Glycosyltransferases A and B: Four Critical Amino Acids Determine

Research: Science and Education edited by

Concepts in Biochemistry

William M. Scovell

Glycosyltranferases A and B: Four Critical Amino Acids Determine Blood Type

Bowling Green State University Bowling Green, OH 43403

Natisha L. Rose and Monica M. Palcic* Department of Chemistry, University of Alberta, Edmonton, Alberta, Canada, T6G 2G2; *[email protected] Stephen V. Evans Department of Biochemistry and Microbiology, University of Victoria, Victoria, British Columbia, Canada, V8W 3P6

Carbohydrates are well researched and understood for their essential roles as energy sources (starches) or as structural components of plant and bacterial cell walls (cellulose and peptidoglycan). More recently, carbohydrate research has expanded to include the areas of molecular mechanisms and carbohydrate–protein recognition. Cell surface carbohydrates, namely glycoproteins and glycolipids, have been shown to be involved in biological and immunological functions, including cell signaling and molecular recognition (1–4). They act as receptors for the binding of other cells, bacteria, viruses, and hormones. These advancements gave rise to the new area of glycobiology (1), the study of carbohydrates in such biological events. Glycosyltransferases, enzymes that catalyze the transfer of monosaccharide units to terminal sites of oligosaccharides, are responsible for the synthesis of the various carbohydrates on animal, plant, and bacterial cell surfaces. Since a different enzyme is required for the addition of each sugar, over 100 mammalian glycosyltransferases are thought to exist given the diversity of sugars and the large number of alternate glycosidic linkages found in mammals (5). Thousands more are required for the production of bacterial and plant oligosaccharide structures (6). Human ABO blood groups are characterized by the presence or absence of distinct carbohydrate structures on red blood cell surfaces. Blood type A and B individuals each display a trisaccharide that differs by only a single substituent, an acetamido group instead of a hydroxyl group on the terminal monosaccharide (Figure 1). However, this small difference can have fatal consequences in the event of a mismatched blood transfusion. Blood type O individuals express only an unsubstituted disaccharide precursor called the H antigen. The terminal sugars on blood type A and B structures are added to the disaccharide precursor by two different glycosyltransferase enzymes, GTA and GTB. They differ by only 4 amino acids of the 354 in full-length enzymes and Table 1. Possible Permutations of Antigens, Antibodies, and Donors for Each of the ABO(H) Blood Types Blood Type/Recipient

Antigens

A B

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Antibodies

Donor

A

Anti-B

A, O(H)

B

Anti-A

B, O(H)

AB

A, B

None

A, B, AB, O(H)

O

None

Anti-A, Anti-B

O

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therefore are a model system for the study of structure–function relationships. The production of E. coli codon-optimized synthetic GTA and GTB genes, in which site-directed mutagenesis can be easily performed, has allowed an in-depth study of the role of these 4 and other amino acids in substrate binding and catalysis. This review will highlight the history of human glycosyltransferases GTA and GTB and our current understanding of how they determine human blood groups. Discovery of ABO(H) Histo-Blood Antigens For centuries there have been reports in which experiments in blood transfusions were performed; however, such experimentation proved fatal to many. First insights into this were demonstrated in 1901, when Karl Landsteiner delineated the A, B, and O(H) blood group classifications (7). He observed that mixing blood from two individuals may lead to blood clumping, or agglutination, and that such agglutination was the result of an immunological reaction that occurred when the receiver of a blood transfusion had antibodies against the donor blood cells. Landsteiner was awarded the Nobel Prize for Medicine or Physiology in 1930 for this work. The fourth and rarest blood group, AB, was discovered in 1902 by his students (8). This initial work demonstrated that a transfusion only succeeds if the blood recipient does not have antibodies against the donor blood antigen. For instance, individuals with type O blood have both anti-A and anti-B antibodies and therefore cannot receive A, B, or AB blood, whereas people with type AB blood do not have either antibody and can receive blood of any type. Table 1 shows the presence of antibodies and antigens and the possible donor blood types for each of the corresponding ABO(H) blood types that can lead to successful blood transfusions. In the 1950s, the chemical structures that define the serological dependence of the ABO(H) blood groups were elucidated, and it was shown that such antigen specificity was determined by the presence or absence of carbohydrates on the red blood cells (9). Chemical characterization of the A and B antigens showed that they differ only by a single monosaccharide unit linked to the end of a carbohydrate chain of a glycoprotein or glycolipid on the surface of the red blood cell, as shown in Figure 1. Type O(H) individuals display a carbohydrate structure on their red blood cells that terminates in the sequence α-Lfucopyranose-(1→2)-β- D -galactopyranose abbreviated αFuc(1→2)βGal (Figure 1) and also known as the H-anti-

