Eur. J. Biochem. 206,269-277 (1992) 0 FEBS 1992

Mouse liver cytidine-5’-monophosphate-N-acetylneuraminic acid hydroxylase Catalytic function and regulation

Lee SHAW I , Petra SCHNECKENBURGER’, Jens CARLSEN ’, Kirsten CHRISTIANSEN‘ and Roland SCHAUER ‘ Biochemisches Institut, Christian-Albrechts Universitat, Kiel, Federal Republic of Germany ’ University of Copenhagen, Panum Institute, Department of Biochemistry C, Copenhagen, Denmark

(Received January 14, 1992) - EJB 92 0048

In this paper, we present the results of an investigation into the catalytic properties of CMP- NeuSAc hydroxylase (NeuSAc: N-acetylneuraminic acid) in high-speed supernatants of mouse liver. The enzyme was most active in Hepes/NaOH pH 7.4 and was markedly inhibited by relatively small increases in ionic strength, though the inhibition was abolished by desalting procedures. Several non- ionic detergents could activate the hydroxylase to various degrees in a concentration-dependent manner. Ionic detergents and a number of phospholipids were, however, generally inert or inhibitory.

The lack of inhibitory influence of a wide range of nucleotides revealed that CMP-NeuSAc hydroxylase binds its sugar-nucleotide substrate with a high degree of specificity. Thus, even millimolar concentrations of several cytidine nucleotides elicited virtually negligible inhibition, though the reaction product, CMP-NeuSGc (Neu5Gc: N-glycoloylneuraminic acid), was a weak inhibitor. The results also indicate that the enzyme is not regulated by any nucleotides or sugar - nucleotides.

Dilution of high-speed supernatants with buffer gave rise to a decrease in the specific activity of the hydroxylase, implicating the involvement of more than one component in catalysis. Activity could be restored by the addition of a heat extract of the supernatant. The active principle in this extract was found to be a heat-stable protein with a molecular mass of about 17 kDa. Immunochemical studies allowed this protein to be identified as cytochrome bs and it was shown that this electron carrier is essential for the activity of CMP-Neu5Ac hydroxylase.

Inhibition studies using iron ligands and activation by exogenous iron salts suggest the involvement of a non-haem iron cofactor in the catalytic cycle of this hydroxylase. Cytochrome bs may thus serve as an electron donor for this postulated cofactor.

Sialic acids constitute a group of about 30 acidic sugars which occur in the oligosaccharide chains of a variety of glycoconjugates, in animals ranging from the echinoderms up to the mammals and in some microorganisms. The structural heterogeneity among sialic acids arises from the variety of biosynthetic modifications that N-acetylneuraminic acid (NeuSAc), the simplest and most ubiquitous sialic acid, can undergo [I].

N-Glycoloylneuraminic acid (Neu5Gc) is a modified sialic acid which is derived by hydroxylation of the N-acetyl group at C-5 of NeuSAc [2]. NeuSGc is very widespread among species possessing sialoglycoconjugates, the relative amount of Neu5Gc expressed being dependent on many factors, most notably the species and tissue [I, 31. Additionally, the stage in development may also determine the extent of sialylation with NeuSGc, as was demonstrated for rat intestinal gangliosides [4] and bovine foetal tissues [ S ] .

Correspondence to R. Schauer, Biochemisches Institut, Christian- Albrechts Universitiit, Olshausenstrasse 40, W-2300 Kiel 1, Federal Republic of Germany

Abbreviations. Buffer A, 50 mM Hepes/NaOH, pH 7.4; NeuSAc, N-acetylneuraminic acid; NeuSGc, N-glycoloylneuraminic acid; Tiron, 4,5-dihydroxy-I ,3-benzenedisulphonic acid; Ferrrozine, 5,6- diphenyl-3-(2-pyridyl)-I ,2,“triazine-(ar)-4’,4”-disulphonic acid.

Enzymes. CMP-N-acetylneuraminic acid : NADH oxidoreductase (N-acetyl hydroxylating) (EC 1.14.99.18); NADH:cytochrome b5 re- ductase (EC 1.6.2.2).

Although it is generally accepted that NeuSGc is absent from normal human glycoconjugates [6 - 81, tiny amounts of this sialic acid have been detected using immunological methods in antigenic gangliosides [9] and glycoproteins [lo] of some human tumours. The existence of these so-called Hanganutziu-Deicher antigens has thus raised the possibility that the gene responsible for the synthesis of NeuSGc is suppressed under normal circumstances in humans, but may be induced in the course of oncogenesis. A detailed knowledge of the enzymology of NeuSGc biosynthesis is thus required in order to understand how this tumour-associated antigen is produced.

Using subcellular preparations of porcine submandibular gland [Ill and mouse liver [12], it has been established that NeuSGc is synthesised by the hydroxylation of CMP-NeuSAc, giving rise to CMP-NeuSGc as the immediate product. The activity of CMP-NeuSAc hydroxylase plays a central role in regulating the expression of this sialic acid by generating a cytoplasmic concentration of CMP-NeuSGc appropriate to the level of NeuSGc to be incorporated into the resulting sialoglycoconjugates. Thus, the magnitude of the hydroxylase activity in high-speed supernatants of rat and mouse liver was found to correlate with the relative amounts of NeuSGc and NeuSAc in the respective total tissue glycoconjugates [I 31. Similarly, the developmentally controlled increase in NeuSGc- containing gangliosides in rat intestine was concomitant with

270

an enhancement of cellular hydroxylase activity [14]. The lat- ter observations suggest that the level of hydroxylase activity is under genetic control, though too little is known about this enzyme to exclude the possible influence of other cellular factors.

