cmp-n-acetylneuraminic acid hydroxylase from mouse liver and pig submandibular glands : interaction...

Eur. J. Biochem. 219, 1001-1011 (1994) 0 FEBS 1994

CMP-N-acetylneuraminic acid hydroxylase from mouse liver and pig submandibular glands Interaction with membrane-bound and soluble cytochrome b5-dependent electron transport chains

Lee SHAW ', Petra SCHNECKENBURGER', Wiebke SCHLENZKA', Jens CARLSEN*, Kirsten CHRISTIANSENZ, Dorthe JURGENSEN' and Roland SCHAUER' ' Biochemisches Institut, Christian-Albrechts Universitat, Kiel, Germany * University of Copenhagen, Panum Institute, Department of Biochemistry C, Copenhagen, Denmark

(Received September 29, 1993) - EJB 93 1480/4

In this report, the nature of the protein components involved in the functioning of cytidine-5'- monophosphate-N-acetylneuraminic acid (CMP-NeuSAc) hydroxylase in high-speed supernatants of mouse liver has been investigated. Fractionation and reconstitution experiments showed that this enzyme system consists of NADH-cytochrome b, reductase, cytochrome b, and a 56-kDa terminal electron acceptor having the CMP-Neu5 Ac hydroxylase activity. This enzyme system is extracted in a soluble protein fraction ; however, the amphipathic, usually membrane-associated, forms of cytochrome b, and the reductase were found to predominate and are presumably the forms which support the turnover of the hydroxylase in vivo. Although the majority of cellular cytochrome b, and cytochrome b, reductase is membrane-bound, the addition of intact microsomes elicited no significant increase in the hydroxylase activity of supernatants. Detergent-solubilised microsomes, however, potently activated the hydroxylase, probably due to the greater accessibility of the cytochrome b,. Accordingly, in reconstitution experiments, pure hydrophilic cytochrome b, interacts more effectively with the hydroxylase than isolated amphipathic cytochrome b,.

Studies on the CMP-Neu5 Ac hydroxylase system in fractionated porcine submandibular glands and bovine liver suggest that the composition of this enzyme system is conserved in all mammals possessing sialoglycoconjugates containing N-glycoloylneuraminic acid.

The sialic acid N-glycoloylneuraminic acid (NeuSGc) occurs in sialoglycoconjugates of most animal groups throughout the deuterostomate lineage [ l , 21. The level of sialylation with Neu5Gc is highly characteristic for each tissue of a par- ticular organism, and can be subject to further variation, de- pending on age and diseased state. Humans and chickens are notable, since Neu5Gc is completely absent from normal tissues and has only been detected in small amounts in can- cerous tissues [3, 41.

Neu5Gc is synthesised as its CMP-glycoside by the ac- tion of a hydroxylase specific for the sugar nucleotide cytidine-5'-monophosphate-N-acetylneuraminic acid (CMP- NeuSAc) [ S , 61. Several metabolic studies suggest that the rate of CMP-Neu5Gc production by CMP-Neu5 Ac hydroxylase is important in regulating the extent of sialylation with NeuSGc [7-101.

Investigations into the properties of CMP-Neu5 Ac hydroxylase from pig [5 , 111, mouse [6, 12, 131 and starfish [14] reveal that the enzyme is an NADH-dependent, cyto-

Correspondence to R. Schauer, Biochemisches Institut, Chri- stian-Albrechts Universitat, Olshausenstr. 40, D-24098 Kiel, Germa- ny

Abbreviations. Neu5 Ac, N-acetylneuraminic acid; NeuSGc, N- glycoloylneuraminic acid.

Enzymes. CMP-N-acetylneuraminic acid : NADH oxidoreductase (N-acetyl hydroxylating) (EC 1.14.13.45); NADH : cytochrome b, oxidoreductase (EC 1.6.2.2).

solic, monooxygenase which may possess a non-haem iron cofactor [12, 141. Several lines of evidence point to the participation of cytochrome b, in the catalytic cycle of CMP- Neu5 Ac hydroxylase from mouse liver [ 12, 13, 151, though this has not been confirmed in any other species.

In vertebrates, cytochrome b, and its reductase occur mainly as membrane-bound proteins, consisting of a hydrophobic membrane-anchoring domain and a hydrophilic catalytic domain [18, 191. In addition, the N-terminal glycine of the membrane-associated domain of cytochrome b, reductase is myristoylated L201. These domains can be separated by proteolytic cleavage, yielding a soluble catalytic fragment and the corresponding hydrophobic polypeptide [21,22]. The amphipathic forms of cytochrome b, and its reductase are associated with the membranes of several cellular compart- ments, mainly the endoplasmic reticulum, with their catalytic domains directed towards the cytoplasm [23 -261.

In addition to the membrane-bound, amphipathic forms of these proteins, soluble, hydrophilic forms are known to exist in vertebrate cells. Erythrocytes, for example, contain large amounts of cytosolic, hydrophilic cytochrome bs and cytochrome b, reductase, where they function in the reduction of adventitiously oxidised haemoglobin [27, 281.

By analogy with other cytochrome-b,-dependent enzymes [17], CMP-Neu5Ac hydroxylase must exist as a separate protein species possessing a CMP-Neu5 Ac binding site

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and the necessary molecular apparatus for oxygen activation. Since the postulated hydroxylase component has not been isolated, this suggestion remains speculative. The participation of cytochrome b, reductase is also presumed, but this has not been unequivocally demonstrated.

The identification of cytochrome b, as an integral component of this enzyme system explains several unusual features of the hydroxylase. For example, inhibition caused by increased ionic strength can be ascribed to a disruption of, ionic interactions between the various components of the system [12, 131. However, the activatory effect of non-ionic detergents [ 121 suggests that hydrophobic interactions may also be involved, though due to lack of information about the proteins participating in the reaction, the nature of these interactions is unknown.

In the work reported here, the molecular forms of cytochrome 6, and cytochrome b, reductase present in high-speed supernatants of mouse liver have been characterised and their involvement in the catalytic activity of CMP-Neu5 Ac hydroxylase has been elucidated. The membrane-bound cytochrome b, system has also been investigated as a possible source of reducing equivalents for the hydroxylase. Experi- ments were also performed to separate the postulated hydroxylase component from cytochrome b, and the reductase and to allow an initial characterisation of this protein. In addition, the role of cytochrome b, in the catalysis of CMP-Neu5Ac hydroxylase from other organisms was investigated.

