THE ATP-DEPENDENT CONCENTRATION OF CALCIUM BY A GOLGI APPARATUS-RICH FRACTION ISOLATED FROM RAT LIVER

Transcription

1 J. Cell Si. 30, (1978) Printed in Great Britain Company of Biologists Limited igys THE ATP-DEPENDENT CONCENTRATION OF CALCIUM BY A GOLGI APPARATUS-RICH FRACTION ISOLATED FROM RAT LIVER STUART HODSON Unit of Eletron Mirosopy, Welsh National Shool of Mediine, Heath Park, Cardiff, CF 4 4XN, U.K. SUMMARY Frations rih in Golgi apparatus were isolated from female rat liver and inubated with different onentrations of MgATP and alium. Calium was sequestered by the fration in the presene but not in the absene of ATP. The kinetis of the uptake proess showed an exponential type aumulation to a maximum value whih ould be explained by a 'pumpleak' mehanism where V max was estimated at 6-7 nmol alium/mg protein/min. Apparent K m for ATP was 1 mm and for alium was 85 fim. The uptake is not inhibited in 5 mm azide, nor is it enhaned in 5 mm oxalate, suggesting that the sequestration is not aused by ontamination from mitohondria or mirosomes. Parallel experiments on a fration rih in plasma membrane show at least a -fold redution of ativity over the Golgi apparatus-rih fration, whih is interpreted as evidene that plasma membrane ontamination in the Golgi apparatus-rih fration is not primarily responsible for the alium sequestration ativity. Morphometri analysis showed that about 85 % of the reognizable membrane profiles in the fration was assoiated with elements of the Golgi apparatus. Further evidene is given whih suggests that at least 90 % of the fration was uniform in its ability to sequester alium. In preliminary experiments it was not found possible to separate galatosyltransferase ativity (a speifi enzyme assoiated with the Golgi apparatus) from the alium-sequestration mehanism. The stoihiometry of the hydrolysis of ATP assoiated with the uptake of alium is the same as that shown by other isolated membrane systems. INTRODUCTION Calium pump ativity has been loalized in mitohondria (Lehninger, Carafoli & Rossi, 1967) and mirosomes (Moore, Chen, Knapp & Landon, 1975) isolated from rat liver ells. Additionally, a alium pump ativity may be inferred to be present in the plasma membrane of rat liver ells (van Rossum, Smith & Cooper, 1974). The polarity in all these systems is suh that alium is pumped out of the ytosol into the extraellular spaes or into the lumen of the subellular fration. Moore et al. (1975) proposed that ATP-dependent alium sequestration systems analogous to the well studied saroplasmi retiulum of skeletal musle (Boland, Martonosi & Tillak, 1974) may be widespread. The present work supports this onept by showing that a Golgi apparatus-rih fration isolated from rat liver ells also has an ATP-dependent mehanism for onentrating alium.