CMP-Neu5Ac hydroxylase is a soluble NAD(P)H-depen- dent inonoxygenase which can be activated by exogenous iron ions 111, 121. This hydroxylase has not been fully character- ised, so the basis of the observed cofactor specificity remains unclear. However, recent evidence implicating the involvement of a soluble form of cytochrome b5 in CMP-Neu5Ac hy- droxylase catalysis provides an interesting new perspective into the functioning of this enzyme [15].

CMP-Neu5Ac hydroxylase thus warrants attention, not only because of its role in biologically and medically important processes, but also because of its enzymologically interesting properties. The work presented in this report was therefore undertaken in order to obtain further information on the regulation and catalytic properties of this enzyme. The report- ed observations also provide a basic characterisation which is essential for the purification of this hydroxylase.

MATERIALS AND METHODS

Reagents

Unless stated otherwise, all purchased reagents were of analytical grade. All salts, KCN and solvents were bought from Merck (Darmstadt, FRG). All buffers, phospholipids, detergents, nucleotides (including sugar-nucleotides), o-phen- anthroline, Tiron, trypsin (bovine pancreas), bovine serum albumin and protein molecular mass standards were pur- chased from Sigma Chemicals (Deisenhofen, FRG). Ferrozine and potassium thiocyanate were obtained from Aldrich (Steinheim, FRG). CMP-[l4C]NeuSAc (approximately 300 mCi/mmol) was supplied by Amersham (Braunschweig, FRG). CMP-Neu5Gc was prepared as previously described [16]. Dialysis tubing was obtained from Serva (Heidelberg, FRG). Cellulose HPTLC plates were supplied by Merck. NAP- 5 and NAP-10 Sephadex G-25 mini-columns, CNBr-activated Sepharose 4B, protein-A - Sepharose, Q-Sepharose, as well as the Superose S.12 (10/30) FPLC column, were obtained from Pharmacia (Freiburg, FRG). Bio-Rad protein reagent was purchased from Bio-Rad (Munich, FRG).

Production of high-speed supernatants and heat extract

Freshly excised livers of male Balb/c mice were homogen- ised at 4°C with an Ultraturrax in three 30-s bursts, allowing 30 s cooling between bursts, into 2 - 5 vol. (ml/g wet mass) of 50 mM Hepes/NaOH pH 7.4 (buffer A). For the experiments in which the effect of pH was determined, the livers were homogenised into 2 vol. 5 mM Hepes pH 7.4. After centrifugation for 3 h at l00000xg (Beckman 60Ti rotor), the clear red supernatant was carefully decanted and stored at - 20 'C until use.

A heat extract was made by heating the supernatant to 95°C for 10 min, followed by removal of precipitated material in a 15-min centrifugation at about 10000 xg . The protein concentration in such heat extracts was generally 1 mg/ml.

Assay for CMP-Neu5Ac hydroxylase

With minor variations, the assay employed in these studies was essentially as described previously [12, 131. Unless stated

otherwise, the incubations were performed in buffer A and contained 2 or 10 pM CMP-['4C]Neu5Ac, 1 mM NADH and 0.5 mM FeS04 in a final volume of 60 pl. The amount of protein, as well as the type and concentration of other sub- stances under investigation, are given in the relevant section of Results. Assays were performed in duplicate at 37 "C over various periods of time (see Results) and were stopped by addition of HCl and analysed by radio-TLC using a Berthold Tracemaster linear analyser (Berthold, Isernhagen, FRG). The reproducibility was generally better than 10%.

Determining the effect of various substances on the hydroxylase activity

All stock solutions of the substances tested were dissolved in buffer A before addition to the incubation mixtures. The final concentrations of each substance type are given in the relevant sections of Results. Phospholipids were suspended by sonication in a sonic bath for 15 min. Where necessary, chloroform was removed from the phospholipids under a stream of nitrogen prior to solubilization.

In experiments on the effects of iron ligands, high-speed supernatants were first subjected to a I-h preincubation at 20°C with the respective substance (1 or 5 mM). FeS04 was subsequently added to the relevant samples to give a final concentration of 5 mM and, after 5 min, the reactions were started by addition of NADH and C:MP-['4C]Neu5Ac and heating to 37°C. The iron chelator o-phenanthroline was dis- solved in 96% ethanol and added such that the ethanol con- centration did not exceed 2% (by vol.) in the enzyme assay. This concentration of ethanol had no detectable effect on the activity of the enzyme.

Effect of buffer type on hydroxylase activity A 1-ml aliquot of high-speed supernatant was dialysed at

4'C for 15 h against 500 ml Hepes, Mops, Tes or Tris, all at 50 mM and adjusted to pH 7.4. A concentration of 50 mM was chosen in order to provide sufficient buffering capacity in the incubation mixtures.

