Acetyltransferase from Guinea Pig Liver

Biol. Chem., Vol. 384, pp. 1035 – 1047, July 2003 · Copyright © by Walter de Gruyter · Berlin · New York

Solubilisation and Properties of the Sialate-4-OAcetyltransferase from Guinea Pig Liver

Matthias Iwersen1, Hiltrud Dora1, Guido Kohla1, Shinsei Gasa2 and Roland Schauer1,* Biochemisches Institut, Christian-AlbrechtsUniversität, Olshausenstr. 40, D-24098 Kiel, Germany 2 Department of Chemistry, Sapporo Medical University School of Medicine, Sapporo 060-8556, Japan 1

*Corresponding author

The O-acetylation of sialic acids turns out to be one of the most important modifications that influence the diverse biological and pathophysiological properties of glycoconjugates in animals and microorganisms. To understand the functions of this esterification, knowledge of the properties, structures and regulation of expression of the enzymes involved is essential. Attempts to solubilise, purify or clone the gene of one of the sialate-O-acetyltransferases have failed so far. Here we report on the solubilisation of the sialate4-O-acetyltransferase from guinea pig liver, the first and essential step in the purification and molecular characterisation of this enzyme, by the zwitterionic detergent CHAPS. This enzyme O-acetylates sialic acids at C-4 both free and bound to oligosaccharides, glycoproteins and glycolipids with varying activity, however, gangliosides proved to be the best substrates. Correspondingly, a rapid enzyme test was elaborated using the ganglioside GD3. The soluble O-acetyltransferase maximally operated at 30 °C, pH 5.6, and 50 – 70 mM KCl and K2HPO4 concentrations. The Km values were 3.6 µM for AcCoA and 1.2 µM for GD3. CoA inhibits the enzyme with a Ki value of 14.8 µM. A most important discovery enabling further enzyme purification is its need for an unknown low molecular mass and heat-stable cofactor that can be separated from the crude enzyme preparation by 30 kDa ultrafiltration. Key words: Guinea pig liver / 5-N-acetyl-4-O-acetylneuraminic acid / Sialate-4-O-acetyltransferase / Sialic acid / Solubilisation.

Introduction Since the discovery of O-acetylated sialic acids by Blix and Lindberg (1960), interest in this modification of sialic acids has increased (Varki, 1992; Schauer et al., 2000; Schauer, 2000a). The frequent finding of such sialic acids, derivatives of both N-acetylneuraminic acid (Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc) (for the nomencla-

ture of sialic acids see Schauer and Kamerling, 1997), is partly due to the recent development of very sensitive analytical techniques that allow a more accurate analysis of sialic acids. Sialate-O-acetylation can be differentiated into two types, one where the glycerol side chain is modified and the other where only the pyranose ring hydroxyl group at C-4 is esterified (Varki, 1992; Schauer and Kamerling, 1997; Schauer, 2000b). All the hydroxyl groups present on the glycerol side chain may be acetylated, either separately or in combination with one another. 9-O-Acetylation, however, prevails. Esterification of the glycerol side chain may also occur together with O-acetylation at C-4. The combinations of O-acetyl groups with other substituents, like 9-O-lactyl (Varki, 1992; Schauer and Kamerling, 1997) or 8-O-methyl groups (Manzi et al., 1990; Bergwerff et al., 1992; Klein et al., 1997), have been reported. This much contributes to the large sialic acid family. Interest in sialate-O-acetylation has so far mainly focused on esterification of the glycerol side chain, since this type of modification seems to be more widely distributed in nature, including man, than 4-O-acetylation (Varki, 1992; Schauer and Kamerling, 1997; Schauer et al., 2000). Nevertheless, the number of animal species expressing 4-O-acetylated sialic acids is growing and these monosaccharides are found in horses and donkeys (Schauer and Kamerling, 1997), the monotreme echidna (Kamerling et al., 1982, and summarised in Schauer, 1982), guinea pigs (Hanaoka et al., 1989), rabbits (Klein and Roussel, 1998), rats (Morimoto et al., 2001), the pit viper Bothrops moojeni (Lochnit and Geyer, 1995), and in the dace Tribolodon hakonensis (Inoue et al., 1989). Due to the fact that mouse hepatitis viruses (MHV) express a sialate-4-O-acetylesterase and are capable of infecting the brain of juvenile mice (Regl et al., 1999), it is reasonable to suggest that these animals contain 4-Oacetylated sialosides. Correspondingly, 5-N-acetyl-4-Oacetyl-neuraminic acid (Neu4,5Ac2) was possibly identified in the mouse parotid gland by lectin staining and saponification experiments (Accili et al., 1996; Menghi et al., 1996). Even the occurrence of 4-O-acetyl-5-N-glycolyl-neuraminic acid (Neu4Ac5Gc) as a component of the ganglioside GM3 (for ganglioside nomenclature see Svennerholm, 1963) in human colon cancer has been reported (Miyoshi et al., 1986), and Zanetta et al. (2001) obtained mass spectrometric evidence for the existence of traces of 4-O-acetylated sialic acids in man. Neu4,5Ac2 has also been reported in young pupae of the insect Galleria mellonella (Karaçali et al., 1999). Little is known about the biological function of 4-Oacetylated sialic acids, although they have been shown

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to prevent the action of catabolic enzymes like sialidases and sialate-pyruvate-lyases (Schauer, 1985; Corfield et al., 1986; Schauer et al., 1988b; Kleineidam et al., 1990; Matrosovich et al., 1998; Smith et al., 1999), which may extend the lifetime of glycoconjugates (Schauer, 1985). These observations led to the discovery of the first enzyme de-esterifying sialic acids, the sialate-4-Oacetylesterase in horse liver (Schauer et al., 1988b). It initiates the catabolic pathway of 4-O-acetylated sialosides prior to the action of sialidases. The finding of Neu4,5Ac2 as a ligand for mouse hepatitis virus has been mentioned above. Furthermore, Neu4,5Ac2-containing glycoproteins inhibit propagation of influenza A virus upon infection (Matrosovich et al., 1998). It can therefore be assumed that this modification is also involved in the regulation of molecular and cellular recognition processes, as is known for sialic acids O-acetylated in their side chain, for example 5-N-acetyl-9-O-acetyl-neuraminic acid (Neu5,9Ac2) (Varki, 1992; Kelm and Schauer, 1997; Schauer and Kamerling, 1997; Schauer et al., 2000; Schauer, 2000b). A wealth of information is now available on the involvement of Neu5,9Ac2 in microbial infections, adhesion processes, immunology, tissue differentiation and tumour growth, as well as in maintaining the physicochemical properties of glycoconjugates. Sialate-O-acetylation is carried out enzymatically by the sialate 4- and the sialate 9(7)-O-acetyltransferases (EC 2.3.1.44/45), which were discovered in equine and bovine submandibular glands, respectively (Schauer, 1970a,b). Since then these membrane-bound enzymes have been shown to be localised in the Golgi apparatus or the trans-Golgi network (Schauer et al., 1988a; Diaz et al., 1989b; Iwersen et al., 1998; Tiralongo et al., 2000; Shen et al., 2002), although a very low soluble sialate 9-Oacetyltransferase activity has also been detected in bovine submandibular glands (Schauer et al., 1988a). However, no sialate-O-acetyltransferase could be isolated or cloned so far (Ogura et al., 1996; Shi et al., 1998; Shen et al., 2002), although detailed knowledge of these enzymes and their genes is required because of their great biological and