2 n8 S. Hodson METHODS Frationation The Golgi apparatus-rih fration was isolated from female Wistar rats, fed ad lib., by a method similar to that desribed by Hodson & Brenhley (1976). All surose solutions were prepared in M K 2 HPO 4 /o-oio5 M itri aid, ph 7-0. The rat was killed by a blow to the nek and 7 g liver were quikly mined with a razor blade and transferred to 15 ml ie-old buffered 14-6 % (w/w) surose. After 20 min of gentle agitation, the solution was deanted and replaed with -5 ml ie-old buffered 14-6% (w/w) surose. Tissue was homogenized with a Polytron PT 20 for 90 s at tap o-6. The homogenate was made up to 24 ml with 146 % (w/w) surose and then entrifuged for min at 0-4 C and 600 g; 6 ml of supernatant were then applied to a step gradient onsisting of 4 ml 237% (w/w) surose and ml 30-8 % (w/w) surose and entrifuged in the 3 x 25-ml swing-out rotor of an MSE Superspeed 50 for 2 h at 0-4 C and g (r ou = 94 mm). The Golgi apparatus-rih fration olleted at the 23-7/30-8% interfae (Hodson & Brenhley, 1976). Sometimes the fration was resuspended in 14-6% (w/w) surose whih was then reapplied to the step gradient and the separation was repeated. Calium sequestration ativity Calium uptake into the Golgi apparatus-rih fration was usually measured at 37 C, but some experiments were onduted at 25 or o C. The ie-old fration was aspirated into a volume of buffer at 37 C in a shaking waterbath usually with no preinubation so that the final surose onentration was 14-6 % (w/w). The final medium normally ontained 6 mm MgCl 2) 40 fim CaCl 2 inluding 0-12 fid 46 CaCl 2 /ml and 1-5 mm ATP (sodium salt). At different times all these onentrations were varied. After the inubation period was ompleted, 500 fi\ of suspension ontaining fig protein were filtered through a o-45-/tm membrane filter (Millipore Corp.). The filters were prepared with a wash of 500 fi\ 14-6 % (w/w) surose. After sample filtration, the filters were washed with 3 x 1 ml buffered surose. Total filtration time inluding the washings was about 20 s. Retained radioativity was determined by air drying the filters and then ounting them under 4-5 ml sintillation fluid, onsisting of 5 g PPO + 02 g POPOP in 1 1. toluene, in a sintillation ounter. Calium ativity was determined by omparison with 20-fd aliquots of the inubation medium dried diretly on to filters. Stoihiometry The Golgi apparatus-rih fration was reated with medium for various times and then ATP hydrolysis was measured by the 'haroal absorption' method (Wood, 1971). The final reation mixture (500 /tl) of buffered 14-6 % (w/w) surose, inluding fig protein, ontained 40 fim CaCl 2) 6 mm MgCl 2 and I-SHIMATP inluding 25 nci [y- 32 P]-ATP, was inubated at 37 C and then quenhed with 1 ml ie-old 7-5 % (w/v) trihloroaeti aid inluding 5 % (w/v) haroal (Norit SX 30). The solution was entrifuged for 2 min at 8000 g (r av = 44 mm) and the supernatant, inluding the liberated 32 P-phosphate, was aspirated and reentrifuged. One millilitre of supernatant was immersed in 9-0 ml of sintillation fluid, onsisting of 5 g PPO+ 0-2 g POPOP and 500 ml of Triton X-0 to 1 1. toluene. The following ontrol experiments were always performed with the main experiment: reating the medium with a boiled Golgi apparatus-rih fration, omitting the alium from the medium and substituting o-i mm EGTA (ethanedioxy bis (ethylamine) tetra-aeti aid), omitting the [y- 32 P]-ATP and substituting 25 nci 32 P-phosphate. Eletron mirosopy Suspensions of the Golgi apparatus-rih frations, either fresh from the entrifuge after dilution to 14-6 % (w/w) surose by the addition of ie-old buffer, or after reating with alium and ATP for 20 min at 37 C, were fixed in 20 vol. of 2-5 % glutaraldehyde in M potassium phosphate, ph 7-0, and proessed for transmission eletron mirosopy as desribed by Hodson & Brenhley (1976).

