cross-linking of membrane proteins of metabolically-depleted and calcium-loaded erythrocytes

BritishJournal of Haematology, 1979,43,375-390.

Cross-Linking of Membrane Proteins of Metabolically-Depleted and Calcium-Loaded

Erythrocytes THERESA L. COETZER AND SOLAM S. ZAIL

Department ofHaematology, School ofPathology of the South African Institute for Medical Research and the University of the Witwatersrand,Johannesburg

(Received 12 September 1978; acceptedforpublication 15 December 1978)

SUMMARY. The membranes of erythrocytes undergoing metabolic depletion or an influx of calcium undergo several changes in structure and function. In erythrocytes incubated without substrate we find extensive cross-linking of membrane proteins by disulphide bonding occurring after 24-48 h, involving all major membrane proteins as well as haemoglobin. Aggregates of mol wt 40 x lo6 or greater are formed. These changes are partially reversible by repletion with adenosine. Rapid introduction of calcium (intracellular concentrations approximately 0.6 mM) into metabolically replete erythrocytes with the ionophore A23187 results in transglutaminase-dependent cross-linking of membrane proteins. Cellular calcium concentrations of approximately 0.3 mM have no cross-linking effect. Cells undergoing metabolic depletion show a progressive loss of transglutaminase activity to undetectable levels at 12 h, so that influx of calcium into such cells cannot cause cross-linking by a transglutaminase-mediated reaction. These studies suggest that the metabolic state of the cell and the rate and degree of calcium influx into erythrocytes are critical factors in determining the type of membrane protein cross-linkage.

Changes in membrane structure and function prior to haemolysis have been studied by several investigators in erythrocytes incubated in vitro under conditions of metabolic depletion (Reed & Swisher, 1966; Weed & Bowdler, 1966; Langley & Axell, 1968; Weed et al, 1969; Sears et al, 1975). Such changes include an increase in membrane-bound protein consisting of both globin and non-haemoglobin protein, as well as a decrease in erythrocyte filterability and deformability (Langley & Axell, 1968; Weed et a l , 1969; Sears et al, 1975). These effects have been correlated with an associated accumulation of calcium in the erythrocyte and wit1 falling ATP levels (Weed et al , 1969; Lichtman & Weed, 1973).

More recently Allen et a1 (1977) have found that such metabolically stressed erythrocytes also show calcium-dependent accumulation of membrane-bound catalase. This enzyme could be identified as a component of the membrane protein 4-5 (mol wt 60 000) on polyacrylamide gel electrophoresis with sodium dodecyl sulphate (SDS-PAGE). Lorand et a1 (1976) found that

Correspondence: Dr S. S. Zail, Department of Haematology, The South African Institute for Medical Research, P.O. Box 1038, Johannesburg 2000, South Africa.

0007-1048/79/1100-0375$02.00 0 1979 Blackwell Scientific Publications

3 75

376 Theresa L. Coetzer and Solam S . Z a i l

erythrocytes incubated in a calcium-containing medium together with the ionophore A23187 showed the presence of high molecular weight nondisulphide-bonded protein polymers in membranes isolated from such cells. Similar changes were found in energy depleted cells. These workers presented evidence that, in the presence of the ionophore, polymerization occurred through y-glutamyl-6-lysine bridges. They suggested that an intrinsic erythrocyte transamidase or transglutaminase is activated and mediates cross-linking of erythrocyte membrane proteins whenever there is an increase in intracellular calcium concentration, and that this might be responsible for the associated loss of deformability of such erythrocytes and predispose to their haemolysis.

In the present study we have also examined the phenomenon of cross-linking of membrane protein in metabolically-depleted erythrocytes. In such cells there is a much slower influx of calcium resulting in increased, although still relatively low levels of intracellular calcium after 48 h incubation (Weed et al, 1969; Lichtman & Weed, 1973; Palek et al , 1976). We have compared such erythrocytes to metabolically-replete erythrocytes into which small amounts of calcium have been rapidly introduced with the ionophore A23187. These studies suggest a model for alteration of erythrocyte membranes by the formation of two different types of cross-linked membrane protein which is dependent on the metabolic state of the cell and the rate and degree of influx of calcium into the erythrocyte. Part of this material has been presented previously in abstract form (Coetzer & Zail, 1978).

METHODS

Materials [I4C]Putrescine and 45CaC12 were obtained from the Radiochemical Centre, Amersham.

Putrescine dihydrochloride was obtained from Sigma Chemical Co., St Louis, and histamine dihydrochloride from E. Merck, Darmstadt. Ionophore A23187 was a generous gift from Eli Lilly and Co. All other standard chemicals were reagent grade.

