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Coupling between non-respiratory energy metabolism and ecto -sialyl-transferase activity of intact ehrlich ascites tumour cell membranes
Pergamon Press
Life Sciences, Vol . 23, pp . 2769-2778 Printed in the U.S .A .
COUPLING BETWEEN NON-RESPIRATORY ENERGY METABOLISM AND ECTO -SIALYLTRANSFERASE ACTIVITY OF INTACT EHRLICH ASCITES TUMOUR CELL MEMBRANES Erik Cervén Institute of Medical and Physiological Chemistry, Biomedical Centre, The University of Uppsala, 751 23 Uppsala, Sweden (Received in final form November 8,
1978)
SUMMARY The sialyltransferase activities associated with intact Ehrlich ascites tumour çells suspended in either nitrogen or oxygen-saturated media were compared . The activity was lower in nitrogen than in oxygen by 40-70% and was insignificantly affected by metabolic inhibitors, but was enhanced to a level observed in an oxygen atmosphere by the addition of glucose, dihydroxy acetone phosphate or phosphoenol pyruvate . The sialyltransferase activity in an oxygen atmosphere was lowered to a level corresponding to that under nitrogen by valinomycin, arsenite, azide or deoxyglucose, but not by cyanide . Studies on the time course of sialyl transfer and of the susceptibility of the incorporated sialic acid to exogenous neuraminidase indicated that similar enzymatic mechanisms were operating under nitrogen and under oxygen . The low activity under nitrogen was restored to the control level observed when incubating under oxygen by transferring the cells to an oxygen atmosphere . It is concluded that the sialyltransferase activity may be coupled to the activity of enzymes involved in glycolytic energy metabolism and associated with the plasma membrane . The effect of oxygen in enhancing the sialyltransferase activity is not due to oxidative phosphorylation but to some other unknown mechanism . During the last decade, evidence has accumulated that glycolytic enzymes may be associated with the plasma membrane of a great variety of animal cells (1-7) . The activity of such enzymes can be detected in subcellular fractions containing plasma membrane (4) as well as at the surface of intact cells (5,6) . The functional implications of these findings are not known although evidence has been obtained that some glycolytic enzymes may be optimally located to fuel the Na + ,K+-stimulated ATPase of intact erythrocyte membranes (7) . The present report gives another example of coupling between different enzyme activities associated with the plasma membrane . These are the newly discovered ecto-sialyltransferase activity of intact Ehrlich ascites tumour cells (8,9) and glycolytic metabolic pathways associated with the surface of these cells . MATERIALS AND METHODS Buffer salts, glucose, potassium cyanide and sodium-meta-arsenite were reagent grade from Merck AG ., Darmstadt, Germany . Neuram~dase (type VI, prepared from Clostridium perfringens ), 2-deoxy-D-glucose, valinomycin, 0300-9653/78/1231-2769$02 .00/0 Copyright (c) 1978 Pergamon Press
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phloridzin, sodium azide, glyceraldehyde-3-phosphate, dihydroxy acetone phosphate, pyruvate, phosphoenol pyruvate, ADP, ATP, NAD, NADH, and UDP-glucose were from Sigma Chemical Co ., St . Louis, Mo . The gas mixtures, composed of 6 .5 * 0,3X COz in oxygen (high oxygen pressure) ; 15 .4 * 0 .08% 0 and 6 .56 t 0 .03% C02 in nitrogen ; 1 .0 t O .1X 02 and 6 .5 * 0 .3% C02 in nitro~en (low oxygen pressure), or 6 .59 ~ 0 .07X CO2 in nitrogen (nitrogen atmosphere were purchased from AGA specialgas, Lidingä, Stockholm . Dinitrophenol (puriss) was from fluky A~~, Chemische Fabrik, Switzerland and Protosol, Aquasol and radioactive CMP- C AcNeu (AcNeu = N-Acetyl-Neuraminic acid) were from NEN Chemicâls, GmbH, Frankfurt-on-Main, Germany . Unlabelled CMP-AcNeu was prepared as described (8-10) from CTP (Sigma) and AcNeu (type IV, Sigma) . Cytochalasin B and dimetylsulphoxide were from Aldrich, Belgium . The experimental details have been described previously (8) . Briefly, cells were grown in the peritoneal cavity of mice, harvested 12-16 days after inoculation and washed in ice-cold Krebs-Ringer bicarbonate buffer . In cases where the sialyltransferase activities in nitrogen and oxygen atmospheres were compared, the cells were always prepared under nitrogen, after which some cells were recentrifuged at 1500 x g followed by suspension in the buffer containing oxygen . Meanwhile, the cell suspension saturated with nitrogen was either used for incubation or kept on ice in tubes covered by plastic and sometimes slightly gassed with a stream of nitrogen . 1 .67 ml of cell suspension (cytocrite 10) was placed in SS-34 propylene plastic centrifuge tubes, which .were put in ice . Water-soluble compounds were added in concentrated aqueous solutions having a volume of 10-40 ul . Scarcely water-soluble compounds were dissolved in buffer, which was subsequently used for suspending 0,15 ml of packed cells (dinitrophenol) or as 10 ul of ethanolic (ethanol/water : 7 :3) solutions (valinomycin) . The activ~~ed sialyl precursor was usually added as 5 ul of commercially prepared CMP- C AcNeu in ethanol/ water (7 :3), and 20 ul of unlabelled CMP-AcNeu in 0 .01 M Tris-HC1, pH 7 .4, to a final concentration of 10 uM . In control experiments, the labelled CMP-AcNeu was freed from ethanol by evaporation and added by solubilization in the cell suspension . The incubations were started by aerating the tubes with the gas mixtures, giving pH 7 .2 and transferring them to a thermostated shaker at 37 ° C, followed by incubation for 30 min or more . After incubation the tubes were transferred to an ice-bath and 25 ml of ice-cold buffer was added ; this was followed by centrifugation for 25 min at 3000 x g . 1 ml of the supernatant was saved for measuring radioactivity and the walls of the ~ubes were rinsed with the buffer, after which the cell pellet was stored at -20 C . After storage, the cell material was washed three times in 3X sulfosalicylic ac~d with the addition of unlabelled AcNeu and then again frozen and stored at -20 C . Three drops of water and ~ ml of Protosol were added, followed by digestion of the cell pellet at 37 C until no particulate material could be seen . The hydrolysate was then chilled on ice and 0 .15 ml of glacial acetic acid was added and 1 ml of the solution transferred to a scintillation vial containing 10 ml of Aquasol followed by measurement of the radioactivity in a Nuclear Chicago Unilux II scintillation equipment . RESULTS The incorporation of 14 C AcNeu from exogenous CMP- 14 C AcNeu was higher in the presence of 93 .5% or 15% oxygen than under nitrogen or under 1X oxygen in nitrogen . The stimulatory effect of oxygen, which increased the incorporation by 50 to 150% of the levels observed under nitrogen or in the presence of 1% oxygen, was more pronounced when the absolute amount of incorporation was low (Fig . 1) . 1% relative to 0% oxygen did not increase the incorporation .