Effect of Hepes concentration A portion of high-speed supernatant (42 mg/ml protein,

from homogenisation in 2 ml buffer A/g wet liver) was dia- lysed against 5 mM Hepes pH 7.4. Aliquots of the dialysed supernatant were added to 2 vol. Hepes pH 7.4 at various concentrations and tested for activity.

Effect of pH on hydroxylase activity The extreme sensitivity of the enzyme activity to changes

in ionic strength (see Results) precluded the use of a simple titration of the buffer, in this case Hepes (free acid), with NaOH. An attempt was therefore made to maintain the ionic strength of the buffer (Z= 0.05 M) at the various pH values employed using NaC1, as previously described [17]. More con- centrated high-speed supernatants (50 mg protein/ml) in 5 mM Hepes pH 7.4 were diluted 10-fold in the Hepes/NaOH/ NaCl mixture of the required pH and tested for activity, the CMP-['4C]Neu5Ac substrate and cofactors being dissolved in the relevant buffer mixture prior to addition.

Influence of protein concentration on hydroxylase activity In these experiments, a high-speed supernatant containing

about 42 mg protein/ml was used (from homogenisation in

27 1

2 ml buffer A/g wet tissue). The supernatant was diluted either with buffer A or with the heat extract described above.

Characterisation of the heat-stable factor

The nature of the heat-stable activatory factor detected in the heat extracts of high-speed supernatants was examined in the following experiments.

Dialysis

Heat extract (1 ml) was dialysed against 300 ml buffer A over a period of 15 h in Visking tubing with a molecular mass cut-off of 8 - 15 kDa.

Ethanol precipitation

The heat extract was treated with 3 vol. pure ice-cold ethanol and left at -20°C for 30 min. The precipitated ma- terial was removed by centrifugation (10000 x g, 10 min), dried under a slow stream of nitrogen and resuspended in buffer A to the starting volume. The supernatant was dried under reduced pressure at room temperature, care being taken to avoid frothing, and the residue was resuspended in buffer A to the original volume.

Determination ojthe molecular mass of the heat-stable factor by gel filtration

A portion of the heat extract was concentrated by ethanol precipitation (for conditions see above) and resuspended in buffer A, containing 0.1 M NaCl, to a protein concentration of 4.7 mg/ml. A 0.2-ml aliquot was applied to a calibrated Superose S.12 (10/30) FPLC gel filtration column (Pharmacia) equilibrated in the above buffer. The column was developed at a flow rate of 0.25 ml/min and 0.5-ml fractions were col- lected. The fractions were first desalted on NAP-5 (Pharmacia) columns equilibrated in buffer A and were then tested for the presence of the heat-stable activatory factor by addition of 1.5 pl intact high-speed supernatant (72 pg protein) to 50 pl desalted fraction. The activity of the hy- droxylase was determined in 3-h incubations with 10 pM CMP-[ 14C]Neu5Ac under the conditions described above. The molecular mass of the factor was determined by compari- son of its elution volume with those of the following standards: cytochrome c (12.4 kDa), carbonic anhydrase (29 kDa), bov- ine serum albumin (66 kDa), alcohol dehydrogenase (150 kDa), which were chromatographed on the same column under identical conditions.

Purification of rat and mouse liver microsomal cytochrome b5

Rat liver microsomal cytochrome b5 was purified as pre- viously described [18]. In order to generate the hydrophilic form, the hydrophobic C-terminus of the native amphiphilic protein was cleaved using trypsin [20].

Cytochrome bs from mouse liver microsomes was purified using a modification of existing procedures [21]. Microsomes from 29.5 g wet mouse liver were prepared as described pre- viously [12] (except that the Tris/HCl buffer was replaced with 50 mM Hepes/NaOH pH 7.4) and solubilized in 80 ml20 mM Tris/HCl pH 8.0 containing 1 YO (by vol.) Triton X-100. After centrifugation at I00000 x g for 30 min, the solubilized protein was applied to a column (1.4 x 26 cm) of Q-Sepharose equili- brated in the solubilization buffer, washed with the same

buffer until no more protein emerged and eluted with a 500-ml linear gradient up to 0.5 M NaCl in the wash buffer. Fractions containing cytochrome bs were tested using their ability to mediate the reduction of cytochrome c by crude microsomal NADH :cytochrome b5 oxidoreductase (present in earlier frac- tions). The pooled cytochrome b5 fractions were dialysed against solubilization buffer and applied to a 4-ml column of Q-Sepharose equilibrated in the same buffer. After extensive washing with Tris/HCl pH 8.0, the bound cytochrome b5 was eluted with 20 mM Tris/HCl pH 8.0, containing 0.25 M potas- sium thiocyanate and 0.23% by mass sodium deoxycholate. The eluate was further purified by FPLC gel filtration on Superose S.12 equilibrated in the latter buffer system. The final preparation revealed one main protein band of molecular mass 18 kDa after analysis with SDSjPAGE followed by stain- ing with Coornassie brilliant blue R-250. The yield of cytochrome bs was not determined.

Prior to use, all cytochrome b5 preparations were thoroughly equilibrated in buffer A by dialysis.