Incubations without Ionophore Heparinized venous blood was obtained from healthy laboratory personnel and immedia-

tely cooled in an ice bath. The cells were washed four times in 10 vol o f09% NaCl at P C , care being taken to remove the residual buffy coat after each centrifugation. One volume ofpacked erythrocytes was suspended in 2.5 vol of fresh autologous plasma or alternatively in 9 vol of phosphate-buffered saline (PBS) prepared according to Dulbecco & Vogt (1954) and containing 0.9 mM CaC12 (final concentration). Glucose, when present, was added in a concentration of30 mM. Penicillin (100 u/ml) and streptomycin (0.1 mg/ml) were added to the flasks which were incubated at 37°C with shaking at 80 oscillations/min. In some experiments venous blood was anticoagulated with ethylene diamine tetraacetic acid (EDTA), washed in saline as above and incubated in PBS, pH 7.4, in which the calcium chloride was replaced by 5 mM EDTA. After incubation one aliquot of cells was taken for preparation of erythrocyte membranes and another for transglutaminase assay. In two experiments in which erythrocytes were incubated in PBS containing 0.9 mM CaC12 an additional aliquot of cells was washed four times in ice-cold 0.9% NaCl and total cellular uptake of calcium determined by atomic absorption spectroscopy in samples prepared by the method of Eaton et al(1973).

Cross-Linking of Erythrocyte Membrane Proteins 377

Incubations with Ionophore In some experiments the ionophore A23187 was added to 10% erythrocyte suspensions

prepared and incubated as above. The ionophore was dissolved in 1 O h dimethylsulphoxide and added in a final concentration of 5 p ~ . In other experiments heparinized venous blood was washed three times in 10 vol of ice-cold medium containing 75 mM NaC1,75 mM KCl, 10 mM Tris (pH 7.5) and 0.1 mM EDTA, followed by four washes in the same solution without EDTA, as described by Ferreira & Lew (1976). 1 ml of packed cells was then added to 9 ml of washing medium (without EDTA) to which had been added 1 mM MgClz, 10 mM inosine, 0.6 p~ ionophore A23187, 7.5 p c i 4sCaC12 (40 pCi/mg) and either 0.2 mM or 0.6 mM CaC12. Penicillin (100 u/ml) and streptomycin (0.1 mg/ml) were added to the flasks which were incubated at 37°C with shaking at 80 oscillationsfmin. The high potassium concentration was used to minimize the effects of an increase in erythrocyte potassium permeability due to the influx of calcium (Lassen et a l , 1974). One aliquot was taken for preparation of erythrocyte membranes and another for 4sCa uptake.

To determine 45Ca uptake, 2.0 ml aliquots were removed at various intervals, washed in an isotonic medium, pH 7.4, containing 2 x M Lac&, extracted with 10% trichloroacetic acid and counted in a Packard Tricarb Scintillation Counter as described by Lake et a1 (1977). A negligible correction was made by subtracting 4sCa uptake of control cells incubated without ionophore. Steady state concentrations of internal cellular calcium in ionophore-treated erythrocytes are reached after about 15 min under these conditions (Ferreira & Lew, 1976). In the presence of ionophore and external calcium concentrations of 0.1 mM or higher, 45Ca uptake under the above conditions in red cells can be used to calculate total cellular uptake of calcium, since Ferreira & Lew (1977) have shown that pump fluxes are then negligible (i.e. calcium uptake is the same as in metabolically-depleted cells where the ATP-dependent calcium pump is inactive). We have in addition confirmed this in ionophore-treated erythrocytes by measuring total cellular uptake of calcium by atomic absorption spectroscopy in samples prepared by the method of Eaton et aI(l973). Calcium uptake was found to be within 5% of those determined by 45Ca uptake.

Preparation of Erythrocyte Menibrunes After incubation packed erythrocytes were obtained after three washes with ice-cold 0.9%

NaCl. Erythrocyte ghosts were prepared by hypotonic lysis in 20 mOsm Tris buffer, pH 7.6, containing 1 mM EDTA and were solubilized in 1.0% sodium dodecyl sulphate (SDS) as described by Fairbanks et a1 (1971) except that mercaptoethanol (final concentration 1% v/v) was used in place of dithiothreitol in the solubilizing solution. The addition of EDTA to the lysing buffer was found to be essential as it inhibits cross-linking but does not reverse it once aggregates have formed (Steck, 1972; Wang & Richards, 1974; Liu et al, 1977). Duplicate membrane preparations were also solubilized in the absence of reducing agents.

