Studies of gastric Ca2+-stimulated adenosine triphosphatase I. characterization and general properties

Gastric microsomes do not contain any significant Ca²⁺-stimulated ATPase activity. Trypsinization of pig gastric microsomes in presence of ATP results in a significant (2–3-fold) increase in the basal (with Mg²⁺ as the only cation) ATPase activity, with virtual elimination of the K⁺-stimulated component. Such treatment causes unmaksing of a latent Mg²⁺-dependent Ca²⁺-stimulated ATPase. Other divalent cations such as Sr²⁺, Ba²⁺, Zn²⁺ and Mn²⁺ were found ineffective as a substitute for Ca²⁺. Moreover, those divalent cations acted as inhibitors of the Ca²⁺-stimulated ATPase activity. The pH optimum of the enzyme is around 6.8. The enzyme has a $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ of 70 μM for ATP and the $\text{K}{}_{\mathrm{<mtext>a</mtext>}}$ values for Mg²⁺ and Ca²⁺ are about $\text{4 · 10}{}^{\mathrm{?4}}\text{M}$ and 10^?7 M, respectively. Studies with inhibitors suggest the involvement of sulfhydryl and primary amino groups in the operation of the enzyme. Possible roles of the enzyme in gastric H⁺ transport have been discussed.     

A Ca2+-activated ATPase specifically released by Ca2+ shock from Paramecium tetraurelia

Deciliation of Paramecium tetraurelia by a Ca²⁺ shock procedure releases a discrete set of proteins which represent about 1% of the total cell protein. Marker enzymes for cytoplasm (hexokinase), endoplasmic reticulum (glucose-6-phosphatase), peroxisomes (catalase), and lysosomes (acid phosphatase) were not released by this treatment. Among the proteins selectively released is a Ca²⁺-dependent ATPase. This enzyme has a broad substrate specificity which includes GTP, ATP, and UTP, and it can be activated by Ca²⁺, Sr²⁺, or Ba²⁺, but not by Mg²⁺ or by monovalent cations. The crude enzyme has a specific activity of 2–3 μmol/min per mg; the optimal pH for activity is 7.5. ATPase, GTPase, and UTPase all reside in the same protein, which is inhibited by ruthenium red, is irreversibly denatured at 50°C, and which has a sedimentation coefficient of 8–10 S. This enzyme is compared with other surface-derived ATPases of ciliated protozoans, and its possible roles are discussed.     

Specific modification of gastric K+-stimulated ATPase activity by thimerosal

Treatment of hog gastric microsomes with the sulfhydryl reagent, thimerosal (ethylmercurithiosalicylate), produced differential effects on the K⁺-ATPase and the K⁺-stimulated p-nitrophenylphosphatase activities. For example, exposure to 2 mM thimerosal for 3 min severely reduced the activity of K⁺-stimulated ATPase, while K⁺-p-nitrophenylphosphatase activity was enhanced 2- to 3-fold. Higher concentration of thimerosal, or longer incubation times, also led to inhibition of K⁺-p-nitrophenylphosphatase. The activated state of p-nitrophenylphosphatase could be sustained by a 20-fold, or greater, dilution of treated membranes, and could be reversed by reduction of membrane SH groups by exogenous thiols. Significant activation of K⁺-p-nitrophenylphosphatase was not produced by p-chloromercuribenzene sulfonate, p-chloromercuribenzoate or mersalyl; however, ethyl mercuric chloride had qualitatively similar activity effects as thimerosal. Kinetics of K⁺-p-nitrophenylphosphatase for thimerosal-treated membranes were altered as follows: V increased; K_m for p-nitrophenylphosphate unchanged for K_a for K⁺ increased. ATP, which is a potent inhibitor of K⁺-p-nitrophenylphosphatase activity in native membranes (K_I ≈ 200 μM). These data suggest that there are multiple SH groups which differentially influence the gastric K⁺-stimulated ATPase activity. Defined treatments with thimerosal are interpreted as an uncoupling of the K⁺-stimulated phosphatase component of the enzyme (for which p-nitrophenylphosphatase is a presumed model reaction). Such differential modifications can be usefully applied to the study of partial reactions of the enzyme and their specific role in the related H⁺-transport reaction.     

