Proteolysis of ankyrin and of band 3 protein in chemically induced cell fusion. Ca2+ is not mandatory for fusion.

Human erythrocytes were fused by incubation with 0.5-2 mM-chlorpromazine hydrochloride at pH 6.8-7.6. Fusogenic preparations of chlorpromazine were cloudy suspensions of microdroplets, and below pH 6.8 chlorpromazine gave clear solutions that were inactive. Unlike control cells, the lateral mobility of the intramembranous particles of the PF-fracture face of chlorpromazine-treated cells was relatively unrestricted, since the particles were partly clustered at 37 degrees C and they exhibited extensive cold-induced clustering. Ca2+ stimulated fusion, but fusion was only very weakly inhibited by EGTA (10 mM) and by N-ethylmaleimide (50 mM); pretreatment of the cells with Tos-Lys-CH2Cl (7-amino-1-chloro-3-L-tosylamidoheptan-2-one) (7.5 mM) markedly inhibited fusion. Changes in the membrane proteins of erythrocytes fused by chlorpromazine, before and after treatment with chymotrypsin to remove band 3 protein, were investigated. The several observations made indicate that the Ca2+-insensitive component of fusion is associated with degradation of ankyrin (band 2.1 protein) to band 2.3-2.6 proteins and to smaller polypeptides by a serine proteinase that is inhibited by Tos-Lys-CH2Cl, and that the component of fusion inhibited by EGTA and N-ethylmaleimide is associated with degradation of band 3 protein to band 4.5 protein by a Ca2+-activated cysteine proteinase. Proteolysis of ankyrin appeared to be sufficient to permit the chlorpromazine-induced fusion of human erythrocytes, but fusion occurred more rapidly when band 3 protein was also degraded in the presence of Ca2+. Since other cells have structures comparable with the spectrin-actin skeleton of the erythrocyte membrane, the observations reported may be relevant to the initiation of naturally occurring fusion reactions in biomembranes. It is also suggested that, should polypeptides with fusogenic properties be produced from integral and skeletal membrane proteins by endogenous proteolysis, their formation would provide a general mechanism for the fusion of lipid bilayers in biomembrane fusion reactions.     

Ca++-dependent disassembly and reassembly of occluding junctions in guinea pig pancreatic acinar cells. Effect of drugs

Incubation of guinea pig pancreatic lobules in Ca++-free Krebs-Ringer bicarbonate solution (KRB) containing 0.5 mM ethylene glycol-bis(beta- aminoethyl ether)N,N,N',N'-tetraacetate (EGTA) results in the progressive fragmentation of the occluding zonulae (ZO) with formation of multiple discrete junctions (fasciae occludentes) localized in the lateral and lumenal plasmalemma. After 1--2 h of such incubation, most ZO appear completely disassembled. This results in the disappearance of the heterogeneity in density of intramembrane particles on the P- fracture faces of the basolateral and lumenal plasmalemma. If Ca++ ions are reintroduced into the incubation fluid at this point, continous zonulae reform around the apices of the cells; in contrast, the density of intramembrane particles (imp) at the lumenal plasmalemma remains the same as in the basolateral region, at least for 3 h after Ca++ reintroduction. When added to the incubation fluid, cycloheximide (at a dose known to inhibit protein synthesis greater than 95%) and cytochalasin B (at doses which disrupt microfilaments and modify the cell shape) had no effect on the organization of ZO, on their disassembly in Ca++-free, EGTA medium, or on their Ca++-dependent reformation. Likewise, the organization and disassembly of ZO were unaffected by colchicine; however, after treatment with the latter drug the reassembly was defective, with formation of strand networks on the lateral surface and incomplete segregation of the lumenal region. Antimycin A, on the other hand, when added to the Ca++-EGTA medium, induced a large proliferation of long, infrequently anastomosed junctional strands, usually arranged to form ribbons, festoons, and other bizarre arrays. The possible relationship of these in vitro findings to the in vivo biogenesis and turnover of occluding junctions is discussed. It is suggested that the impairment of reassembly of zonulae by colchicine might be correlated with the disorder induced by the drug on the general organization of pancreatic exocrine cells. Moreover, antimycin A could act by promoting the aggregation of a pool of free junctional strand components (or precursors) that might exist normally in pancreatic exocrine cells.     