Vol . 23, Nos . 27 Fr 28, 1978
&ctosialyltransferase
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w
10 5 INCORPORATION OF fuC)AcNeu UNDER NITROGEN OR UNDER i/, CIXYGEN IN NITROGEN
(pmoles/ 108 oelis) FIG. 1
The stimulatory effect of high (93 .5%) compared with low (~~ or 0%) partial pressure of oxygen on the amount of ~acorporated C AcNeu plotted against the amount of incorporated C AcNeu under the non-stimulated condit~~ns (1% or 0% oxygen) . The duration of the C AcNeu was 30 min . incubation with CMPThe relative susceptibility of the incorporated 14 C AcNeu to exogenous neuraminidase, which is a criterion for location of the label at the ce~~ surface (8), was not significantly different after incubation with CMPC AcNeu under nitrogen and under oxygen . In one ~~periment, for example, 68 .2 0 .9% was hydrolyzed by neuraminidase when the C AcNeu had been incorporated under 1% oxygen while 65 .7 * 1 .1% was hydrolyzed from ~~e same cell preparation under identical conditions following incorporation of C AcNeu under 93 .5% oxygen . As illustrated in Fig . 2, the sialyltransferase activity was reversibly depressed under nitrogen and could be increased by i~~ubating with carbon dioxide in oxygen . The velocity of Incorporation of C AcNeu under these conditions was comparable to that initially observed when the cells were incubated under 93 .5% oxygen, No difference of incorporation under oxygen and under nitrogen was detected when the ~~115 were washed in oxygen-containing buffer prior to incubation with CMPC AcNeu (Data not shown) . Metabolic inhibitors generally differed in their effect on the sialyl transfer under nitrogen and under oxygen (Table I) . Arsenite (1 or 2 mM) and azide. (10 mM) only slightly diminished the incorporation under nitrogen while they diminished the incorporation under oxygen to a level corresponding to that
277 2
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°ô w
m
~ 20 z
0 F O
O
I
30
I
60
I
90
I
120
TIME OF INCUBATION
I
150
Iminl
1
180
FIG . 2
The time-dependency of incorporation of 14 C AcNeu from CMP- 14 C AcNeu under 93 .5% nitrogen (filled circles) and 93 .5% oxygen (open circles) . After 90 min of incubation, tubes were transferred from a nitrogen to an oxygen atmosphere and incubated further with (squares) or without (open circles) the addition of glucose, 5 .6 mM . under nitrogen . No further inhibition of the incorporation was detected at higher concentrations of arsenate and the inhibition observed with Z mM of this compound was considered maximal . Cyanide in moderate concentrations (0 .01 - 1 mM) slightly stimulated the incorporation under low oxygen pressure and slightly inhibited that under high oxygen pressure . In some experiments, the presence of 10 mM of cyanide resulted in an inhibition of the incorporation comparable to that observed with arsenate, while i9 others only about 10% inhibition could be detected . Valinomycin (5 x 10 - M) diminished the incorporation under oxygen to a level corresponding to that in the presence of arsenate, in contrast to its weak effect under nitrogen . Dinitrophenol (0 .25 mM) inhibited the sialyltransferase activity under oxygen by about 40% . The addition of glucose (to 5 .6 mM) did not influence, or slightly increased the incorporation of sialic acid under oxygen, and increased the incorporation under nitrogen to a level corresponding to that under oxygen (Table II) . This latter effect was reversed by 2-deoxy-D-glucose . Deoxyglucose, in contrast to its weak effect on the incorporation under nitrogen (without added glucose) diminished the sialyltransferase activity under oxygen to a level corresponding to the activity under nitrogen . The stimulatory effect of glucose, reproducible at concentrations above 5 mM, upon the incorporation under nitrogen was not mimicked by UDP-glucose (0 .1 mM) or by the glycolytic intermediates glyceraldehyde-3-phosphate or pyruvate (2 .5 mM) . However, the sialyl transfer was significantly enhanced in the presence of two other intermediates, dihydroxy acetone phosphate (2 .5 mM) or phosphoenoT pyruvate (2 .5 mM) . At 1 mM or lower concentrations these compounds did not influence the ecto-sialyltransferase activity . Phloridzin and cytochalasin B, both of w~~ch inFi'6i"t glucose transport (11) slightly inhibited the incorporation of C AcNeu under oxygen . It is noteworthy that these compounds were effective without exogenously added glucose . At a
Vol . 23, Nos . 27 8 28,
1978
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TABLE I Sialyl transfer in the presence of various metabolic inhibitors . Mean of two determinations in typical experimenif~ . In each expériment, 100% refer to the incorporation of C AcNeu without additive . The incubations lasted for 30 min . Type of incubation
Incorporation (%)
(Total cmp)
Expt . no. 1 (1% 02 ), control NaAsO (1 mM NaN3 2(10 mM ; Valinort~ycin (5 x lÔ 7 M) 93 .5% 02
100 89 .4 96 .9 85 .8 148
* * * * *
4% 0 .9% 4 .0% 7 .1% 9%
189
Expt . no . 2 (93 .5% 02 ), control NaAs02 (1 mM) NaN3 (10 mM) Yalinomycin (5 x lÔ 7M) 1% 02
100 64 .7 67 .7 64 .4 54 .6
* * * * *
2% 2 .8% 4 .8% 1 .8% 0 .6%
408
3 (1% 02) and KCN (1 mM) 119 * 3 93 .5% 02 , control 196 * 4 93 .5% O2 ; and KCN (1 mM) 91 .0 * 4 (93 .5% 02 ) and NaAs0 2 (1 mM),control 57 .6 *
5% 8% 0.4% 1 .5%
71
Expt . Expt . Expt . Expt .
no . no . n o. no.