K+-stimulated ATPase in purified microsomes of bullfrog oxyntic cells

Differential centrifugation of oxyntic cell homogenates yielded microsomal fractions which contained large amounts of mitochondrial membrane. The presence of marker enzymes (succinate dehydrogenase and cytochrome c oxidase) indicated that mitochondrial contamination of crude microsomes ranged from 20 to 60% in different preparations. A discontinuous sucrose density gradient procedure was developed for the routine preparation of purified oxyntic cell microsomes. A K⁺-stimulated, Mg²⁺-requiring ATPase was localized in these purified membranes and coincided with the presence of a K⁺-stimulated p-nitrophenylphosphatase. Na⁺ and ouabain had no effect on the K⁺ stimulation of the microsomal ATPase. The apparent activation constant for K⁺ was approximately 1 mM at pH 7.5, the optimal pH for stimulation.An anion-sensitive ATPase has been widely studied in gastric microsomal preparations. We found that the basal microsomal ATPase (i.e. without K⁺) and the mitochondrial ATPase were inhibited by SCN^? and enhanced by HCO₃^?, however, the K⁺-stimulated component of the microsomal ATPase was virtually unaffected by these anions.     

ATP/ADP exchange activity of gastric (H+ + K+)-ATPase

The ATP/ADP exchange is shown to be a partial reaction of the $\text{(}\text{H}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ by the absence of measurable nucleoside diphosphokinase activity and the insensitivity of the reaction to $\text{P}{}^1$, $\text{P}{}^5$ -di(adenosine-5′) pentaphosphate, a myokinase inhibitor. The exchange demonstrates an absolute requirement for Mg²⁺ and is optimal at an ADP/ATP ratio of 2. The high ATP concentration ($\text{K}{}_{0.5}\text{= 116 μ}\text{M}$) required for maximal exchange is interpreted as evidence for the involvement of a low affinity form of nucleotide site. The ATP/ADP exchange is regarded as evidence for an ADP-sensitive form of the phosphoenzyme. In native enzyme, pre-steady state kinetics show that the formation of the phosphoenzyme is partially sensitive to ADP while modification of the enzyme by pretreatment with 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB) in the absence of Mg²⁺ results in a steady-state phosphoenzyme population, a component of which is ADP sensitive. The ATP/ADP exchange reaction can be either stimulated or inhibited by the presence of K⁺ as a function of pH and Mg²⁺.     

Lysophosphatidylcholine-activated,vanadate-inhibited,Mg2+-ATPase from radish microsomes

An orthovanadate-inhibited, nitrate-insensitive, phospholipid-requiring Mg²⁺-ATPase has been partially purified (approx. 40-fold) from microsomal preparations from 24 h germinated radish seedlings. The specific activity obtained was 10–13 μmol P_i · min^?1 per mg protein, namely by 4- to 10-fold higher than that reported for the known similar enzyme preparations from corn and oat roots, and by 3- to 10-fold lower than that of the extensively purified plasmalemma enzymes from Neurospora and yeast. The partially purified activity was fairly specific for ATP, other nucleotide triphosphates being hydrolysed at less than 10% the rate with ATP; no activity was present towards ADP, AMP, p-nitrophenyl phosphate and other phosphate esters. The activity was strongly dependent on the presence of phospholipids with a marked preference for lysophosphatidylcholine, and showed an absolute requirement for Mg²⁺ or some other divalent cations (CO²⁺, Mn²⁺, Mg²⁺, Ni²⁺, Zn²⁺ in order of decreasing effectiveness); Ca²⁺ could not substitute for Mg²⁺ and was strongly inhibitory in its presence. K⁺, Rb⁺ and Na⁺ and also to a lesser extent NH₄⁺ and Li⁺ were significantly stimulatory, while the anions NO₃^?, H₂PO₄^?, Cl^? and SO₄^2? were ineffective. Orthovanadate, N,N′-dicyclohexylcarbodiimide, diethylstilbestrol, p-chloromercuribenzensulfonate, tetraiodofluorescein and tetrachlorotetraiodofluorescein were strongly inhibitory. The coincidence of the K_m for ATP with that for Mg²⁺ suggested that ATP-Mg is the true substrate. Accordingly, the enzyme showed a normal Michaelis-Menten kinetics for ATP-Mg with an apparent K_m of approx. 0.5 mM. The similarity of the characteristics of this enzyme with those of the plasmalemma enzymes from lower plants suggests its location at the plasma membrane, while some data ‘in vivo’ and in native sealed vesicle systems indicate its involvement in active proton transport.     