Redistribution of intramembrane particles of human erythrocytes induced by HVJ (Sendai virus): A prerequisite for the virus-induced cell fusion

Aggregation of intramembrane particles of human erythrocytes was found to be induced by HVJ (Sendai virus) under conditions which lead to cell fusion. Degree of polyerythrocyte formation was compared under a variety of conditions with extent of cluster formation observed with the same preparations. Both structural changes of the membranes, ie, fusion and clustering of the particles, behaved very similarly under widely different virus-to-cell ratios and over the time course of cell fusion. Furthermore, by inclusion of high concentrations of antispectrin antibodies within the ghosts, inhibition of clustering of intramembrane particles and hindrance of virus-induced cell fusion were found to occur simultaneously. Antibodies by themselves did not induce aggregation of particles under isotonic conditions, whereas particle clustering could be induced under hypotonic conditions at antibody concentrations causing partial cross-linking of spectrin molecules. In conclusion, clustering of intramembrane particles seems to be required for virus-induced fusion of human erythrocytes.     

Rapid increases in inositol trisphosphate and intracellular Ca++ after heat shock

Heat shock (45 degrees C) caused a rapid (less than 1 min) release of inositol trisphosphate from the membranes of HA-1 CHO fibroblasts. The rise in inositol trisphosphate concentration was followed by an increase in intracellular free Ca++. In addition to the heat induced rise in intracellular free Ca++, we observed an increase in 45Ca++ influx following nonlethal heat shock (45 degrees C/10 min). The heat-induced increase in 45Ca++ influx was linearly related to membrane accumulation of phosphatidic acid, phosphoinositide metabolite that may be involved in Ca++ gating. These results suggest that the membrane may be the proximal target of heat shock; stimulation of rapid breakdown of polyphosphoinositides and subsequent increases in intracellular free Ca++ may provide a mechanistic insight into the pleiotropic effects of heat. In addition, the large increases in Ca++ influx could initiate a Ca++ dependent mechanism of thermal cell killing.     

Use of chlortetracycline fluorescence for the detection of Ca storing intracellular vesicles in normal human erythrocytes

The uptake of chlortetracycline (CTC) and the nature of the fluorescence of CTC was studied in intact human erythrocytes from apparently healthy donors. The uptake of CTC at 22 degrees C proceeded with a t1/2 of about 3 min, and after 15 min a stable equilibrium was achieved with an intracellular accumulation by a factor of 5-6 relative to the medium concentration. The accumulation did not change in the range of CTC concentrations tested (20-500 microM). The Ca specificity of the CTC fluorescence spectrum was confirmed by Ca depletion of red cells using A23187 in the presence of EGTA and 0.2 mM Mg. This procedure decreased the total intracellular calcium content by about 70% and reduced the fluorescence intensity to one-fourth. Fluorescence microscopy of red cells incubated with 100 microM CTC at 22 degrees C showed that the fluorescence originated mainly from the red cell membrane. In addition, in about 15% of erythrocytes one or more fluorescent dots (diameter greater than 0.2 less than 1 microns) were detected. The fluorescence of the dots and membranes was related to calcium, as evidenced by the reduction of their intensity in Ca depleted cells. The number of erythrocytes with fluorescent dots and the frequency of the dots per cell was largely unaffected by lowering the incubation temperature to 0 degrees C, indicating that the dots most probably do not represent endocytotic artifacts induced by CTC. The number of dots was increased in erythrocytes preincubated with primaquine, demonstrating that CTC fluorescence can be applied to monitor the appearance of intracellular Ca storing vesicles. It is concluded that in (at least) 15% of erythrocytes obtained from apparently healthy donors intracellular vesicles containing Ca can be detected by CTC fluorescence microscopy.     