Fig. 2 Radio Thin-Layer Chromatogram of Neu4,5Ac2 Formed by Soluble Sialate-4-O-Acetyltransferase. Panel (A): The enzyme was released from Golgi membranes of guinea pig liver and incubated with 300 µM free Neu5Ac and [3H]AcCoA. Panel (B): control incubation without exogenous Neu5Ac. Both samples were treated exactly under the same conditions of Test B. The nature of the radioactive product formed was confirmed by co-migration with authentic Neu4,5Ac2 purified from horse submandibular glands and staining with the orcinol reagent. For further details see the text.

pathophysiological significance. In a previous study we characterised the membrane-bound sialate 4-O-acetyltransferase from guinea pig liver (Iwersen et al., 1998). Here we report on the solubilisation of this enzyme by the zwitterionic detergent 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonic acid (CHAPS), where ‘solubilised enzyme’ is used for detergent-solubilised proteins in micelles, since all work was done above the critical micellar concentration of CHAPS. The O-acetyltransferase was characterised with regard to substrate specificity, kinetic properties, inhibitors and activators.

Results Solubilisation of the Sialate-4-O-Acetyltransferase from Guinea Pig Liver Golgi Membranes Fig. 1 Solubilisation of the Sialate-4-O-Acetyltransferase. The optimal concentration of CHAPS for solubilisation of the enzyme from the Golgi membranes of guinea pig liver was 20 mM. For details of enzyme Test B see the Materials and Methods section.

Different detergents were tested for their ability to solubilise the 4-O-acetyltransferase from guinea pig liver Golgi membranes. The success of solubilisation was monitored by the incorporation of radioactive acetate into free Neu5Ac (enzyme Test B). In this respect only the zwit-

Sialic Acid 4-O-Acetylation in Guinea Pig Liver

terionic detergent CHAPS was successful and resulted in 80 – 90% solubilisation at 16 mM concentration. Up to that amount, soluble enzyme activity increased almost linearly, but then rapidly decreased (Figure 1). With the exception of Tween 20, which solubilised incompletely and left 50 – 60% of enzyme activity in the Golgi pellet, all other detergents tested completely destroyed enzyme activity. To confirm that the enzyme product of this reaction is the expected one, an analysis of sialic acids was performed, revealing only the mono-O-acetylated sialic acid form Neu4,5Ac2 (Figure 2). Substrate Specificity To set up an appropriate enzyme test for purification of the 4-O-acetyltransferase from guinea pig liver Golgi membranes, various sialic acid-containing acceptors were tested with the solubilised enzyme. Figure 3 shows that the solubilised enzyme can modify a large variety of free or glycosidically bound sialic acids, although to a rather different degree. Only Neu4,5Ac2, equine submandibular gland mucin (ESM), and a neo-glycolipid, which contains the trisaccharide Neu4,5Ac2-α2,6-Galβ1,4-GlcNAc glycosidically β1-1 linked to an octadecanyl residue, which all are already O-acetylated at the site of enzyme attack, as well as the ganglioside GM1b and the non-sialylated control substance lactosyl ceramide, were inactive. Collocalia mucin, not shown in Figure 3, is also a relatively good acceptor. When comparing the degree of 4-O-acetylation of low molecular weight compounds such as free sialic acids and sialyloligosaccharides, sialyllactoses and sialyl Lex are better acceptors than free

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Neu5Ac, which on the other hand is more active than Neu5Gc. When comparing the sialyllactose and sialyllactosamine isomer pairs, there is a slight tendency that the α2,6 isomer is more easily O-acetylated. Interestingly, Neu5,9Ac2 can also be 4-O-acetylated to the same degree as Neu5Ac. The Neu5Ac dimer with α2,8 linkages is a weak substrate, too. Remarkably, also CMP-Neu5Ac can be O-acetylated. However, the 4-O-acetylation of these acceptors was still too low to use them in a fast and sensitive assay. The application of gangliosides, on the other hand, as exogenous substrates resulted in a significant increase in sialate-4-O-acetyltransferase activity. Among the gangliosides proven and shown in Figure 3, GD3, GT1b and GQ1b were the most successful acceptors, while GM1b was unable to act as a substrate at all. Interestingly, no 4-O-acetylated gangliosides could be detected in guinea pig liver; the only ganglioside was GM3 (data not shown). As was the case with free Neu5Ac and Neu5Gc, Neu5Ac-GM3 was a better O-acetyl acceptor than Neu5Gc-GM3. Similarly, Neu5,9Ac2-GD3 was 4-Oacetylated to the same extent as GD3, a result also observed for free Neu5Ac and Neu5,9Ac2. Also the enzyme still bound to Golgi membranes is capable of accepting exogenous sialic acids or sialyllactoses as substrates, although this was not investigated further. As an explanation for this observation we suggest this to be due to an access of the membrane-bound enzyme to the low-molecular mass substrates by the formation of inside-out vesicles or of membrane fragments formed during homogenisation procedures. Maximum activity was present in the sucrose gradient fraction that

Fig. 3 Substrate Specificity of Solubilised Sialate-4-O-Acetyltransferase. Nearly all substrates could be 4-O-acetylated. Gangliosides are obviously the best substrates while free Neu5Gc is the weakest. Only GM1, Neu4,5Ac2 and other substances bearing this sialic acid were inactive.