128
TABLE II The influence of glucose metabolism on sialyl transfer . Mean of two determinations in typical exper~~ents . In each experiment, 100% refer to the incorporation of C AcNeu without additive . Type of incubation Expt . Expt . Expt . Expt, Expt .
no . no. no . no . n o.
Incorporation (%)
1 1 2 3 3
(1% O2) with glucose (5 .6 mM) 144 (93 .5% 02) 148 (93 .5% O2) with glucose (5 .6 mM) 100 (93 .5% N2) with glucose (1 mM) 171 (93 .5% N2) with glucose (1 mM) and deoxyglucose (0 .05 mM) 101 Expt . no. 4 (93 .5% 02) with deoxyglucose (5 m!!) 64 .0 Expt . no . 4 (93 .5% 02 ) with deoxyglucose (10 mM) 64 .5
* * * *
Expt . no . 5 (93 .5% N2), control UDP-glucose (0 .1 mM) Glucose (5 .6 mM Glucose (5 .6 mM ; and UDP-glucose (0 .1 mM)
* * * *
Expt . no. 6 (93 .5% N2), control Dihydroxy acetone phosphate (2 .5 mM) Phosphoenolpyruvate (2 .5 mM)
100 95 .7 145 160
4% 9% 2% 14%
* 4% * 0.0% * 2 .5% 3% 0% 2% 4%
100 * 2% 138 * 3% 149 * 12%
(Total cpm) 272 408 170 124 627
501
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Vol . 23, Nos . 27 $ 28, 1978
TABLE II, tont . Type of incubation Expt, no . 7 (93 .5% 0 2 ) with with with Expt . n o . 7 (93 .5% 0 2 ) with
Incorporation (%) phloridzin (1 mM) 90 .5 phloridzin (5 mM) 88 .5 phloridzin (10 mM) 75 .1 NaAs0 2 (2 mM),contro156 .0
(Total cpm)
* = * *
2 .5% 1 .5% 1 .0% 0 .2%
175
Expt . n o . 8 (93 .5% 0~ ), control 100 * Dimethylsulfoxide (DMSO), 6 .3% v/v 97 .1 t -5 DMSO (6 .3%, v/v) and cytochalasi~ B (10 M) 92 .6 * DMSO (6 .3%), cytochalasin B (10 - M) and glucose (5 .6 mM) 106 =
4% 1 .3% 1 .6%
197
2%
concentration of 10 mM, phloridzin caused 25% inhibition of sialyl transfer . The inhibitory effect of cytochalasin B (10 - 5 M) was reversed by adding glucose . Since the incorporation of 14 C AcNeu under nitrogen was significantly enhanced by adding glucose, the influence of glycolysis and residual ATP on the ecto-sialyltransferase activity under nitrogen was investigated (Fig . 3) . DeoxygTcose, an inhibitor of glucose metabolism and glycolysis (12) and valinomycin, which lowers the ATP content of Ehrlich ascites tumour cells by short-circuiting the sodium-potassium pump or by uncoupling the respiratory activity f~m phosphorylation (13) were added, and the subsequent incubation with CMPC AcNeu was performed under nitrogen . Neither deoxyglucose nor valinomycin significantly altered the sialyltransferase activity under nitrogen . The stimulatory effect of glucose under nitrogen was confirmed and shown to be roughly proportional to the sialyltransferase activity without added glucose during the first three hours of incubation (Fig . 3) . DISCUSSION The present data show that energy metabolism influences the ecto-sialyltransferase activity of Ehrlich estates tumour cells . The coupling between sialyl transfer and energy metabolism was detected only at high partial pressure of oxygen and/or in the presence of glucose . ATP generated from mitochondrial respiration cannot be essential for sialyl transfer, for the following reasons : 1 . 2-deoxy-D-glucose, an inhibitor of glucose metabolism including glycolysis, diminished the incorporation under oxygen to a level corresponding to that under nitrogen . 2 . The sialyltransferase activity was depressed even at 1% oxygen, which is sufficient to maintain respiration (cf . 14) . 3 . Cyanide only slightly inhibited iFe sialyl transfer at high oxygen pressure . These data imply that the inhibitory effects of valino~ycin, dinitrophenol, arsenate and azide under oxygen are not related to the mitochondrial production of ATP . Since the sialyl transfer occurs on the cell surface, it is reasonable to assume that the inhibitory effect of valinoigycin is related to its capability to short-circuit the sodium-potassium pump (cf . 13), thus dissipating the electrochemical potential across the cell me~m rane . Dinitrophenol, a proton ionophore, may have a similar effect . Arsenate inhibited the
Vol . 23, Nos . 27 $ 28, 1978
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~ 20 0 o. 0
30
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90
120
150
TIME OF INCIrBATION Im~1
180
FIG . 3 The time-dependency of incorporation of 14 C AcNeu from CMP- 14C AcNeu under nitrogen without an additive (closed circles) ; with glucose, 5 .6 mM (open circles) ; wi~h deoxyglucose, 5 mM (closed squares) or with valinort~ycin, 5 x 10 - M (closed triangles) . Each coordinate was obtained from two determinations which are illustrated separately when distinguishable . sialyl transfer under high oxygen pressure while under nitrogen, the activity was only slightly affected, indicating that some transferases are inherently resistant to this dithiol reagent . The effective target of the metabolic inhibitor arsenite could be glycolytic biochemical pathways (15) or oxidative biochemical pathways (16) . The evidence cited above excludes any role for mitochondrial oxidative phosphorylation in sialyl transfer . This conclusion is further corroborated by two lines of evidence : 1 . Dihydroxy acetone phosphate and phosphq~nol pyruvate, two glycolytic intermediates, enhanced the incorporation of C AcNeu under nitrogen . 2 . Glucose but not deoxyglucose significantly stimulated the activity under nitrogen, while glucose only slightly or not at all stimulated the activity under oxygen . The possibility that glucose and deoxyglucose influence the sialyltrans-
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Vol, 23, Nos . 27 $ 28, 1978