Purification and some properties of an Mg2+-, Ca2+- and calmodulin-stimulated ATPase from spinach chloroplast envelope membranes

Spinach chloroplasts display an ATPase activity which is associated with the envelope. This envelope-bound activity is stimulated by Ca²⁺, Mg²⁺ and calmodulin (Nguyen, T.D. and Siegenthaler, P.A. (1983) FEBS Lett. 164, 67–70). The Triton X-100-solubilized enzyme was retained specifically on a calmodulin-Sepharose affinity column in the presence of calcium. The fractions eluted by EGTA contained two proteins characterized by pI values of 7.3 and 6.0 (isoelectric focusing). Both proteins, separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-polyacrylamide gel electrophoresis), were resolved into a single polypeptide having and identical apparent M_rmr of 65 000. This suggests that the two initial proteins might be isoelectric variants. However, the amount of the enzyme fraction obtained by the calmodulin-Sepharose column was small and the ATPase activity was very labile. A linear glycerol gradient allowed the recovery of a greater amount of the enzyme which was, however, only partially purified, but the activity of which was much more stable. Electrophoresis of the ATPase-containing fractions in a native polyacrylamide gradient gel permitted the separation of a 260 kDa protein which was resolved by SDS-polyacrylamide gel electrophoresis into a single polypeptide of 65 kDa. Thus, the chloroplast envelope-bound ATPase might be a tetramer (260 kDa) consisting of 4 identical monomers (65 kDa). The purified ATPase had properties similar to that of the envelope-bound enzyme. TheK_m value for ATP was 0.45 mM. The activity was stimulated by Ca²⁺ and Mg²⁺, and further enhanced by calmodulin. The physiological significance of the chloroplast envelope-bound ATPase is discussed.     

The K+- and HCO3−-stimulated phosphatase activities in renal microsomes of rats

The K⁺-stimulated phosphatase activity of microsomes from rat kidney was not inhibited by l-phenylalanine, but the HCO₃^?-stimulated phosphatase activity was markedly inhibited by l-phenylalanine. Valinomycin enhanced the HCO₃^?-stimulated phosphatase activity, but did not enhance the K⁺-stimulated phosphatase activity. Ouabain did not inhibit the HCO₃^?-stimulated phosphatase activity, but inhibited the K⁺-stimulated phosphatase activity.The renal K⁺-stimulated phosphatase activity was suppressed to 40% of the control values by adrenalectomy, but the renal HCO₃^?-stimulated phosphatase activity was little suppressed by adrenalectomy. The renal K⁺-stimulated phosphatase activity in intact and adrenalectomized rats was found to be significantly elevated, in a manner similar to the elevation of the renal (Na⁺ + K⁺)-ATPase activity by aldosterone treatment (P < 0.02).     