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exhibited sialyltransferase activity with exogenous asialofetuin in the absence of detergent. Characterisation of the Enzyme Products Test E was used to characterise the enzymatically modified gangliosides and thus the specificity of the enzyme reaction; O-acetylated GD3 formed (Figure 4A) was treated in a number of different ways. Loss of enzyme activity due to heat treatment (Figure 4B) or to the addition of 1 mM para-chloromercuribenzoate (p-CMB) demonstrates the enzymatic nature of the reaction. Radioactive AcCoA alone without the addition of solubilised enzyme did not result in labelling of the ganglioside fraction (Figure 4C). The radiolabelled gangliosides were nearly 100% de-O-acetylated when saponified (Figure 4E), and asialo-GD3 (lactosylceramide) could not serve as an acceptor (Figure 4F, for further descriptions of this Figure see the legend). Surprisingly, the hydrolysis of 4-O-Ac-GD3 performed with 2 M propionic acid did not yield significant quantities of free Neu[3H]4,5Ac2. It appears likely that the majority of the radiolabel was lost as free acetate during hydrolysis and subsequent lyophilisation, or that lactone formation has occurred. Further characterisation of the enzyme product was therefore afforded by the use of various sialidases. Arthrobacter ureafaciens sialidase and Vibrio cholerae sialidase, as expected, had no effect on the hydrolysis of 4-O-Ac-GD3, whereas Newcastle disease virus sialidase, which is capable of hydrolysing 4-O-acetylated sialic acids, although at a reduced rate (Kleineidam et al., 1990), released all radiolabel from 4O-[3H]Ac-GD3. A direct enzymatic product identification was possible with Neu5Gc-GM3. This O-acetyl acceptor allowed the identification of the correctly radiolabelled product by cochromatography with authentic Neu4Ac5Gc-GM3 isolated from horse erythrocytes, which itself, of course, is not a substrate for the 4-O-acetyltransferase (Figure 5). Fur-

Fig. 4 O-Acetylation of GD3 by the Sialate-4-O-Acetyltransferase. Solubilised enzyme was incubated (Test E): (A) in the presence of exogenous GD3 and [3H]AcCoA; (B) with heat-treated enzyme and [3H]AcCoA; (C) without solubilised enzyme but with [3H]AcCoA; (D) with [3H]AcCoA but without exogenous GD3; (E) in the presence of exogenous GD3 and [3H]AcCoA after saponification of the reaction product with ammonia vapour overnight; (F) in the presence of lactosylceramide and [3H]AcCoA. From these experiments it is obvious that (i) GD3 is a real substrate for the 4-Oacetyltransferase (panel D) and the glycosidically bound sialic acid is essential for enzyme activity (panel F); (ii) GD3 is Oacetylated (panel E), and (iii) the reaction is of enzymatic origin (panels B and C). Peak denomination: 1, residual [3H]AcCoA; 2, [3H]Neu4,5Ac2GD3; 3, residual [3H]acetate. For experimental details see the text.

Fig. 5 Enzymatic 4-O-Acetylation of Neu5Gc-GM3. The radioactive product formed by incubation of Neu5Gc-GM3 with [3H]AcCoA and soluble sialate-4-O-acetyltransferase was co-chromatographed with authentic Neu4Ac5Gc-GM3 and stained by the orcinol/Fe(III)/HCl-reagent after radio-scanning.


thermore, we could demonstrate by the more sensitive fluorescent HPLC analysis that the amount of Neu4,5Ac2, following incubation of the enzyme with GD3 and non-radiolabelled AcCoA and release by propionic acid hydrolysis, was increasing in comparison to the control reaction without AcCoA (Figure 6). However, the amount was not as high as expected, which might be due to the loss of Oacetylation under these acidic conditions as described previously (Manzi et al., 1990). These studies with GD3 and analysis of the reaction product by radio-HPTLC enabled easy, rapid, sensitive and specific testing of the 4-O-acetyltransferase. Intracellular localisation of the Sialate-4-OAcetyltransferase The enzyme is located in the Golgi apparatus or the transGolgi network, as has been described for all mammalian sialate-O-acetyltransferases investigated (Schauer et al., 1988a; Diaz et al., 1989b; Varki, 1992; Schauer and Kamerling, 1997; Iwersen et al., 1998; Tiralongo et al., 2000; Schauer, 2000b; Shen et al., 2002) including guinea pig liver. Having found a very suitable substrate for the enzyme test (GD3), we studied the subcellular membrane localisation of the enzyme with this ganglioside and compared it with the solubilised 4-O-acetyltransferase. Both the endogenous and the GD3-dependent sialate-4-Oacetyltransferase activity were located concordantly with the sialyltransferase activity (Figure 7). Enzyme activity could be neither detected in the supernatant of homogenised Golgi membranes without detergent nor in the supernatant of homogenised fresh liver tissue even with

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GD3, underlining the integration of the enzyme in the Golgi membrane. Enzyme Properties To evaluate whether the solubilised 4-O-acetyltransferase behaves similarly to the previously described membrane-bound enzyme (Iwersen et al., 1998), its dependence on temperature, pH, salt and buffer concentration, as well as kinetic parameters, were determined using a protein concentration of 7 – 15 µg. Protein-dependent sialate 4-O-acetylation was shown to be linear in a range of 1 – 300 µg and up to 20 min. The CHAPS-solubilised 4-O-acetyltransferase activity was optimal at 30 °C (Figure 8A); this temperature optimum is the same as that observed for the membrane-bound system. All other parameters tested showed differences to the membrane-bound system. Both the optimal KCl and phosphate buffer concentrations decreased to 50 mM (Figure 8B, C) in comparison to 90 mM and 70 mM, respectively, for the Golgi membrane-bound system. Higher KCl concentrations decreased enzyme activity, which, however, could be recovered following dilution of the salt to its optimum concentration of 50 mM. The pH optimum for the solubilised enzyme, pH 5.7 (Figure 8D), showed a significant shift from that observed in the membranebound system, where an optimum at pH 6.7 was observed earlier. The reaction catalysed by the solubilised 4-O-acetyltransferase was found to be linear for time points up to 20 min. In contrast, the membrane-bound enzyme was linear up to only 5 min (Iwersen et al., 1998). The apparent Km for both systems reflects the high affinity

Fig. 6 Identification of Neu4,5Ac2 as Reaction Product with Gangliosides. Sialic acids were hydrolytically released after incubation of GD3 with unlabelled AcCoA and soluble enzyme and analysed by fluorimetric HPLC. The Neu4,5Ac2-peak increases after incubation of GD3 with the co-substrate compared to the incubation of GD3 without AcCoA, underlining the correctness that Neu4,5Ac2-GD3 is the enzymatic product.