MATERIALS AND METHODS

Reagents and chromatographic media

Unless stated otherwise, analytical grade reagents from Sigma Chemicals Ltd, Merck and Boehringer Mannheim were used throughout this study. CMP-[4,5,6,7,8,9-'"C]- Neu5 Ac (250- 3OOCi/mol) was purchased from Amersham. The Superose S.12 10/30, NAP-5 and NAP-10 columns, Q- Sepharose, as well as Sephadex G-75 (medium) were purchased from Pharmacia. AMP- Sepharose and Cibacron- blue-3GA- agarose (type 3000-CL-L) were supplied by Sigma Chemicals Ltd.

Affinity chromatographic media

Production of anti-(cytochrome b,) antibody and coupling to Sepharose

The production of rabbit anti-(rat liver cytochrome b,) IgG fraction (titre, 2.9nmol cytochrome b,lmg IgG) and coupling to agarose was performed as previously described c121.

Coupling of cytochrome b, to Sepharose

Amphipathic rat liver microsomal cytochrome b, was purified and coupled to CNBr-activated Sepharose as described elsewhere 1121.

Protein determination

Protein concentrations were quantified according to the method of Bradford [29] using the Bio-Rad reagent with bovine serum albumin as standard.

Preparation and fractionation of concentrated supernatant of mouse liver

Freshly excised livers from male Balblc mice were homogenised with eight strokes of a Potter-Elvehjem homogeniser into precooled 50 mM HepesNaOH, pH 7.4 (5 mVg wet tissue) and centrifuged at 1OOOOOXg for 1 h at 4°C. The clear portion of the supernatant was carefully removed and stored frozen at -20°C until use.

Production of heat extract A heat extract was made by heating the concentrated su-

pernatant to 95 "C for 10 min followed by removal of precipitated protein by centrifugation at lOOOOXg for 15 min [12].

Removal of cytochrome b, from high-speed supernatants High-speed supernatants lacking cytochrome b, were

produced by repeated (three times) passage of supernatant over a column of immobilised anti-(cytochrome b,) described above 1121.

Enzyme assays Assay for cytochrome b, reductase

The test used here is a modification of the ferricyanide reduction assay described by several authors [30]. Briefly, the reduction of 0.6 mM potassium ferricyanide was measured photometrically (420 nm) at 37°C in 50 mM Hepes/ NaOH pH 7.4 in the presence of 0.5 mM NADH in a total volume of 0.9-1.0 ml. The sample volume was generally 5 -50 pl.

Measurement of cytochrome c reductase activity of cytochrome b, reductase in the presence of cytochrome b,

The cytochrome-b,-mediated reduction of cytochrome c by cytochrome b, reductase was measured photometrically in various cell fractions and in systems consisting of purified cytochrome b, and its reductase at 37°C in the presence of 0.5 mM NADH and 0.5 mglml cytochrome c (horse heart type 111, Sigma) in 50 mM Hepes pH 7.4. Details of samples under investigation are given in Results and Discussion.

Assay for CMP-NeuSAc hydroxylase Unless stated otherwise, CMP-Neu5 Ac hydroxylase ac-

tivity was determined in 50 mM Hepes/NaOH pH 7.4 at 37°C in a final volume of 30-36 pl with the following sub- strates and cofactors at the concentrations given in parenthe- sis: CMP-[4,5,6,7,8,9,-'"C]NeuSAc (10 pM, 0.0125 pCi), NADH (1 mM) and FeSO, (0.5 mM). Reactions were stopped by addition of 8 p11 M trichloroacetic acid and, after removal of precipitated material by centrifugation, the re- leased ['"Clsialic acids were quantitatively analysed by radio thin-layer chromatography [ 121.

Immunochemical identification of cytochrome b, in high-speed supernatants of mouse liver Purification of cytochrome b, from native high-speed supernatant using immunoafinity chromatography

A 7-ml portion of concentrated high-speed supernatant was applied to the immunoaffinity medium described above

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at a flow rate of 0.07 ml/min. The effluent was reapplied and the column was washed with 16 ml50 mM Hepes/NaOH pH 7.4. The hydroxylase activity in the effluent was reduced to 10% of the starting activity. Bound material was eluted with 6 ml 0.1 M sodium citrate pH 3.0. After concentration and desalting on a NAP-10 column, the eluate was lyophilised and resuspended in 120 pl water.

Partial purification of the heat-stable factor

The heat-stable factor capable of activating CMP- Neu5 Ac hydroxylase was enriched from a heat extract of concentrated mouse liver high-speed supernatant by immunoaffinity chromatography [12]. A 6.7-ml portion of heat extract of concentrated high-speed supernatant (6 mg protein) was applied to a 0.8-ml column of anti-(cytochrome b,)- Sepharose at a flow rate of 0.07 ml/min. The effluent was reapplied to the column under the same conditions and the column was washed with 8 ml 50 mM Hepes/NaOH pH 7.4. Using the previously described assay [12], it was shown that all of the heat-stable factor had bound to the immunoaffinity medium. Bound material was eluted with 4 mlO.1 M sodium citrate pH 3.0 and, after concentrating to 1 ml by ultrafiltration (membrane cut-off 5 m a ) , the sample was desalted on a NAP-10 column equilibrated in 5 mM Hepes/NaOH pH 7.4. The resulting material was lyophilised and resuspended in 60 p1 water.

Immunoblotting procedure for characterisation of cytochrome b, forms in the above preparations

The indicated amounts of the following samples were analysed by SDSPAGE on 15% acrylamide gels 1311. Immunoaffinity-purified heat-stable factor (15 p1, representing a quarter of the total material from 6.7 ml heat extract), 10 p1 immunoaffinity-purified cytochrome b, from whole supernatant (representing 1/12 of material isolated from 7 ml supernatant), 1 pg each of purified amphipathic and hydrophilic rat liver cytochrome b, and amphipathic mouse liver cytochrome b,. All samples were denatured in 0.5 % SDS and 1% 2-mercaptoethanol at 95°C. The separated proteins in the gel were transferred onto a cellulose nitrate membrane (Schleicher & Schuell) which was dried and washed with 50mM TrisMCl pH7.5 containing 150mM NaCl and 0.05% (by vol.) Tween 20. The membrane was incubated at 4°C overnight with 30 p1 anti-(cytochrome b,) IgG (titre, 2.9 nmol cytochrome b,/ml) diluted in 30 ml of the latter buffer and excess antibody was removed by repeated washing in the same buffer. Bound antibody was detected with goat anti-(rabbit IgG) coupled to peroxidase (Sigma) followed by staining with a solution of 5 mM H202 and 1.8 mM 4-chloro-1-naphthol in 50 mM Tris/HCl pH 7.5 containing 10% methanol.