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Research: Science and Education OH

HO

O HO

OH

HO

O

OH

OR

O

OR

O H3C

O

OH

HO

O

NH

OR

HO

O HO

OH

HO

O

OH

HO

O

O

O

O

O

O

OH

OH

OH

HO HO

HO

HO HO

HO

αFuc(1→2)βGal-OR O(H) precursor antigen

αGal(1→3)[αFuc(1→2)]βGal-OR blood group B determinant

αGalNAc(1→3)[αFuc(1→2)]βGal-OR blood group A determinant

Figure 1. Terminal structures of blood group O, A, and B antigen determinants. The blood group O(H) precursor antigen is αFuc(1→2)βGalOR. The blood type A antigen terminates in the sequence αGalNAc(1→3)[αFuc(1→2)]βGal-OR and the blood type B antigen terminates in the sequence αGal(1→3)[αFuc(1→2)]βGal-OR. In natural acceptors R is a protein or lipid; for kinetic studies R is (CH2)7CH3.

HO

OH O OR

HO O O OH HO HO O(H) precursor

HO

O

OH O

O

HO

H3C

HO

O

NH

O

O

OH

O

HO

NH P P O O O ⴚ ⴚ O O O

N

O

OH

O

HO

P O

GTA

P ⴚ

O

O

HO

N

O

OH

UDP-Gal

OH

OH

HO O HO

O HO

OH

HO

O

OH

OR

O

OH

HO

O

NH H3C

ⴚ

O

OH

UDP-GalNAc

HO

O

O

GTB

NH

O

OR

O

O

O

O

O

O OH

OH

HO

HO HO

HO

Figure 2. Enzymatic synthesis of blood group A and B determinants. Human glycosyltransferase type A catalyzes the transfer of GalNAc from the donor UDP-GalNAc to the O(H) precursor to give the A antigen αGalNAc(1→3)[αFuc(1→2)]βGal-OR. Human glycosyltransferase type B catalyzes the transfer of Gal from the donor UDP-Gal to the same precursor to give the B antigen αGal(1→3)[αFuc(1→2)]βGal-OR.

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gen. This stands for heterogenetic as it is a common substance for the majority of red blood cells independent of blood type. The H-antigen is tethered to the red blood cells via integral membrane proteins or membrane associated lipids. Type A and B individuals also display some H-antigen precursor; however, they differ solely by the addition of either α-N-acetylgalactosamine (GalNAc) or α-galactose (Gal), respectively. Human blood group AB exhibits both the type A and type B antigens; whereas type O(H) exhibits neither. The same A and B carbohydrate structures have been found on other cells, primarily epithelial cells, and changes in the antigens may be associated with cell development of oncogenesis (10–13). Knowledge of the antigen structures helped to elucidate the nature of the biosynthesis of the ABO(H) blood groups that was first proposed by Watkins and Morgan (14). It was suggested that blood group A and B determinants were added to the same blood group O(H) substance by genetically encoded glycosyltransferase enzymes, as shown in Figure 2. This activity was first demonstrated by Tuppy and Staudenbauer (15) when they observed the transfer of [14C]-labeled Nacetylgalactosamine from UDP-[14C]-GalNAc to terminal positions of blood group O(H) substance in the presence of gastric microsomal preparations of type-A hogs, while microsomes from type-O(H) hogs did not transfer radiolabel from the donor. Furthermore, an N-acetyl-D-galactosaminyl transferase from human submaxillary glands of group A and AB donors, but absent from group O(H) and B donors was also described as the enzyme responsible for the transfer of N-acetylgalactosamine to the acceptor substrate (16). Similar studies have also demonstrated the transfer of galactose from UDP-Gal to the same O(H) acceptor unit (17, 18). These studies confirmed that the A antigen is formed by the addition of N-acetylgalactosamine from the UDP-GalNAc donor to the O(H) acceptor αFuc(1→2)Gal-β-R by Nacetylgalactosaminyl transferase A. The B antigen is formed by the addition of galactose from the UDP-Gal donor to the same O(H) acceptor by galactosyl transferase B. GTA and GTB are widespread in mammalian systems, as they have also been found in human serum, human milk, ovarian cyst fluid and erythrocyte membranes (19–23). Since the biosynthetic pathway of the determinants became established in the late 1960s, research attempts have focused on elucidating the chemical nature and properties of the A and B glycosyltransferases, with more recent efforts on understanding the structure–function relationships of these enzymes.

