

SUMMARY

The product of the A blood group gene in the erythrocyte membrane and serum from ‘weak A’ variants was investigated using low and high molecular weight acceptors and compared with common A, and A, blood samples. A reduced enzyme activity was detected, but only in some of the A variants, namely the A, (eight out of eleven), A, or A, samples. Other sera from A, (three out of eleven), A,, Aend or A,, individuals were apparently devoid of enzyme activity.

A kinetic study by temperature inactivation of A blood group sera also showed that A enzymes are more labile than B enzymes from B, or B,, sera. Moreover, in one case (A, sample Del.), a very fast inactivation of the A enzyme was observed, suggesting the occurrence of a variant enzyme qualitatively different from the others so far studied.

The erythrocyte membrane preparations from all A variants contained no detectable A enzyme activity except for the A, and A, samples, the former being three to five times more active than the latter. The B enzyme activities from four samples of B RBC tested were comparatively stronger than the A enzyme activity of A, RBC.

The results were discussed and it was suggested that the synthesis and/or the secretion of the A enzyme in the organism is not uniform from one tissue to another, but could depend on which of the ‘weak A’ alleles or modifier genes is involved.

I N T R O D U C T I O N

The occurrence of soluble A, B and H blood group gene-dependent glycosyltransferases in human sera allowed studies of these enzymes among normal blood donors with common ABH phenotypes (Schachter et al., 1971; Chester, 1971; Sawicka, 1971) and among

Correspondence: Dr J. P. Cartron, INSERM U76, Service d’Immunologie du Centre National de transfusion Sanguine, 6 Rue Alexandre Cabanel, F75739 Paris, Cedex 15, France.

0305-181 1/78/04004107$02.00 0 1978 Blackwell Scientific Publications

107

108 J . P. Cartron et al. exceptional blood samples such as 'Bombay' (Race & Watkins, 1972a,b), cis AB (Kogure, 1975; Pacuszka et al., 1975; Badet, 1976), A,, A,, (Cartron et al., 1975; Cartron, 1976) and B, phenotypes (Simmons & Twaitt, 1975; Koscielak et al., 1976).

Such studies are of considerable value to biochemical genetics, because they demon- strate that the synthesis of the ABH substances is actually controlled not only by numerous ABO alleles but also by several modifier or suppressor genes not necessarily linked to the ABO locus.

Therefore other infrequent variants of the ABO locus, namely A,, A,, Aend and A,, phenotypes were investigated with respect to their serum and red cell membrane a-N- acetylgalactosaminyltransferase and compared to A,, A,, A, and A, blood samples. Furthermore, thermal inactivation of the A enzymes in these sera is reported and com- pared to the B enzyme (a-D-galactosyltransferase) in blood group B individuals.

M A T E R I A L S A N D M E T H O D S Blood samples

Red cell samples from normal blood donors (Al, A,, B and 0 RBC) and 'weak A' individuals (A3, A,, Aend, A,, A, and A,, RBC) were kept frozen in liquid nitrogen until used. Sera from these people had been stored at -2OOC before glycosyltransferase studies.

Nucleotide sugars UDP-N-acetyl-D-[ l-14C]-galactosamine (49.5 mCi/mmole) and UDP-~-['~C]galactose

(28 1.5 mCi/mmole) were purchased from NEN Chemicals (Frankfurt) and respectively used in 50% and 2% ethanol solution. Unlabelled UDP-N-acetyl-D-galactosamine was synthesized according to Carlson et al. (1964) and Roseman et al. (196 1).

A cceptors fo r glycosyltransferases N-acetyl-D-galactosamine (Sigma) was used in 10 mg/ml solution in distilled water. Fucosyllactose (2'FL), or o-a-L-fucopyranosyl( 1 -+ 2)-o-~-~-galactopyranosyl( 1 4 4 ) - ~ -

glucopyranose was extracted from human milk according to Whisler & Durso (1950). The purified human blood group H substance from ovarian cyst fluid was generously

supplied by Dr W. M. Watkins. The N-acetyllactosamine obtained by chemical synthesis was a gift from Professor

Sinay.

A and B blood group glycosyltransferase assays in sera 18 hr incubation process with 2'FL. The amount of A or B [14C]tetra~accharide*

synthesized after 18 hr of incubation at 37°C by 10 p1 of A or B human sera in the presence of 2'FL in standard conditions of assay has already been described (Badet et al., 1974; Cartron et al., 1975).