3 Golgi apparatus-rih fration homogeneity Calium pump of the Golgi apparatus 119 Investigations were made into whether the alium sequestration ativity of the fration was aused by ontaminating partiles suh as mirosomes, mitohondria or vesiles originating from the plasma membrane rather than elements of the Golgi apparatus, here taken to onsist of the staked isternae, the vesiles of diameter fim filled with very low density lipoproteins and the 50-nm diameter vesiles (Hodson & Brenhley, 1976). Morphometri analyses of setions ut through the pelleted frations were made by the method of Weibel, Kistler & Shrle (1966). Mirosomes isolated from rat liver exhibit the ' oxalate effet' of trapping the sequestered alium (Moore et al. 1975). Experiments were made in whih 5 mm ammonium oxalate was added to the inubation medium in order to see whether the Golgi apparatus-rih fration showed the ' oxalate effet' harateristi of mirosomes. In other experiments 5 mm sodium azide, whih ompletely inhibits the uptake of alium by mitohondria isolated from rat liver (Moore et al. 1975) was added to the inubation medium. Experiments on alium sequestration were repeated on a plasma membrane-rih fration isolated from rat liver (Hodson & Brenhley, 1976) whih has little or no galatosyltransferase ativity - a speifi marker enzyme for the Golgi apparatus in rat liver ells (Fleisher, Fleisher & Ozawa, 1969). If it were possible to separate the galatosyltransferase ativity from the alium-sequestration ativity in the Golgi apparatus-rih fration, this would suggest that the alium-sequestration ativity of the fration was a property of a ontaminating partile rather than the Golgi apparatus. The fration was repurified by resuspending in buffered 14-6 % (w/\v) surose and then rapplying it to the gradient for periods of 30, 60 or 90 min. Only a fration of the total applied protein olleted at the 23-7/30-8% interfae. Eah fration was tested for total protein (Lowry, Rosebrough, Farr & Randall, 1951), galatosyltransferase ativity (Freilih, Rihmond, Repui & Silbert, 1975) and alium-sequestration ativity. In a seond method, the Golgi apparatus-rih fration was reated with the inubation medium and then filtered through membrane filters (Millipore Corp.) of nominal pore sizes 0-3, 0-45, 0-65, o-8, 1-2 or 3 /»m, where different portions of the partiulate omponent were retained or passed through the filter. Total protein of the filter and filtrate were measured and ompared with the alium retained on the filter. RESULTS Calium was sequestered by the Golgi apparatus-rih fration in the presene of ATP but not in its absene (Fig. 1). The time ourse of uptake was not linear but levelled off towards a maximum value. This did not reflet a deterioration in the ativity of the preparation beause if the fration was inubated at 37 C in the medium exluding only the marker ^CaC^ for min and then the marker 45 CaCl 2 was added, uptake was as vigorous (Fig. 1). Clearly, alium is being released from the preparation as well as being taken up. The preparation maintains its ability to sequester alium for at least 20 min at 37 C, but after 30 min shows a signifiant deterioration. The presene of ATP at 37 C is essential not only for the uptake of alium but also to stabilize the preparation. If the preparation is preinubated at 37 C in the absene of ATP and marker ^CaClg and then inubated in the presene of ATP and marker 45 CaCl 2 for a fixed interval, the uptake of alium is signifiantly redued (Fig. 2). However, the ability to sequester alium at 37 C is not impaired by storing the fration at o C C from between 0-4 h after reovery from the entrifuge. The uptake of alium is a funtion of ATP onentration, temperature and alium onentration (Fig. 3). Uptake is redued by about 60 % at 25 C and abolished

4 120 S. Hodson Fig. i. The sequestration of alium by the Golgi apparatus-rih fration in the presene and absene of ATP. The fration was inubated at 37 C in 146 % (w/w) buffered surose inluding 6 mm MgCl 2 and 40 /*M CaCl 2. Calium is aumulated by the fration in the presene of r5mmatp(#), but not in the absene of ATP (O), vvhere it is indistinguishable from bakground, measured with a preboiled fration ( ) The time ourse of alium aumulation with ATP {%) is not linear but this does not reflet a deterioration of the preparation beause if the fration is preinubated for min at 37 C, and then the marker 46 CaCl 2 is added, the uptake proess shows similar kinetis ( ). r CD O a. CO O " Time, min Fig. 2. The loss of alium sequestration ativity of the Golgi apparatus-rih fration inubated at 37 C in the absene of ATP. The fration was preinubated at 37 C in 14-6% (w/w) buffered surose inluding 6 mm MgCl 2 and 40 /*M CaCl 2 in the absene of ATP for the times shown and then inubated for a standard period of 6 min in the presene of 1-5 mm ATP. The ability to aumulate alium fell with inreasing preinubation times suggesting that ATP is neessary to stabilize the fration at 37 C.

5 Calium pump of the Golgi apparatus r 15 B oa O).E to O "5 Time, min Fig. 3. Calium sequestration at 37 C as a funtion of external alium onentration. The fration was inubated at 37 C in 14-6 % (w/w) buffered surose inluding 6 mm MgCl 2, 1-5 mm ATP and either (O) /tm CaCl 2, ( ) 2o/tMCaCl 2) (A) 30 /tm CaCl 2, ( ) 40 fim CaCl 2, or ( ) 60 fim CaCL. 06 r _E o Q. ro O S~\ (MM-) Fig. 4. The relationship between alium onentration and initial influx into the Golgi apparatus-rih fration. The fration was inubated at 37 C in 14-6 % (w/w) surose inluding 6 mm MgCl 2 and 4 mm ATP. Reiproal plots of alium onentration (S) against initial influx (V) gave a linear relationship. 006