ferase activity by contributing to biosynthesis of glycoproteins and glycolipids is not likely since in the present experimental system, the site of sialylation (the cell membrane) is physically dissociated from the intracellu1ar endoplasmic reticulum where the early stages of glycoprotein synthesis, such as incorporation of glucose and deoxyglucose, are generally believed to occur (cf . 17-19) . If the stimulating effect of glucose were due to an enhanced glucosyTfransferase activity at the cell surface, then a comparable effect of exogenous UDP-glucose should be expected, contrary to the experimental results obtained . The evidence thus jointly indicates that non-respiratory, notably glycolytic energy metabolism, probably including electro-chemical components, plays a part in sialyl transfer at the cell surface . This implies that other mechanisms are not responsible for the effects observed . Thus, the low incorporation of sialic acid under nitrogen was restored by oxygen to control levels bserved when the same cell preparation was immediately incubated with CMP- 1 ~C AcNeu under oxygen . These and other data suggest that elution of inhibitors, activators or enzyme into the medium, and hydrolysis of the added CMP-AcNeu, are not factors involved in the different levels of incorporation under nitrogen and under oxygen . Aq~ther important observation is that the susceptibility of the incorporC A Neu to exogenous neuraminidase was not different after incubation ated with CMP- ~4 C AcNeu under oxygen and nitrogen . The susceptibility of the 14C AcNeu after incorporation under these conditions was about 2/3, which is a criterion for location of all label at the cell surface in the present experimental system (B) . These latter data, combined with the observation of similar (rate-limited) time-dependency of incorporation under nitrogen and under oxygen and of proportionality between the sialyltransferase activities observed in the presence and absence of glucose under nitrogen, serve as indicators that similar enzymatic mechanisms are operating irrespective of the degree of stimulation caused by energy metabolism . The stimulatory effect of oxygen as compared with nitrogen as roughly inversely proportional to the absolute amount of incorporated 1 ~C AcNeu under the non-stimulated conditions . This may indicate that the sialyltransferase activity in each cell preparation may be optimized by incubating under oxygen and that the degree of depletion of fuel for non-respiratory energy metabolism in the intraperitoneally grown cells cf . 20, 21) may be critical for the magnitude of the stimulatory effect . Seemingly, endogenous fuel for these biochemical pathways only enhances the sialyltransferase activity under oxygen . The effect of high oxygen pressure in bringing about coupling between ectosialyltransferase activity and glycolytic biochemical pathways is interesting in view of data from other experimental systems indicating that oxygen tension may affect cellular differentiation (22) and neoplastic transformation (23,24) . Both these processes involve cell surface glycoconjugates . Glycolytic enzymes have long been known to be associated with the plasma membrane of various animal cells (1-7) including the Ehrlich ascites tumour cells (6 ) . The present finding that glycolytic biochemical pathways and not oxidative phosphorylation may stimulate the ecto-sialyltransferase activity of intact plasma membranes most probably indica~hat precisely this pool of membrane-bound glycolytic enzymes is involved in the effects observed . This extreme compartmentalization is surprising since inhibitors of energy metabolism which lower the intracellular level of ATP are known to affect amino acid transport and the function of Na+ - and IC+ -stimulated ATPase of intact plasma membranes in this and other experimental systems (25,26) . Apparently, the enzymes and acceptor molecules involved in ecto-sialyltransferase activity are
Vol . 23, Nos . 27 $ 28,
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located exterior to any influence that the intracellular level of ATP may exert, but at the same time they are affected by membraneous energy metabolism . ACKNOWLEDGEMENTS This work was generously supported by Dr . Gunner Ronquist and Dr . Gunner Agren through grants from the Swedish Medical Research Council (Project B75-13X-228-11), by Svenska Sällskapet fdr Medicinsk Forskning, The University of Uppsala, Fortia AB and Uddeholms AB . REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10 . 11 . 12 . 13 . 14 . 15 . 16 . 17 . 18 . 19 . 20 . 21 . 22 . 23 . 24 . 25 . 26 .
G. RONQUIST, Acta Ph siol . Scand. 74 594-605 (1968) . an tJTER, Bioc him. Biophys . Acta 382 (1975) . W. TILLMAN, A, T . L . STECK, J . Cell Biol . 62 1-19 (197~H . R. KNOLL, oc m. o h Acta 522 1-9 (1978) . Tands WESTERMARK, J . Cell . Physiol . 77 G. AGREN, J . , . 331-336 (1971) . C . 0. WERNSTEDT, G . K. AGREN and G . RONQUIST, Cancer Res . .35 1536-1541 (1975) . J . C. PARKER and J . F. HOFFMAN, J . Gen . Ph siol . 50 893-916 (1967) . E . CERVEN, Biochim. Bio h s . Acta 7j E . CERVEN, ~âÔl an sala J. Med . Sci . 80 58-60 (1975) . E .' L. KEAN, in Methods Enz ~1 . Vol , ar , p . 41422 (1972) . M. P. CZECH, J . o . em . J . L. WEBB, nzyme an e aboT~c Inhibitors , Vol . I . Academic Press, New York, (1966) . C . deCESPEDES and H . N . CHRISTENSEN, Biochim. Biophys . Acta 339 139-145 (1974) . J . KIELER, N . I . NISSEN and W . BICZ, Acta Unio Int . Cancrum 118 228-233 (1962) . J . F. KUO, I. K . DILL and C. E. HOLMLUND, Biochim. Biophys. Acta 148 683688 (1967) . W. BARTLEY and B. DEAN, Biochim: J . 115 903-912 (1969) . R. G. SPIRO, New En 1 . f$T 9~T--1001 (1969) . M. W. MYERS an . . LT;Biochim. Biophys . Res . Commun . 63 164-171 (1975) . R . C. HUGHES, A. MEAGER and R. NAIRN, Eur. J . Biochim. 72 265-273 (1977) . 0 . WARBURG and E. HIEPLER, Z . f. Natur orsc . _ (1~3) . G. KLEIN, Z . f. Krebsforsch . J . M. PAW ve . o . 1 52-72 (1969) . 0 . WARBURG, XVITÔIIôq:des-Physiol . Chem . (Mosbach, Baden ), Berlin, 1 (1966) . H . GOLDBLATT, L. FRIEDMAN and R . L . LECHNER, Biochim. Med . 7 241-252 (1973) . H . N . CHRISTENSEN, C deCESPEDES, M. E . HANDLO an . OAQUIST, Ann . N .Y . Acad . Sci . 22 7 255-379 (1974) . A-OWEIP~S .îß CAPLAN and A. ESSIG, Biochim. Biophys . Acta 394 438-448 (1975) .
s.