Preparation of amphipathic and hydrophilic cytochrome b, from rat and mouse liver microsomes

The amphipathic and hydrophilic forms of rat liver cytochrome b, were prepared as described previously [12].

Mouse liver amphipathic cytochrome b, was isolated from microsomes by a modification of the procedure described in [12], whereby gel filtration on Superose S.12 was replaced by chromatography on a column (1.4X90 cm) of Sephadex G-75 (medium) in 20 mM Tris/HCl pH 8.0 containing 0.5 mM EDTA and 1 % (by mass) sodium deoxycho-

late [32]. Before use in experiments, the cytochrome b, was dialysed against 50 mM HepesNaOH and desalted on NAP- 10 columns equilibrated in the same buffer, The hydrophilic tryptic fragment was produced by digesting microsomal cytochrome b, (0.16 mg) with 4 pg bovine pancreatic trypsin (Sigma) on ice over 20 h followed by the addition of a stoi- chiometric amount of bovine lung aprotinin (Sigma). Analy- sis of the product by SDS/PAGE revealed one stained band with a molecular mass of 14200Da.

Purification of cytochrome b, reductase Unless stated otherwise, all procedures were camed out

at 4°C using precooled buffers. Protein solutions were concentrated by ultrafiltration on Sartorius membranes with a molecular mass cut-off of 10kDa. The purity of the reductase preparations was examined by SDSPAGE on 15% acrylamide gels followed by staining with Coomassie bril- liant blue R-250.

Purification of microsomal cytochrome b, reductase The method employed here is a modification of pre-

viously published procedures [19, 331. Mouse liver microsomes were isolated from about 30 g tissue and solubilised as previously described [12]. The clear solubilised protein extract was applied to a column (1.4X26 cm) of Q-Sepharose equilibrated in 20 mM Tris/HCl pH 8.0 containing 0.5 mM EDTA and 1% (by mass) Triton X-100. The column was washed with the same buffer until no more protein emerged and was then developed with a 500-ml linear gradient up to 0.5 M NaCl in the starting buffer. Cytochrome b, reductase activity eluted as a single peak at about 0.1 M NaC1. Peak fractions were pooled and dialysed for 15 h against 2 1 of the starting buffer mentioned above. The dialysed protein was applied at 0.2 ml/min to a 5-ml column of AMP-Sepharose, equilibrated in 20 mM Tris/HCl pH 8.0 containing 0.1 % Tri- ton X-100, washed with the equilibration buffer and eluted with the same buffer containing 0.2 mM NADH. The final reductase preparation exhibited one main protein band of molecular mass 33 kDa after SDSPAGE. The preparation described gave a yield of about 10% reductase activity with an apparent enrichment of about 40-fold over solubilised microsomal protein.

Purification of cytochrome b, reductase from high-speed supernatants in the absence of Triton X-100

20 livers from male Balb/c mice were homogenised with 8 strokes of a Potter-Elvehjem homogeniser into 9 vol. (mVg wet tissue) 50 mM HepesMaOH pH 7.4 containing 0.25 M sucrose. After removal of particulate material by centrifugation at 1OOOOXg (20 min) and 1OOOOOXg (60 min; this latter microsomal pellet was used in later experiments, see below), the supernatant was applied to a column (1.3X20 cm) of Q- Sepharose equilibrated in 10 mM Hepes/NaOH pH 7.4. Un- bound protein was washed out with the starting buffer and the column was developed with a 1.6-1 linear gradient of NaCl up to 0.5 M, collecting 8-ml fractions. Fractions from the single peak of reductase activity were pooled, concentrated 2.5-fold and dialysed overnight against 2 1 50 mM Hepes/NaOH pH 7.4 containing 0.25 mM EDTA. The dialysed material was applied to a 2-ml column of AMP-Seph- arose equilibrated in the latter buffer at a flow rate of 0.2 ml/ min and unbound material was washed out with the same

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buffer. The reductase was eluted from the column with 0.3 mh4 NADH dissolved in equilibration buffer, concentrated to about 1.5 ml and desalted on a NAP columns equilibrated in 10 mM HepesNaOH pH 7.4. The protein was sub- sequently applied to a 1-ml column of Sepharose covalently modified with rat microsomal cytochrome 6, at a flow rate of 0.1 ml/min [34]. On washing the column with 3 ml of the equilibration buffer, about 13 % of the activity leached from the column suggesting that the reductase was retarded rather than tightly bound. The remaining activity could be eluted in 6 ml of the equilibration buffer containing 1 M NaCl. The reductase was finally concentrated to about 0.5 ml and equilibrated in 50 mM HepesNaOH pH 7.4.

Purification of cytochrome b, reductase from high-speed supernatants in the presence of Triton X-100

The procedure employed here is essentially identical to the one described above, with the following modifications. Triton X-100 was added to the high-speed supernatant to give a final concentration of 1% (by mass). This detergent was included in order to eliminate the formation of protein aggregates with potentially anomalous chromatographic properties should an amphipathic form of the reductase be present. The buffers used in the chromatography on Q-Seph- arose also contained 1% (by mass) Triton X-100. Fractions containing reductase activity from this chromatographic step were pooled, dialysed without concentration and applied di- rectly to the AMP- Sepharose column, which was equilibrated in the same buffer described above, containing 0.3% (by mass) Triton X-100. Triton X-100 (0.1 %) was also added to the NADH-containing elution buffer. The eluate was concentrated to 2 ml and applied to and eluted from the cytochrome-b5-Sepharose column as outlined above in the absence of detergent. A certain fraction of activity (16%) did not bind to the column. All buffers in this latter step contained 0.05% Triton X-100. The final preparation was concentrated by ultrafiltration to about 1 ml and equilibrated in 0.1% Triton X-100.

Preparation of mouse liver microsomes for enzymatic tests

A microsomal pellet was produced as described above under the section titled: Purijication of cytochrome b, reduc- rase from high-speed supernatant in the absence of Triton X - 100. The pellet was washed free of soluble protein by resuspension in the original volume of homogenisation buffer followed by centrifugation at 1OOOOOXg for 1 h. The final pellet was resuspended in 50 mM Hepes pH 7.4 giving a protein concentration of about 14 mg/ml and stored at -20°C until use.