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Fig. 7 Co-Localisation of the Sialate-4-O-Acetyltransferase with Sialyltransferase in the Golgi-Fraction from Guinea Pig Liver. Relative specific activities of the membrane-bound O-acetyltransferase (gray bars: tested with endogenous substrate), solubilised Oacetyltransferase (white bars: tested with GD3), and membrane-bound sialyltransferase activity (black bars: tested with asialofetuin and CMP-Neu5Ac). Fractions A-D correspond to the sucrose density gradient interfaces 0.8/1.8 M, 1.2/1.4 M, 1.4/1.6 M and 1.6/2.3 M sucrose, respectively.

Fig. 8 Dependence of the Solubilised Sialate-4-O-Acetyltransferase Activity on pH, KCl and Phosphate Concentrations, and Temperature Tested Using Enzyme Test E. The optimal conditions for enzyme incubation are 30 °C, 50 mM potassium phosphate and 50 mM KCl, respectively, at a pH of 5.6.

of the enzyme toward its co-substrate AcCoA, with the membrane-bound enzyme (Km of 0.6 µM) having a higher affinity for AcCoA than the solubilised system (Km of 3.6 µM) (Figure 9A). The affinity of the solubilised enzyme to the ganglioside GD3 is strong as well (1.2 µM) (Fig-

ure 9B). The Vmax values for AcCoA and GD3 were 215 and 1310 pmol/min × mg protein, respectively. The Km values for GM3, GD1a and GD1b were 8.4, 2.2 and 2.5 µM, respectively, and the corresponding Vmax values 235, 2136 and 1385 pmol/min × mg protein. Thus, the


catalytic efficiency was highest with GD3 and GD1a (1.82×10– 5 and 1.72×10– 5 l/mol × s × mg protein). The determination of the inhibitor constant for CoA resulted in a Ki of 14.8 µM, which is slightly higher than that reported earlier (Iwersen et al., 1998) for the membranebound enzyme (4.2 µM). Furthermore, desulfo-CoA and 3’-dephospho-CoA inhibited the enzyme by 50% at 13 µM and 22 µM, respectively. At higher concentrations of the latter compound, the inhibitory effect did not increase further and the 3’-dephospho-CoA became radioactively acetylated probably at the thiol group, as shown by TLC analysis. Another effective inhibitor of both this soluble and the membrane-bound enzyme is pCMB. Weaker inhibitors of the solubilised enzyme were

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several pyridine nucleotides, pyridoxalphosphate, 4,4’dithioisocyano-2,2’-stilbene-disulfonic acid (DIDS), and the triazine dye Cibacron Blue F3G-A (Table 1). The stability of the enzyme during the purification procedures is of highest relevance; we observed that the soluble enzyme preparation is stable after various freezethaw cycles and also for more than 72 h at 4 °C without significant loss of activity. Almost total enzyme activity could be recovered after lyophilisation of the solubilised enzyme. The addition of stabilising and anti-oxidising agents, such as 10% glycerol and 1 mM dithiothreitol (DTT), respectively, to the solubilisation mixture did not notably affect enzyme activity, whereas 200 mM sucrose slightly enhanced it.

Fig. 9 Study of the Kinetic Properties of the Solubilised Sialate-4-O-Acetyltransferase. The apparent Km and Vmax values for AcCoA (3.6 µM and 215 pmol/min × mg protein, respectively) with GD3 as acceptor (A), and GD3 (1.2 µM and 1310 pmol/min × mg protein, respectively) (B) were calculated using Test E. For the kinetic data of other substrates see the corresponding Results section.

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Co-Factor Requirement of the 4-O-Acetyltransferase In order to prepare the enzyme for purification by chromatographic methods, it was necessary to concentrate the enzyme samples, which was possible by 30 kDa ultrafiltration but not by 10 kDa ultrafiltration, since the latter did not let the CHAPS micelles pass. Curiously, the retentate, thought to contain the enzyme, and the filtrate were both inactive. However, when the filtrate and retentate were combined, full activity was restored. Furthermore, after heat treatment of the filtrate for 15 min at 96 °C and lyophilisation, the filtrate was still able to activate the enzyme, suggesting that the possible cofactor is not a protein (Figure 10). Assays performed in the presence of EDTA, and various cations and nucleotides, indicated that the cofactor probably is neither a metal ion nor a nucleotide.

Table 1 Substances and Concentrations Inhibiting the Soluble Sialate 4-O-Acetyltransferase from Guinea Pig Liver Golgi Membranes. Inhibitor

Solubilised enzyme

p-CMB (1 mM) CoA (13 µM) Desulfo-CoA (13 µM) 3’-Dephospho-CoA (≥ 22 µM) 3’,5’-ADP (1 mM) IMP (1 mM) IDP (1 mM) ITP (1 mM) AMP (1 mM) ATP (1 mM) NADH (1 mM) NADPH (1 mM) Pyridoxal phosphate (1 mM) DIDS (125 µM) Cibacron Blue F3G-A (400 µM)

100% 50% 50% 50% 80% 16% 30% 58% 0% 20% 0% 18% 75% 50% 65%

Discussion Golgi membrane-bound sialate-4-O-acetyltransferase from guinea pig liver, the solubilisation of which was achieved in the present study, is the first sialic acid-specific O-acetyltransferase described to be released from subcellular membranes. This is a remarkable development on the way to purify this enzyme using further techniques. The most striking feature of the solubilised enzyme from guinea pig liver is its high substrate specificity toward gangliosides, especially the tetrasialoganglioside GQ1b, the trisialoganglioside GT1b and the disialogangliosides GD3, GD1a and GD1b. GM1b is not a substrate for the enzyme, probably because of the internal position of the sialic acid moiety. The good acceptance of gangliosides is in contrast to glycoproteins, oligosaccharides and especially free sialic acids (Figure 3), which are substrates but to an appreciably lower degree. Free Neu5Gc, oligosaccharides and GM3 containing this sialic acid are weaker substrates than Neu5Ac and the corresponding glycoconjugates. Neu5,9Ac2 and compounds containing this sialic acid are as good acceptors as the according Neu5Ac-containing compounds. This is in contrast to 4-O-acetylated sialic acids, which both in free form or bound to glycoprotein and glycolipids cannot accept O-acetyl groups by this enzyme and therefore serve as excellent control substances to prove the specificity of this O-acetyltransferase. With regard to the possible natural substrates of this O-acetyltransferase in guinea pig liver, only 4-O-acetylated α2-macroglobulin occurring in blood serum of this animal is known (Hanaoka et al., 1989). Research in this direction includes knowledge of the O-acetylation mechanism in the Golgi apparatus, the natural location of the 4-O-acetyltransferase studied. According to a hypothesis put forward by (Diaz et al., 1989a) for the biosynthesis of 9-O-acetylated sialic acids in rat liver, sialic acids bound to nascent glycoconjugates are O-acetylated by a

