The association of the copper derived from ceruloplasmin with cytocuprein

'Biochimica et Biophysica Acta, 328 (1973) 351-358 (D Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands

BBA

36585

T H E ASSOCIATION OF T H E C O P P E R D E R I V E D FROM CERULOPLASMIN W I T H CYTOCUPREIN

N. MARCEAU AND N. ASPIN

The Departments of Medical Biophysics and Paediatrics, University of Toronto and the Research Institute, The Hospital for Sick Children, Toronto (Canada) (Received March I9th, 1973) (Revised manuscript received August 28th, 1973)

SUMMARY

The nature of the cytoplasmic protein which receives copper from ceruloplasmin in the plasma has been examined. Ceruloplasmin bound 67Cu was injected intravenously into rats and the soluble fraction from liver and spleen homogenates was fractionated by gel filtration, and subsequently on preparative electrophoresis. 2-3 days after injection, the 67Cu activity appeared in a protein fraction of about 35 000 mol. wt which exhibited superoxide dismutase activity, a property of eytocuprein. The injection into rats of doubly E3Hlvaline - or IaHlleucine-labelled~e7Culceruloplasmin demonstrated that the "7Cu appeared in the cytocuprein fraction of the cytoplasm while the 3H lable was associated with the amino acid fraction. This suggests that this intracellular copper protein is cytocuprein and that the copper from ceruloplasmin is not detected in the cell associated with a significant portion of the ceruloplasmin molecule.

INTRODUCTION

Earlier studies in rats have shown that the copper derived from ceruloplasmin in plasma appears in a cytoplasmic protein fraction which has a tool. wt of 3 ° 0004 ° 000 and in the cytochrome C oxidase associated with mitochondria 1. Cytocuprein, a copper protein isolated from human liver, brain and red blood cells z,3 and which exhibits superoxide dismutase activity 4 has a tool. wt of 32 000-33 000. The possibility that the previously described cytoplasmic copper protein 1 is cytocuprein was first investigated. Rats were injected intravenously with E67Cu~ceruloplasmin and at later times the soluble fractions of the liver and spleen homogenates were subjected to gel filtration and polyacrylamide electrophoresis. The different fractions were assayed for 6~Cu and superoxide dismutase activity. Second, ceruloplasmin doubly labelled with 67Cu and I3H]valine or E3H~leucine was iniected and cytoplasmic protein fractions obtained by gel filtration were assayed for 67Cu and ZH activity within the cytoplasm.

352

N. MARCEAU, N. ASPIN

MATERIALS AND METHODS

Labelling of ceruloplasrnin In experiments to measure superoxide dismutase activity, ceruloplasmin was singly labelled with 67Cu as previously described 1. The ceruloplasmin was doubly labelled in vivo for the studies of the catabolism of the molecule. A 1.5-ml aliquot of an isotonic solution containing 5 ° #Ci 67CUC12 (ref. 5) plus I mCi [aHivaline or laH] leucine (NEN, 3o-5o Ci/mM; o-o2 mg in 5 ml o.oi M HC1) was injected into a tail vein of a donor rat. 8 h later the rat received a second injection of 1.5 mCi of the same labelled amino acid. After a further io h the rat was exsanguinated and approx. 4 ml of serum was separated from the packed cells.

Partial purification of [3H yeucine - or S3Hjvaline-labelled[67CuSeeruloplasmin The 4 ml of serum obtained from the donor rat was diluted i :5 with 0.02 M potassium pyrophosphate buffer at pH 7.5- This volume was applied to a column (15 cm × 0.5 cm), packed with 0.5 cc DEAE-Sephadex A-5o equilibrated in the above buffer. A blue band containing ceruloplasmin was retained at the top of the column. The column was washed with 5° m l 0.02 M pyrophosphate buffer and ceruloplasmin was eluted with 2.5 ml 0.25 M NaC1 in 0.08 M sodium pyrophosphate buffer at pH 7-5.

Soluble fractions Soluble fractions from rat liver and spleen homogenates were prepared 1 from Wistar rats maintained for IO weeks on a copper-deficient diet G.

A nalytical electrophoresis Tests for purity were performed by polyacrylamide electrophoresis 7. A 7% polyacrylamide gel was made in o.oi M Tris, 0.05 M glycine buffer, pH 8. 4. A 0.005 M Tris, 0.03 M glycine buffer, pH 8.3, was used in the chambers. Proteins were stained with amido black and the excess stain was washed out of the gel with a solution of methanol-acetic acid-water (5 :I :5, by vol.).

Preparative electrophoresis on polyacrylamide A transistorized version of Brownstone's apparatus s was built to obtain a protein preparation of high purity. A 7% polyacrylamide gel (4 cm × 9 cm) was prepared in the same buffering system as for analytical electrophoresis. The applied protein sample was contained in a 4-7 ml volume. The eluting buffer which was the same as that in the chamber was composed of 0.005 M Tris, 0.03 M glycine at pH 8.3. Electrophoresis was run for IO min at 250-275 volts, 60-80 mA, and the polarity was reversed for 15 s before collection. The volume of each fraction was 2.6 ml.