Polyacrylamide Gel Electrophoresis In one-dimensional separations the membrane polypeptides were separated and stained with

Coomassie Blue after electrophoresis in 3.0% polyacrylamide gels containing 0.1 % SDS (SDS-PAGE) as described by Fairbanks et a1 (1971). 50 pg of membrane protein were loaded on all gels. Protein was measured by the method of Lowry et al (1951) using bovine serum

378 Theresa L. Coetzer and Solam S . Zai l

albumin as standard. Estimates of molecular weights of large membrane protein aggregates were made by linear extrapolation of plots of log molecular weight versus mobility curves, using as standards the values assigned to the major membrane proteins by Steck (1972). Two-dimensional separations in polyacrylamide gels were performed essentially as described by Wang & Richards (1974). After the separation of non-reduced membrane proteins in 3% cylindrical gels as described above the proteins were separated in the second dimension in a 5.6% slab polyacrylamide gel after initial passage through a 20 mm layer of 1% agarose containing 10% mercaptoethanol on top of the slab gel.

Gel-@ration Chromatography in S D S Erythrocyte membranes (4-10 mg protein) were solubilized in 0.01 M Tris buffer, pH 7.8,

containing 4% SDS and 10% sucrose, layered on a 1 x 30 cm Sepharose-2B column and eluted at 25°C with 0.01 M Tris buffer, pH 7.8, containing 1% SDS a t a flow rate of 6 ml/h. The absorbance of the eluate was monitored continuously a t 280 nm.

Transglutaminase Assay Packed erythrocytes were obtained after incubation and washed three times with ice-cold

0.9% NaCI. One volume of packed cells was lysed in 2 vol of water and frozen and thawed once before assay. Transglutaminase activity was determined by measuring the incorporation of ['4C]putrescine (60 mCi/mmol) into N,N'-dimethylcasein. The reaction conditions were those of Dvilansky et al (1970), except that thrombin was omitted and N,N'-dimethylcasein (prepared according to Lin et al, 1969) was used in place of casein. Alkylation of the free amino groups of lysine prevents casein from acting as an amine donor and cross-linking itself (Curtis & Lorand, 1976). The reaction mixture contained 0.3 ml of N,N'-dimethylcasein (10 mglml), 0-3 ml of 0.5 M Tris buffer, pH 7.5, and 0.1 ml of each of the following: ['4C]putrescine 7.3 x M; EDTA (in Tris) 0.001 M; CaC12 (in Tris) 0.1 M; mercaptoethanol (in Tris) 0.5 M;

haemolysate prepared as above. Blank assays contained 0.1 M EDTA in place of CaC12. All assays were done in duplicate. The mixture was incubated at 37°C for 30 min, the reaction being linear until at least 60 min, and then terminated by precipitation with 4 ml ice-cold 7.5% trichloroacetic acid (TCA). The precipitate was washed once with 5 ml ethanol/ether (l/l), dissolved in 0.4 ml1-0 M NaOH and precipitated with 3 ml5% TCA. After redissolving again in NaOH and reprecipitating in TCA, the pellet was digested with 0.2 ml60% perchloric acid and 0.4 ml30% H 2 0 2 and incubated in stoppered tubes at 70°C for 2 h. After cooling, 10 ml Instagel was added, transferred to glass vials and counted in a Packard Tricarb Scintillation Spectrometer.

RESULTS

Membrane Proteins of Substrate-Depleted Erythrocytes The membrane proteins, solubilized in the absence and presence of mercaptoethanol, of

control erythrocytes prior to incubation are shown in Figs l(a) and l(b) respectively. The polypeptides are numbered 1-7 according to the convention of Steck (1972). The most anodal band is globin. Several very faint bands could be seen between band 1 and the top of the gel in control membranes (Fig la) which disappeared after mercaptoethanol reduction (Fig 1 b).