6 122 S. Hodson Time, min (j -20 o L Fig. 5. The exponential inrease of alium sequestration by the Golgi apparatusrih fration in the presene of ATP. When the fration was inubated at 37 C in 14-6 % (w/vv) buffered surose and 6 mm MgCl 2, then in the range of onentrations -120 /im CaCl 2 and o-8-4'o mm ATP, the linear relationship shown above holds, where C,- is the onentration of alium in the fration (nmol alium/mg protein) at the time t(min) and C mos is the maximum value of C. I o Q. 20 I f 0. "5 E Time, min Fig. 6. The rate of hydrolysis of ATP by the Golgi apparatus-rih fration in the presene and absene of alium. The fration was inubated at 37 C C in 14-6 % (w/w) buffered surose inluding 1-5 mm ATP and 6 mm MgCl 2 plus either (#) 40 fim CaCl 2 or ( ) less than 01 /(M CaCl

7 Calium pump of the Golgi apparatus 133 i V Fig. 7. Morphology of the Golgi apparatus-rih fration after sequestration of alium. The fration was inubated at 37 C in 14-6 % (w/vv) buffered surose inluding 1-5 mm ATP, 6 mm MgCl 2 and 40 fim CaCl 2 for 15 min and then prepared for transmission eletron mirosopy (Hodson & Brenhley, 1976). The stak of ysternae (single arrow), very low density lipoprotein ontaining vesiles (double arrows) and 50-nm vesiles (ringed), all of whih are assoiated with the Golgi apparatus of intat liver ells (Hodson & Brenhley, 1976) are all present in the alium-enrihed fration and show no obvious strutural alteration. Bar represents 1 /tm.

8 124 $' Hodson at o C. Applying Mihaelis-Menten kinetis, the apparent K m for ATP is about i-omm. When the fration was inubated in 4 mm ATP, the apparent K m for alium was /tm and F max was estimated at 6-7 nmol Ca/mg protein/min (Fig. 4). The period for half-maximal aumulation of alium into the fration Table 1. Morphometri analysis of the Golgi apparatus-rih fration as perentage membrane length present Golgi apparatus ys* VLDLP ves 50-nm ves RER SM Unidentified no Abbreviations: ys = ysternae, VLDLP ves very low density lipoprotein-ontaining vesiles, 50-nm ves vesiles of diameter about 50 nm, RER = rough endoplasmi retiulum, SM = smooth membranes whih ould inlude fragments of plasma membrane, smooth endoplasmi retiulum and individual membranes from lysed mitohondria. + Sampling from 12 profiles of different preparations (see Fig. 7). The standard error on eah mean is less than 1 %. Table 2. The homogeneity of the Golgi apparatus-rih fration Golgi apparatus-rih Repurified (30 min) # Repurified (60 min)* Repurified (90 min)* dpm mg [ 14 C]galatose nmol alium protein/g transferred/mg sequestered/mg Ratio liver protein/min protein/min olumns 3: i±3 9i±7 2-8 ± ±02 30 ± ± ±o-5 The fration was repurified and its galatosyltransferase ativity and ability to sequester 40 /tm alium in the presene of 15 mm ATP at 37 C were ompared to the original. * Repurifiation onsisted of entrifuging the Golgi apparatus-rih fration, resuspended in 14-6% (w/w) buffered surose on to a surose gradient at iosooog and olleting the fration whih appeared after the indiated times at the % (w/w) interfae. See Methods for details. was invariant through the onentration ranges mm ATP, -120 /<M alium (Fig. 5) and ould be measured at 3-5 min. The interept on the absissa, magnitude 1 min, orresponded with the time for the inubation medium to warm up to 37 C Calium uptake into the Golgi apparatus-rih fration was always aompanied by an inreased rate of hydrolysis of ATP (Fig. 6). When little or no alium was present, the base rate of ATP hydrolysis was 8-9 ± 1-3 nmol ATP/mg protein/min (n = 5), when the fration was inubated in 1-5 mm ATP, 6 mm MgCl 2. When 40 /tm CaCl 2 was added to the fration, the rate of hydrolysis of ATP was inreased by nmol ATP/mg protein/min (n = 5). Under these onditions of inubation, the uptake of alium is estimated at nmol alium/mg protein/min