The effects of deuterium oxide (2H2O) on the polymerization of tubulin in vitro

The initial rate and final extent of polymerization of both bovine brain tubulin and sea urchin egg tubulin were enhanced in the presence of ²H₂O. The yields were increased in association with the elevation of the ²H₂O concentration. ²H₂O also reduced the critical concentration for polymerization of brain tubulin. Thermodynamic analysis was attempted using the temperature dependence of the critical concentration for polymerization in the presence of ²H₂O. We obtained linear van 't Hoff plots and calculated thermodynamic parameters which were positive and were increased with the elevation of the ²H₂O concentration. The enhancement of the polymerization of tubulin by ²H₂O could, therefore, be the result of the strenghening of intra-and/or inter-molecular hydrophobic interactions of the tubulin molecules. We believe that the increase in lenghth and number of microtubules of the mitotic spindles in the dividing cells of the eukaryotes with ²H₂O may be caused by the direct involvement of ²H₂O in the polymerization of tubulin.     

An electrogenic Na+/Ca2+ antiporter in addition to the Ca2+ pump in cardiac sarcolemma

Vesicles isolated from rat heart, particularly enriched in sarcolemma markers, were examined for their sidedness by investigation of side-specific interactions of modulators with the asymmetric (Na⁺ + K⁺)-ATPase and adenylate cyclase complex. The membrane preparation with the properties expected for inside-out vesicles showed the highest rate of ATP-driven Ca²⁺ transport. The Ca²⁺ pump was stimulated 1.7- and 2.1-fold by external Na⁺ and K⁺, respectively, the half-maximal activation occurring at 35 mM monovalent cation concentration. In vesicles loaded with Ca²⁺ by pump action in a medium containing 160 mM KCl, a slow spontaneous release of Ca²⁺ started after 2 min. The rate of this release could be dramatically increased by the addition of 40 mM NaCl to the external medium. In contrast, 40 mM KCl exerted no appreciable effect on vesicles loaded with Ca²⁺ in a medium containing 160 mM NaCl. Ca²⁺ movements were also studied in the absence of ATP and Mg²⁺. Vesicles containing an outwardly directed Na⁺ gradient showed the highest Ca²⁺ uptake activity. These findings suggested the operation of a Ca²⁺/Na⁺ antiporter in addition to the active Ca²⁺ pump in these sarcolemmal vesicles. A valinomycin-induced inward K⁺-diffusion potential stimulated the Na⁺- Ca²⁺ exchange, suggesting its electrogenic nature. If in the absence of ATP and Mg²⁺ the transmembrane Na_i⁺/Na_o⁺ gradient exceeded 160/15 mM concentrations, Ca²⁺ uptake could be stimulated by the addition of 5 mM oxalate, indicating Na⁺ gradient-induced Ca²⁺ uptake to be a translocation of Ca²⁺ to the lumen of the vesicle. A sarcoplasmic reticulum contamination, removed by further sucrose gradient fractionation, contained rather low Na⁺-Ca²⁺ exchange activity. This result suggests that the activity can be entirely accounted for by the sarcolemmal content of the cardiac membrane preparation.     

Spontaneous inactivation of the Ca2+-sensitive K+ channels of human red cells at high intracellular Ca2+ activity

Ionophore A23187-mediated Ca²⁺-induced oscillations in the conductance of the Ca²⁺-sensitive K⁺ channels of human red cells were monitored with ion specific electrodes. The membrane potential was continuously reflected in CCCP-mediated pH changes in the buffer-free medium, changes in extracellular K⁺ activity were followed with a K⁺-selective electrode, and changes in the intracellular concentration of ionized calcium were calculated on the basis of cellular ⁴⁵Ca content. An increased cellular ⁴⁵Ca content at the successive minima of the oscillations where the K⁺ channels are closed indicates that the activation of the channels might be a $\text{(}\text{dCa}{}^{\mathrm{2+}}\text{/}\text{d}\text{t)-}\text{sensitive}$ process and that accommodation to enhanced levels of intracellular free calcium may occur. An incipient inactivation of the K⁺ channels at intracellular ionized calcium levels of about 10 μM and a concurrent membrane potential of about ?65 mV was observed. At a membrane potential of about ?70 mV and an intracellular concentration of about 2·10^?4M no inactivation of K⁺ channels took place. Inactivation of the K⁺ channels is suggested to be a compound function of the intracellular level of free calcium and the membrane potential. The observed sharp peak values in cellular ⁴⁵Ca content support the notion that a necessary component of the oscillatory system is a Ca²⁺ pump operating with a significant delay in the activation/inactivation process in response to changes in cellular concentration of ionized calcium.     