These were probably artefactual and appeared to be due to spontaneous cross-linking of membrane proteins by disulphide bonds (Liu et al, 1977) even in the presence of EDTA in the lysing buffer. The membrane polypeptides of cells incubated for 48 h in autologous plasma without added glucose are shown in Fig l(c) (non-reduced) and Fig l(d) (reduced). The corresponding patterns for erythrocytes incubated in the absence of substrate in calcium-containing PBS and calcium-free PBS are shown in Figs l(e), l(f), l(g) and l(h) respectively. In all three incubation systems a similar pattern of non-reduced membranes was found. This consisted of a high molecular weight aggregate (minimum molecular weight 700 000) which remained at the top of the gels, the presence of two faint bands corresponding to apparent molecular weights of approximately 400 000 and 350 000, a band present just cathodal to band 1 of apparent molecular weight 250 000 and a diminution in the intensity of staining of globin and bands 1-7, including minor components such as 2.1, 2.2, 2.3 and 4.5. Addition of mercaptoethanol to the solubilizing solution resulted in restoration of the original pattern of fresh membranes, except that there was a greater accumulation of globin as well as a slight increase in staining intensity of the ill-defined 4.5 area, and the presence of a new component between band 7 and globin (apparent molecular weight 24 000). Incubation of erythrocytes with the alkylating agent N-ethylmaleimide (5 mmol/l erythrocytes) completely prevented the formation of aggregates (not shown). These findings were highly reproducible in three experiments and indicated partial cross-linking of almost all membrane components as well as globin into a high molecular weight aggregate with a minimum molecular weight of 700 000.

SDS-PAGE of membranes isolated after 24 h incubation in calcium-containing PBS was similar to the 48 h pattern (Figs 2c and 2d), but the degree of cross-linking of membrane proteins was more variable than that found at 48 h and usually less pronounced than that depicted. Similar findings were obtained in calcium-free incubations. In two experiments total erythrocyte calcium concentration in cells incubated in PBS containing 0.9 mM CaClz increased from a mean basal concentration of 5 p~ to 32 p~ at 24 h and 68 PM at 48 h.

EJects of Substrate Repletion Fig 2(e) shows that addition of 30 mM adenosine to ATP-depleted cells partially reversed the

formation of the high molecular weight aggregate after an additional 4 h incubation. Although large amounts of the high molecular weight aggregate were still present, there was an increased intensity of staining of bands 1, 2,2.1, 2.2,4.1 and 4.2 when compared to Fig 2(c). Reduction with mercaptoethanol resulted in complete reversal of aggregation (Fig 2f). Incubation of cells ab initio with substrate (30 mM glucose or adenosine) completely prevented aggregate formation (not shown).

Two-Dimensional SDS-PAGE The extensive cross-linking of membrane proteins after 24 h incubation in calcium-contain-

ing PBS is also shown in a two-dimensional separation of SDS-solubilized membranes with reduction prior to separation in the second dimension (Fig 3a). In three experiments bands 1 and 2 (spectrin) formed the major components of the high molecular weight aggregate at the top of the first dimension. A protein of apparent molecular weight 450 000 seen in the first dimension gel (solid arrow) appears to consist of spectrin. The composition of the complex of apparent molecular weight of 250 OOO in the first dimension gel (dotted arrow) could not be

380 Theresa L. Coetzer and Solam S . ZaiJ

FIG 1. SDS-PAGE of membrane proteins of erythrocytes incubated for 48 h. (a) and (b) Non- reduced and reduced membranes of control (unincubated) erythrocytes; (c) and (d) non-reduced and reduced membranes of erythrocytes incubated in plasma; (e) and (f) non-reduced and reduced membranes of erythrocytes incubated in PBS containing calcium (0.9 mM); (9) and (h) non-reduced and reduced membranes of erythrocytes incubated in calcium-free PBS. Membrane proteins were reduced with 1 % (v/v) mercaptoethanol in the solubilizing solution.

FIG 2. Effects of substrate repletion on SDS-PAGE of erythrocyte membrane proteins. (a) and (b) Non-reduced and reduced membrane proteins of control (unincubated) erythrocytes; (c) and (d) non-reduced and reduced membrane proteins of erythrocytes incubated for 24 h in PBS containing calcium (0.9 mM); (e) and (f) non-reduced and reduced membrane proteins of erythrocytes incubated for 24 h in PBS plus calcium and then for an additional 4 h after addition of adenosine (30 mM). Membrane proteins were reduced with 1% (v/v) mercaptoethanol in the solubilizing solution.

Cross-Linking of Erythrocyte Membrane Proteins

FIG 3. Two-dimensional SDS-PAGE of erythrocyte membranes. (a) Membranes of erythrocytes incubated for 24 h in PBS containing calcium (0.9 mM); (b) membranes oferythrocytes incubated for 24 h in PBS plus calcium and then for an additional 4 h after addition ofadenosine (30 mM). Control (unincubated) membranes are shown a t the left of the slab gels. The agarose layer containing 10% mercaptoethanol through which the membrane proteins are electrophoresed after separation on the

381

first dimension is not shown.

382 Theresa L. Coetzer and Solam S. Zail

completely resolved and appears to consist a t least partially ofspectrin (band 1 or 2). The partial reversal of membrane protein aggregation after additional incubation for 4 h with 30 mM adenosine is also seen in the two-dimensional separation shown in Fig 3(b).