72 hr incubation process with 2'FL or human H substance. A more sensitive assay of A enzyme activity was developed by increasing the amount of serum and substrate in

* A tetrasaccharide: GalNAc-a(l -P 3)-Gal-[~-Fuc-ar-(l + 2)1-/3(1 + 4)Gl. B tetrasaccharide: Gal-dl - 3)- Gal-[~-Fuc-lr-( 1 -P 2)]-/l( 1 -+ 4)Gl.

Transferases in human A subgroups 109 assay and also by raising the incubation time to 72 hr. The following components in a total volume of 90 pl were used: 4.0 pmol MnCl,; 4 -0 pmol of a sodium cacodylate buffer containing 0.5 g/l N,Na (pH = 6.0 for A,, A:' sera and A, (Del.), or pH = 7.0 for other A variant sera); 0.4 pmol ATP; 20 pl serum as source of enzyme; 2.0 nmol UDP-N-acetyl-D-[ l-14C]gdactosamine (approximately 1.6 x lo5 ct/min); 0.6 pmol 2'FL or 100 pg of human H substance. Controls without H substrate were simultaneously run.

Ident8cation of reaction products. Following the incubation process of 18 to 72 hr, the A and/or B tetrasaccharides synthesized from 2'FL were isolated and identified as pre- viously described (Badet et al., 1974; Cartron et al., 1975). The N-acetyl-~-[-l-~~C]galact- osamine incorporated onto the H substance was estimated after high voltage electro- phoresis in 0.1 M ammonium formate buffer, pH 3-5 (Whatman paper No. 40; 3000 V, 200 mA), and descending chromatography in ethylacetate-pyridine-acetic acid-water

In all cases, the endogenous activities estimated from incubation mixtures devoid of the H substrate were deduced. For the A enzyme tests, these endogenous activities are always very weak (100 to 500 ct/min), even for prolonged incubation times.

( 5 : 5 : 1 : 3).

A and B blood group glycosyltransferase assay in erythrocyte membranes The red cell ghosts from unthawed cells were prepared according to Dodge et al. (1963).

The protein content of the red cell membrane suspension was then estimated by the Folin ciocalteu reagent (Lowry et al., 1951). The A or B blood enzymes of the preparation were measured in incubation mixtures containing the following components: 400 to 600 pg erythhrocyte membrane proteins; 6.0 pmol MnC1,; 10 pl of Triton X-100 5% (v/v); 6.0 pmol of sodium cacodylate buffer, pH 7.0, containing 0.5 g/l N,Na; 0.6 pmol 2'FL; 2.0 nmoles UDP-N-acetyl-D-[ l-'4Clgalactosamine (approximately 1-6 x lo5 ct/ min) or 0.7 nmol UDP-[14C]galactose (approximately 3 x lo5 ct/min). Negative controls devoid of 2'FL were simultaneously run. After standing 72 hr at 37°C the A and B [ ''C]tetrasaccharides have been isolated and estimated as described above.

Thermal inactivation of A and B blood group enzymes from human sera 100 p1 of unfractionated sera were introduced in a series of glass test tubes of 13 mm

diameter. The tubes were closed and heated in a temperature-controlled water bath (k0.1 "C) for a period of time ranging from 10 to 60 min. At the end of the heating period, the sera were allowed to cool by standing for 1 hr at room temperature. The remaining A and B enzyme activities were then measured by the 18 hr incubation process and the results expressed as the percentage residual activity of a non-heated sample. All sera were run in duplicate at 5OoC, 56°C and 62°C.

Assay of Pgalactosyltransferase in human sera The /3-galactosyltransferase of sera was estimated using the following components in a

total volume of 50 pl: 1.0 pmol MnCl,; 2.0 pmol of a sodium cacodylate buffer, pH 6.5; 0.5 pmol ATP; 10 pl serum as enzyme source; 0.2 nmol UDP-[14Clgalactose (approxim- ately 9 x lo4 ct/min); 0.45 pmol N-acetylglucosamine.

The mixture was incubated for 30 min at 37°C and the N-acetyl-[14Cllactosamine was identified by paper chromatography after high voltage electrophoresis in a method similar to that described for the identification of A and B tetrasaccharides.