Inhibition of membrane fusion by suppression of lateral movement of membrane proteins.

Chicken erythrocytes were fused either by Sendai virus or by the combination of Ca2+ and ionophore A23187. Intramembrane particles and external anionic sites of cells undergoing fusion were found to acquire the ability to undergo a process of cold-induced clustering (thermotropic separation). Cationized ferritin (200 microgram/ml 5% (v/v) cell suspension) inhibited both the fusion process and the thermotropic separation of intramembrane particles and external anionic sites. The correlation between the mobility of membrane proteins and the fusion process is discussed. It is suggest that an increase in the lateral mobility of membrane proteins is a prerequisite for initiation of membrane fusion.     

Calpain activation is essential for membrane fusion of erythrocytes in the presence of exogenous Ca2+.

The membrane mobility agent, 2-(methoxyethoxy)ethyl-cis-8-(2-octylcyclopropyl)octanoate (A2C) promotes the fusion of rat, rabbit, and human erythrocytes in the presence of exogenous Ca2+. Under these conditions, the high sensitivity form of calcium-activated neutral protease (mu-calpain) in erythrocytes is activated autolytically. mu-Calpain is activated in accordance with fusion; that is, both erythrocyte fusion and autolytic activation of mu-calpain are induced in rat erythrocytes at 30 min, in rabbit erythrocytes at 150 min, and in human erythrocytes at 240 min after the addition of A2C and Ca2+. When erythrocytes are preincubated with the Ca2+ ionophore A23187, both fusion and autolytic activation start earlier. A leupeptin analogue, Cbz-Leu-Leu-Leu-aldehyde (ZLLLal), inhibits both the autolytic activation of mu-calpain and fusion induced by A2C and Ca2+. These results indicate that treatment of erythrocytes with A2C and Ca2+, results in first an influx of Ca2+ into the cells, followed by autolytic activation of mu-calpain, proteolysis of membrane proteins, exposure of fusion-sites, and, finally, fusion of erythrocytes.     

Effects of calcium ions and the bivalent cation ionophore A23187 on the agglutination and fusion of chicken erythrocytes by Sendai virus.

1. Ca2+ (0.4-16 mM) had no detectable action on the agglutination of hen erythrocytes by Sendai virus. 2. Pretreatment of the cells with Ca2+ (0.1-8 mM) in the presence of the bivalent cation ionophore A23187 led, however, to a significant decrease in the subsequent agglutination of the cells by the virus. 3. It thus appears that the entry of Ca2+ into the interior of these cells decreases cellular agglutination by Sendai virus; possible interpretations of this phenomenon are discussed in terms of the movement of intramembranous particles. 4. With a small number of virions, maximum cell fusion by Sendai virus occurred in the presence of EGTA [ethanedioxybis(ethylamine)tetra-acetate]. 5. Virus-induced cell fusion was significantly decreased by Ca2+, even at a concentration of 0.2 mM; it is suggested that this may result from diminished interactions between virus particles and erythrocyte membranes.     

Ca++-ionophore-induced dissipation of intracellular proton gradients in rat thymocytes