Fractionation of hydroxylase in high-speed supernatant of mouse liver on Q-Sepharose

High-speed supernatants of mouse liver were fractionated on Q-Sepharose as described above in the purification of cytochrome b, reductase from high-speed supernatants in the absence of detergents. Cytochrome b, reductase was detected using the femcyanide reduction test. The relatively low concentrations of cytochrome b5 in the fractions precluded the use of the cytochrome c reduction assay for its detection. The presence of cytochrome b, was therefore tested by the reconstitution of hydroxylase activity in high-speed superna-

tants depleted of cytochrome b,. Mouse liver supernatants lacking cytochrome b, were generated by adsorption of concentrated high-speed supernatants on to a column of Ciba- cron-blue-3GA-agarose (5 mg protein/ml blue agarose). Af- ter washing with 50mM HepesNaOH pH7.4, a fraction containing negligable hydroxylase activity was eluted with 1 M NaC1, dissolved in the equilibration buffer. The hydroxylase activity could be reconstituted specifically by addition of purified cytochrome b, or heat extract described previously [12] (results not shown). This material was concentrated, desalted, and 20 pg used together with 15 pl desalted fraction in a standard hydroxylase test described above.

The hydroxylase component was detected after desalting the fractions on NAP-5 columns (Pharmacia) followed by a standard CMP-Neu5 Ac hydroxylase test described above, performed in the presence of mouse liver microsomes (45 pg protein) solubilised in 0.5% (by mass) Triton X-100. The small background hydroxylase activity of the microsomes (generally about 8pmol . min-' mg protein-') was ac- counted for in all calculations. Each fraction was pooled and concentrated by ultrafiltration (10 kDa cut off) to give protein concentrations of 9, 2.6 and 4.2mg/ml for pools I, 11 and 111, respectively. These pools were used reconstitution experiments as described in Results.

Determination of the molecular mass of the monooxygenase component

The molecular mass of the terminal monoxygenase component enriched in pool I1 was determined by gel filtration on a Superose S.12 column (Pharmacia) calibrated with the following proteins : bovine serum albumin, 67 kDa; oval- bumin, 45 kDa; carbonic anhydrase, 29 kDa; cytochrome c, 12.4 kDa. The mobile phase was 50mM HepesNaOH pH7.4 containing 100 mM NaCl and the flow rate was 0.25 mumin. A 100-pl aliquot of the concentrated Q-Sephar- ose eluate, containing 1.4 mg protein, was applied to the column and fractions of 0.25 ml were collected. The hydroxylase activity was determined in the presence of Triton-X- 100-solubilised microsomes, using the same test as described for the fractionation on Q-Sepharose.

Homogenisation and fractionation of pig and bovine liver and pig submandibular glands

Pig submandibular glands, freshly obtained 1-2 h before processing, were minced and dispersed in 50 mM Hepes/ NaOH pH 7.4 (5 ml/g wet tissue) for 3 min in an Ultraturrax and further homogenised by four strokes of a Potter-Elveh- jem homogeniser. The resulting homogenate was centrifuged at lOOOXg for 15 min to sediment tissue debris, which was discarded. The supernatant was centrifuged at lOOOOXg for 30min giving rise to a pellet, which was discarded, and a supernatant, which was further centrifuged at lOOOOOXg for 1 h to yield a microsomal pellet and a high-speed supernatant. The microsomes were washed by resuspension in homogenisation buffer (5 mug wet starting material) and recentri- fugation at lOOOOOXg and were finally suspended in the same buffer (0.5 mllg original wet tissue). Pig and bovine liver were treated essentially as above, with the exception that the tissue was not minced and the homogenisation buffer contained 0.25 M sucrose.

1 2 3 4 5 6 7 1 2 3

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Fig. 1. Immunochemieal identification of cytochrome b , in intact high-speed supernatants of mouse liver. Cytochrome b,, isolated by immunoaffinity chromatography of mouse liver high-speed supernatant on anti-(cytochrome b,) - Sepharose, was resolved by SDSPAGE and blotted onto a nitrocellulose filter. The cytochrome b, bands were stained using peroxidase-conjugated anti-(rabbit IgG), as described in Materials and Methods. Lanes 1-3, immunoaffinity purified cytochrome b, from three different high-speed Supernatants ; lane 4, immunoaffinity fractionated heat extract; lane, 5 mouse liver amphipathic cytochrome b,; lane 6, rat liver hydrophilic cytochrome b,; lane 7, amphipathic cytochrome b, from rat liver.

Influence of microsomes, cytochrome b, and anti-(cytochrome b,) antibody on CMP-NeuSAc hydroxylase from pig submandibular gland

Using the standard assay, the effect of microsomes from pig submandibular gland, pig liver and mouse liver as well as of purified cytochrome b, from mouse liver was determined on the activity of the hydroxylase in high-speed supernatants of pig submandibular gland. In addition, the influence of anti-(rat cytochrome b,) on the hydroxylase activity in pig submandibular gland supernatants was also tested. The amounts of each component is given for the individual experiments in Results and Discussion.

RESULTS AND DISCUSSION Characterisation of cytosolic cytochrome b, Cytochrome b, in mouse liver supernatant

Cytochrome b, was enriched from whole high-speed supernatants by immunoaffinity chromatography on anti-(cytochrome b,) - Sepharose. After separation on SDSPAGE, the cytochrome b, was visualised by immunoblotting. One heavily stained band migrating at the same position as native microsomal cytochrome b, was visible (Fig. 1). A very faint band of staining coinciding with the trypsinised form was also observed at higher loadings (results not shown).

Heat-stable factor Cytochrome b, was enriched from heat supernatants of

mouse liver by immunoaffinity chromatography on anti-(cytochrome b,)-Sepharose as described in Materials and Methods. After separation by SDSPAGE followed by immunoblotting, it was observed that only one band of staining co-migrating with the trypsinised cytochrome b, could be visualised, clearly showing that only the low-molecular-mass form of cytochrome b, was present in heat supernatants (Fig. 1). A comparison of the staining density of the heat- stable factor (lane 4, Fig. 1) with that of the cytochrome b, isolated from whole supernatants (lanes 1-3, Fig. 1) revealed that the amphipathic form of cytochrome b, predomi- nates in high-speed supernatants of mouse liver.