Gel Filtration Chromatography in SDS Since it was possible that membrane protein aggregates of very high molecular weight

(> 7 x lo5 daltons) might not enter the 3% polyacrylamide gels, membrane proteins obtained after 48 h incubation were dissolved in SDS and subjected to gel filtration chromatography using Sepharose-2B (Fig 4b). Control membranes are shown in Fig 4(a). A peak appearing coincident with the void volume indicated a considerable amount of cross-linked material of molecular weight 40 x lo6 or greater in the membranes of the incubated erythrocytes. When subjected to SDS-PAGE in 3% gels, this fraction (or part thereof) remained at the top of the

02 0

N m

0

b

031

6 12 18 2 4 30 Volume mi

FIG 4. Gel filtration chromatography of non-reduced SDS-solubilized membrane proteins. (a) Control (unincubated) membranes (10 mg); (b) membranes of erythrocytes incubated for 48 h in plasma (7 mg); (c) membranes of erythrocytes incubated in PBS (0.9 mM calcium) with 5 pmol A23187 per litre cell suspension (4 mg). Vo represents the void volume.


gel, but after reduction with mercaptoethanol completely entered the gel and gave a pattern similar to that seen for the cross-linked aggregate of approximately 7 x lo5 daltons depicted in the 2-D separation in Fig 3(a). This strongly suggested that the detection of the high molecular weight aggregate at the top of the 3% gels was a valid indicator for the formation of membrane protein aggregates of much higher molecular weight in substrate-depleted erythrocytes.

Efects of High Concentrations of Ionophore on Erythrocyte Membrane Proteins The effects of high concentrations of the ionophore A23187 (5 pmol/l cell suspension) on

erythrocytes incubated in PBS under various conditions was determined in three separate experiments, one of which is depicted in Fig 5. The changes induced in the membrane proteins were similar in all three experiments and are summarized as well in Table I. At this concentration of ionophore approximately 80% of the calcium in the medium is taken up by the erythrocytes (Kirkpatrick et al, 1975), resulting in an intracellular calcium concentration of approximately 7 mM. Addition of this concentration of the ionophore A23187 to erythrocytes incubated for 6 h in PBS containing calcium (0.9 mM) without added substrate, resulted in the formation of a high molecular weight aggregate with a minimum molecular weight of 700 000 which remained at the top of the gel (Fig 5h) and which was resistant to mercaptoethanol reduction (Fig 5i). Small amounts of three aggregates of apparent molecular weight 440 000,400 000 and 290 000 were also formed and there was an increased intensity of staining of band 2.3. These changes were associated with the disappearance of bands 2.2 and 4.1 and with marked diminution in intensity of staining of band 6. In addition the intensity of staining of bands 1 and 2 and 2.1 was decreased. Similar changes were noted in the presence of substrate (Figs 51 and 5m). Gel filtration chromatography of membranes of such ionophore-treated erythrocytes (Fig 4c) showed that, like metabolically-depleted cells, very high molecular weight aggregates of 40 x lo6 daltons or greater are formed. These aggregates, however, could not be dissociated by mercaptoethanol. Addition of histamine (10 mM), an alternate substrate for transglutaminase (Lorand et al, 1972), to the medium prevented the formation of the high molecular weight aggregates (not shown), indicating that their formation was transglutaminase dependent, findings similar to those of Lorand et al(1976).

It is of interest that the ionophore itself caused perturbation of the membrane proteins. Addition of ionophore to cells incubated in the absence of substrate and calcium resulted in the formation of a small amount of high molecular weight aggregate which remained at the top of the gel (Fig 5j) as well as faint bands of apparent molecular weight 440 000 and 400 000. This was associated with some reduction in the intensity of staining of bands 4.2, 5 and 6. These changes were completely reversible by reduction with mercaptoethanol (Fig 5k). The presence of substrate (30 mM glucose) completely prevented the ionophore-induced aggregate of minimum molecular weight of 700 000, but not the lower molecular weight aggregates (Fig 5 4 , although these disappeared after mercaptoethanol reduction (Fig 50). In the absence of ionophore, cells incubated with or without calcium or glucose for 6 h showed minimal changes (Figs 5b-5g). A consistent slight decrease in intensity of staining of bands 4.2 and 5 was present in the non-reduced membranes which was reversed after reduction with mercaptoethanol.