Life Sciences, Vol . 23, pp . 2769-2778 Printed in the U.S .A .
COUPLING BETWEEN NON-RESPIRATORY ENERGY METABOLISM AND ECTO -SIALYLTRANSFERASE ACTIVITY OF INTACT EHRLICH ASCITES TUMOUR CELL MEMBRANES Erik Cervén Institute of Medical and Physiological Chemistry, Biomedical Centre, The University of Uppsala, 751 23 Uppsala, Sweden (Received in final form November 8,
1978)
SUMMARY The sialyltransferase activities associated with intact Ehrlich ascites tumour çells suspended in either nitrogen or oxygen-saturated media were compared . The activity was lower in nitrogen than in oxygen by 40-70% and was insignificantly affected by metabolic inhibitors, but was enhanced to a level observed in an oxygen atmosphere by the addition of glucose, dihydroxy acetone phosphate or phosphoenol pyruvate . The sialyltransferase activity in an oxygen atmosphere was lowered to a level corresponding to that under nitrogen by valinomycin, arsenite, azide or deoxyglucose, but not by cyanide . Studies on the time course of sialyl transfer and of the susceptibility of the incorporated sialic acid to exogenous neuraminidase indicated that similar enzymatic mechanisms were operating under nitrogen and under oxygen . The low activity under nitrogen was restored to the control level observed when incubating under oxygen by transferring the cells to an oxygen atmosphere . It is concluded that the sialyltransferase activity may be coupled to the activity of enzymes involved in glycolytic energy metabolism and associated with the plasma membrane . The effect of oxygen in enhancing the sialyltransferase activity is not due to oxidative phosphorylation but to some other unknown mechanism . During the last decade, evidence has accumulated that glycolytic enzymes may be associated with the plasma membrane of a great variety of animal cells (1-7) . The activity of such enzymes can be detected in subcellular fractions containing plasma membrane (4) as well as at the surface of intact cells (5,6) . The functional implications of these findings are not known although evidence has been obtained that some glycolytic enzymes may be optimally located to fuel the Na + ,K+-stimulated ATPase of intact erythrocyte membranes (7) . The present report gives another example of coupling between different enzyme activities associated with the plasma membrane . These are the newly discovered ecto-sialyltransferase activity of intact Ehrlich ascites tumour cells (8,9) and glycolytic metabolic pathways associated with the surface of these cells . MATERIALS AND METHODS Buffer salts, glucose, potassium cyanide and sodium-meta-arsenite were reagent grade from Merck AG ., Darmstadt, Germany . Neuram~dase (type VI, prepared from Clostridium perfringens ), 2-deoxy-D-glucose, valinomycin, 0300-9653/78/1231-2769$02 .00/0 Copyright (c) 1978 Pergamon Press
277 0
Ectosialyltransferase
Vol, 23, Nos . 27 & 28, 1978
phloridzin, sodium azide, glyceraldehyde-3-phosphate, dihydroxy acetone phosphate, pyruvate, phosphoenol pyruvate, ADP, ATP, NAD, NADH, and UDP-glucose were from Sigma Chemical Co ., St . Louis, Mo . The gas mixtures, composed of 6 .5 * 0,3X COz in oxygen (high oxygen pressure) ; 15 .4 * 0 .08% 0 and 6 .56 t 0 .03% C02 in nitrogen ; 1 .0 t O .1X 02 and 6 .5 * 0 .3% C02 in nitro~en (low oxygen pressure), or 6 .59 ~ 0 .07X CO2 in nitrogen (nitrogen atmosphere were purchased from AGA specialgas, Lidingä, Stockholm . Dinitrophenol (puriss) was from fluky A~~, Chemische Fabrik, Switzerland and Protosol, Aquasol and radioactive CMP- C AcNeu (AcNeu = N-Acetyl-Neuraminic acid) were from NEN Chemicâls, GmbH, Frankfurt-on-Main, Germany . Unlabelled CMP-AcNeu was prepared as described (8-10) from CTP (Sigma) and AcNeu (type IV, Sigma) . Cytochalasin B and dimetylsulphoxide were from Aldrich, Belgium . The experimental details have been described previously (8) . Briefly, cells were grown in the peritoneal cavity of mice, harvested 12-16 days after inoculation and washed in ice-cold Krebs-Ringer bicarbonate buffer . In cases where the sialyltransferase activities in nitrogen and oxygen atmospheres were compared, the cells were always prepared under nitrogen, after which some cells were recentrifuged at 1500 x g followed by suspension in the buffer containing oxygen . Meanwhile, the cell suspension saturated with nitrogen was either used for incubation or kept on ice in tubes covered by plastic and sometimes slightly gassed with a stream of nitrogen . 1 .67 ml of cell suspension (cytocrite 10) was placed in SS-34 propylene plastic centrifuge tubes, which .were put in ice . Water-soluble compounds were added in concentrated aqueous solutions having a volume of 10-40 ul . Scarcely water-soluble compounds were dissolved in buffer, which was subsequently used for suspending 0,15 ml of packed cells (dinitrophenol) or as 10 ul of ethanolic (ethanol/water : 7 :3) solutions (valinomycin) . The activ~~ed sialyl precursor was usually added as 5 ul of commercially prepared CMP- C AcNeu in ethanol/ water (7 :3), and 20 ul of unlabelled CMP-AcNeu in 0 .01 M Tris-HC1, pH 7 .4, to a final concentration of 10 uM . In control experiments, the labelled CMP-AcNeu was freed from ethanol by evaporation and added by solubilization in the cell suspension . The incubations were started by aerating the tubes with the gas mixtures, giving pH 7 .2 and transferring them to a thermostated shaker at 37 ° C, followed by incubation for 30 min or more . After incubation the tubes were transferred to an ice-bath and 25 ml of ice-cold buffer was added ; this was followed by centrifugation for 25 min at 3000 x g . 1 ml of the supernatant was saved for measuring radioactivity and the walls of the ~ubes were rinsed with the buffer, after which the cell pellet was stored at -20 C . After storage, the cell material was washed three times in 3X sulfosalicylic ac~d with the addition of unlabelled AcNeu and then again frozen and stored at -20 C . Three drops of water and ~ ml of Protosol were added, followed by digestion of the cell pellet at 37 C until no particulate material could be seen . The hydrolysate was then chilled on ice and 0 .15 ml of glacial acetic acid was added and 1 ml of the solution transferred to a scintillation vial containing 10 ml of Aquasol followed by measurement of the radioactivity in a Nuclear Chicago Unilux II scintillation equipment . RESULTS The incorporation of 14 C AcNeu from exogenous CMP- 14 C AcNeu was higher in the presence of 93 .5% or 15% oxygen than under nitrogen or under 1X oxygen in nitrogen . The stimulatory effect of oxygen, which increased the incorporation by 50 to 150% of the levels observed under nitrogen or in the presence of 1% oxygen, was more pronounced when the absolute amount of incorporation was low (Fig . 1) . 1% relative to 0% oxygen did not increase the incorporation .