Although amphipathic cytochrome b, is normally firmly bound to cellular membranes, in purified form monomers

- 20.1

- 14.2

Fig. 2. Gel electrophoresis of cytochrome b, reductase species isolated from mouse liver. Lane 1, cytochrome b, reductase from mouse liver microsomes ; lane 2, cytochrome b, reductase from high- speed supernatant isolated in the absence of detergent; lane 3 cytochrome b, reductase from high-speed supernatant isolated in the presence of Triton X-100. The numbers on the right are the molecular mass (in kDa) of standard proteins.

may associate to give stable octomeric aggregates of micelle- like structure at physiological ionic strength and in the absence of detergents [35]. Interestingly, it has been established that the cytochrome b, protein is translated on cytosolic ribosomes [36,37] and that the cytochrome in vitro inserts spon- taneously into cellular membranes [38], a process that may be preceded by an initial interaction with a cytochrome-b, - lipid complex [39]. The existence of a small transient pool of amphiphilic cytochrome b, in the cytosol is thus possible.

The results of this study also show that a small amount of the low-molecular-mass hydrophilic form of cytochrome b, is present in heat extracts of the high-speed supernatant, suggesting that the amphipathic form of cytochrome b, is co-precipitated with other proteins by the rather harsh heat treatment. The hydrophilic form may originate from erythrocytes, which are known to possess hydrophilic cytochrome b, or by proteolysis of microsome-bound amphipathic cytochrome b, [32].

Characterisation of cytosolic cytochrome b, reductase Molecular mass of the purified reductase

The purification of the reductase in the presence of Triton X-100 is summarised in Table 1. On a polyacrylamide gel, the final preparation exhibited one main band of staining with a molecular mass of about 33 kDa (Fig. 2). The stained band migrated the same distance as the amphipathic cytochrome b, reductase isolated from microsomes, suggesting that this form of the reductase is present in the supernatant.

In contrast, the reductase purified from a high-speed supernatant (Table 1) in the absence of Triton X-100 yielded a protein with a molecular mass of about 29 kDa (Fig. 2), some 4 kDa smaller than the form isolated in the presence of detergent. This suggests that the soluble, hydrophilic form of the reductase had been isolated, a conclusion that was confirmed by kinetic data (see below). A 30-kDa cytochrome b, reductase was also detected in mouse liver supernatants by Kawano et al. [46]. The soluble cytochrome b, reductase from mouse erythrocytes has a molecular mass of 30kDa [40], very close to the value reported here. The preferential enrichment of the hydrophilic reductase in the absence of detergent may be due to anomalous chromatographic behavi-

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Table 1. Purification of cytochrome b, reductase from mouse liver high-speed supernatant in the presence and absence of Triton x-loo. Fraction Total activity Specific activity

+ Triton - Triton + Triton - Triton

Supernatant Q-Sepharose eluate AMP- Sepharose eluate Cyt. b, -Sepharose eluate

pmol . min-'

813 600 0.64 0.32 498 180 1.16 2.95 328 139 88.2 122 210 62 913 177

pmol . min-' . mg protein-'

our of the incompletely solubilised, possibly aggregate-borne cytochrome 6, reductase, though no further peaks of reductase activity were eluted from the Q-Sepharose column employed in the isolation of this enzyme, even after washing with 1 M NaCl dissolved in the equilibration buffer.

Since cytochrome t7, reductase is translated on cytosolic ribosomes, in the same way as cytochrome b,, followed by a signal-recognition-particle-independent insertion into various cellular membranes [41], a transient amount of the enzyme might exist in the cytosol, possibly in the form of stable high- molecular-mass aggregates [ 19, 421.

The higher yield of the amphipathic reductase suggests that this is the main form of cytochrome b, reductase in supernatants of mouse liver. Contamination of the liver tissue with erythrocytes or proteolysis may account, at least in part, for the presence of the hydrophilic form.

Table 2. Activity of various purified forms of mouse liver cytochrome b, reductase in the cytochrome-b,-mediated cytochrome- e-reduction assay. The ferricyanide reductase activities employed in each experiment were 26 nmol . min-' with purified cytochrome b, reductase and 9nmol . min-l in the crude supernatant. Cyto- chrome c reductase activity was determined under the conditions outlined in Materials and Methods, with 8.5 nmol purified amphipathic cytochrome b, from mouse liver being added to produce the full activity.

Cytochrome b5 reductase type Cytochrome c reductasel ferricyanide reductase activity

Purified microsomal 1.92 Cytosolic purified

with Triton X-100 0.57 Cytosolic purified

without Triton X-100 0.006 Unfractionated supernatant 0.23

lnteraction of the purijied cytochrome b, reductases with cytochrome b, in a cytochrome c reduction assay

As a further test of the identity of the two cytochrome b, reductase species described above, the enhancement of electron transfer from the reductase to cytochrome c by amphipathic cytochrome b, was determined as described in Ma- terials and Methods. The ferricyanide reduction test was used to determine the cytochrome b, reductase turnover in isolation, while the cytochrome-b,-mediated cytochrome c reduction assay gives an impression of the rate of electron transfer between the reductase and cytochrome b,. In all experiments, similar amounts of reductase activity (about 26 nmol ferricyanide reducedfmin), were added in 5 pl to cytochrome c. The rate of cytochrome c reduction was determined after addition of 8.5 nmol native mouse liver microsomal cytochrome b, in 5 pl. The ratio of cytochrome c reductase/ferricyanide reductase activities was determined for the two forms of the reductase isolated from supernatants, for the amphipathic reductase purified from microsomes and for the reductase activity detected in whole supernatants.

The results in Table 2 show that the most efficient cytochrome-b,-mediated electron transfer occurs with the amphipathic, microsomal cytochrome b, reductase. The cytochrome c reductase turnover observed with the low-molecular-mass form of cytochrome b, reductase was some 300-fold lower. This result suggests that the hydrophobic domain has a considerable influence on the behaviour of the reductase in the cytochrome c reduction assay, in accordance with previous observations [42, 431. The cytochrome c reductase activity of the high-molecular-mass form of the cytochrome b, reductase, isolated from supernatants in the presence of Tri- ton X-100, was considerably greater than that of the low-

molecular-mass form, though it was not as efficient as the microsomal, amphipathic reductase. The reason for this

. discrepancy is not known, though modifications of the hydrophobic domain either in vivo or in the course of the purification may be responsible.

The cytochrome-b,-dependent stimulation of cytochrome c reductase activity of high-speed supernatants lay between that of the two purified supernatant cytochrome b, reductase forms (Table 2). This may be a result of the mixture of the two cytochrome b, reductase forms in the supernatant.