Effect of antibodies against cytochrome b5 on CMP-NeuSAc hydroxylase activity and immunochemical identification of the heat-stable factor

Preparation of cytochrome b5 antibody

An antiserum against rat liver microsomal cytochrome b5 was raised in rabbits as previously described [18]. The IgG fraction of this antiserum was isolated using protein-A - Sepharose [19]. The IgG fraction was stored in phosphate- buffered saline containing 15 mM sodium azide and dialysed extensively against buffer A prior to use. For some exper- iments, the IgG fraction was further purified by application to an affinity column consisting of rat cytochrome bs immobilized on CNBr-activated Sepharose (3.5 mg cyto- chrome b,/g unswollen gel). Bound IgG was eluted with 0.1 M glycine pH 3.0, followed by immediate neutralization and di- alysis against phosphate-buffered saline containing 3 mM so- dium azide. The resulting crude and affinity-purified IgG preparations had titres of 0.2 and 2.9 nmol cytochrome b5/mg IgG, respectively, as determined by immunoelectrophoresis.

Ejfect of cytochrome bs antibody on the activity of CMP-NeuSAc hydroxylase

Aliquots of mouse liver high-speed supernatant containing 300 pg protein were preincubated with varying amounts of crude IgG in a total volume of 30 pl for 20 min at 20°C. The hydroxylase activity was determined as described above after addition of cofactors and substrate to a final volume of 36 pl.

Covalent coupling of cytochrome bs antibody to Sepharose

The IgG fraction (10mg crude IgG or 5 mg affinity- purified IgG/ml settled gel) was coupled to CNBr-activated Sepharose 4B in 0.2 M sodium citrate pH 6.5, as described by the manufacturer. The media from crude and affinity-purified IgG had binding capacities of 0.25 and 2.0 nmol cytochrome bJml gel, respectively.

Adsorption of cytochrome b5 in native high-speed supernatant onto an immunoajyinity medium

1.5 ml supernatant was applied onto a 2-ml column of anti- (cytochrome bs ) - agarose equilibrated in buffer A (capacity

272

% Actlvl ty

l Z O 1

1 0 4 I

% Act lv l ty

120-------

0 20 40 60 80 100 120 140 160 Salt concen t ro t ion (mM)

Fig. 1. lnfluence of several salts on the activity of CMP-Neu5Ac hy- droxylase. High-speed supernatant (20 mg protein/ml) was diluted with various amounts of a 1.5 M solution of each salt, dissolved in buffer A, to give the concentrations indicated in the figure. The hydroxylase activity was determined in 10-min incubations, under the conditions described in Materials and Methods. The salts employed were as follows: (V-V) NH4CI; (0----0) KCI; (0-0) NaCl; (+----+) Na2HP04; (V----V) Na2S04; (+-+) MgC12; ( A ) Na4P207.

0.25 nmol/ml gel) at a flow rate of 0.1 ml/min. The effluent (0.17 mg protein) was tested for CMP-NeuSAc hydroxylase activity in the presence of various factors: heat extract (0.015 mg protein), native and trypsin-cleaved cytochrome b5 from rat liver microsomes (0.15 nmol) and native cytochrome b5 from mouse liver microsomes (0.15 nmol).

Effect of anti-(cytochrome b,) - Sepharose on the activatory potential of the heat extract

A 6-ml portion of the heat extract (6mg protein) was applied to an 0.8-ml column of the anti-(cytochrome b5) immunoaffinity gel (capacity, 2.0 nmol/ml gel), equilibrated in buffer A. at a flow rate of 0.1 ml/min. The effluent was collected for further experiments and the column was washed with 15 ml buffer A. The bound material was eluted with S ml 0.1 M sodium citrate pH 3.0 and collected directly into a vessel containing 2 ml 1 .S M Hepes/NaOH pH 7.4 in order to effect rapid neutralization. After extensive dialysis against buffer A, the eluate was concentrated to a volume of 1 ml in an Amicon pressure dialysis cell using an ultrafiltration membrane with a 5-kDa cut-off limit (Sartorius, Gottingen, FRG). The pres- ence of the heat-stable factor was tested for in the various fractions using the effluent obtained after application of whole supernatant to the anti-(cytochrome b5) immunoaffinity column.

RESULTS

Effect of buffer type

The highest CMP-Neu5Ac hydroxylase activity was de- tected in the Hepes buffer. The relative activities in Hepes, Tes, Mops and Tris (all at SO mM, pH 7.4) were loo%, 99%,

6.6 6.8 7 7.2 7.4 7.6 7.8 8

PH

Fig. 2. Influence of pH on the activity of CMP-Neu5Ac hydroxylase at constant ionic strength. The hydroxylase activities were measured in Hepes buffers adjusted to the desired pH with NaOH and kept at constant ionic strength by addition of NaC1, as described in Materials and Methods. The duration of the incubations was 80 min.

89% and 50%, respectively. All subsequent experiments were therefore performed in Hepes/NaOH pH 7.4. Increasing the concentration of this buffer exercised a significant inhibitory effect on the enzyme, the concentration of Hepes giving SO% inhibition (Z50) being 55 mM. In order to investigate whether this inhibition was a consequence of increased ionic strength or an effect specific to Hepes, the influence of a number of different salts was tested.