proteolytically sensitive stem region (35–62 aa) that functions as a flexible tether to the large catalytic domain that included the carboxy terminus (∼300 aa), as shown in Figure 3 (5, 6, 25, 27). Proteolysis of GTA and GTB in the stem region occurs naturally and produces soluble and active enzymes as found in blood and milk. Work in our laboratory has demonstrated that the minimal catalytic domain of GTB is comprised of amino acids 74–354; removal of additional amino acids from the N-terminus or any amino acids from the Cterminus of recombinant GTB and GTB results in loss of enzyme activity. Cloning and sequencing of complementary DNA from cell lines of different ABO status demonstrated both sequence and structural differences between the ABO genes (25). GTA and GTB were found to be among the most homologous of the glycosyltransferases, showing 99% homology (25, 28). The two enzymes differ by seven single base substitutions characterizing A and B allelic cDNAs. Three of the substitutions result in silent mutations, whereas four result in amino acid substitutions at residues 176, 235, 266, and 268. Given that they utilize a common acceptor, the differences of Arg176Gly, Gly235Ser, Leu266Met, and Gly268Ala between GTA and GTB, respectively, were assumed to be responsible for the donor specificity difference between the enzymes, with the last two substitutions thought to be the most critical (28, 29). The naturally low abundance of GTA and GTB in human sera is a limitation in studying human glycosyltransferases because of the inability to purify the enzymes from natural sources to homogeneity and in sufficient quantities. GTA was initially isolated from the plasma of type A individuals with 1000–100,000 fold purification (30, 31) and has since been purified to greater than 95% homogeneity from porcine submaxillary glands, human plasma, human stomach, and duodenal tissues (32–35). The reported molecular weight of purified GTA enzymes ranged from 36,000 to 46,000 with an optimum pH between 7.0 and 7.4. Purification to homogeneity from a natural source for GTB has

soluble glycosyltransferase C C

Properties and Sequences of Glycosyltransferases A and B

N

Despite the discovery of the ABO(H) blood groups in the early 1900s, it was not until 1990 when the nucleotide sequence of the ABO gene was determined and until 1995 when its genomic organization was elucidated (24–26). Based on these sequences, the mammalian GTA and GTB glycosyltransferases were confirmed to be type II integral membrane proteins containing 354 amino acids. Type II integral membrane proteins typically have a short aminoterminal cytoplasmic tail (6–27 aa), a hydrophobic membrane-anchoring domain (16–20 aa), and a short 1848


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proteolytic cleavage

Golgi lumen

C

Golgi membrane cytosol N

N

Figure 3. Schematic of the structure of glycosyltransferases showing how proteolytic cleavage of the membrane-tethered glycosyltransferase results in a soluble glycosyltransferase.


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yet to be achieved (36). A cloning strategy using synthetic genes with codons optimized for expression of human GTA and GTB in E. coli yields 50–100 mg of purified enzyme per liter of bacterial culture (37). This facilitates site-directed mutagenesis for the production of mutant hybrid enzymes (38) and allows in-depth studies of GTA and GTB using enzyme activity assays for kinetic characterization and crystallography to probe structure–function relationships. Kinetic Analysis of Glycosyltransferases A and B Reactions Through mutagenesis, we are able to manipulate the glycosyltransferase enzymes to alter their structure and behavior. Enzyme kinetics, the study of the rates and mechanisms of chemical reactions catalyzed by enzymes, is one tool that can assist in quantifying the effect of the change and in trying to determine the exact mechanism of GTA and GTB action. A detailed review on assay methods for glycosyltransferases has been reported (39). Radiochemical assays are commonly used for kinetics because of their sensitivity and the availability of UDP-[3H]Gal and UDP-[3H]-GalNAc donors. Co-incubation of the acceptor and radio-labeled donor results in the transfer of the labeled monosaccharide to the acceptor. The unreacted radio-labeled donor is removed from the labeled reaction product by taking advantage of the hydrophobic nature of our synthetic acceptor and products (Figure 1), which bind to reverse-phase (C18 ) SepPak cartridges. The unreacted radio-labeled donor is removed by washing the cartridge with water, then the radio-labeled product is eluted with an organic solvent (e.g., methanol) and quantitated by counting in a liquid scintillation counter (40). All kinetic parameters presented in this article were obtained by radiochemical assays with six different concentrations of donor and acceptor. Data were analyzed for a two-substrate system that can be described by eq 1 (41).