Interaction of the cytosolic cytochrome 6, electron transport system with CMP-Neu5 Ac hydroxylase

The above results demonstrate that the amphipathic forms of cytochrome b, and cytochrome b, reductase make up a considerable proportion of these proteins in high-speed supernatants of mouse liver, and as such may have a bearing on the turnover of CMP-Neu5 Ac hydroxylase system in vivo. One of the unusual and, as yet, unexplained characteristics of this soluble enzyme system is its activation by non-ionic detergents [12]. In view of the amphipathic nature of the cytochrome b, and the reductase in the supernatant, the presence of detergents may influence the dispersion of these electron carriers, so affecting their interaction with each other and the hydroxylase. Since previous experiments [12] indicate that the level of cytochrome b, in supernatants may be severely limiting the turnover of the hydroxylase, any factors rendering cytochrome 6, more accessible to the postulated hydroxylase component would enhance its activity. This was investigated in the following experiment.

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l o o 7 300

0 0.0 0.5 1.0 1.5 2.0 2.5

[Cytochromm ba] (BY)

Fig. 3. Reconstitution of CMP-NeuSAc hydroxylase activity in cytochrome-b,-depleted supernatants with native and trypsinised cytochrome b, both in the presence and absence of Triton X-100. Cytochrome-b,-depleted mouse liver high-speed supernatant (0.25 mg protein) was incubated in the standard CMP-NeuSAc hydroxylase assay with various concentrations of amphipathic (V, 0) and hydrophilic (0, 0) mouse microsomal cytochrome b, in the presence (V, 0) and absence (0, 0) of 1.4% (by mass) Triton X- 100.

High-speed supernatant, depleted of cytochrome b, by passage over an anti-(cytochrome b,) immunoaffinity column, exhibited 3% of its starting CMP-Neu5 Ac hydroxylase activity. The hydroxylase in this supernatant was reconstituted with the amphipathic and hydrophilic forms of mouse liver cytochrome b, both in the presence and absence of 1.4% (by mass) Triton X-100. The results in Fig. 3 show that, in the absence of Triton X-100, the hydrophilic form of cytochrome b, was far more effective at restoring the hydroxylase activity than the amphipathic cytochrome b,. The presence of Triton X-100, however, only caused a significant increase in the hydroxylase activity supported by the amphipathic cytochrome b,. The same experiment performed with cytochrome-b,-depleted mouse liver supernatant and purified amphipathic and hydrophilic forms of cytochrome b, from rat liver yielded a similar result. Thus, with 0.25 pM hydrophilic rat cytochrome b,, 1.4% Triton X-100 activated the hydroxylase by 13% and at the same concentration of amphipathic cytochrome b5, an enhancement of 11 6% was elicited by Tri- ton X-100.

The influence of other detergents was also tested. Using cytochrome-b,-depleted supernatant and mouse liver amphipathic cytochrome b, at 0.25 pM, Nonidet P-40 (30mM), Chaps (10 mM), and octyl glucoside (50 mM), increased the activity of the hydroxylase by 55%, 52% and 33%, respectively. These detergents, however, had no effect on the hydroxylase activity supported by hydrophilic rat liver cytochrome b,.

These data clearly demonstrate that the hydrophilic form of cytochrome b, is more effective at reconstituting hydroxylase activity than the amphipathic form, suggesting that the former cytochrome b, type interacts more efficiently with either the reductase or the hydroxylase. Given the signifi- cance of the membrane binding domains for the interaction between cytochrome b, and the reductase (Table 2) [42], it is tempting to suggest that the interaction with the hydroxylase, rather than with the reductase, is facilitated. The fact that the level of cytochrome b, reductase activity in the high-speed supernatants is more than sufficient to support the relatively

250 -

0 20 40 SO 80 100 120

Uicrosomd Protein ( f ig )

Fig. 4. Effect of microsomes in the activity of CMP-Neu5 Ac hydroxylase. Mouse liver high-speed supernatant (200 pg protein) was incubated with varying amounts of mouse liver microsomes in the presence (0) and absence (0) of 1.4% (by mass) Triton X-100.

low CMP-Neu5 Ac hydroxylase turnover lends weight to this hypothesis. Using the ferricyanide reduction assay, a specific reductase activity of 0.5 pmol a min-' . mg protein-' was measured. This is considerably greater (about 8000-fold greater, assuming that the hydroxylase reaction is a two- electron process) than the activity of the hydroxylase which is normally in the region of 15-30 pmol . min-' . mg protein-'. The activating effect of detergents on the CMP- Neu5 Ac hydroxylase system in intact high-speed supernatants [ 121 and in cytochrome-b,-depleted supernatants reconstituted with amphiphilic cytochrome b, may therefore result from a dissolution of cytochrome b5 aggregates, rendering the catalytic sites more accessible to the hydroxylase.

These results also indicate that the level of cytochrome b, is limiting the turnover of CMP-Neu5Ac hydroxylase in high-speed supernatants of mouse liver. Whether this is the case in vivo could be called into question, since the concentration of the various components of this system in a supernatant is considerably lower than in the cytosol and the activity of the hydroxylase is sensitive to dilution [12]. Nevertheless, the interaction of the hydroxylase with other cellular pools of cytochrome b, must first be investigated.

Interaction of CMP-Neu5 Ac hydroxylase with the microsomal cytochrome b, electron transport system

Since the cytosolic cytochrome b, system makes up only a small proportion of the total cellular cytochrome b, and its reductase, the possibility that the turnover of the hydroxylase could be supported by the microsome-associated cytochrome b, system must be considered. The influence of mouse liver microsomes on the activity of the hydroxylase in high-speed supernatants was therefore determined both in the presence and absence of Triton X-100.