Vol . 23, Nos . 27 Fr 28, 1978
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100
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w
10 5 INCORPORATION OF fuC)AcNeu UNDER NITROGEN OR UNDER i/, CIXYGEN IN NITROGEN
(pmoles/ 108 oelis) FIG. 1
The stimulatory effect of high (93 .5%) compared with low (~~ or 0%) partial pressure of oxygen on the amount of ~acorporated C AcNeu plotted against the amount of incorporated C AcNeu under the non-stimulated condit~~ns (1% or 0% oxygen) . The duration of the C AcNeu was 30 min . incubation with CMPThe relative susceptibility of the incorporated 14 C AcNeu to exogenous neuraminidase, which is a criterion for location of the label at the ce~~ surface (8), was not significantly different after incubation with CMPC AcNeu under nitrogen and under oxygen . In one ~~periment, for example, 68 .2 0 .9% was hydrolyzed by neuraminidase when the C AcNeu had been incorporated under 1% oxygen while 65 .7 * 1 .1% was hydrolyzed from ~~e same cell preparation under identical conditions following incorporation of C AcNeu under 93 .5% oxygen . As illustrated in Fig . 2, the sialyltransferase activity was reversibly depressed under nitrogen and could be increased by i~~ubating with carbon dioxide in oxygen . The velocity of Incorporation of C AcNeu under these conditions was comparable to that initially observed when the cells were incubated under 93 .5% oxygen, No difference of incorporation under oxygen and under nitrogen was detected when the ~~115 were washed in oxygen-containing buffer prior to incubation with CMPC AcNeu (Data not shown) . Metabolic inhibitors generally differed in their effect on the sialyl transfer under nitrogen and under oxygen (Table I) . Arsenite (1 or 2 mM) and azide. (10 mM) only slightly diminished the incorporation under nitrogen while they diminished the incorporation under oxygen to a level corresponding to that
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°ô w
m
~ 20 z
0 F O
O
I
30
I
60
I
90
I
120
TIME OF INCUBATION
I
150
Iminl
1
180
FIG . 2
The time-dependency of incorporation of 14 C AcNeu from CMP- 14 C AcNeu under 93 .5% nitrogen (filled circles) and 93 .5% oxygen (open circles) . After 90 min of incubation, tubes were transferred from a nitrogen to an oxygen atmosphere and incubated further with (squares) or without (open circles) the addition of glucose, 5 .6 mM . under nitrogen . No further inhibition of the incorporation was detected at higher concentrations of arsenate and the inhibition observed with Z mM of this compound was considered maximal . Cyanide in moderate concentrations (0 .01 - 1 mM) slightly stimulated the incorporation under low oxygen pressure and slightly inhibited that under high oxygen pressure . In some experiments, the presence of 10 mM of cyanide resulted in an inhibition of the incorporation comparable to that observed with arsenate, while i9 others only about 10% inhibition could be detected . Valinomycin (5 x 10 - M) diminished the incorporation under oxygen to a level corresponding to that in the presence of arsenate, in contrast to its weak effect under nitrogen . Dinitrophenol (0 .25 mM) inhibited the sialyltransferase activity under oxygen by about 40% . The addition of glucose (to 5 .6 mM) did not influence, or slightly increased the incorporation of sialic acid under oxygen, and increased the incorporation under nitrogen to a level corresponding to that under oxygen (Table II) . This latter effect was reversed by 2-deoxy-D-glucose . Deoxyglucose, in contrast to its weak effect on the incorporation under nitrogen (without added glucose) diminished the sialyltransferase activity under oxygen to a level corresponding to the activity under nitrogen . The stimulatory effect of glucose, reproducible at concentrations above 5 mM, upon the incorporation under nitrogen was not mimicked by UDP-glucose (0 .1 mM) or by the glycolytic intermediates glyceraldehyde-3-phosphate or pyruvate (2 .5 mM) . However, the sialyl transfer was significantly enhanced in the presence of two other intermediates, dihydroxy acetone phosphate (2 .5 mM) or phosphoenoT pyruvate (2 .5 mM) . At 1 mM or lower concentrations these compounds did not influence the ecto-sialyltransferase activity . Phloridzin and cytochalasin B, both of w~~ch inFi'6i"t glucose transport (11) slightly inhibited the incorporation of C AcNeu under oxygen . It is noteworthy that these compounds were effective without exogenously added glucose . At a
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TABLE I Sialyl transfer in the presence of various metabolic inhibitors . Mean of two determinations in typical experimenif~ . In each expériment, 100% refer to the incorporation of C AcNeu without additive . The incubations lasted for 30 min . Type of incubation
Incorporation (%)
(Total cmp)
Expt . no. 1 (1% 02 ), control NaAsO (1 mM NaN3 2(10 mM ; Valinort~ycin (5 x lÔ 7 M) 93 .5% 02
100 89 .4 96 .9 85 .8 148
* * * * *
4% 0 .9% 4 .0% 7 .1% 9%
189
Expt . no . 2 (93 .5% 02 ), control NaAs02 (1 mM) NaN3 (10 mM) Yalinomycin (5 x lÔ 7M) 1% 02
100 64 .7 67 .7 64 .4 54 .6
* * * * *
2% 2 .8% 4 .8% 1 .8% 0 .6%
408
3 (1% 02) and KCN (1 mM) 119 * 3 93 .5% 02 , control 196 * 4 93 .5% O2 ; and KCN (1 mM) 91 .0 * 4 (93 .5% 02 ) and NaAs0 2 (1 mM),control 57 .6 *
5% 8% 0.4% 1 .5%
71
Expt . Expt . Expt . Expt .
no . no . n o. no.