v =

K ia K B

Vmax [ A ][ B] + KB [ A ] + K A [B ] + [ A ][ B]

(1)

Equation 1 relates the reaction velocity (v) to the maximum velocity (Vmax) and the concentration of the acceptor [A] or donor [B] substrate, respectively. KB is the Michaelis–Menten constant (Km) for the donor; it is the concentration of donor that gives a velocity of Vmax2 when the acceptor concentration is saturating (Figure 4). KA is the Michaelis–Menten constant for the acceptor; it is the concentration of acceptor that gives a velocity of Vmax2 when the donor concentration is saturating. Kia is the dissociation constant for acceptor that is independent of the donor concentration. A low KA or KB value means the enzyme operates well with a low substrate concentration, whereas, a high KA or KB means enzyme is functional with a high concentration of substrate. When the experimental Vmax is divided by enzyme concentration, kcat, the catalytic rate constant is obtained. This is the maximum number of substrate molecules that can be converted to product per enzyme active site per unit time. These kinetic parameters were determined for the GTA and GTB enzymes and mutants to give insight into the roles of the four critical amino acids in substrate discrimination. www.JCE.DivCHED.org

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Reaction Velocity / (µmol Lⴚ1 sⴚ1)


3

Vmax

2

1

0 0

Km

400

800

1200

1600

2000

2400

ⴚ1

[Substrate] / (µmol L ) Figure 4. Michaelis–Menten plot of the reaction velocity (v) as a function of the substrate concentration.

Kinetic Constants for Wild-Type and Mutant GTA and GTB In the early 1980s, it was demonstrated that GTB had the capacity to utilize UDP-GalNAc for the synthesis of blood group A structures at a reduced rate relative to UDP-Gal (42). Slow cross-reactions of GTA with UDP-Gal can also be detected (43). These cross-reactions can be exploited in characterizing mutants in which the four critical amino acids have been interchanged to produce hybrid A and B transferase enzymes (44, 45). The parent enzymes have been designated as AAAA (GTA) and BBBB (GTB) and the mutant enzymes are denoted by combination of the A and B letters (e.g., BBAA), where A and B represent the corresponding critical amino acid of GTA and GTB, respectively. Table 2 has kinetic constants for wild-type and mutants using both UDPGal and UDP-GalNAc as donor substrates. True hybrid enzymes would possess equal A and B transferase activity analogous to the rare cis-AB phenotype of blood type AB individuals that have a single protein utilizing both donors (46). Compared to the parent GTA and GTB enzymes, the catalytic activity of mutants is significantly affected with as little as a single amino acid substitution with the observed changes being primarily due to differences in kcat rather than Km (44). The substitution of GTB residues into the first and second critical amino acid positions of GTA did not result in any GTB activity; but rather an enhanced reaction with the GTA donor UDP-GalNAc. In particular, the hybrid BAAA showed an 11-fold increase in the kcat compared with the wildtype AAAA enzyme, which is among the largest increases in kcat reported for a single amino acid change (44). Despite weaker acceptor binding, the double mutant BBAA also exhibited an increase in enzyme turnover with a 5-fold increase in kcat over the wild-type GTA. Hybrid A–B enzymes with the third and fourth amino acids consistent with those of GTA (i.e., AAAA, BAAA, BBAA, and ABAA), strongly catalyzed the A reaction with B activity being less than 1% of the wildtype activity, based on kcat results (44, 45). Whereas hybrids AABA, ABBA, BBBA, and BABA, which all have Met266 and Gly268 from GTB and GTA, respectively, possess dual A and B specificity and can therefore catalyze both reactions.