110 J. P. Cartron et al.

R E S U L T S Galactosyltransferase activities of human sera

As could be expected, a Pgalactosyltransferase activity was found in all the sera tested, whatever their ABO blood group, unless the sera had previously been heated, destroying the heat-labile components of complement. The synthesized product was destroyed by a ,&galactosidase from the jack bean, being specific for the /I( 1 --* 4) linkage. Its RBs, (0.48 to 0.50) after chromatography in n-butanol-ethanol-water (10 : 1 : 2) was similar to the chemically pure N-acetyl-lactosamine (Rga, = 0.50). The results are reported in Table 1 for A,, A,, By AB and 0 human sera as well as for ‘weak A’ (A,, A,, Aend, A,, AJ samples.

TABLE 1. P-galactosyltransferase activity of human sera from common A and ‘weak A’ individuals expressed as the amount of N-acetyl- [‘4Cllactosamine synthesized according to experimental conditions

described in the text

Sera N-acetyl-[ 14Cllactosamine

Mean activity of total Percentage

Number Type (ct/min) counts

5 A1 45600 0.506 5 A2 4 1500 0.46 1

14 B(I, 11,111) 5 1390 0.571 6 0 39730 0.441 6 AB 41770 0.464

4 1 ‘Weak A’ 40600 0.45 1

The amount of radiolabelled N-acetyl-lactosamine synthesized was very similar for each group, and more sera should be tested before statistically demonstrating a higher activity in B sera.

From these results it was considered that the ‘weak A’ samples were suitable for further studies of the A blood group gene-specified glycosyltransferase. Indeed, some sera have been kept frozen for several years and these results mean that such sera have not been altered or, above all, been heat-inactivated before storage.

A enzyme activities of the A variant sera In the standard 18 hr incubation process no a-N-acetylgalactosaminyltransferase

activity was found among most of the A variants sera, except for A,,, and some A, individuals (Cartron et al., 1975). By increasing the incubation time to 72 hr and the amount of reagents in the incubation mixtures, more reliable enzyme activities were obtained for these blood samples (Table 2). Two main A, groups (I and 11) were schemati- cally distinguished according to the amount of labelled product formed. In the group I, 1 to 17% of the N-acetyl-D-[ l-14C]galactosamine was incorporated onto the acceptor. Sera from group 11 seem to be devoid of A enzyme activity, but it is more likely that these individuals belong, in fact, to the trailing edge of group I sera. With one A, blood (Del.) a high A enzyme activity optimum at pH 6.0 was detected.

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112 J. P. Cartron et al. Further studies of A, blood samples, using more sensitive assays, have also demon-

strated a weak but definitive A serum enzyme activity, which suggests that despite a double dose of the hypothetical ‘yAy recessive modifier, the A gene may not be completely silent.

All the sera from A,, Aend and A,, individuals, which were examined are apparently devoid of A enzyme activity, even when the incubation time was extended to 6 days, We also verified that these sera, as well as the A, group I1 sera, had neither inhibitory activity towards the A enzyme from normal A, and A, sera nor glycolitic activity towards a purified A-specific [‘4Cltetra~accharide. For that purpose mixtures (v/v) of A, or A, sera and A,, Aend, A,, or A, (group 11) sera were incubated with 2’FL, UDP-N-acetyl-D- [ l-14Clgalactosamine and appropriate cofactors under experimental conditions similar to the usual assays. In all cases, an expected enzyme activity half that of saline controls was observed. The absence of glycolytic activity in all the ‘weak A’ samples was deduced from experiments in which the incubation at 37OC for 72 hr of A,, A,, Aend, Ae,, A,,, or A, sera with the purified A-specific 14Cltetrasaccharide or UDP-N-acetyl-D-[ l-I4C]galac- tosamine did not lead to a significant release of N-acetyl-D-[ 1 -‘4Clgalactosamine (results not shown).

When the A enzyme activities were tested with H glycoprotein from ovarian cyst fluid as an acceptor, only A,, A, and A$‘ sera gave clear positive results (Table 2). The A, enzymes were on average eight times more active than the A, enzymes. In agreement with previous results, A$, enzyme activity is only 1/3 to 1/2 of that found in A, control sera (Cartron et al., 1975). Approximately 0.4% of the total N-acetyl-~-[ l-14C]galactos- amine was conveyed onto the H glycoprotein precursor by the A, (Del.) serum. Such a weak activity is, however, similar to that obtained with common A, samples.