128
TABLE II The influence of glucose metabolism on sialyl transfer . Mean of two determinations in typical exper~~ents . In each experiment, 100% refer to the incorporation of C AcNeu without additive . Type of incubation Expt . Expt . Expt . Expt, Expt .
no . no. no . no . n o.
Incorporation (%)
1 1 2 3 3
(1% O2) with glucose (5 .6 mM) 144 (93 .5% 02) 148 (93 .5% O2) with glucose (5 .6 mM) 100 (93 .5% N2) with glucose (1 mM) 171 (93 .5% N2) with glucose (1 mM) and deoxyglucose (0 .05 mM) 101 Expt . no. 4 (93 .5% 02) with deoxyglucose (5 m!!) 64 .0 Expt . no . 4 (93 .5% 02 ) with deoxyglucose (10 mM) 64 .5
* * * *
Expt . no . 5 (93 .5% N2), control UDP-glucose (0 .1 mM) Glucose (5 .6 mM Glucose (5 .6 mM ; and UDP-glucose (0 .1 mM)
* * * *
Expt . no. 6 (93 .5% N2), control Dihydroxy acetone phosphate (2 .5 mM) Phosphoenolpyruvate (2 .5 mM)
100 95 .7 145 160
4% 9% 2% 14%
* 4% * 0.0% * 2 .5% 3% 0% 2% 4%
100 * 2% 138 * 3% 149 * 12%
(Total cpm) 272 408 170 124 627
501
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TABLE II, tont . Type of incubation Expt, no . 7 (93 .5% 0 2 ) with with with Expt . n o . 7 (93 .5% 0 2 ) with
Incorporation (%) phloridzin (1 mM) 90 .5 phloridzin (5 mM) 88 .5 phloridzin (10 mM) 75 .1 NaAs0 2 (2 mM),contro156 .0
(Total cpm)
* = * *
2 .5% 1 .5% 1 .0% 0 .2%
175
Expt . n o . 8 (93 .5% 0~ ), control 100 * Dimethylsulfoxide (DMSO), 6 .3% v/v 97 .1 t -5 DMSO (6 .3%, v/v) and cytochalasi~ B (10 M) 92 .6 * DMSO (6 .3%), cytochalasin B (10 - M) and glucose (5 .6 mM) 106 =
4% 1 .3% 1 .6%
197
2%
concentration of 10 mM, phloridzin caused 25% inhibition of sialyl transfer . The inhibitory effect of cytochalasin B (10 - 5 M) was reversed by adding glucose . Since the incorporation of 14 C AcNeu under nitrogen was significantly enhanced by adding glucose, the influence of glycolysis and residual ATP on the ecto-sialyltransferase activity under nitrogen was investigated (Fig . 3) . DeoxygTcose, an inhibitor of glucose metabolism and glycolysis (12) and valinomycin, which lowers the ATP content of Ehrlich ascites tumour cells by short-circuiting the sodium-potassium pump or by uncoupling the respiratory activity f~m phosphorylation (13) were added, and the subsequent incubation with CMPC AcNeu was performed under nitrogen . Neither deoxyglucose nor valinomycin significantly altered the sialyltransferase activity under nitrogen . The stimulatory effect of glucose under nitrogen was confirmed and shown to be roughly proportional to the sialyltransferase activity without added glucose during the first three hours of incubation (Fig . 3) . DISCUSSION The present data show that energy metabolism influences the ecto-sialyltransferase activity of Ehrlich estates tumour cells . The coupling between sialyl transfer and energy metabolism was detected only at high partial pressure of oxygen and/or in the presence of glucose . ATP generated from mitochondrial respiration cannot be essential for sialyl transfer, for the following reasons : 1 . 2-deoxy-D-glucose, an inhibitor of glucose metabolism including glycolysis, diminished the incorporation under oxygen to a level corresponding to that under nitrogen . 2 . The sialyltransferase activity was depressed even at 1% oxygen, which is sufficient to maintain respiration (cf . 14) . 3 . Cyanide only slightly inhibited iFe sialyl transfer at high oxygen pressure . These data imply that the inhibitory effects of valino~ycin, dinitrophenol, arsenate and azide under oxygen are not related to the mitochondrial production of ATP . Since the sialyl transfer occurs on the cell surface, it is reasonable to assume that the inhibitory effect of valinoigycin is related to its capability to short-circuit the sodium-potassium pump (cf . 13), thus dissipating the electrochemical potential across the cell me~m rane . Dinitrophenol, a proton ionophore, may have a similar effect . Arsenate inhibited the
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w ~ 60
~ 20 0 o. 0
30
60
90
120
150
TIME OF INCIrBATION Im~1
180
FIG . 3 The time-dependency of incorporation of 14 C AcNeu from CMP- 14C AcNeu under nitrogen without an additive (closed circles) ; with glucose, 5 .6 mM (open circles) ; wi~h deoxyglucose, 5 mM (closed squares) or with valinort~ycin, 5 x 10 - M (closed triangles) . Each coordinate was obtained from two determinations which are illustrated separately when distinguishable . sialyl transfer under high oxygen pressure while under nitrogen, the activity was only slightly affected, indicating that some transferases are inherently resistant to this dithiol reagent . The effective target of the metabolic inhibitor arsenite could be glycolytic biochemical pathways (15) or oxidative biochemical pathways (16) . The evidence cited above excludes any role for mitochondrial oxidative phosphorylation in sialyl transfer . This conclusion is further corroborated by two lines of evidence : 1 . Dihydroxy acetone phosphate and phosphq~nol pyruvate, two glycolytic intermediates, enhanced the incorporation of C AcNeu under nitrogen . 2 . Glucose but not deoxyglucose significantly stimulated the activity under nitrogen, while glucose only slightly or not at all stimulated the activity under oxygen . The possibility that glucose and deoxyglucose influence the sialyltrans-