The results shown in Fig. 4 clearly demonstrate that the hydroxylase is not activated by the microsome-associated cytochrome b, system. The potential rate of electron delivery from microsome-bound cytochrome b, measured with the cytochrome c reduction assay is 1.6 pmol . mit-' . mg protein-', while that of the supernatant is 0.03 pmol . min-' . mg protein-' (for comparison, the cytochrome b, reductase activity in microsomes measured by the femcyanide test is 10 pmol . mini . mg protein-'). Thus, in the experiment employing

1008

1.5 7 - E 0

1.0 v

0 u C

; 0.5 o

4 n

0.0

0 200 400 600 800

Elutton Volume (rnl)

Fig. 5. Resolution of cytochrome b, reductase, CMP-NeuSAc hydroxylase and cytochrome b, from high-speed supernatants by chromatography on Q-Sepharose. High-speed supernatant of mouse liver, containing 1436 mg protein was applied to a column (2.4X22 cm) of Q-Sepharose. After washing out unbound material, the column was developed with a 1.3 -1 gradient up to 0.5 M NaCl; 8-ml fractions were collected. The reductase was detected using the ferricyanide (FeCy) reduction assay and cytochrome b, was tested for by reconstitution of CMP-Neu5 Ac hydroxylase activity in cytochrome-&-depleted supernatants. Cytochrome b, is expressed/volume of reconstituted hydroxylase activity. The activity of the CMP- Neu5 Ac hydroxylase component was assayed after reconstitution with Triton-X-100-solubilised mouse liver microsomes.

the highest concentration of microsomal protein, a 27-fold excess of cytochrome c reductase activity had been added to the supernatant. In the presence of Triton X-100, however, the turnover of the hydroxylase was stimulated by almost 10- fold on addition of microsomes. This result suggests that the cytochrome b, system of intact microsomes is accessible to a small macromolecular acceptor, such as cytochrome c, but is not available to the hydroxylase in the supernatant. Since cytochrome b, and the reductase are orientated with their catalytic domains directed into the solvent (i.e. originally on the cytoplasmic side of the endoplasmic reticulum) [23 -261 and cytochrome b, has lateral freedom of movement within the microsomal membranes [44], there may be some sort of hinderance to the approach of the hydroxylase, possibly as a result of the size or charge of the hydroxylase and the limita- tions imposed by the membrane and other microsomal proteins. It is tempting to draw a parallel with the behaviour of the hydroxylase towards the amphipathic cytochrome b, in the presence and absence of detergent described in the previous section.

In this connection, it is worth noting that virtually all cytochrome-b,-dependent enzymes so far described, for ex-

Table 3. Reconstitution of CMP-NeuSAc hydroxylase activity with various fractions from chromatography of mouse liver supernatant on Q-Separose as well as with purified cytochrome b, and cytochrome b, reductase. 10-pl portions of the following pools from the fractionation of mouse liver high-speed supernatant on Q- Sepharose (see Materials and Methods and Fig. 6) were combined as indicated: peak I, cytochrome b, reductase (90 pg); peak 11, hydroxylase (20 pg) and peak 111, cytochrome b, (42 pg). The masses represent the amount of protein added to each incubation.

Fraction Activity/assay

pmol . min-' Q-Sepharose peak I 0 Q-Sepharose peak I1 0 Q-Sepharose peak 111 0.02 Peaks I and I1 0.16 Peaks I and 111 1.1 Peaks 11 and I11 0.13 Peaks I, I1 and I11 7.95

ample stearoyl-CoA desaturase, d7-sterol desaturase and cytochrome P450 [17, 451, are firmly associated with micro- soma1 membranes. Their effective functioning is heavily dependent on the association of the enzyme and cytochrome b, with the membrane via hydrophobic membrane anchor domains. This presumably ensures that the interacting surfaces of cytochrome b, and the acceptor enzymes are brought into a catalytically productive orientation. This is evidently not the case for the hydroxylase described here. CMP-Neu5Ac hydroxylase is therefore an unusual cytochrome-b,-dependent enzyme, since it is not membrane-bound and does not function effectively with the membrane-associated cytochrome b, electron transport system.

Separation and reconstitution of the CMP-Neu5 Ac hydroxylase enzyme system in mouse liver supernatants

As discussed above and in previous publications [12, 131, the current hypothesis for the functioning of CMP-Neu5 Ac hydroxylase is a system composed of cytochrome b, reductase, cytochrome b, and a postulated hydroxylase component, or terminal electron acceptor, which is responsible for the binding of CMP-Neu5 Ac and oxygen activation. In previous sections of this report, the cytochrome b, reductase and cytochrome b, species that may be involved in the CMP-Neu5Ac hydroxylase reaction in vivo have been characterised. How- ever, in order to confirm the present model, the existence of the hydroxylase component must be more unequivocally demonstrated. Adsorption of mouse liver high-speed supernatant onto Q-Sepharose followed by elution with a gradient of NaCl permitted almost complete separation of all three postulated components. Cytochrome b, reductase and cytochrome b, were well separated (Fig. 5 ) at 0.15 M and 0.45 M NaCl, respectively. None of the fractions exhibited any hydroxylase activity when assayed alone. However, when tested in the presence of solubilised microsomes (see Materials and Methods), a single peak of CMP-Neu5 Ac hydroxylase activity could be observed, eluting at about 0.3 M NaC1.

The peaks of cytochrome b, reductase, hydroxylase and cytochrome b, (termed peaks I, I1 and 111, respectively) were pooled separately and reconstitution experiments performed. From the results presented in Table 3, it is clear that the greatest hydroxylase activity is obtained when all three fractions are mixed. The small amounts of activity detected on

1009

Table 4. Reconstitution of hydroxylase activity with peak I1 and solubilised microsomes or purified microsomal cytochrome b, reductase and cytochrome b,. Incubations were performed using the standard hydroxylase assay with 140 pg hydroxylase (peak I1 from Q-Sepharose chromatography) in the presence of 0.75% Triton X-100, 2 pM native cytochrome b,, 8.6 nmol ferricyanide min-' . cytochrome b, reductase and 84 pg microsomal protein.

Combination Activityhcubation

pmol . min-' Peak I1 0 Peak I1 and cyt. b, Peak I1 and reductase 0 Peak 11, cyt. b,

Peak I1 and solubilised

1 .o

and reductase 12.8

microsomes 15.3

mixing fractions I and I11 presumably arise from overlap with the hydroxylase under peak 11.

Similarly, reconstituting the same hydroxylase pool from a different preparation with purified amphiphathic forms of cytochrome b, reductase and cytochrome b, isolated from microsomes revealed that the hydroxylase was most active in the mixture of all three components, though there was evidence for contamination with the reductase (Table 4). Since similar activities were observed with solubilised microsomes as well as with purified cytochrome b, and reductase, these latter two proteins are the only microsomal components supporting the activity of the hydroxylase.

Determination of the molecular mass of the monooxygenase component

The molecular mass of the monooxygenase enriched from mouse liver supernatant on Q-Sepharose was determined using a calibrated Superose S.12 gel filtration column. The hydroxylase eluted as a single peak of activity in a volume corresponding to a molecular mass of 56 kDa. This cor- responds well with the molecular mass reported for a component of this system by Kawano et al. [46].