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Table 2. Kinetic Parameters of Wild-Type and Mutant Glycosyltransferases A and B Enzyme Residue Examineda 176, 235, 266, 268

UDP-N-Acetylgalactosamine b

KA /µM

KB /µM

UDP-Galactose

kcat/s

᎑1

KB/µM 6.3

Overall Activity

15

13

0.020

A

43

126

55

68

43

0.037

A

BBAA

206

51

24

708

36

0.030

A

ABAA

44

22

5.8

529

18

0.010

AABA

35

75

7.4

30

ABBA

251

184

12

292

BBBA

348

253

10

420

BBBB P234Sd

23

kcat/s᎑1

BAAA

BGlyBSerBMetBAla

4.9

KA/µM

AArgAGlyALeuAGly

BABA

5.6

A

0.95

A+B

87

4.4

A+B

56

2.1

A+B A+B

67

508

5.5

100

55

3.4

281

285

0.3

54

34

6.5

B

3740

167

0.088

A

a

Data from ref 45.

b

Michaelis–Menten constant for acceptor.

c

c

14

67

3.2

Michaeli–-Menten constant for donor.

d

The designation BBBB P234S indicates that proline 234 has been mutated to serine in the wild-type GTB enzyme (38).

It was previously thought that residues 266 and 268 were the most critical amino acids in determining both activity and nucleotide sugar-donor specificity (47). However, these data now suggest that residues 176 and 235 may also have important functions. Increased catalytic activity of BAAA and BBAA mutants show that Gly176 is involved in enzyme turnover; whereas Gly235 is involved in favorable binding of the acceptor substrate since BBAA, ABBA, and BBBA mutants with Ser235 showed weaker acceptor binding (44, 45). Further efforts to understand the structure and function of GTA and GTB requires information obtained from crystal structures that give molecular details of substrate interactions to determine the role played by each of the four critical amino acids in determining donor specificity (48).

X-Ray Crystal Structures Reveal Basis for Substrate Discrimination In 2002 we reported the crystal structures of soluble forms of cloned GTA and GTB (aa 63–354) (48). The general topology of GTA and GTB enzymes showed a polypeptide chain that was organized in two domains separated by a cleft that contained all four critical amino acid residues, as shown in Figure 5. The N-terminal domains recognized the nucleotide donor and the amino acid residues in the C-terminal domain formed the disaccharide acceptor binding site. The cleft housing the four critical amino acids also contains the DXD motif (defined by the residues Asp211, Val212, and Asp 213) that coordinate a manganese ion that interacts with

Figure 5. Crystal structure of α-(1→3)-galactosyltransferase (GTB).

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the β-phosphate group of the UDP-donor and is essential for catalysis. There are two disordered regions in the structure, the final 10 amino acids on the C-terminus and a loop consisting of residues 179–194. The crystal structure shows that only two of the four critical residues can make contact with the donor. Arg176Gly and Gly235Ser are the two critical amino acid residues that do not make contact with the donor. The Arg176Gly residue is the most distant from the substratebinding site; however, it is located in close proximity to the disordered loop that is often seen in structures of glycosyltransferases determined in the absence of donor. Although Gly235Ser does have direct contact with the donor, it is responsible for a change in conformation of the aliphatic tail on the acceptor in the presence of either GTA or GTB; this may account for the significant decrease in acceptor Km for GTA compared to GTB (Figure 6, top and center). The critical amino acids Leu266Met and Gly268Ala occupy positions in the active sites of GTA and GTB and, therefore, come into direct contact with the donor nucleotide sugar residues (Figure 6, top and center). This agrees with the early kinetic studies that suggested that residues 266 and 268 are involved in donor specificity (28, 29). The Leu266Met residue contacts the acetamido or hydroxyl group of the respective donor and, therefore, distinguishes between UDP-GalNAc and UDP-Gal. The larger acetamido group in UDP-GalNAc is accommodated by the smaller Leu266 in GTA. Similarly, the smaller hydroxyl group in UDP-Gal is accommodated by the larger Met266 in GTB. The role of Gly268Ala in donor specificity is unclear from the crystal structure because it is positioned to contact only the region of the donor nucleotide sugars that are identical in both UDP-GalNAc and UDP-Gal. However, it can be argued that Met266 and Ala268 in GTB are both bulkier than the corresponding residues in GTA that results in the floor of the active site being raised in GTB compared to GTA, as shown in Figure 6, top and center. This region in GTB is significantly smaller and is, therefore, more conformationally restricted in the active site cleft, which serves to position only UDP-Gal. Current efforts in this area are focused on obtaining crystal structures on mutants with other amino acids in the four critical positions. Conclusions and Future Work Despite such slight differences between GTA and GTB, other amino acids besides the four critical ones play significant roles in the specificity of the enzymes. These are often identified in blood banking laboratories when weak agglutination reactions are observed during blood typing (46, 49). One such mutatation having an effect on donor specificity in GTB-P234S was reported in France in 2002 (50). The GTB-P234S mutant was produced using site-directed mutagenesis of GTB and kinetic analysis show reversed donor preference and decreased acceptor binding (Table 2) (38). Crystal structure results suggested that the reversal of donor specificity was due to a void at the proline C-γ position that resulted in the alteration of the side chain orientation of M266 (Figure 6, bottom). Such a change in orientation was unfavorable to UDP-Gal, but provided sufficient space to accommodate the NAc moiety and so allow www.JCE.DivCHED.org