Effect of various salts on hydroxylase activity The influence of a variety of salts, consisting of several

different ion combinations at a number of concentrations, was tested. The results depicted in Fig. 1 clearly illustrate that mouse liver CMP-Neu5Ac hydroxylase is very sensitive to increases in the concentration of all the salts tested. The ap- proximate I s0 values, under the conditions employed in this study, were as follows: NaC1, 21 mM; KCl, 21 mM; NH4Cl, 25 mM; Na2HP04, 18 mM; Na2S0,, 16 mM; MgC12, 13 mM; Na4Pz07, < 5 mM. Although the inhibitory potency of all the salts tested falls within the same order of magnitude, there is a tendency for salts containing multivalent ions to give the greatest inhibition.

The reversibility of this inhibition was investigated as fol- lows. High-speed supernatant was treated with 200 mM NaCl over a period of 100 min followed by the removal of the salt by dialysis against 250 ml buffer A in the course of 15 h or by rapid gel filtration of 0.5 ml supernatant on Sephadex G-25 (NAP-S columns) equilibrated in the same buffer. A significant proportion of the hydroxylase activity was recovered after both desalting procedures (dialysis, 85% ; gel filtration, 96%) suggesting that the enzyme is not irreversibly inactivated by increased ionic strength.

Effect of pH on CMP-Neu5Ac hydroxylase activity The effect of pH on the activity of the hydroxylase was

measured under conditions of constant ionic strength, ( I = 0.05 M) as shown in Fig. 2. In the pH range tested, the enzyme

273

exhibited no clearly defined pH optimum, only a plateau of optimal activity stretching from pH 6.8 to about pH 7.4, a pH region which is generally accepted to be physiological.

Influence of detergents and lipids on CMP-NeuSAc hydroxylase

Although CMP-Neu5Ac hydroxylase is only detectable in the particle-free supernatant of fractionated mouse liver, preliminary results suggest that this hydroxylase is activated in the presence of Triton X-100 [12]. This somewhat unexpected finding was therefore examined in detail using a range of ionic and non-ionic detergents. The results of this investigation, depicted in Fig. 3, clearly demonstrate that a number of deter- gents are capable of activating this enzyme. The non-ionic detergents Nonidet P-40, Triton X-100 and octyl glucoside were by far the most effective activators, eliciting a 4.5-, 2.8- and 2.4-fold increase in activity, respectively (Fig. 3 a). Decyl glucoside was more effective at lower concentrations, giving rise to a 2.2-fold activation. Interestingly, hexyl glucoside was without effect, suggesting that a certain hydrophobicity is necessary in order to manifest this effect.

In contrast, both anionic and cationic detergents were generally inhibitory at higher concentrations (Fig. 3 b), though both SDS and octanoic acid were capable of inducing a modest activation at concentrations around 1 mM, suggesting that some sort of amphiphilic effect was prevailing under these conditions. To what extent the observed inhibition was a result of the increase in ionic strength, which undoubtedly occurred at the higher levels of these detergents (50mM), or due to enzyme denaturation cannot be unequivocally determined. Unexpectedly, the zwitterionic detergent Chaps behaved in essentially the same manner as the non-ionic detergents, eliciting a maximal twofold increase in activity.

In view of the considerable activatory potential exhibited by a number of the detergents tested, the influence of several phospholipids was examined. At 3 mM, the following phospholipids were either inert or inhibitory: phosphatidic acid (80%), phosphatidylethanolamine (100%0), phosphatid- ylcholine (97 YO), cardiolipin (85%) and phosphatidylinositol (32%) (numbers in parenthesis indicate percentage activity relative to activity in the absence of lipid). Only phosphatidylserine ( 3 mM) activated the enzyme, producing a 1.4-fold stimulation of activity.

Influence of nucleotides on CMP-NeuSAc hydroxylase

The influence of a number of nucleotides and sugar- nucleotides on the activity of CMP-NeuSAc hydroxylase was examined over a wide range of concentrations. In all exper- iments, CMP-[14C]Neu5Ac was used at 2 pM. This concen- tration is slightly greater than the apparent K , of 1.3 pM [ 3 2 ] and should thus allow the detection of any significant inhibitory effects. Briefly, the 5'- mono-, di- and triphosphates of adenosine, guanosine, inosine, uridine, thymidine and cytidine as well as 2'CMP, 3'CMP, 3',5'cAMP and 3',5'cCMP, tested at 2 mM, exhibited onIy a weak inhibitory influence, amounting to no more than 30% inhibition. The same was true for the following sugar-nucleotides : GDP-mannose, UDP- glucose, UDP-N-acetylglucosamine, UDP-galactose and UDP-N-acetylgalactosamine. Only CMP-NeuSGc, the prod- uct of the hydroxylase reaction, exhibited any significant inhi- bition. This latter substance elicited a 50% inhibition at about 40 pM (Fig. 4).

The fact that both Na2HP0, and Na4P20, were inhibi- tory (86% and 54% of control values, respectively) at 2 mM,

a)

Nonidet P-40

0 ' I I I 0 20 40 60 80 100 120

Detergent concentration (mM)

0 5 10 15 20 25 30 Detergent concentration (mM)

Fig.3. Influence of detergents on the activity of CMP-Neu5Ac hy- droxylase. The effect of several concentrations of neutral (a) and ionic (b) detergents was elucidated in 6-min incubations using 0.59 mg protein as described in Materials and Methods. The detergent is indicated adjacent to the respective curve. CPC, cetylpyridinium chloride.

suggests that the inhibition observed for all nucleotides other than CMP-Neu5Gc was due mainly to non-specific, possibly ionic, effects.