Rat thymocytes incorporate large amounts of acridine orange at concentrations of approximately 10 microM in about 10 minutes at 37 degrees C. The addition of (NH4)2SO4 (at a final concentration of several mMolars) releases about 30% of the incorporated dye into the medium. The NH4+-releasable dye uptake is almost completely abolished by 20 minutes' incubation with 4 microM of the Ca++-ionophore A23187. Dye uptake is associated with an absorbance change at 492 mm and thus may be followed spectrophotometrically. However, NH4+ at the above concentrations or nigericin (0.5 mg/ml) completely annul this change in absorbance, indicating that it reflects only the accumulation of the dye within the acidic cellular compartments. In a Ca++-containing physiological saline, the addition of A23187 (at a final concentration of 8 microM) at the end of the dye uptake phase initiated a reversal in the absorbance change; in the absence of Ca++, reversal occurred at a much lower rate. Incubation of cells for 30 minutes with 2 microM A23187 in Ca++-containing saline completely abolished NH4+-sensitive dye uptake; less A23187 (0.5 microM) and like or a longer incubation period brought about a striking decrease in NH4+-sensitive dye uptake. Similar results were obtained with cells suspended in RPLM 1640 medium. In the absence of external Ca++, A23187 impaired cell capacity to incorporate dye in a delta pH-dependent manner, but a longer incubation time or higher concentrations of the ionophore were required to obtain a comparable effect. It is thus concluded that the ionophore dissipates intracellular pH gradients by an intracellular divalent cation (Ca++ or also Mg++)-H+ exchange.     

Rearrangement of intramembranous particles and fusion promoted in chicken erythrocytes by intracellular Ca2+.

Ca2+ was introduced into fresh and ATP-depleted chicken erythrocytes through the aid of the inophore A23187. Intracellular Ca2+ (10-40 mM) induced fusion in ATP-depleted cells after 30-60 min incubation at 37 degrees C, but not in fresh cells. Fresh cells underwent a higher degree of haemolysis than ATP-depleted cells after accumulation of Ca2+. Uptake of Ca2+ was the same in these two systems. Intracellular Ca2+ induced rearrangement of intramembranous particles, as revealed by freeze-etching studies. The intramembranous particles in the protoplasmic face of fractured membranes obtained from fresh cells incubated with 1 mM of Ca2+ were more scattered and their density was lower than in control cells. Incubation with higher concentrations of Ca2+ (10-40 mM) induced transient changes in the intramembranous particles' density with the appearance of protrusions and depressions on the protoplasmic and exoplasmic faces of the fractured membranes, respectively. These effects were reversible upon removal of Ca2+ by washing the cells with ethyleneglycol bis(alpha-aminoethylether)-N,N'-tetraacetic acid; rearrangement of intramembranous particles was less evident after accumulation of Ca2+ in ATP-depleted cells, whose fractured membranes did not contain any protrusions or depressions. Transferring Ca2+-loaded cells to the cold caused the formation of large smooth areas devoid of intramembranous particles in the protoplasmic face of the fractured membranes. Cells containing Ca2+ appeared spherical, and removal of Ca2+ restored the normal oval shape of chicken erythrocytes.     

Possible involvement of Ca++ ions, protein kinase C and Na(+)-H+ antiporter in insulin-induced endogenous dopamine release from tuberoinfundibular neurons

Insulin (63 microM) stimulated endogenous dopamine (DA) release from tuberoinfundibular neurons. This effect was independent on the presence of extracellular glucose and did not involve the outward transport of DA, mediated by its membrane carrier. By contrast this effect was completely prevented by the removal of extracellular Ca++ ions in presence of the Ca(++)-chelator ethyleneglycol-2-(2-aminoethyl)-tetracetic acid (EGTA). Furthermore 1-(5-isoquinolinyl-sulfonyl)-2-methyl-piperazine (H7), a compound which behaves as a putative inhibitor of protein kinase C (PK-C) (10 microM), completely counteracted the stimulation of endogenous DA release induced by insulin. Amiloride (300 microM) and its 5-amino nitrogen atom-substituted derivative, 5-(N-methyl-N-(guanidinocarbonylmethyl) amiloride (MGCMA) (10 microM), a highly selective inhibitor of the Na(+)-H+ membrane antiporter, were both able to prevent the stimulatory action exerted by insulin on endogenous DA release. Collectively, these results suggest that the transductional events by which insulin stimulated endogenous DA release from TIDA neurons may involve the activation of PK-C, the enhancement of Ca++ influx and the stimulation of the Na(+)-H+ exchange system.     