Effects OJLOW Concentrations oflonophore on Erythrocyte Membrane Proteins At the concentrations of ionophore used in the above experiments intracellular calcium

384 Theresa L. Coetzer and Solam S . Zai l

FIG 5. SDS-PAGE of membrane proteins of erythrocytes incubated for 6 h without and with high concentrations of ionophore A23187 (5 pmol/l cell suspension). (a) Reduced membranes of control (unincubated) erythrocytes; (b) and (c) non-reduced and reduced membranes of erythrocytes incubated for 6 h in PBS containing calcium (0.9 mM); (d) and (e) non-reduced and reduced membranes oferythrocytes in calcium-free PBS; (0 and (g) non-reduced and reduced membranes of erythrocytes incubated in PBS containing calcium (0.9 mM) and glucose (30 mM); (h) and (i) non-reduced and reduced membranes of erythrocytes incubated in PBS containing calcium (0.9 mM) and A23187; (j) and (k) non-reduced and reduced membranes of erythrocytes incubated with A23187 in calcium-free PBS; (1) and (m) non-reduced and reduced membranes of erythrocytes incubated with A23187 in PBS containing calcium (0.9 mM) and glucose (30 mM); (n) and (0) non-reduced and reduced membrane proteins of erythrocytes incubated with A23187 in calcium- free PBS containing glucose (30 mM). Membrane proteins were reduced with 1% (v/v) mercaptoethanol in the solubilizing solution.

Cross-Linking of Erythrocyte Membrane Proteins 385 concentrations are found which are far in excess of that occurring in physiological or pathological changes in erythrocytes. In addition, using such concentrations of ionophore, we found at least 50% haemolysis even after short periods of incubation. Accordingly, we introduced smaller amounts of calcium into erythrocytes using the experimental conditions of Ferreira 81 Lew (1976) in which much lower concentrations of ionophore (0.6 ,MM) were employed. At external medium calcium concentrations of 0.6 mM and 0.2 mM, cellular uptake of calcium was 575 and 302 pmol/l of packed erythrocytes respectively after 3 h incubation. Haemolysis was always less than 1 YO under these conditions. Fig 6 represents the results of two such experiments which are also summarized in Table I. Only mercaptoethanol-reduced membranes are shown, but the findings were identical in non-reduced membranes. Erythro- cyte calcium uptake ofapproximately 300 pmol/l of cells did not cause detectable cross-linking (Fig 6d), but there was a slight accentuation in the intensity of staining of band 2.3, which was not affected by the presence ofhistamine (10 mM) in the incubation of medium (Fig 6e). With a calcium uptake of 575 pmol/l of erythrocytes, small amounts of a high molecular weight aggregate were found a t the top of the gel (arrowed in Fig 6f) and there was an intensification in the staining of band 2.3 and disappearance of band 4.1. Globin binding was also increased. Addition of histamine to the incubation medium prevented the formation of the high molecular weight aggregate but band 4.1 did not reappear and there was no effect on the increased staining of band 2.3 or the binding of globin (Fig 6g). Cells incubated in the absence of ionophore (Figs 6b and 6c) did not show these changes when compared to an unincubated control (Fig 6a). In three experiments, the usual pattern noted in the presence of ionophore and 0.6 mM calcium in the incubation medium was the formation of the high molecular weight aggregate of minimum molecular weight of 700000 at the top of the gel as in Fig 6(f). However, on one occasion, the disappearance of band 4.1 (molecular weight 78 000) was associated with the appearance of a new band anodal to band 2.3 (Fig 6h, side arrow), of apparent molecular weight 150 000. Band 2.2 also showed a decreased intensity of staining. Addition of histamine to the medium led to the reappearance of bands 4.1 and 2.'2 and prevented the formation of the protein of apparent molecular weight 150 000 indicating these were transglutaminase-dependent processes.

Transglutaminase Activity of Incubated Erythrocytes Transglutaminase activity of red cell lysates prepared from washed cells after incubation

without substrate in a calcium-containing and calcium-free medium is shown in Table 11. There was a progressive fall in the activity of this enzyme to undetectable levels after 12 h incubation, when measured in the presence of 10 mM Ca++ and N,N'-dimethyl-casein in the assay reaction mixture. Incubation for 24 h in the presence of substrate (30 mM glucose) resulted in a fall in activity of approximately 25% (not shown).