Gel filtration Fractionation of the proteins contained in the soluble fraction of the cell homogenates was achieved by gel filtration on Sephadex G-Ioo (ref. I), with 2.6-Inl fractions being collected. Both 67Cu and ~H activities were determined in each fraction.

67Cu and 3tl counting The 67Cu activity in the eluted fractions was first measured in an automatic

353

C u FROM CERULOPLASMIN

y-well counter. The same fractions were then prepared for determination of 3H activity by mixing 2 ml of their volume with io ml aquasol (NEN) in 2o-ml counting vials. The activity was measured in a 3 channel liquid scintillation counter after the ~TCu activity had decayed.

Superoxide dismutase assay The method of McCord and Fridovich 4 was used with minor modifications. The tetrabutylammonium superoxide produced in the cathode chamber of the electrolytic cell was used to reduce cytochrome c (ref. 4)- The rate of reduction was measured in a Zeiss spectrophotometer equipped with a magnetic stirrer. The superoxide solution was infused into the cuvette at a constant flow of 13 #l/rain. The reduction of cytochrome c with the addition of the superoxide solution was measured at 3o-s intervals by the change in absorbance at 550 nm. The presence of superoxide dismutase activity was assumed whenever the rate of inhibition of the cytochrome c was reduced by more than 500/0 . EXPE RIMENT AL RESULTS

A normal rat was injected intravenously with ceruloplasmin bound STCu; 56 h later the rat was killed, its liver perfused and the soluble fraction separated. 6 ml of the soluble fraction were fractionated on a Sephadex G-Ioo column. The results in Fig. I show that as before the 67Cu activity eluted as a sinle peak 1. The fractions were assayed for superoxide dismutase activity b y adding IOO #1 of each fraction to the cuvette of the spectrophotometer before starting the infusion of the superoxide solution. Fig. i shows that superoxide dismutase activity was detected only in those fractions which contained s~Cu activity. The fractions numbered 63-65 were pooled, dialysed against 0.0o5 M Tris, 0.03 M glycine buffer, p H 8.3, and processed by prepa•

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354

N. MARCEAU, N. ASPIN

rative electrophoresis. The results plotted in Fig. 2 show the svCu radioactivity eluted as a single peak (solid line). A 2oo/~1 aliquot from each collected fraction was assayed for superoxide dismutase activity. It was found to be positive only in the region of the peak as indicated in Fig. 2. The same results were found when spleen soluble fraction was assayed for superoxide dismutase activity. These findings suggest very strongly that the 30 ooo-4o ooo mol. wt protein containing 67Cu derived from ceruloplasmin and found in the soluble traction of rat liver and spleen is cytocuprein. 10

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Catabolism of ceruloplasmin E3H]leucine - or [3Hlvaline-labelledE6vCulceruloplasmin was partially purified on DEAE-Sephadex. The purity of this material was tested by analytical electrophoresis. Fig. 3 shows at least 5 or 6 plasma proteins in the gel after staining with amido black. The leading band corresponds to ceruloplasmin. Further purification by preparative electrophoresis was considered. Fig. 4 shows a typical elution pattern of the ~TCu and 3H activity obtained after electrophoresis of o.o5-o.2 ml partially purified EaHlleucine-labelledE67Culceruloplasmin mixed with 4 ml of buffer. The 67Cu activity eluted as a single peak whereas the 3H activity eluted in two peaks in this region. The first peak corresponds to ceruloplasmin, the second one probably represents a fl-globulin. However, the overall yield of labelled protein was relatively low and the procedure involves treatments which can disrupt the labile properties of ceruloplasmin 9-al. For these reasons, preparative electrophoresis was used only to obtain the ratio of 3H to n~Cu activity in ceruloplasmin.

C u FROM CERULOPLASMIN

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Fig. 3- Polyacrylarnide gel stained for proteins with amido black after analytical electrophoresis of partially purified EaH~leucine-labelled[e7Cu~ceruloplasmin. The leading band corresponds to ceruloplasmin. Two rats were injected by tail vein with 2 ml of partially purified 67Cu and 3Hlabelled ceruloplasmin. One sample of ceruloplasmin was labelled with I3Hlvaline, the other with [3Hlleucine. Microsamples of blood were taken from the retro-orbital venous plexus 7 at IO min, 32 and 34 h after the injection. The animals were exsanguinated later and the plasma samples were assayed for 67Cu activity. Table I shows that the 67Cu activity disappears quite slowly from the plasma. These results compare well with those obtained for the disappearance of plasma 67Cu activity following an intravenous injection of whole plasma containing ceruloplasmin bound 67Cu (ref. 6). This suggests that the DEAE-Sephadex procedure used for partial purification of ceruloplasmin did not change significantly the properties of the molecule. The 3-4 ml of plasma, obtained when the rats were killed, were processed as follows: ceruloplasmin was partially purified on DEAE-Sephadex and 0.2 ml of the eluant were diluted with 4 ml of the Tris-glycine buffer and processed by preparative electrophoresis in order to measure the ratio of ~H to 67Cu activity. These results are also shown in Table I. In both animals, the ratio of activities in plasma ceruloplasmin at 57 or 7 6 h after injection were the same as those in the iniected ceruloplasmin samples. This would suggest that ceruloplasmin breakdown occurs concomitantly with the transfer of its copper into the tissues.