Fig. 10 Cofactor Requirement of the Soluble Sialate-4-O-Acetyltransferase. Ultrafiltrated (30 kDa) solubilised protein from guinea pig liver Golgi membranes did not show any 4-O-acetyltransferase activity in the filtrate and a negligible activity in the retained protein, while the combination of both restored more than 90% of the initial enzyme activity. The nature of this cofactor is unknown, but it is heat-stable and smaller than 10 kDa.


membrane-bound complex in which the acetyl-CoAtransporter, the sialyltransferase and the O-acetyltransferase cooperate, perhaps also by participation of an acetylated intermediate. Since sialoglycoconjugates are the best substrates of the 4-O-acetyltransferase, a similar mechanism may be assumed to operate in guinea pig liver. Whether the highly active gangliosides play the role of such an intermediate can only be hypothesised presently. Since CMP-Neu5Ac, the substrate of sialyltransferases, was also found to be a substrate in the present studies, its role as a natural substrate of the Golgi 4-O-acetyltransferase cannot be excluded. Similar observations were made with sialate-7(9)-O-acetyltransferases from Golgi membranes of bovine submandibular gland (Lrhorfi et al., unpublished results) and human colon (Shen et al., 2002), pointing to the possibility of O-acetylation also before the transfer of sialic acids to Golgi glycoconjugates. For most conditions concerning the incubation parameters, the difference between the membrane-bound (Iwersen et al., 1998) and the solubilised enzyme is not pronounced, except for the pH shift from 6.7 for the membrane-bound to 5.6 for the solubilised enzyme, which may reflect the conditions within the Golgi apparatus. Both enzyme forms can be inhibited by CoA in the low micromolar range. Interestingly, the thioester linkage or the free thiol group seem not to be of main importance for substrate binding but rather the nucleotide part of the molecule, since also desulfo-CoA and 3´,5´ADP had a relatively strong inhibitory effect on the soluble enzyme. In this respect the inhibitory effect of inosin phosphates increasing linearly from IMP to ITP, although weaker, suggests participation of the phosphate groups in binding of the substrate to the protein. Why the adenosine phosphates are less effective in this respect remains unclear. Crucial for further purification of the enzyme is the requirement of a cofactor for activity, which was elucidated by ultrafiltration experiments. Its nature has not yet been identified, although bi- or trivalent metal ions as well as the possible proteinacious nature were largely excluded. However, since the co-factor could be eluted with the aqueous washing phase in reversed-phase chromatography, its nature seems to be hydrophilic. In forthcoming purification experiments of the O-acetyltransferase, this cofactor obtained by 30 kDa-ultrafiltration of the solubilisate has to be added to the enzyme assays, either in the form of the filtrate or as a defined substance after its future isolation and identification. Some enrichment of the O-acetyltransferase was already achieved by ion-exchange and affinity chromatography. Purification and analysis of peptide sequences of the sialate-4-O-acetelyltransferase appear feasible now and may be the safe way to clone the gene encoding the enzyme and elucidate its primary structure. This approach may also be useful in solubilising and purifying of the corresponding sialate-9(7)-O-acetyltransferase(s) for molecular biological studies, which has not yet been possible, in spite of many efforts. Various attempts to pu-

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rify this enzyme have been described (Diaz et al., 1989b; Varki, 1992; Schauer and Kamerling, 1997; Schauer et al., 2000; Schauer, 2000b). Expression cloning also did not result in the 9(7)-O-acetyltransferase protein, as described by Ogura et al. (1996), Kanamori et al. (1997) and Shi et al. (1998). Success in this respect would be an important step toward the understanding of the regulation of sialate-O-acetylation and its evident biological and pathophysiological significance.

Materials and Methods Reagents Sialidase from Vibrio cholerae was purchased from Behringwerke, Marburg, Germany, that from Arthrobacter ureafaciens and Newcastle disease virus from Boehringer, Mannheim, Germany. Neu5Ac and the ganglioside Neu5Ac-GD3 (corresponding to GD3 containing Neu5Ac; in the case of other sialic acids occurring in GD3 or other gangliosides, for example Neu5,9Ac2, this terminology is also used) were a gift from Snow Brand Milk Products Co. Ltd., Tokyo, Japan. All other free sialic acids used in these studies were purified from horse or bovine submandibular glands by standard methods described elsewhere (Reuter and Schauer, 1994). CMP-Neu5Ac was kindly provided by Dr. U. Kretschmer, Forschungszentrum Jülich, Germany. CMP[4,5,6,7,8,9-14C]Neu5Ac (30 mCi/mmol) was from Amersham, Braunschweig. α2,3- and α2,6-sialyllactoses, Neu5Ac-α2,8dimer and -tetramer and disialoganglioside GD2 were obtained from Calbiochem, Bad Soden, Germany. All other gangliosides were purchased from Matreya Incorporation, Tokyo, Japan. Lactosylceramide was kindly provided by Dr. Bernhard Kniep, Institut für Immunologie der Technischen Universität Dresden, Germany. GM3, containing Neu4Ac5Gc as terminal sialic acid (Neu4Ac5Gc-GM3), was prepared according to Gasa et al. (1983). A part of this substance was de-O-acetylated to probe as substrate. The neoglycolipid Neu4,5Ac2-α2,6-Gal-β1,4-GlcNAc-β1,1-OC18H37 was a kind gift of Dr. T. Yamamoto, Osaka, Japan. For the preparation of asialofetuin, 10 mg fetuin were dissolved in 5 ml 0.1 M HCl and heated for 1 h at 80 °C. The solution was afterwards exhaustively dialysed against water at 4 °C and the content of the dialysis tubing lyophilised. The yield of the colourless powder was 95%. Mucin from bovine and equine submandibular glands was prepared as described elsewhere (Schauer and Kamerling, 1997). HPLC solvents of gradient grade, ammonium acetate, chloroform, ethanol, methanol, orcinol, Na-cacodylat, FeCl3, HCl, NaOH, NH3, propionic acid, TLC plates and HPLC columns were obtained from Merck, Darmstadt, Germany. Ion-exchange resins, Dowex 2×8 (200 – 400 mesh) and Dowex 50W×8 (20 – 50 mesh) were purchased from Pharmacia Biosystems, Freiburg, Germany. 1,2-Diamino-5,6-methylene dioxybenzene (DMB) was either obtained from Dojindo Laboratories, Tokyo, Japan, or Fluka, Buchs, Switzerland. Pyridoxalphosphate (PLP), fetuin, galactose, Tris, AcCoA and CoA were from ICN, Aurora, Ohio, USA. [3H]AcCoA (4.9 Ci/mmol) was obtained from Moraveck Biochemicals, Brea, California, USA. Nucleotides, adenine, adenosine, primulin, p-CMB, colominic acid, human α2-macroglobulin, bovine transferrin and bovine serum albumin (BSA) were purchased from Sigma, Deisenhofen, Germany. Scintillation cocktail and sucrose were from Carl Roth GmbH, Karlsruhe, Germany. The protease inhibitors Pefabloc™ and Complete™, as well as the sialidase inhibitor Neu2en5Ac, were purchased from Roche, Mannheim, Germany. Centrex tubes