Effect of iron ligands on CMP-NeuSAc hydroxylase activity

In the absence of exogenously added iron, all substances tested suppressed the hydroxylase activity, (Table 1). The inhi- bition caused by o-phenanthroline and ferrozine could be partly overcome by the addition of an excess of FeSO,. How-

274

% Act iv l ty

I Z 0 1

Rate (pmol/mln)

35 I

0 50 100 150 200 250 300 ICMP-Neu5Gcl (uM)

Fig. 4. Effect of CMP-Neu5Gc on the activity of CMP-Neu5Ac hy- droxylase. The effect of various concentrations of CMP-NeuSGc was tested under the conditions described in Materials and Methods.

0 2 4 6 8 I0 12 14 16 Proteln c o n c e n t r a t l o n (rng/ml)

Fig. 5. Effect of protein concentration on the activity of CMP-Neu5Ac hydroxylase. High-speed supernatant was diluted to various degrees using buffer A (*) or a heat extract of the supernatant (0). The hydroxylase activity was determined in incubations ranging over 4 - 180 min.

Table 1. Effect of various iron ligands on the activity of CMP-Neu5Ac hydroxylase. High-speed supernatants were preincubated for 1 h with the respective substances at the indicated concentrations. FeS04 was added to S mM and the samples tested for activity as described in Materials and Methods. The inhibition is expressed as a percentage of that of untreated controls in the presence or absence of FeSO,.

Treatment Inhibition

o-Phenanthroline (1 mM) o-Phenanthroline (1 mM) + FeS0, o-Phenanthroline ( 5 mM) o-Phenanthroline (5 mM) + FeS0,

KCN ( 5 mM) KCN ( 5 mM) + FeS0, Tiron ( 5 mM) Tiron ( 5 mM) + FeSO,

Ferrozine (0.7 mM) Ferrozine (0.7 mM) + FeS0,

Yo

66 28

100 46

49 55 82

100

82 2s

ever, instead of reversing the effects of Tiron and cyanide, the presence of exogenous iron gave rise to further inhibition.

Effect of protein concentration on CMP-Neu5Ac hydroxylase activity

As expected, dilution of the high-speed supernatani with buffer gave rise to a decrease in CMP-Neu5Ac hydroxylase activity. However, a plot of protein concentration against activity exhibited a marked curvature (Fig. S), the specific activity of the hydroxylase at lower protein concentrations being significantly smaller than at higher protein concen- trations. Even at the lowest protein concentrations, the reac- tion progress curves were linear with respect to time, suggesting that the enzyme was not being de-activated by the low protein concentrations or by the rather long periods of incubation (2 - 3 h) required to detect such a low turnover.

Dilution of the native supernatant with a particle-free heat- treated preparation of the same supernatant gave rise to a less curved plot of activity against protein concentration, with a significant increase in the specific activity of the highly diluted hydroxylase (Fig. 5). The heat extract itself was completely devoid of any hydroxylase activity.

Characterization of the heat-stable factor

The heat-stable factor present in the extract was further characterized as described in Materials and Methods. The factor was not dialysable and was precipitable by addition of ethanol at - 20 "C, with full recovery of activatory potential after redissolving the precipitated material in buffer A. The non-precipitated material had no effect on the hydroxylase activity.

Gel filtration chromatography of the heat extract on Superose S.12 revealed that a number of substances were present in this extract. However, only material eluting between 14.5- 15.5 ml was capable of activating the hydroxylase in highly diluted supernatants (Fig. 6). From the elution volume of the factor, an approximate molecular mass of 17 kDa was determined.

These experiments therefore establish that high-speed supernatants of mouse liver contain a heat-stable 17-kDa protein which is capable of restoring the anomalously low activity of CMP-NeuSAc hydroxylase in highly diluted super- natants.

Effect of cytochrome b5 antibody on CMP-NeuSAc hydroxylase and immunochemical identification of the heat-stable factor

Antibody directed against rat microsomal cytochrome bs proved to be a very potent inhibitor of CMP-NeuSAc hy- droxylase in mouse liver supernatants (Fig. 7).

Accordingly, passage of intact mouse liver supernatant over an anti-(cytochrome b5) immunoaffinity column resulted in a significant reduction of the hydroxylase activity in the

275

A b s o r b a n c e (280nrn) A c l l v a t l o n factor

0.08 -

0.06 -

0.04 -

Oo2L 0

3.8

3.3

2.8

2.3

I .a

I .3

1.8 0 5 10 15 20 25 30

Elution volume (ml) Fig. 6. Gel filtration chromatography of the heat-stable factor on Superose S.12. A 0.2-ml aliquot of concentrated heat extract (0.95 mg protein) dissolved in 50 mM Hepes pH 7.4, containing 0.1 M NaCI, was applied to a Superose S.12 (10/30) column equilibrated in the same buffer and eluted at a flow rate of 0.25 ml/min. The eluate was monitored for absorbance at 280 nm (solid curve) and the fractions collected were desalted and tested for their ability to activate CMP- NeuSAc hydroxylase in diluted high-speed supernatants (dashed curve) as described in Materials and Methods. Numbers at the top indicate molecular masses of marker proteins in kDa.