Finally, it is observed that 2’fucosyllactose acts as a much better substrate, compared to the H glycoprotein; this reflects a difference on a molar basis between the number of available H-determinants.

Glycosyltransferases in A and B erythrocyte membranes The presence of A and B enzymes was demonstrated in erythrocyte membrane prepara-

tions from A,, A, and B individuals respectively, but Triton X-100 was, however, needed for maximal enzyme activity (Table 2). The A, red cell enzyme was found to be three to five times less active than the A, enzyme. When B erythrocytes were tested, a large difference between B, and B,, individuals was noticed, the latter having the highest enzymatic activity. It is interesting to recall that B, and B,, categories were defined on the basis of a significant difference between their serum a-D-galactosyltransferase activity (Badet et al., 1974). However, more B samples have to be analysed before a strict correlation could be established between the two phenomena.

In the experimental conditions used, the ghost preparations from all ‘weak A’ RBC were devoid of A enzyme activity.

This observation is not surprising for A,,, and A, individuals, known to have only a small number of A receptors; the latter very likely arise from either infrequent ABO alleles or modifying genes inhibiting the A enzyme synthesis, at least in the erythroblasts.

The absence of A enzyme in the red cell membranes from A, people was rather unexpected because they are characterized by an A antigen site density ranging between 30,000 and 100,000 receptors per RBC. The very low A antigenic content of A, and Aend

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114 J. P. Cartron et al. and A,, RBC may explain the negative results observed, but other hypotheses could be formulated (see the Discussion section).

Thermal inactivation of A and B glycosyltransferases For most of the proteins, the range of suitable temperatures for kinetic studies of heat

inactivation is rather small, usually between 50 and 65°C. In this work, the blood group A and B enzyme activities of whole sera have been

measured after exposure at 50"C, 56°C and 62°C for various periods of time, the shortest being 10 min. Control experiments were simultaneously run with the sera kept at room temperature. The results were expressed as the percentage of residual enzyme activity of heated vs non-heated samples and are plotted in Fig. la, b and c. Eleven sera were investigated, including three B (1, I1 and HI), two A,, two A,, two A, (group I and 111) and two A, samples.

It can be seen that the loss of A and B catalytic activity is irreversible and proceeds according to a first order kinetics reaction, which suggests that this step is independent of the protein concentration.

From the reported data, it can be concluded that A,, A,, A, (group I) and A,,, enzymes are more thermolabile than B enzymes. Indeed, half of the A catalytic activity is destroyed after 10 to 17 min at 62°C while the same decrease is observed only after 24 to 28 min with B enzymes.

A fast heat denaturation was observed with the A, enzyme from Del., the only sample of the so-called A, group 111, which suggests that this peculiar enzyme molecule is somewhat different from the other A enzymes investigated. This difference is obvious when the temperature increases from 50°C to 62°C.

D I S C U S S I O N

In this study, the product of the A gene has been investigated in the sera and red cell membranes from numerous A blood samples, including A,, A,, A,, A,, Aend, A,,, A, and A, variants. The properties of the A, and A, enzymes were found to be in agreement with qualitative and quantitative studies previously reported (Schachter et al., 197 1 ; Schachter et al., 1973). Furthermore, it was shown that the A enzyme activity is three to five times higher in A, than in A, red cell membranes, but in our hands the two enzymes did not display a different optimum pH for their activity as demonstrated with A, and A, whole sera.

The clear-cut difference between A, and A, membrane enzymes was not found by Kim et al. (1971), probably because these authors used a sialidase-treated porcine submaxillary mucin rather than 2'fucosyllactose as the blood group H active acceptor.

The a-D-galactosyltransferase activity (B blood group enzyme) of some B erythrocyte membrane preparations was also investigated. Higher enzyme activities were noticed, together with a clear distinction between B, and B,, blood samples, the latter having the highest enzyme activity, a phenomenon also demonstrated in the respective sera (Badet et al., 1974).