DISCUSSION

In the present study we find no evidence in support of the premise that cross-linking of membrane proteins in energy-depleted erythrocytes is a transglutaminase-dependent process. Such cells, whether incubated in autologous plasma or in protein-free buffer, develop high molecular weight aggregates ranging from 250 000 to approximately 40 x lo6 daltons or

386 Theresa L. Coetzer and Solam S. Zail

FIG 6. SDS-PAGE of membrane proteins of erythrocytes incubated for 3 h without and with low concentrations of ionophore A23187 (0.6 pmol/l cell suspension). Only membrane preparations reduced with 1% (v/v) mercaptoethanol are shown as the non-reduced preparations were identical. The incubation medium is that of Ferreira & Lew (1976). (a) Membranes of control (unincubated) erythrocytes; (b) and (c) membranes of erythrocytes incubated without ionophore; calcium concentration was0.6 KIM; (d) and (e) membranesoferythrocytes incubated with A23187 without and with histamine (10 mM); calcium concentration was 0.2 mM; (0 and (g) membranes of erythrocytes incubated with A23187 without and with histamine; calcium concentration was 0.6 mM; (h) and (i) same conditions as (0 and (g) in a separate incubation. The arrow at the top of gel findicates a high molecular weight aggregate at the top of the gel. The arrow at the right side of the figure indicates the position of a new protein in gel h (see text for explanation).

Cross-Linking of Erythrocyte Membrane Proteins 387 TABLE I . Effect of various concentrations of intracellular calcium on membrane proteins of

metabolically-replete erythrocytes (for conditions of incubation see Methods)

Intracellular Transglutaminase cafcium cross-linked high

concentration molecular weight aggregate (PM) (minimum mol wt 700 OW) Band 4.1 Band 2.3 Other effects

302 575

7 000

-* + t Moderate increase in globin binding + - Marked increase in globin binding.

Variable formation of probable dimer of 4.1. Variable decrease in 2.2.

+ Formation of aggregates of mol wt 440 000,400 ooo, 290 000. Absence of 2.2. Diminished staining of 1,2,2.1,6. Marked increase in globin binding.

* -=absent; +=present; ?=accentuated staining.

TABLE 11. Transglutaminase activity of lysates of incubated erythrocytes. 10% erythrocyte suspensions in PBS were in-

cubated at 37°C.

['4C]Putrescine incorporated (nmollhlmg protein)

Time (h) Calcium in medium (0.9 mM) Calcium-free medium

0 2 4 6 8

10 12 14 16 18 20

4.20 3.50 1.92 0.86 0.14 0.10 -

4.40 2.30 1 5 0 1.08 0.24 0.15 -

greater. These aggregates are completely reversible after reduction with mercaptoethanol and their formation is prevented by incubating cells with the alkylating agent, N-ethylmaleimide, suggesting that they are disulphide-bonded. Their formation in whole erythrocytes is at least partially reversible as evidenced by the effects of reincubation of metabolically-depleted cells with adenosine.

We find also that the formation of these membrane aggregates in incubations of cells which have undergone metabolic depletion is not dependent on the presence of calcium in the external medium, as incubation in a calcium-free medium has no effect on this process. Basal

388 Theresa L. Coetzer and Solam S. Zai l

erythrocyte calcium concentration has been variously estimated at between 1 ,UM (Schatzman, 1973) and 15 ,UM (Harrison & Long, 1968; Lichtman & Weed, 1973; Palek et al , 1976). Cellular calcium concentrations in metabolically depleted erythrocytes incubated in plasma may rise to approximately 30 and 70 ,UM after 24 and 48 h incubation respectively (Lichtman & Weed, 1973; Palek et a l , 1976), concentrations which are considerably less than earlier estimates (Weed et a l , 1969). We found almost identical low values in erythrocytes incubated in PBS containing 0.9 mM calcium. In pathological erythrocytes such as in homozygous sickle cell disease, erythrocyte calcium concentrations ranging from 110 to 302 ,UM have been reported (Eaton et a l , 1973). Lower levels have been found by Palek et a1 (1976) rising to approximately 100 ,UM

after 4 h incubation under deoxygenated conditions. Such concentrations of calcium could conceivably give rise to cross-linking of membrane proteins by activation of erythrocyte transglutaminase. However, information on the dependence of cross-linking on cellular calcium concentration in intact erythrocytes is lacking. We find no evidence of protein cross-linking in reduced or non-reduced membranes of erythrocytes in which calcium concentration has been rapidly elevated by the ionophore A23187 to approximately 300 ,UM. At approximately 600 ,UM cellular calcium concentration, transglutaminase-dependent cross-linking of membrane protein can be detected, a figure which agrees closely with the concentration of calcium (500 ,UM) which is necessary to activate transglutaminase in erythrocyte lysates (Lorand et al , 1976).