Involvement of inositol 1,4,5-trisphosphate in Ca2+ influx in thrombin stimulated human platelets

By incubating platelets at low temperature (10 degrees C), the relationship between Ca2+ mobilization and formation of inositol 1,4,5-trisphosphate (IP3) in thrombin stimulated platelets could be precisely investigated. In the presence of 1 mM EGTA, time dependent changes in the intracellular free calcium concentration [( Ca2+]i) were closely related to those in IP3 formation. Time course of the influx of external Ca2+, estimated by delta [Ca2+]i obtained by subtracting [Ca2+]i in the presence of 1 mM EGTA from that in the presence of 1 mM CaCl2 was also very similar to that of IP3 formed. Furthermore, the increase in delta [Ca2+]i was extremely well correlated with the amount of IP3 formed (Y = 49X - 34, r = 0.99). Thus, these data indicate that IP3 might be involved not only in intracellular Ca2+ mobilization but in Ca2+ influx of human platelets stimulated by thrombin.     

The interaction of Ca2+ with the calmodulin-sensitive adenylate cyclase from Bordetella pertussis

Bordetella pertussis, the etiologic agent of whooping cough, produces a calmodulin-sensitive adenylate cyclase which elevates intracellular cAMP in a variety of eucaryotic cells. Exogenous calmodulin added to the partially purified adenylate cyclase has been shown to inhibit invasion of animal cells by this enzyme (Shattuck, R. L., and Storm, D. R. (1985) Biochemistry 24, 6323-6328). In this study, several properties of the calmodulin-sensitive adenylate cyclase are shown to be influenced by Ca2+ in the absence of calmodulin. The presence or absence of Ca2+ during QAE-Sephadex ion exchange chromatography produced two distinct chromatographic patterns of adenylate cyclase activity. Two different forms of the enzyme (Pk1 and Pk2EGTA) were isolated by this procedure. Pk1 adenylate cyclase readily elevated intracellular cAMP levels in mouse neuroblastoma cells (N1E-115) while Pk2EGTA adenylate cyclase had no effect on cAMP levels in these cells. Gel exclusion chromatography of Pk1 adenylate cyclase gave apparent Stokes radii (RS) of 43.5 A (+/- 1.3) in the presence of 2 mM CaCl2 and 33.8 A (+/- 0.94) in the presence of 2 mM EGTA [( ethylenebis (oxyethylenenitrilo)]tetraacetic acid). These Stokes radii are consistent with molecular weights of 104,000 (+/- 6,400) and 61,000 (+/- 3,600), respectively. Pk2EGTA adenylate cyclase had an apparent RS of 33.0 (+/- 1.2) (Mr = 60,600 (+/- 2,800] in the presence of Ca2+ or excess EGTA. At 60 degrees C, Pk1 adenylate cyclase exhibited a Ca2+-dependent heat stability with a half-life for loss of enzyme activity of 10.3 min in 5 mM CaCl2 and a half-life of 2.8 min in the presence of 0.1 microM CaCl2. The stability of Pk2EGTA adenylate cyclase was not affected by changes in free Ca2+. The adenylate cyclase preparations described above were submitted to sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, and enzyme activity was recovered from gel slices by extraction with detergent containing buffers. The catalytic subunit isolated from SDS-polyacrylamide gels was activated 7-fold in the presence of Ca2+ with maximum activity observed at 1 microM free Ca2+. With both preparations, the apparent molecular weight of the catalytic subunit on SDS gels was 51,000 in the presence of 2 mM CaCl2 and 45,000 in the presence of 2 mM EGTA. The catalytic subunit of the enzyme was purified to apparent homogeneity by preparative SDS-polyacrylamide gel electrophoresis and resubmitted to SDS gel electrophoresis in the presence or absence of free Ca2+. The purified catalytic subunit also exhibited a Ca2+-dependent shift in its mobility on SDS gels.(ABSTRACT TRUNCATED AT 400 WORDS)     

The lateral mobility of cell membrane components is not altered following cell fusion induced by Sendai virus