0

$ Y

20 , I

15

10

5

0 0 5 10

pg antibody

Fig. 7. Inhibition of CMP-Neu5Ac hydroxylase by antibody against rat microsomal cytochrome b5. High-speed supernatant (300 pg) was preincubated with varying amounts of anti-(cytochrome b,) as de- scribed in Materials and Methods and tested for hydroxylase activity.

resulting effluent. A large proportion of the activity could, however, be restored by addition of heat extract, strongly suggesting that the heat-stable factor is in fact cytochrome b5. Significantly, the addition of cytochrome b5 purified from rat or mouse liver microsomes as well as tryptically cleaved cytochrome b5 from rat liver gave rise to a dramatic increase in the hydroxylase activity of this effluent (Table 2).

As expected, passage of the heat extract over an anti- (cytochrome b5) immunoaffinity column completely abolished its ability to reactivate the hydroxylase in the effluent de- scribed above (Table 3). Elution of the bound material with

Table 2. Effect of anti-(cytochrome b,) immunoaffinity chromatography on the activity of CMP-Neu5Ac hydroxylase in the high-speed super- natant. 1.5 mi high-speed supernatant was applied to a 2-ml column of anti-(cytochrome b5) - Sepharose as described in Materials and Methods. The effluent from the column was tested for hydroxylase activity in the presence of heat extract or 0.15 nmol purified microsomal cytochrome b5.

Fraction Hydroxylase activity

pmol. min- ' mg protein-'

Supernatant 11.8 Effluent + buffer 1 .o Effluent + heat extract 7.1 Effluent + cytochrome b5 (mouse) Effluent + cytochrome b5 (rat) Effluent + trypsinised cytochrome b5 (rat)

151 117 147

Table 3. Fractionation of the active component in a heat extract of high- speed supernatant on anti-(cytochrome b5) - Sepharose. Heat extract was applied to a 0.7-ml column of anti-(cytochrome b5)- Sepharose. The presence of the heat-stable factor was tested for using the cytochrome-b,-depleted supernatant (see Table 2), denoted by efflu- ent, i.e. effluent from application of high-speed supernatant to anti- (cytochrome b5) - Sepharose.

Fraction Hydroxylase activity

pmol . min-l . mg protein-'

Effluent + buffer 0.8 Effluent + non-bound fraction 0.6 Effluent + eluate 3.4 Effluent + heat extract 6.2

sodium citrate pH 3.0, with immediate neutralization, led to a partial recovery of the active principle (Table 3).

DISCUSSION

The results presented in this report demonstrate that the CMP-NeuSAc hydroxylase from mouse liver exhibits a num- ber of unusual properties. With regard to the sialic acid sub- strate specificity, this enzyme only appears to recognize CMP- NeuSAc. The low apparent K,,, of the hydroxylase for this substrate (1.3 pM) [12] suggests that CMP-NeuSAc is bound with a very high affinity. Any deviation from this structure leads to a drastic decrease in the strength of interaction with the enzyme. Thus, neither CMP (and other cytidine nucleotides) nor free NeuSAc were capable of competing with CMP-NeuSAc, even at considerably higher (up to 1000-fold greater) concentrations than the substrate. Accordingly, none of the other purine and pyrimidine nucleotides and sugar- nucleotides tested were able to influence the activity of the hydroxylase to any significant degree under conditions where the CMP-NeuSAc concentration was limiting. The results also suggest that these intermediates of general and glycoconjugate metabolism are not involved in regulating this enzyme in vivo.

The relatively poor inhibition caused by CMP-NeuSGc, the product of the hydroxylase reaction, demonstrates that the substrate binding site of the hydroxylase is not able to tolerate a hydroxyl group on the N-acetyl function, despite

276

the identity of the rest of this molecule with the substrate. This result also indicates that the hydroxylase is unlikely to be subjected to any feed-back inhibition by the reaction product, as indicated by the linearity of the reaction progress curves, even after the turnover of a considerable proportion of the substrate [ll, 121.

The lack of activity of this enzyme towards the cx-glyco- sidically bound NeuSAc in the glycan chains of glycoproteins [12] and the absence of inhibition by the methyl fl-glycoside of NeuSAc 1221 underline the stringent structural and stereochemical requirements of the hydroxylase for its sub- strate and the relevance of this specificity to its biological function.

Although several enzymes can be activated by artificial surfactants 1231, the reason for the extraordinary activation of the hydroxylase by non-ionic detergents is not immediately apparent. Since the enzyme is mainly detectable in high-speed supernatants with only traces of activity present in the particu- late fractions [12], the possibility that the hydroxylase is en- trapped in membrane vesicles can be excluded. The fact that phospholipids generally had little influence on the enzyme activity suggests that the detergents were not simply mimick- ing these naturally occurring amphiphilic compounds. Fur- thermore, the effects of a series of chemically similar alkyl glucosides suggests that a hydrophobic portion of optimal (C,) size is required for activation. It is additionally worth noting that with all activatory detergents, maximal enhancement of activity was achieved at concentrations exceeding the critical micellar concentration [24], possibly implicating the involve- ment of some micellar structure in activation. The possibility that detergents imitate a hitherto unidentified amphiphilic regulator must also be considered.