Only a few of the A variants, namely some A,, A, and A,, individuals, had a detectable A enzyme activity which was, however, always reduced compared to common A, or A, blood samples. The heat lability of these enzymes was investigated, to see if such a property may be used to distinguish betweeneach gene product.

Transferases in human A subgroups 115 The heat inactivation of the glycosyltransferases probably results from the unfolding

of peptide chains following the breakdown of non-covalent bonds. The consecutive loss of enzyme activity is irreversible, and it was shown that A enzymes are more susceptible to the effects of temperature than B enzymes. However, the effects observed may partly be due to other serum components and the behavior of A or B enzymes should be checked with purified preparations. In the case of A, (Del.) serum, the presence of a very heat- labile enzyme may indicate the occurrence of a defective enzyme qualitatively different from the other glycosyltransferases.

The results presented in this paper are in accord with the difference between A, and A, individuals previously reported (Cartron et al., 1975). Furthermore, it is now demon- strated in a more sensitive assay that A, people have a very weak A enzyme activity, which suggests that the A blood group gene is not completely silent, despite the presence of the hypothetical ‘yA’ suppressor gene.

A similar situation was encountered by Chester (1971), who found a weak 4 - m ~ - fucosyltransferase activity among Le(a - b-) individuals. In both cases, however, it cannot be excluded that a normal amount of catalytically inactive enzyme was present.

At variance with what is observed with A,, people, the apparent blockage of A enzyme synthesis could be restricted, in A, individuals, to the erythroblasts, as is suggested by the absence of the A enzyme in A, ghosts. Our results in this field are in agreement with those of Koscielak et al. (1976), who failed to detect any B enzyme activity in B, erythrocyte preparations. However, it is not known if other cells, in addition to erythro- blasts, are devoid of A or B enzyme activity, but this is probably the case since A,,, and B, sera have only 30 to 50% of the enzyme activity found in common A or B blood samples. The submaxillary glands are obviously not affected, since normal amounts of A or B blood group substances were found in A, and B, saliva (Race & Sanger, 1975). Furthermore, Kogure & Furukawa (1976) have recently shown that the B enzyme activity of B, and common B saliva investigated by enzymatic conversion of 0 into B RBC are similar. The same result is expected for the A enzyme of A,, A, or A, saliva. However, preliminary studies in our laboratory revealed that the A enzyme activity of all A saliva is very weak and quite difficult to quantify with sufficient accuracy (Cartron, unpublished data).

With some exceptions among A, blood samples, no a-N-acetylgalactosaminyltrans- ferases could be found either in the sera or erythrocyte membranes from A, (group II), A , Aend and A,, individuals. These corresponding sera have no inhibitory activity towards A enzyme activity and no splitting activity towards A-specific tetrasaccharide or UDP- GalNAc.

In our opinion, the absence of detectable A enzyme activity in these sera probably means that the synthesis of A-reactive structures is limited to a very few cellular lines.

We suggest not only that each ‘weak A’ variant leads to a variable number of A re- ceptors on the cell membranes, as a result of more or less active a-N-acetylgalactosaminyl- transferase, but also that the distribution of cells (or tissues) which are capable of synthesiz- ing or secreting the A enzyme is not necessarily identical from one variant to another. This would explain why among A, blood samples, all characterized by a similar number of A red cell receptors, only some of them (A, group I) have a detectable A enzyme activity in their sera. Similarly, A, blood samples, despite their very low RBC antigenic content, have a small but easily detectable serum enzyme, whereas Ax, Aend or A,, variants with a higher A RBC antigenic density are apparently devoid of enzyme activity. Such a situation

116 J. P. Cartron et al. argues for the possible role of still unknown regulator genes, which would control the ABO blood group enzyme synthesis or secretion.

R E F E R E N C E S

BADET, J. (1 976) Activites glycosyltransferasiques seriques associees a la biosynthese des antigenes de groupes sanguins A et B. Application a I’ktude de sujets B normaux et cis AB. Revue Francaise de Transfusion et d’Immuno-hbmatologie, 19, 105.

BADET, J., ROPARS. C., CARTRON, J.P. & SALMON, CH. (1974) Groups of a-D-galactosyltransferase activity in sera of individuals with normal B phenotype. Biomedicine, 21,230.