9 Calium pump of the Golgi apparatus r a <o '5 2 a E O % applied protein retained by filter Fig. 8. Calium retained versus protein retained by Millipore filters of various nominal pore size. The fration was inubated at 37 C in 146% (w/w) buffered surose in the presene of 15 mm ATP, 6 mm MgCl 2 and 40/tM CaCl 2 and then filtered through nominal pore sizes, in/tm: ( ) 045; {%) 065; (O) '8; (A) i"2; and ( ) 3-0. The retained alium per unit of protein retained was linear. (Interept on the absissa was 9 %, determined by regression analysis.) 0 15 r o a o> m o "6 E Time, min Fig. 9. Evidene against ontaminating partiles in the Golgi apparatus-rih fration being responsible for the alium-sequestration ativity. The fration was inubated at 37 C in 1 4 6% (w/w) buffered surose in the presene of 15 mm ATP, 6 mm MgCl 2 and 40 /*M CaCl 2 (#). Addition of 5 mm ammonium oxalate ( ), or 5 mm sodium azide (A) to the inubation medium had no readily detetable effet. Sequestration by a plasma membrane-rih fration inubated under similar onditions was low (T). CEL 30

10 126 S. Hodson {n = 31). The stoihiometry of alium ions transported per ATP moleule hydrolysed is alulated to be Morphometri analysis showed that the membrane in the fration was derived mainly from elements of the Golgi apparatus (Table 1 and Fig. 7). When the fration was repurified by steps whih lost as muh as 80 % of the total protein, the repurified fration nevertheless showed the same ability to sequester alium as a funtion of its total galatosyltransferase ativity (Table 2). This experiment shows it to be diffiult to separate the galatosyltransferase ativity (a marker enzyme for the Golgi apparatus) from the alium-sequestration ativity. When the reated fration was filtered through different pore-size niters, as the amount of protein retained by the filter was redued so was there a orresponding redution of retained alium (Fig. 8). The interept on the absissa is 9%, suggesting that the remaining 90% of the fration is fairly uniform in its ability to sequester alium. The presene of 5 mm sodium azide or 5 min ammonium oxalate in the inubation medium aused no readily detetable alteration in the kinetis of the uptake proess (Fig. 9). When a plasma membrane-rih fration, whih ontained little or no galatosyltransferase ativity, was prepared from the same homogenate (Hodson & Brenhley, 1976), alium aumulation was very little greater than that of the bakground (Fig. 9). THEORY The sequestration of alium by the Golgi apparatus-rih fration in the presene of ATP may be desribed by (Fig. 5) ^ ) -fa, (1) where C t is the onentration of alium in the fration (nmol alium/mg protein) after t min of inubation, C max is the maximal onentration (2->-oo) and k is an independent onstant of 0-19 min" 1. Differentiating with respet to time gives f = r-k t, (2) where V (nmol alium/mg protein/min) is the initial influx used to alulate the Mihaelis-Menten kinetis (Fig. 4) and is numerially equal to kc max. Whereas V varies with ATP and alium onentrations, k is onstant. Equation 2 is formally idential to the situation where alium is pumped into the lumen of the Golgi apparatus and passively permeates aross the membrane, whih is desribed by ^f- w p ~w k ' where C" is the free alium onentration in the lumen (mol/1.), A is the total surfae area of the fration (m 2 ), W is its total lumenal volume (ml), P is the pump (3)