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were obtained from Schleicher & Schuell, Dassel, Germany. The detergents CHAPS and Triton X-100 were purchased from Biomol, Hamburg, Germany; n-octyl-D-glucopyranoside, Tween 20 and Zwittergent 3 – 14 from Fluka, Buchs, Switzerland, and desoxycholate and Lubrol PX from Sigma.

Sialate-4-O-Acetyltransferase Assays

Guinea Pigs

Test A In order to find a suitable detergent, approximately 100 µg protein of homogenised Golgi membranes containing endogenous substrate were suspended in 190 µl buffer containing 70 mM potassium phosphate, pH 6.7, 90 mM potassium chloride, 2 mM Pefabloc™, 2 mM PMSF or 30 µl Complete mini™ and the corresponding detergent. After shaking and centrifugation of the mixture (see above), the clear supernatant was incubated with 10 µM [3H]AcCoA (4.6 kBq) for 30 min at 30 °C. The reaction was stopped by the addition of 400 µl perchloric acid (PCA) yielding a final concentration of 400 mM PCA. The tubes were cooled for 10 min on ice and centrifuged for 10 min at 14 000 g. The supernatant was discarded, the pellet sonicated in 1 ml water and centrifuged for 10 min at 14 000 g. After repeating this procedure twice, the pellet was sonicated for 5 s in 200 µl water. Fifty µl of the suspension were hydrolysed in 400 µl 1 M NaOH for 15 min at 80 °C, cooled for 5 min on ice and quenched by the addition of 200 µl 2 M HCl. Five hundred µl of the solution were added to 1 ml of scintillation cocktail and counted in a liquid scintillation counter. In the case where incorporation of radioactivity was measured, the remaining 150 µl of the suspension underwent sialic acid analysis (see corresponding section below).

Adult animals of either sex were obtained from the Pharmacological Institute, University of Kiel, Germany. The liver was prepared immediately after death and stored not longer than 1 h in ice-cold 0.01 M Tris-buffer, pH 7.4, containing 0.25 M sucrose. Preparation and Solubilisation of Golgi Membranes All procedures were carried out at 0 – 4 °C. The preparation of microsome fractions followed the technique described in (Iwersen et al., 1998) with slight modifications. Twenty to 30 g of guinea pig liver were minced in 30 ml 0.01 M Tris-buffer, pH 7.4, containing 0.25 M sucrose and homogenised in a Potter-Elvehjem homogeniser with three strokes. 27 ml of the homogenate were gently mixed with 81 ml of 0.01 M Tris-buffer, pH 7.4, containing 2.3 M sucrose, yielding 108 ml homogenate with a final sucrose concentration of 1.4 M. Golgi membranes were then prepared by discontinuous sucrose gradient centrifugation using the following procedure: a Beckman SW28 tube was layered successively with 3 ml 2.3 M sucrose, 3 ml 1.8 M sucrose, 9 ml homogenate, 9 ml 1.2 M sucrose and 9 ml 0.8 M sucrose; all sucrose solutions were prepared in 0.01 M Tris-buffer, pH 7.4. 6 tubes prepared in this way were centrifuged in a Beckman SW28 rotor for 90 min at 90 000 g. The Golgi membrane fractions at the 1.2 M/0.8 M sucrose interfaces were carefully harvested with a Pasteur pipette and diluted in a final volume of 200 ml with 0.01 M Tris-sucrose buffer, pH 7.4. The Golgi membrane suspension was further centrifuged at 100 000 g for 20 min in a Beckman 45Ti rotor at 4 °C. The resulting Golgi membrane pellets were transferred with 1 ml of 50 mM phosphate buffer pH 5.7 into Beckman TLA45 tubes and centrifuged for 5 min at 100 000 g. The final supernatants were discarded and the tubes containing the membrane pellets frozen at – 80 °C until required. The enrichment of Golgi membranes in this fraction was controlled by electron microscopy. Golgi membranes (50 – 100 µl) were thawed at 30 °C and rehomogenised in 100 µl water using a mini-Potter-Elvehjem homogenizer. One hundred µl of the homogenate were added to 1 ml solubilisation buffer containing 50 mM KCl, 50 mM potassium phosphate (pH 5.7), 2 mM Pefabloc™, 150 µl Complete™ (1 tablet Complete mini™ per 1.5 ml water) and as detergent either CHAPS or desoxycholate, Lubrol PX, n-octyl-D-glucopyranoside, Triton X-100, Tween 20, or Zwittergent 3 – 12. The final concentrations of these detergents were adjusted 3- to 4-fold higher than their critical micellar concentrations. In routine preparations and enzyme assays 16 mM CHAPS was used. The suspensions were shaken vigorously for 5 min at room temperature and ultracentrifuged in a Beckman TLA45 rotor at 100 000 g for 20 min at 4 °C. The resulting clear supernatant was collected and either directly used for the enzyme tests or frozen at – 80 °C. The solubilisation efficiency of the various detergents was judged by the liberation of enzyme activity with ‘endogenous substrate’ from Golgi membranes, the determination of which is described by the first O-acetyltransferase assay below. Proteins were determined in the membrane fractions and in the solubilisate (Peterson, 1977). Sialyltransferase activity was estimated according to (Vandamme et al., 1992) in the membrane fractions obtained by density gradient centrifugation using asialofetuin and separation of the reaction product from radioactive CMP-Neu5Ac by cellulose thin layer chromatography.