The interaction of Sendai virus glycoproteins with cell membranes was proposed to increase the lateral mobility of membrane proteins, enabling membrane fusion and the aggregation of intramembrane particles by thermotropic separation (Volsky, DJ & Loyter, A, Biochim biophys acta 514 (1978) 213 [13]; Maeda, T et al. Exp cell res 123 (1979) 333 [15]; and Kim, J & Okada, Y, Exp cell res 132 (1981) 125 [44]). In order to test this hypothesis, we employed fluorescence photobleaching recovery to investigate the effects of Sendai virus-induced fusion on the lateral mobility of membrane proteins and lipids in a variety of cell types (human erythrocytes, BHK21, HeLa, 3T3 NIH, and mouse spleen lymphocytes). The results of the lateral diffusion measurements demonstrate that no significant alterations occur in the lateral motion of membrane proteins or a fluorescent phospholipid on all the cell types examined, including cells which revealed high susceptibility to the virally mediated fusion (human erythrocytes and BHK21 cells). These findings suggest that a permanent increase in the lateral mobility of cell surface components does not generally occur during Sendai virus-induced cell fusion, and thus cannot play a role in the fusion mechanism. The possible involvement of transient alterations in the lateral mobility of membrane components in the fusion mechanism is discussed.     

Ca++-induced fusion of proteoliposomes: dependence on transmembrane osmotic gradient.

The fusion of cytochrome oxidase liposomes with liposomes reconstituted with mitochondrial hydrophobic protein is dependent on the presence of an acidic phospholipid in the liposomes and on the addition of Ca++ions. Liposomes which have grown, by fusion, to diameters in excess of 1000 A lose the ability to fuse further, unless an osmotic gradient across the liposome membrane is established, with the internal osmotic pressure higher than the external. At a given Ca++ concentration, the extent to which this second fusion step takes place is determined by the ratio of internal to external osmolarity. Single-walled liposomes with diameters exceeding 1 mumM have been produced by this technique. The data suggest that the thermodynamic driving force for the Ca++-induced fusion is an excess surface free energy which can be supplied by membrane curvature or transmembrane osmotic gradients.     

Leucine enkephalin antagonizes norepinephrine-induced 45Ca++ accumulation in rat atria

Exposure of rat atrial slices to 10(-5) M norepinephrine (NE) for 10 minutes increases 45Ca++ accumulation from 1.64 +/- 0.10 to 2.23 +/- 0.06 nmol/mg tissue. In the presence of leucine enkephalin (10(-8) M), NE-stimulated 45Ca++ uptake is reduced to 1.44 +/- 0.10 nmol/mg tissue. The effect of leu-enkephalin is reversed in the presence of 10(-7) M naloxone, NE-stimulated 45Ca++ uptake being increased to 2.17 +/- 0.15 nmol/mg tissue. The results support a direct interaction of leu enkephalin with beta-agonist-stimulated Ca++ flux in rat atria, and correlate with the previously reported enkephalin antagonism of NE-induced positive chronotropy in the same tissue.     

The immediate induction of extensive cell fusion by Ca2+ addition in Dictyostelium discoideum

In mated cultures (NC4 X V12) of Dictyostelium discoideum containing 1.0 mM CaCl2, cell fusion generates large numbers of binucleate cells which develop into zygote giant cells. In the absence of Ca2+, binucleate formation does not occur. When 1.0 mM CaCl2 is added to Ca2+-deficient cultures at 18 h, 50% of the cells fuse within 45 min producing large multinucleate syncytia. Small, presumptive gametes appear in Ca2+-deficient cultures and reach a peak of about 20% of the cell population by 10 h, but they maintain this plateau and do not fuse. Upon the addition of CaCl2, the presumptive gametes immediately fuse, producing binucleate cells which develop rapidly into morphologically distinct giant cells. Cell fusion continues, resulting in the formation of extremely large (40-80 microns diameter) multinucleate syncytia by 45 min. The induction of this extensive, synchronous cell fusion does not occur in the presence of other chloride salts and EGTA inhibits it, revealing that Ca+ is the regulatory ion.     