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The livers of rats similarly labelled with [aH]leucine-labelled[67Cu]ceruloplas min were perfused and homogenized. The soluble fraction, obtained by ultracentrifugation, was fractionated on a Sephadex G-Ioo column and a typical result shown in Fig. 5- The nTCu activity appears as a single peak, which is very distant from the 3H peak. The latter elutes at the same position as that obtained when [3HJleucine was passed through the same Sephadex column as a marker. Similar results were obtained in rats following the injection of [3HJvaline-labelledE67Cu]ceruloplasmin. When the TABLE THE

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357

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soluble fraction of spleen cells were assayed in this way, essentially the same results were obtained. These findings indicate that ceruloplasmin-copper does not remain in the cell attached to a significant part of the molecule. DISCUSSION

These studies present additional information about a cytoplasmic protein which receives copper from ceruloplasmin in the plasma 1 2-3 days following its injection. This particular protein has a mol. wt of 30 0o0-40 000 and exhibits superoxide dismutase activity, both properties of cytocuprein. Sternlieb et al. 12 have studied the disappearance of the oxidase activity of ceruloplasmin from human plasma. Their results show that the copper label disappears from the plasma at the same rate as the enzyme activity. From this they have inferred that the copper remains bound to the ceruloplasmin molecule until the protein breaks down. Holtzman and Gaumnitz~3, ~4 measured the disappearance from rat plasma of [~4Clceruloplasmin by immune precipitation techniques. Their data show that the molecule disappears from the plasma with the same half-time as the radioactivity from 67Cu-labelled ceruloplasmin ~. The constant ratios of ~TCu and 3H activities found in the plasma of rats injected with doubly labelled ceruloplasmin lend further support to the hypothesis that the eeruloplasmin molecule is catabolized at the time that its copper is transferred into the cells. The present studies illustrate the distribution within the cytoplasm of the cell of the 67Cu and [3Hlleucine or [~H]valine derived from doubly labelled ceruloplasmin in plasma. These data show that the 3H appears exclusively with the amino acid markers in the soluble fraction of the liver and spleen, thus showing that the copper does not remain in the cell attached to a significant portion of the ceruloplasmin molecule.

35 8

N. MARCEAU, N. ASPIN

ACKNOWLEDGEMENTS T h e a u t h o r s w i s h t o t h a n k D r s A. S a s s - K o r t s a k a n d B. S a r k a r for t h e i r c o n s t a n t a d v i c e a n d M r K. G. S t e i m a n n for t e c h n i c a l a s s i s t a n c e . T h i s w o r k w a s s u p p o r t e d b y t h e J o h n A. H a r t f o r d F o u n d a t i o n I n c .

REFERENCES I 2 3 4 5 6 7 8 9 IO ii 12 13 14

Marceau, N. and Aspin, N. (1973) Biochim. Biophys. Acta 328, 338-35 ° Hartz, J. w . and Deutsch, H. F. (1969) J. Biol. Chem. 244, 4565-4572 Carrico, R. J. and Deutsch, H. F. (1969) J. Biol. Chem. 244, 6087-6093 McCord, J. M. and Fridovich, I. (1969) J. Biol. Chem. 244, 6049-6055 Marceau, N., Kruck, T. P. A., McConnell, D. B and Aspin, N (197 o) Int. J. Appl. Radioacl. Isot. 21, 667-669 Marceau, N. and Aspin, N. (1972) Am. J. Physiol. 222, lO6-11o Marceau, N., Aspin, N. and Sass-Kortsak, A. (197 o) Am. J. Physiol. 218, 377-383 Brownstone, A. D. (1969) Anal. Biochem. 27, 25-46 Kasper, C. B. and Deutsch, H. F. (1963) J. Biol. Chem. 238, 2325-2337 Kasper, C. B., Deutsch, H. F. and ]3einert, H. (1963) J. Biol. Chem. 238, 2338 2342 Kasper, C. B. and Deutsch, H. F. (1963) J. Biol. Chem. 238, 2343-2350 Sternlieb, I., Morell, A. G., Tucker, W. D,. Grene, M. W. and Scheinberg, I. H. (1961) J. Clin. Invest. 4 o, 1834-184o Holtzman, N. A. ad Gaumnitz, B. M. (197 o) J. Biol. Chem. 245, 2350-2353 Holtzman. N. A. and Gaumnitz, ]3. M. (197 o) J. Biol. Chem. 245, 2354-2358