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Figure 6. Location of substrates in the active sites of GTA (top), GTB (center) and P234SGTB (bottom).


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the transfer of sugar from UDP-GalNAc, resulting in reversed donor specificity (i.e., the production of a GTB enzyme with a subsequent increase in A and a decrease in B transferase activity; ref 38). This demonstrates that conserved neighboring amino acids can strongly influence the specificity of the A and B transferases, which in turn means that there are many more areas to explore in understanding our human ABO(H) blood types. The review stresses that the four critical amino acids in type A and B glycosyltransferases play a crucial role in determining donor specificity; however, it also demonstrates that a single point mutation completely changes the donor specificity. We are only beginning to understand how these enzymes interact with their respective donors. Current work is focused on continuing to study the kinetic parameters of mutated forms of the enzymes and obtaining crystal structures of these enzymes in efforts to understand the complexities of our human ABO(H) blood system. Acknowledgments The authors would like to thank all of the numerous individuals who contributed to this multidisciplinary project. This research has been funded by the Natural Sciences and Engineering Research Council of Canada, the Canadian Institutes of Health Research, and the Alberta Ingenuity Centre for Carbohydrate Science. Literature Cited 1. Taylor, M. E.; Drickamer, K. Introduction to Glycobiology; Oxford University Press: Oxford, 2002. 2. Biology of Carbohydrates; Ginsberg, V., Phillips, W. R., Eds.; JAI Press Ltd.: London, 1991; Vol. 13. 3. Varki, A. Glycobiology 1993, 3, 97–130. 4. Dwek, R. A. Chem. Rev. 1996, 96, 683–720. 5. Paulson, J. C.; Colley, K. J. J. Biol. Chem. 1989, 264, 17615– 17618. 6. Hu, Y.; Walker, S. Chem. Biol. 2002, 9, 1287–1296. 7. Landsteiner, L. Wein Klin. Wschr. 1901, 14, 1132–1134. 8. Decastello, A.; Sturli, A. Muen. Med. Wschr. 1902, 49, 1090– 1095. 9. Watkins, W. M.; Morgan, W. T. J. Nature 1957, 180, 1038– 1040. 10. Clausen, H.; Hakomori, S. Vox Sang. 1989, 56, 1–20. 11. Davidsohn, I.; Stejskal, R. Haematologia 1972, 6, 177–184. 12. Singhal, A.; Hakomori, S. Bioessays. 1990, 12, 223–230. 13. Fiezi, T. Nature 1985, 314, 53–57. 14. Watkins, W. M.; Morgan, W. T. J. Vox Sang. 1959, 4, 91–119. 15. Tuppy, H.; Staudenbauer, W. L. Nature 1966, 210, 316–317. 16. Hearn, V. M.; Smith, Z. G.; Watkins, W. M. Biochem. J. 1968, 109, 315–317. 17. Kobata, A.; Grollman, E. F.; Ginsburg, V. Biochem. Biophys. Res. Commun. 1968, 32, 272–277. 18. Race, C.; Ziderman, D.; Watkins, W. M. Biochem. J. 1968, 107, 733–735. 19. Kim, Y. S.; Perdamo, J.; Bella, A.; Nordberg, J. Proc. Natl. Acad. Sci. U.S.A. 1971, 68, 1753–1756. 20. Ko, G. K. W.; Raghupathy, E. Biochem. Biophys. Res. Commun. 1972, 46, 1704–1712. 21. Kobata, A.; Ginsburg, V. J. Biol. Chem. 1970, 245, 1484–1490.

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