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ferase activity by contributing to biosynthesis of glycoproteins and glycolipids is not likely since in the present experimental system, the site of sialylation (the cell membrane) is physically dissociated from the intracellu1ar endoplasmic reticulum where the early stages of glycoprotein synthesis, such as incorporation of glucose and deoxyglucose, are generally believed to occur (cf . 17-19) . If the stimulating effect of glucose were due to an enhanced glucosyTfransferase activity at the cell surface, then a comparable effect of exogenous UDP-glucose should be expected, contrary to the experimental results obtained . The evidence thus jointly indicates that non-respiratory, notably glycolytic energy metabolism, probably including electro-chemical components, plays a part in sialyl transfer at the cell surface . This implies that other mechanisms are not responsible for the effects observed . Thus, the low incorporation of sialic acid under nitrogen was restored by oxygen to control levels bserved when the same cell preparation was immediately incubated with CMP- 1 ~C AcNeu under oxygen . These and other data suggest that elution of inhibitors, activators or enzyme into the medium, and hydrolysis of the added CMP-AcNeu, are not factors involved in the different levels of incorporation under nitrogen and under oxygen . Aq~ther important observation is that the susceptibility of the incorporC A Neu to exogenous neuraminidase was not different after incubation ated with CMP- ~4 C AcNeu under oxygen and nitrogen . The susceptibility of the 14C AcNeu after incorporation under these conditions was about 2/3, which is a criterion for location of all label at the cell surface in the present experimental system (B) . These latter data, combined with the observation of similar (rate-limited) time-dependency of incorporation under nitrogen and under oxygen and of proportionality between the sialyltransferase activities observed in the presence and absence of glucose under nitrogen, serve as indicators that similar enzymatic mechanisms are operating irrespective of the degree of stimulation caused by energy metabolism . The stimulatory effect of oxygen as compared with nitrogen as roughly inversely proportional to the absolute amount of incorporated 1 ~C AcNeu under the non-stimulated conditions . This may indicate that the sialyltransferase activity in each cell preparation may be optimized by incubating under oxygen and that the degree of depletion of fuel for non-respiratory energy metabolism in the intraperitoneally grown cells cf . 20, 21) may be critical for the magnitude of the stimulatory effect . Seemingly, endogenous fuel for these biochemical pathways only enhances the sialyltransferase activity under oxygen . The effect of high oxygen pressure in bringing about coupling between ectosialyltransferase activity and glycolytic biochemical pathways is interesting in view of data from other experimental systems indicating that oxygen tension may affect cellular differentiation (22) and neoplastic transformation (23,24) . Both these processes involve cell surface glycoconjugates . Glycolytic enzymes have long been known to be associated with the plasma membrane of various animal cells (1-7) including the Ehrlich ascites tumour cells (6 ) . The present finding that glycolytic biochemical pathways and not oxidative phosphorylation may stimulate the ecto-sialyltransferase activity of intact plasma membranes most probably indica~hat precisely this pool of membrane-bound glycolytic enzymes is involved in the effects observed . This extreme compartmentalization is surprising since inhibitors of energy metabolism which lower the intracellular level of ATP are known to affect amino acid transport and the function of Na+ - and IC+ -stimulated ATPase of intact plasma membranes in this and other experimental systems (25,26) . Apparently, the enzymes and acceptor molecules involved in ecto-sialyltransferase activity are
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located exterior to any influence that the intracellular level of ATP may exert, but at the same time they are affected by membraneous energy metabolism . ACKNOWLEDGEMENTS This work was generously supported by Dr . Gunner Ronquist and Dr . Gunner Agren through grants from the Swedish Medical Research Council (Project B75-13X-228-11), by Svenska Sällskapet fdr Medicinsk Forskning, The University of Uppsala, Fortia AB and Uddeholms AB . REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10 . 11 . 12 . 13 . 14 . 15 . 16 . 17 . 18 . 19 . 20 . 21 . 22 . 23 . 24 . 25 . 26 .
G. RONQUIST, Acta Ph siol . Scand. 74 594-605 (1968) . an tJTER, Bioc him. Biophys . Acta 382 (1975) . W. TILLMAN, A, T . L . STECK, J . Cell Biol . 62 1-19 (197~H . R. KNOLL, oc m. o h Acta 522 1-9 (1978) . Tands WESTERMARK, J . Cell . Physiol . 77 G. AGREN, J . , . 331-336 (1971) . C . 0. WERNSTEDT, G . K. AGREN and G . RONQUIST, Cancer Res . .35 1536-1541 (1975) . J . C. PARKER and J . F. HOFFMAN, J . Gen . Ph siol . 50 893-916 (1967) . E . CERVEN, Biochim. Bio h s . Acta 7j E . CERVEN, ~âÔl an sala J. Med . Sci . 80 58-60 (1975) . E .' L. KEAN, in Methods Enz ~1 . Vol , ar , p . 41422 (1972) . M. P. CZECH, J . o . em . J . L. WEBB, nzyme an e aboT~c Inhibitors , Vol . I . Academic Press, New York, (1966) . C . deCESPEDES and H . N . CHRISTENSEN, Biochim. Biophys . Acta 339 139-145 (1974) . J . KIELER, N . I . NISSEN and W . BICZ, Acta Unio Int . Cancrum 118 228-233 (1962) . J . F. KUO, I. K . DILL and C. E. HOLMLUND, Biochim. Biophys. Acta 148 683688 (1967) . W. BARTLEY and B. DEAN, Biochim: J . 115 903-912 (1969) . R. G. SPIRO, New En 1 . f$T 9~T--1001 (1969) . M. W. MYERS an . . LT;Biochim. Biophys . Res . Commun . 63 164-171 (1975) . R . C. HUGHES, A. MEAGER and R. NAIRN, Eur. J . Biochim. 72 265-273 (1977) . 0 . WARBURG and E. HIEPLER, Z . f. Natur orsc . _ (1~3) . G. KLEIN, Z . f. Krebsforsch . J . M. PAW ve . o . 1 52-72 (1969) . 0 . WARBURG, XVITÔIIôq:des-Physiol . Chem . (Mosbach, Baden ), Berlin, 1 (1966) . H . GOLDBLATT, L. FRIEDMAN and R . L . LECHNER, Biochim. Med . 7 241-252 (1973) . H . N . CHRISTENSEN, C deCESPEDES, M. E . HANDLO an . OAQUIST, Ann . N .Y . Acad . Sci . 22 7 255-379 (1974) . A-OWEIP~S .îß CAPLAN and A. ESSIG, Biochim. Biophys . Acta 394 438-448 (1975) .
s.