Interaction of CMP-Neu5 Ac hydroxylase from pig submandibular glands with cytochrome b,

Since the involvement of cytochrome b, has only been demonstrated for CMP-Neu5 Ac hydroxylase from mouse liver, its possible participation in the catalytic cycle of CMP- Neu5 Ac hydroxylase from pig submandibular glands and bovine liver was investigated.

Anti-(rat cytochrome b,) antiserum was a potent inhibitor CMP-Neu5 Ac hydroxylase in a high-speed supernatant of pig submandibular gland (Fig. 6). The same antibody inhibited the hydroxylase in a high-speed supernatant of bovine liver (430 pg protein) where 42 pg anti-(cytochrome b,) (0.2 nmol/mg IgG) inhibited by 74%. The requirement of the pig salivary gland enzyme for this electron carrier was further corroborated by the fact that the addition of amphipathk cytochrome b, purified from mouse liver microsomes to a concentration of 0.9 yM gave rise to a dramatic increase in the specific activity of the hydroxylase from 1.1 pmol - mit-' . mg protein-' to 105 pmol . min-' mg protein-'. As with mouse liver enzyme, this result also shows that the level of

O . A I \

O.' I n v

0.0 0 20 40 60 80 100 120

Antibody (pg)

Fig. 6. Inhibition of CMP-NeuSAc hydroxylase from pig submandibular glands with anti-(cytochrome b,). High-speed supernatant from pig submandibular glands (108 pg protein) was preincu- bated with varying amounts of anti-(rat cytochrome b,) and tested for hydroxylase activity as described in Materials and Methods.

Table 5. Influence of microsomes and cytochrome b, on the activity of CMP-Neu5Ac hydroxylase in high-speed supernatant of pig submandibular glands. Hydroxylase activity was determined using the standard assay described in Materials and Methods. All incubations had a final volume of 30 pl. Assays contained 65 pg supernatant protein and 20 pg microsomal protein was added where indicated. In the case of purified cytochrome b,, 32 pmol was added. The final concentration of Triton X-100 was 1.3%.

Addition Specific hydroxylase activity

None Triton X-100 Pig liver microsomes Pig liver microsomes

+ Triton X- 100 Pig submandibular gland

microsomes Pig submandibular gland

microsomes + Triton X-100 Mouse liver microsomes Mouse liver microsomes

Mouse liver cytochrome b, + Triton X-100

pmol . min-' . mg supernatant protein-'

1.1 5.1 7.4

151

1.2

11.9 16.4

177 105

cytochrome b, in the supernatant is limiting the turnover of the hydroxylase.

The CMP-Neu5 Ac hydroxylase from pig salivary gland supernatants had several properties in common with the mouse liver enzyme, as presented in Table 5. The addition of Triton X-100 to a concentration of 1.3% (by mass), for example, led to a fivefold stimulation of the hydroxylase turnover. Moreover, the hydroxylase from salivary glands could be activated by the addition of microsomes from several sources (Table 5). As with the mouse liver hydroxylase, optimal stimulation of activity by microsomes occurred in the presence of Triton X-100. The relative stimulation of hydroxylase activity in the presence of Triton X-100 corres-

1010

ponded with the respective cytochrome c reductase activities of the various microsome preparations (pig liver, 1.2 pmol, pig submandibular gland 0.7 pmol and mouse liver 3 pmol cytochrome c reduced min-' . mg protein-'). Nevertheless, in contrast to the mouse system, microsomes could stimulate hydroxylase activity in the absence of this detergent.

The CMP-Neu5 Ac hydroxylase activity detected in SU- pernatants of this tissue was generally low (1.1 pmol . mi^' . mg protein-', compared with about 20pmol . min-' . mg protein-' in mouse liver supernatants). Moreover, the activation of the hydroxylase by microsomes from the same tissue was modest in comparison with the activation by microsomes from other tissues, again suggesting that the membrane- bound cytochrome b, system does not support the activity of CMP-Neu5 Ac hydroxylase to a large extent. The possibility that the measured activity was artificially low due to the conditions prevailing in vitro must nevertheless be considered.

GENERAL CONCLUSIONS The results reported here clearly demonstrate that mam-

malian CMP-Neu5 Ac hydroxylase is a soluble enzyme system consisting of cytochrome b, reductase, cytochrome b, and a separate 56-kDa monooxygenase component. Cyto- chrome b, reductase and cytochrome b, deliver reducing equivalents from NADH to this latter component which is postulated to bind CMP-Neu5 Ac and reductively activate oxygen. In contrast to the majority of cytochrome-b,-dependent enzymes, CMP-Neu5 Ac hydroxylase is a soluble enzyme that accepts electrons at a very low rate from the intact microsomal cytochrome 6, electron transport chain and ap- pears to be more dependent on the more disperse cytosolic, amphipathic cytochrome b, system, though differences between the mouse and pig enzymes were seen. Since amphipathic cytochrome b,, which exists as an aggregate in an aqueous environment, is the most abundant species in high- speed supernatants, it is likely to be the main participant in electron transfer to the hydroxylase. However, the fact that the monomeric hydrophilic form of cytochrome b, is the most effective electron donor suggests that the hydroxylase is dependent on the accessibility of the acceptor docking sur- face of cytochrome b,.

The results in this report indicate that the concentration of cytochrome b, in supernatants of at least two tissues is limiting the activity of the hydroxylase. Although turnover could be stimulated to a degree by the addition of microsomes, the full activity of the hydroxylase could only be real- ised under artificial conditions, i.e. after addition of detergent-solubilised microsomes or purified cytochrome b, from other organs. One could, therefore, speculate that the conditions in the cell, for example higher protein concentrations, more suitable substrate and cofactor levels as well as compartmentalisation, are such that CMP-Neu5 Ac hydroxylase can function optimally.

Since cytochrome b, and its reductase are involved in a large number of cellular processes, the regulation of Neu5Gc biosynthesis presumably occurs via alterations in the level of the hydroxylase component. Measurement of changes in hydroxylase activity during development, for example, would therefore be best carried out in the presence of an excess of cytochrome b, and its reductase. From a method- ological point of view, these results show that solubilised microsomes are a suitable crude preparation of both cytochrome b, and its reductase for assaying the hydroxylase in tissue extracts and in the course of its purification.

The authors thank the Deutsche Forschungsgemeinscha~r (pro- jects Scha 202115-2 and Sh 34/1-11 and Fonds der chernischen In- dustrie for their financial support.

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cmp-n-acetylneuraminic acid hydroxylase from mouse liver and pig submandibular glands : interaction...

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