Control of mitochondrial Mg++-efflux]

Inorganic phosphate stimulates the release of Mg++ from liver mitochondria, depending on concentration; a concentration as low as 0.1 mM phosphate is already effective. The process is dependent on the electron transfer of the respiratory chain, and its rate is highest under conditions of endogenous respiration and with ascorbate and TMPD as substrates, respectively. The phosphate stimulated release of Mg++ is followed, with a pronounced delay, by a Ca++ efflux and a swelling of mitochondria. Addition of EGTA strongly reduced the rate of Mg++ liberation in the presence and absence of inorganic phosphate. Exogenous Ca++ is able to abolish the EGTA effect. ADP and ATP inhibit the phosphate stimulated release of Mg++. Phosphoenol pyruvate and free fatty acids enhance the rate of Mg++ and Ca++ efflux from the mitochondria. The results permit the conclusion that inorganic phosphate, Ca++ and various metabolites of the cell metabolism influence the Mg++ distribution between the extra- and intramitochondrial space, thus controlling the permeability of the mitochondrial inner membrane for monovalent cations.     

Cross-linking of the (Ca2+ + Mg2+)-ATPase protein.

The addition of cupric-1,10,-phenanthroline, a cross-linking catalyst, to sarcoplasmic reticulum membranes caused protein sulfhydryl groups to form disulfide bridges. Following a short exposure to the catalyst (15 s, 22 degrees C) most of the protein was in a dimeric form (Mr = 248 000). Longer exposure times resulted in the formation of trimers, tetramers and other oligomers too large to enter the gel. At low temperatures (4 degrees C) dimer formation predominates even for exposure times as long as 5 min. Cross-linking in the presence of 7.5 mM Triton X-100 (a concentration that resulted in clearing of the membrane suspension and thus solubilization of the membrane components) showed the appearance of a considerable dimer fraction, however, most of the (Ca2+ + Mg2+)-ATPase protein appeared as a monomer. Following 1 min of cross-linking at 22 degrees C, freeze-etched membranes showed no alteration in the number or appearance of 80 A intramembranous particles. Thus extensive cross-linking of the (Ca2+ + Mg2+)-ATPase protein can occur without disruption of the normal position of the intramembrane portion of the molecule.     

Difference in kinetic properties of phosphorylated intermediates formed in the forward and backward reactions of solubilized Ca2+-ATPase of sarcoplasmic reticulum

Solubilized Ca2+-ATPase (SSR) was prepared by solubilizing fragmented sarcoplasmic reticulum (FSR) with a nonionic detergent (C12E8) then displacing the detergent with Tween 80, using a DEAE-cellulose column. The kinetic properties of the phosphorylated intermediate (EP) formed by the reaction of SSR with ATP were compared with those of EP formed by the reaction with Pi. The time course of decay of E32P formed with 4 microM AT32P in the presence of 19 mM CaCl2 and 10 mM MgCl2 (forward reaction) was measured by adding 0.4 mM unlabeled ATP and 10 mM Pi at pH 6.0 and 30 degrees C. The rate of E32P decay was accelerated by 0.4 mM ADP. On the other hand, when the time course of decay of E32P formed with 10 mM 32Pi in the presence of 5 mM EGTA and 10 mM MgCl2 (backward reaction) was measured by adding 0.4 mM unlabeled ATP and 15 mM CaCl2, the rate of E32P decay was unaffected by 0.4 mM ADP. AT32P was produced on adding ADP to E32P formed with AT32P in the presence of 10 mM CaCl2 and 10 mM MgCl2, while no AT32P was produced on adding ADP to E32P formed with 32Pi in the presence of 5 mM EGTA and 10 mM MgCl2, even when 15 mM CaCl2 was added simultaneously with ADP.