11 Calium pump of the Golgi apparatus 127 ativity per unit area of the membrane (mol/s/m 2 ) and k' is the permeability of the membrane to alium (m/s). Although the formal identity of equations 2 and 3 suggest a 'pump-leak' mehanism for the sequestration of alium by the Golgi apparatus, it annot be taken as proof of the atual mehanism, whih may well be quite different. DISCUSSION The Golgi apparatus-rih fration isolated from rat liver will, in the presene of MgATP, sequester alium. The kinetis are onsistent with a 'pump-leak' mehanism but this has not been established in the present series of experiments. Comparison of the kinetis of the uptake proess shows V max to be about half that found for a rude mirosomal fration and about 6 times faster than a smooth endoplasmi retiulum fration (Moore et al. 1975). The apparent K m for alium is estimated at 85 /6M. The stoihiometry of the proess (about 2 mol alium sequestrated as 1 mol ATP is hydrolysed) is similar to that found in other preparations. It is not known what element or elements in the fration are responsible for the sequestration of alium, but experiments desribed here suggest that mitohondria, mirosomes and vesiles originating from the plasma membrane whih are present in low onentrations in the fration (Table 1 and Hodson & Brenhley, 1976) are not primarily responsible for the sequestration mehanism. This evidene is summarized in Fig. 9. Sodium azide, whih is an inhibitor of rat liver mitohondrial sequestration of alium (Moore et al. 1975), has no obvious effet on the alium sequestration of the Golgi apparatus-rih fration. The presene of ammonium oxalate in the medium auses a linear uptake of alium by mirosomes prepared from rat liver, presumably by trapping the alium and inhibiting its efflux from the mirosomes (Moore et al. 1975). No suh effet was noted on the Golgi apparatusrih fration (Fig. 9), whih suggests that the ontribution by mirosomes to the sequestration mehanism is low. When a plasma membrane-rih fration was isolated from rat liver (Hodson & Brenhley, 1976), it showed little or no tendeny to aumulate alium (Fig. 9). No matter how the fration was subfrationated, it seemed to be reasonably uniform in its ability to sequester alium. In partiular, the inability to separate galatosyltransferase ativity from the alium-sequestration ativity and the fat that about 90% of the fration showed a uniform rate of alium uptake permg protein, taken together with the morphometri evidene that more than 85% of the reognizable membrane in the fration belongs to one or other of the elements whih make the Golgi apparatus, ould be interpreted as evidene for the involvement of the Golgi apparatus itself in the sequestration of alium. It is not easy to attribute a physiologial signifiane to any alium sequestration mehanism that may be loated in the Golgi apparatus of rat liver. If the property were widespread and found in the Golgi apparatus of other ell types, then it ould provide an intriguing link between seretion and alium metabolism. 9-2

12 128 S. Hodson REFERENCES BOLAND, R., MARTONOSI, A. & TILLACK, T. W. (1974). Developmental hanges in the omposition and funtion of saroplasmi retiulum. J. biol. Chem. 249, FLEISCHER, B., FLEISCHER, S. & OZAVVA, H. (1969). Isolation and haraterisation of Golgi membranes from bovine liver. J. Cell Biol. 43, FREILICH, L. S., RICHMOND, M. E., REPUCCI, A. C. & SILBERT, J. (1975). A miromethod for simultaneous determination of galatosyltransferase and s'-nuleotidase in ell frations. Biohem. J. 146, 741^743. HODSON, S. & BRENCHLEY, G. (1976). Similarities of the Golgi apparatus membrane and the plasma membrane in rat liver ells. J. Cell Si. 20, LEHNINGER, A. L., CARAFOLI, E. & Rossi, C. S. (1967). Energy-linked ion movements in mitohondrial systems. Adv. Enzymol. 29, LOWRY, O. H., ROSEBROUGH, N. J., FARR, A. L. & RANDALL, R. J. (1951). Protein measurement with the Folin phenol reagent. J. biol. Chem. 193, MOORE, L., CHEN, T., KNAPP, H. R. & LANDON, E. J. (1975). Energy-dependent alium sequestration ativity in rat liver mirosomes. J. biol. Chem. 250, VAN ROSSUM, G. D. V., SMITH, K. P. & COOPER, P. B. (1974). Partiipation of mitohondria in the ontrol of liver-slie alium. Fedn Pro. Fedn Am. Sos exp. Biol. 33, WEIBEL, E. R., KISTLER, G. S. & SCHERLE, W. F. (1966). Pratial stereologial methods for morphometri ytology. J. Cell Biol. 30, WOOD, T. (1971). Breakdown of adenosine triphosphate by musle preparations in the presene of perhlori aid. Analyt. Biohem. 43, {Reeived 24 August 1977)