A number of assays for the investigation of soluble or membrane-bound enzyme, and especially its substrate, specificity were developed:

Test B This assay was set up for exogenous, low molecular weight acceptors such as free sialic acids and sialyllactoses. Golgi membranes (100 µg protein) were solubilised in 190 µl of the above phosphate buffer containing 16 mM CHAPS and 30 µl Complete mini™. The clear supernatant (‘solubilisate’) was transferred to reaction tubes containing 300 µM lyophilised acceptor and incubated with 10 µM [3H]AcCoA (4.6 kBq) for 30 min at 30 °C. The reaction was stopped by shock freezing in liquid nitrogen and after thawing diluted with 1 ml ice-cold water. The solution was directly applied to a 1 ml column of Dowex 2×8 (200 – 400 mesh, formate form) and washed with 10 ml water. Sialic acids were eluted with 10 ml 0.8 M formic acid and the column washed with 10 ml water. The eluent was collected, lyophilised and transferred with 3 times 400 µl water into an Eppendorf tube. After drying in a speed-vac concentrator, the residue was dissolved in 20 µl water. The incorporation of radioactivity was quantified in 5 µl by scintillation counting, while the remaining 15 µl were applied to 20×20 cm cellulose TLC plates and developed overnight in butan-1-ol/propan-1-ol/0.1 M HCl (1:2:1, v:v:v). The plate was carefully dried under a cold stream of air and analysed via a TLC-linear analyser (Tracemaster 40, Berthold, Wildbad, Germany). Co-migration of standard Neu4,5Ac2 and the staining of sialic acids with the orcinol/ Fe(III)/HCl reagent confirmed the nature of the radioactive product. Test C To detect enzymatic 4-O-acetylation of free sialic acids, sialosaccharides and sialoglycoproteins by both the membranebound and solubilised enzyme, we extended enzyme test B by the addition of a 10 kDa ultrafiltration step. Golgi membranes (100 µg protein) were homogenised in 100 µl of the above buffer and used for incubation either directly or after solubilisation in the presence of 16 mM CHAPS. The membranes or the solubilisate were incubated with 10 µM [3H]AcCoA (4.6 kBq) for 30 min at 30 °C in the presence of 300 µM of free or glycosidically bound sialic acids. The reaction was stopped by freezing in liquid nitrogen. The unsolubilised sample was ultracentrifuged for 10 min at 100 000 g, and the supernatant, as it was also performed with the


solubilised mixture, diluted with 2 ml ice-cold water. The diluted reaction mixtures were applied to 10 kDa ultrafiltration tubes. Sialic acid analysis was performed on both the filtrate and the retentate. In the case where glycosidically bound sialic acids were used as acceptors, sialic acid analysis was performed following hydrolysis and lyophilisation (see below). Test D Solubilisate (90 µl) from 100 µg Golgi protein was incubated in the above buffer with 150 µM of gangliosides in the presence of 10 µM [3H]AcCoA (4.6 kBq) for 20 min at 30 °C. The reaction was stopped by shaking vigorously with 400 µl chloroform/methanol (1:2, v:v). After cooling the samples on ice for 30 min and centrifugation at 14 000 g for 10 min, the supernatant was dried under vacuum in a speed-vac concentrator and resuspended in 50 µl chloroform/methanol (1:2, v:v). The suspension, which contained some detergent and salts, was centrifuged at 14 000 g. Five µl of the resulting supernatant were used for liquid scintillation counting and 10 – 20 µl applied to 10×10 cm silica gel 60 HPTLC plates. These were developed in chloroform/methanol/25 mM CaCl2 (50:40:10, v:v:v) for 45 min. Analysis of radioactive spots was carried out by a TLClinear analyser. Test E In a modification of Test D, 7 – 15 µg of solubilised protein were incubated in 30 µl of the phosphate buffer, however, having a pH of 5.7 and only 50 µM KCl, with 30 µM [3H]AcCoA (4.6 kBq) and 70 µM gangliosides. The reaction was stopped by shock freezing in liquid nitrogen and 10 µl were, after thawing on ice, analysed on silica gel thin layer plates as above. Characterisation of Enzyme Products Sialidase Treatment of O-Acetylated-GD3 GD3 (65 µM) and 5 µM radioactive O-Ac-GD3 (1000 dpm) were incubated in 100 µl 50 mM sodium acetate buffer (pH 5.5), and either 20 mU Arthrobacter ureafaciens sialidase, 20 mU Vibrio cholerae sialidase, or 10 mU Newcastle disease virus sialidase. For optimal activity Vibrio cholerae sialidase also required 5 mM CaCl2. The reaction was stopped after 1 h at 37 °C by shock freezing in liquid nitrogen and the sample lyophilised. The residue was dissolved in 20 µl water, centrifuged at 14 000 g and 15 µl of the supernatant applied to a 10×10 cm silica gel 60 HPTLC plate, which was developed in chloroform/methanol/25 mM CaCl2 (50:40:10, v:v:v) for 45 min. Plates were scanned by a TLC linear analyser and stained initially with the fluorescent dye primulin and then with the orcinol/Fe(III)/HCl reagent (below). Sialic Acid Saponification O-Acetylated samples developed on TLC plates were saponified overnight at room temperature in TLC chambers saturated with ammonia vapour. The probes could also be saponified prior to TLC analysis either by treatment with 0.1 M NaOH for 30 min at 37 °C followed by quenching with 0.2 M HCl, or by suspending a lyophilised sample in 25% ammonia for 8 h at 37 °C. Fluorescence Staining with Primulin Gangliosides developed on silica gel plates were sometimes stained with the fluorescent dye primulin (Skipski, 1975). Dried HPTLC plates were sprayed with a solution of 0.01% primulin in acetone/water (4:1, v:v), dried and detected under UV light at 280 nm. Since primulin is not specific for gangliosides, a further staining with the orcinol/Fe(III)/HCl reagent is recommended. Orcinol staining does not interfere with the prior staining procedure. Staining with the Orcinol/Fe(III)/HCl Reagent Free sialic acids or sialic acid-containing compounds developed on TLC