The unusually potent inhibition caused by increased ionic strength poses a number of problems, in particular assaying the enzyme under conditions where increased salt concen- trations are unavoidable, for example in certain chromatogra- phic fractionation procedures. It is therefore of paramount importance to control the ionic composition of the reaction medium in order to make quantitative measurements of hy- droxylase activity. This inhibition can be overcome by des- alting procedures such as dialysis or gel filtration on Sephadex

The acute sensitivity of this enzyme to ionic strength is difficult to reconcile with its proposed cytosolic location [I2 - 141, since the observed inhibition occurred at essentially physiological salt concentrations. Although the greater ac- tivity at lower ionic strengths might be an artefact of the in vitro assay conditions, it is tempting to propose that, in the cell, this enzyme is exposed to an environment of lower ionic strength.

Increases in the ionic strength of a reaction medium fre- quently lead to enzyme inhibition, by causing conformational changes or altering the charge status of ionic groups involved in substrate binding or catalysis. In addition, changes in ionic strength may disrupt specific protein - protein interactions necessary for catalytic function. Many monooxygenases are dependent on several protein components interacting as an electron-transport chain, in which electrons from a reduced coenzyme, usually NAD(P)H, are transferred via one or more electron carriers to a terminal monooxygenase where they are used in the reductive activation of molecular oxygen 1251. Interactions between electron carriers are frequently mediated by complementary charges on their interacting surfaces and as such can be influenced by the ionic strength of the reaction medium [26].

G-25.

The deleterious effect of dilution on the specific activity of the hydroxylase suggests that the hydroxylation of CMP- NeuSAc may be catalysed by a multi-component enzyme sys- tem [27]. A very similar dilution-dependent decrease in the specific activity of methane monooxygenase, an enzyme con- sisting of three components, including an NADH-dependent reductase, has also been observed [28].

The discovery of a soluble heat-stable factor, capable of activating CMP-NeuSAc hydroxylase in highly diluted super- natants, provides more evidence for the existence of a multi- component enzyme system. Several lines of evidence suggest that this heat-stable factor is cytochrome b,.

Firstly, its molecular mass (17 kDa) is the same as cytochrome bs [29]. Moreover, microsomal cytochrome bs is known to be very thermostable, resulting from its ability to adopt an active conformation after thermal denaturation 130, 311. The most convincing proof, however, emerges from the immunochemical studies using anti-(cytochrome b5) anti- bodies. The acute sensitivity of CMP-NeuSAc hydroxylase to the addition of antibody against cytochrome b5 suggests that this electron carrier is essential for the hydroxylase activity. Thus, passage of a high-speed supernatant over an anti- (cytochrome b5) immunoaffinity column resulted in a con- siderable decrease in hydroxylase activity. Addition of either heat extract or purified microsomal cytochrome b5 from either rat or mouse liver to this cytochrome-b5-depleted supernatant allowed the reconstitution of hydroxylase activity. These re- sults also demonstrate that the hydroxylase can function with amphiphilic microsomal cytochrome b, as well as with the hydrophilic proteolytically cleaved form. Interestingly, the purified cytochrome bs species elicited activities which were some tenfold greater than the original activity, suggesting that the level of cytochrome b, in the high-speed supernatant is severely limiting the turnover of the hydroxylase. This might be a reason for the sensitivity of the hydroxylase to dilution.

The requirement for NADH, together with the finding that cytochrome b5 participates in the reaction cycle of CMP- NeuSAc hydroxylase, suggests the involvement of cytochrome bs reductase. The disruption of complementary charge-pair interactions between cytochrome b5 reductase and cyto- chrome b5, as well as between cytochrome b5 and several electron acceptors, caused by increased ionic strength [32 - 351 could explain the effect of higher salt concentrations on CMP-NeuSAc hydroxylase.

Although the physiological role of cytochrome b5 is still incompletely understood, it seems to be largely responsible for the delivery of reducing equivalents to the iron prosthetic groups of certain proteins, for example, the haem group of methaemoglobin [32] or cytochrome P-450 [36], the cyanide- sensitive factor of the microsomal fatty acid desaturase and A7-sterol 5-desaturase [37, 381 and the non-haem iron pros- thetic group of methaemerythrin [39]. In view of the sensitivity of CMP-NeuSAc hydroxylase to iron ligands such as CN- ions as well as o-phenanthroline, ferrozine and Tiron, it is tempting to propose that this monooxygenase possesses a prosthetic group, with a dissociable iron component, that undergoes a cytochrome-b5-mediated reduction in the course of its catalytic cycle. The fact that the hydroxylase is stimu- lated by exogenously added Fez+ and Fe3+ [12] ions lends further weight to this hypothesis.

Since the hydroxylase preparations employed in these in- vestigations were impure, the exact nature of the molecular interactions, responsible for the phenomena reported here, cannot be defined. Further research on the purified enzyme system is therefore required to clarify the questions raised

by these experiments and the results of other workers [15]. Nevertheless, the observations in this paper provide a useful basis for optimizing the assay and handling conditions of this enzyme system. In addition, they provide some insight into the specificity and regulation of the CMP-Neu5Ac hydroxylase as well as some clues to its catalytic mechanism.

The authors would like to thank Dorthe Jurgensen and Sabine Stoll for their excellent technical assistance. The financial assistance of the Deutsche For.~chun~sgemeinschaft (project number, Scha 2021 15-l), Fonds der Chernischen Industrie and the Sialinsauregesellschuft is gratefully acknowledged.

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