The perturbations of membrane proteins induced at low cellular calcium concentrations require further comment. At calcium concentrations of 300 ,UM the only change observed was an intensification of staining of band 2.3, which was not transglutaminase-dependent, as histamine had no effect on this process. At a concentration of 600 ,UM the usual pattern was a further intensification of staining of band 2.3, the development of a high molecular weight aggregate a t the top of the gel and a complete disappearance of band 4.1. The formation of the high molecular weight aggregate only appeared to be transglutaminase-dependent as histamine reversed only this process. These findings suggest that the intensification of staining of band 2.3 (apparent molecular weight 160 000) may be due to dimer formation of band 4.1 (apparent molecular weight 78 000). However, in the experiment depicted in Figs 6(h) and 6(i), the formation of a new protein of apparent molecular weight 150 000 is also associated with the disappearance of 4.1 and could be due to dimer formation of4.1. Its formation, as well as the disappearance of 2.2 and 4.1, appears to be transglutaminase-dependent in that histamine reverses this process. The possibility that this new protein could be derived from 2.2 (e.g. by proteolysis) is unlikely as the intensity of staining of 2.2 in the cells incubated with histamine is less than the intensity of staining of the new protein. At present we can offer no reasonable explanation for the variation in the properties of 2.2 and 4.1 in these experiments.

These findings indicate that considerable increases in erythrocyte calcium are required to initiate transglutaminase-dependent membrane protein cross-linking. Such concentrations are approximately 7-8 times higher than that found in metabolically-depleted erythrocytes and 2-3 times that found in sickle cells, which at present represents the only well-documented pathological condition associated with a rise in erythrocyte calcium. However, a t lower concentrations of cellular calcium (300 ,UM) some perturbation of membrane protein is discernible in the slight intensification of staining of band 2.3, and probably represents the earliest change in calcium-loaded erythrocytes.

Cross-Linking of Erythrocyte Membrane Proteins 389 A finding of some interest in this study was the progressive loss of transglutaminase activity

in cells undergoing ATP depletion. A recent report by Brenner & Wold (1978) that ATP is required as a stabilizing agent during purification of erythrocyte transglutaminase may have some bearing on this observation. Since it was found that the requirement for ATP became less pronounced as the enzyme was purified, they suggested that ATP may act as a negative effector for an enzyme which could inactivate erythrocyte transglutaminase. Conceivably a similar mechanism could be operative in ATP-depleted cells. Whatever the explanation, an influx of calcium into such erythrocytes resulting in concentrations that could activate transglutaminase, would have no effect if this process occurred after 10-12 h ofATP-depletion, as there is no detectable transglutaminase activity at this time (Table 11). This finding, together with those outlined above, has some implications in suggesting a model for perturbations in erythrocyte membrane proteins which is dependent on the metabolic state of the cell and the rate and degree of calcium influx into such cells. Under conditions of ATP-depletion, cross-linking of erythrocyte membrane protein occurs only by disulphide linkages. This process is not dependent on the presence of calcium in the incubation medium. If an influx of calcium occurs into metabolically-replete erythrocytes or in the early stages of incubation of erythrocytes without substrate, cross-linking of membrane proteins occurs by a transglutaminase-mediated reaction, provided cellular calcium concentrations of the order o f 0 5 mM are attained. Influx of calcium into erythrocytes in the late stages of metabolic depletion cannot cause transglutaminase-dependent cross-linking of membrane proteins. If these findings can be extrapolated to the in vivo situation, they may lead to a better understanding of some of the properties of membranes in erythrocytes which may undergo ATP-depletion or calcium influx, e.g. in the process of erythrocyte senescence or in pathological situations such as the congenital non- spherocytic haemolytic anaemias where ATP generation may be compromised.

After this work was completed two reports appeared relating to the present study. A report appeared by Palek et al(1978) in which disulphide-bonded membrane protein aggregates very similar to those described above were found in ATP-depleted erythrocytes. A similar lack of dependence of aggregate formation in such cells on calcium in the external medium was found. A study also appeared by Anderson et al (1977) confirming the observations of Lorand et al (1976) on the effects of calcium on erythrocyte transglutaminase activity. In relation to this study, they too found that aggregate formation is not significant until the calcium concentration approaches 0.5 mM, and presented convincing evidence that disappearance of band 2.2 and accentuation of 2.3 is due to calcium-dependent proteolysis of 2.2. Disappearance of band 4.1 was correlated grossly with aggregate formation, but specific evidence that this could be a transglutaminase-dependent process was not presented.

ACKNOWLEDGMENTS

These studies were supported by grants from the Medical Research Council of South Africa and The Atomic Energy Board.

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