CARLSON, D.M., SWANSON, A. & ROSEMAN, S. (1964) Preparation of crystalline D-galactose-1-phosphoric acid and its conversion of UDP-N-acetylgalactosamine. Biochemistry, 3,402.

CARTRON, J.P. (1 976) Etude des proprietes a-N-acetylgalactosaminyltransferasiques des serums de sujets A et ‘A faible’. Revue Francaise de Transfusion et d’lmmuno-himatologie, 19, 67.

CARTRON, J.P., GERBAL, A., ROPARS, C. & SALMON, CH. (1975) Assay of a-N-acetylgalactosaminyl- transferases in human sera. Vox Sanguinis, 28, 347.

CHESTER, M.A. (1971) The role of fucosyltransferases in the biosynthesis of blood group substances. Thesis for Ph.D. degree, University of London.

DODGE, J.T., MITCHELL, C. & HAWAHAN, D.J. (1963) The preparation and chemical characteristics of hemoglobin free ghosts of human erythrocytes. Archives of Biochemistry and Biophysics, 100, 119.

KIM, Y .S., PERDOMO, J. BELLA, A. & NORDBERG, J. (197 1) N-acetylgalactosaminyltransferase in human serum and erythrocyte membranes. Proceedings of the National Academy of Sciences (Washington), 68, 1753.

KOGURE, T. (1975) The action of group Bm of cis AB sera on group 0 red cells in the presence of UDP-rGalactose. Vox Sanguinis, 29, 5 1.

KOGURE, T. & FUKUKAWA, K. (1976). Enzymatic conversion of human group 0 red cells into group B active cells by a-D-galactosyltransferases of sera and salivas from group B and its variant types. Journal of Immunogenetics, 3, 147.

KOSCIELAK, J., PACUSKA, T. & DZIERZKOWA-BORODEJ, W. (1976) Activity of B gene specified galactosyl- transferase in individuals with B, phenotypes. Vox Sanguinis, 30,58.

LOWRY, O.H., ROSENBROUGH, N.J., FAKR, A.L. & RANDAL, R.L. (1951) Protein measurements with the Folin reagent. Journal of Biological Chemistry, 193, 250.

PACUSZKA, T., KOSCIELAK, J., SEYFRIED, H. & WALEWSKA, I. (1975). Biochemical, serological and family studies in individuals with cis AB phenotypes. Vox Sanguinis, 29, 292.

RACE, C. & WATKINS, W. (1972a) The enzymic products of the human A and B blood group genes in the serum of the ‘Bombay’ 0, donors. FEBS Letters, 27, 125.

RACE, C. & WATKINS, W.D. (1972b) The action of the blood group gene specified a-galactosyltransferase from human serum and stomach mucosal extraits on group 0 and ‘Bombay’ 0, erythrocytes. Vox Sanguinis, 23, 380

RACE, R.R. & SANGER, R. (1968) Blood groups in man, 5th edn, p. 18. Blackwell Scientific Publications Ltd, Oxford.

ROSEMAN, S., DISTLER, J.J., MOFFATT, J.G. & KHORANA, H.G. (1961) Nucleosides polyphosphates of nucleotide coenzymes. XI. An improved general method for the synthesis of uridine-5’, cytidine-5’ and guanosine-5’ diphosphate derivatives. Journal of American Chemical Society, 83,659.

SAWICKA, T. (1971) Glycosyltransferases of human plasma. FEBS Letters, 16,346. SCHACHTER, H., MICHAELS, M.A., CROOKSTON, M.C., TILLEY, C.A. & CROOKSTON, J.H. (1971) A

quantitative difference in the activity of blood group A specific a-N-acetylgalactosaminyltransferase in serum from A, and A, human subjects. Biochemical and Biophysical Research Communications, 45, 101 1.

SCHACHTER, H., MICHAELS, M.A., TILLEY, C.A., CROOKSTON, M.C. & CROOKSTON, J.H. (1973) Qualitative differences in the a-N-acetyl-D-galactosaminyltransferases produced by human A, and A, genes. Proceedings of the National Academy of Sciences (Washington), 10,220.

SIMMONS, A, & TWAITT, J. (1975) Another example of a B variant. Transfusion (Philadelphia), 15,359. WHISLER, R.L. & DURSO, D.F. (1950) Chromatographic separation of sugars and charcoal. Journal of the

American Chemical Society, 12,677.