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plates were stained with the orcinol/Fe(III)/HCl reagent (Reuter and Schauer, 1994). After spraying, the TLC plates were baked in closed glass chambers for 20 min at 120 °C. Typical purple bands characterised the sialic acid-containing substances. Analysis of Radioactive Sialic Acids Analysis of glycosidically bound, radioactive sialic acids from the reaction products was performed by hydrolysis with 2 M propionic acid at 80 °C for 4 h. After ultrafiltration (MWCO = 10 kDa), propionic acid was removed by lyophilisation and the sialic acids were dissolved in 100 µl water. When the samples contained high protein concentrations, the ultrafiltration step was omitted and they were ultracentrifuged instead after hydrolysis, followed by lyophilisation. Further purification and analysis followed the method described by Reuter and Schauer (1994) with slight modifications. Briefly, the sialic acid-containing sample dissolved in 0.5 – 1 ml of water was loaded onto a 1 ml column of Dowex 2×8 (200 – 400 mesh, formate form) and washed with 10 ml water. Bound sialic acids were eluted with 10 ml 0.8 M formic acid followed by rinsing with 10 ml water. The eluate was lyophilised, dissolved in 2× 400 µl water, and dried in a speed vac concentrator. The residue was dissolved in 20 µl water, applied to cellulose TLC plates developed in butan-1-ol/propan-1-ol/0.1 M HCl (1:2:1, v:v:v), followed by radio-scanning. Analysis of Non-Radioactive Sialic Acids by Fluorimetric HPLC For some samples like serum from guinea pig or for quality control of the substrates used, the purification procedure was abbreviated by direct analysis after propionic acid hydrolysis and lyophilisation of sialic acids using fluorimetric HPLC (Hara et al., 1989). These were dissolved in 10 µl 2 M acetic acid and 49 µl of DMB reagent (7 mM DMB, 0.75 M β-mercaptoethanol, and 18 mM sodium hydrogensulfite) and incubated for 1 h at 56 °C in the dark. After 14 000 g centrifugation for 5 min, 5 – 20 µl of the sialic acid quinoxalines were separated on an RP18-column (Merck, RP18, Lichrospher 100, particle size 5 µm, 250×50 mm) and detected with a fluorescence detector at wavelengths for excitation of 373 nm and absorbance at 448 nm. Analysis of Neu4,5Ac2 after Enzymatic 4-O-Acetylation of GD3 Using O-acetyltransferase Test D in a non-radioactive assay, 230 µg of solubilised Golgi membrane protein were incubated with 50 µM unlabelled AcCoA with or without 150 µM GD3 in a final volume of 450 µl phosphate buffer for 2 h at 30 °C. The reaction was stopped by cooling on ice, the mixtures dialysed overnight against 3× 70 ml water, with the water being changed after 2 and 4 hours, the retentates lyophilised, dissolved in 500 µl 2 M propionic acid and hydrolysed at 80 °C for 4 h. After lyophilisation, the residues were dissolved in 500 µl water and dialysed as described above. The dialysates were collected, lyophilised and the residues taken up in 100 µl water. 10 µl were lyophilised and derivatised with DMB for RP-HPLC analysis. Study of Enzyme Properties Most kinetic assays were carried out with GD3, since this ganglioside was revealed to be an excellent substrate and its analysis is easy. Enzyme Test D was applied for study of the time dependence of the sialate 4-O-acetylation of GD3. Solubilised protein (14 µg) was incubated in 5 to 10 min intervals for maximal 50 min. The temperature dependence of the enzyme reaction was determined by using enzyme Test E; 30 µg solubilised protein were incubated with 70 µM GD3 between 0 °C and 55 °C for 30 min at 20 °C. Investigation of the pH optimum for GD3-sialate-4-O-

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acetylation was carried out with the same assay (E) by using 140 mM potassium phosphate buffer solutions with pH values between 4.9 and 8.2 and incubation times of 20 min at 30 °C. For estimation of the KCl optimum, various amounts of KCl in water were lyophilised and mixed in the same tube with the substances and soluble enzyme of Test E, yielding end concentrations between 0 and 90 mM KCl, followed by incubation at 20 °C for 30 min. In order to determine the apparent Km and Vmax values of AcCoA, GD3, and other gangliosides, the following procedures were carried out: AcCoA at a given concentration was lyophilised and dissolved in 5 µl [3H]AcCoA (4.6 kBq), yielding end concentrations between 0.6 and 23.6 µM in the 30 µl assay of Test E with 15 min incubation at 30 °C. In the case of gangliosides, given concentrations between 0 and 150 µM per 30 µl incubation volume were lyophilised before mixing with the enzyme test solution. The inhibitory potency of CoA on the sialate-4-O-acetyltransferase was investigated by using enzyme Test E at the AcCoA concentrations of 5, 10, 15, 20, and 40 µM with each different concentration of CoA (0, 10 and 30 µM) and 150 µM GD3. Plotting the reciprocal velocities versus reciprocal substrate concentrations led to three linear graphs from which the inhibitory constant was calculated. Furthermore, the influence of the following substances on enzyme reaction was studied by Test E: 3´-dephospho-CoA at 0, 1, 5, 10, 30, and 100 µM concentrations and adenine, adenosine, AMP, ATP, 3´,5´-ADP, c-AMP, IMP, IDP, ITP, CMP, NAD, NADH, PLP, pantothenic acid, p-CMB, EDTA, DTT, and β-mercaptoethanolamine, each at 1 mM concentration. Stability of the solubilised enzyme was tested by storage of 130 µl of the optimally solubilised enzyme at 4 °C for 120 h. At 24 h intervals, 25 µl of the mixture were incubated for 20 min under conditions of enzyme Test E. For freezing and thawing cycles, 5 of 6 100 µl-aliquots of soluble enzyme were frozen at – 80 °C for half an hour. Ninety µl of the unfrozen aliquot were used for the 4-O-acetyltransferase Test D. After half an hour, the frozen samples were thawed at 30 °C, 4 samples frozen at – 80 °C again, and the fifth incubated as described above. This procedure was repeated unless all samples were tested for 4-Oacetyltransferase activity. Isolation of a Natural Enzyme Activator by Ultrafiltration CHAPS-solubilised enzyme was ultrafiltered using Centrex UF2 filtration devices (MWCO 30 kDa). The filtrate was collected and the residual protein filtered, following dilution with appropriate solubilisation buffer, for a second time. The filtrate and residue were tested individually or after mixing for 4-O-acetyltransferase enzyme activity, using enzyme Test E. The filtrate or the residue were heat-inactivated by incubating at 96 °C for 15 min and checked for enzyme respective activator activity. Various substances mentioned in the Results section were tested for possible cofactor activity.

Acknowledgements The authors thank Annika Dorst, Micheline Neubert and Marion Pauer, Pharmakologisches Institut, Christian-Albrechts-Universität Kiel, and Marzog El Madani, Biochemisches Institut, Christian-Albrechts-Universität Kiel, for technical help. We thank Dr. Friedrich Paulsen, Anatomisches Institut, Christian-AlbrechtsUniversität Kiel, for electron microscopy of the Golgi fraction. Valuable advice given by Dr. Lee Shaw and Dr. Joe Tiralongo, Biochemisches Institut, Christian-Albrechts-Universität Kiel, is

gratefully acknowledged. These studies were financially supported by the Sialic Acids Society, Kiel, and the Fonds der Chemischen Industrie, Frankfurt.

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