Effects of adenosine diphosphate on Ca2+ fluxes and Ca2+ accumulation of sarcoplasmic reticulum

Ca²⁺ transport by sarcoplasmic reticulum vesicles was examined by incubating sarcoplasmic reticulum vesicles (0.15 mg/ml) at 37°C in, either normal medium that contained 0.15 M sucrose, 0.1 M KCl, 60 μM CaCl₂, 2.5 mM ATP and 30 mM Tes at pH 6.8, or a modified medium for elimination of ADP formed from ATP hydrolysis by including, in addition, 3.6 mM phosphocreatine and 33 U/ml of creatine phosphokinase. In normal medium, Ca²⁺ uptake of sarcoplasmic reticulum vesicles reached a plateau of about 100 nmol/mg. In modified medium, after this phase of Ca²⁺ uptake, a second phase of Ca²⁺ accumulation was initiated and reached a plateau of about 300 nmol/mg. The second phase of Ca²⁺ accumulation was accompanied by phosphate uptake and could be inhibited by ADP. Since, under these experimental conditions, there was no significant difference of the rates of ATP hydrolysis in normal medium and modified medium, extra Ca²⁺ uptake in modified medium but not in normal medium could not be explained by different phosphate accumulation in the two media. Unidirectional Ca²⁺ influx of sarcoplasmic reticulum near steady state of Ca²⁺ uptake was measured by pulse labeling with ⁴⁵Ca²⁺. The Ca²⁺ efflux rate was then determined by subtracting the net uptake from the influx rate. At the first plateau of Ca²⁺ uptake in normal medium, Ca²⁺ influx was balanced by Ca²⁺ efflux with an exchange rate of 240 nmol/mg per min. This exchange rate was maintained relatively constant at the plateau phase. In modified medium, the Ca²⁺ exchange rate at the first plateau of Ca²⁺ uptake was about half of that in normal medium. When the second phase of Ca²⁺ uptake was initiated, both the influx and efflux rates started to increase and reached a similar exchange rate as observed in normal medium. Also, during the second phase of Ca²⁺ uptake, the difference between the influx and efflux rates continued to increase until the second plateau phase was approached. In conditions where the formation of ADP and inorganic phosphate was minimized by using a low concentration of sarcoplasmic (7.5 μg/ml) and/or using acetyl phosphate instead of ATP, the second phase of Ca²⁺ uptake was also observed. These data suggest that the Ca²⁺ load attained by sarcoplasmic reticulum vesicles during active transport is modulated by ADP accumulated from ATP hydrolysis. ADP probably exerts its effect by facilitating Ca²⁺ efflux, which subsequently stimulates Ca²⁺ exchange.     

Localization of (Ca2+ + Mg2+)-ATPase,Ca2+ pump and other ATPase activities in cardiac sarcolemma

N-Ethylmaleimide was employed as a surface label for sarcolemmal proteins after demonstrating that it does not penetrate to the intracellular space at concentrations below 1·10^?4 M. The sarcolemmal markers, ouabain-sensitive (Na⁺ + K⁺)-ATPase and Na⁺/Ca²⁺-exchange activities, were inhibited in N-ethylmaleimide perfused hearts. Intracellular activities such as creatine phosphokinase, glutamate-oxaloacetate transaminase and the internal phosphatase site of the Na⁺ pump (K⁺-p-nitrophosphatase) were not affected. Almost 20% of the (Ca²⁺ + Mg²⁺)-ATPase and Ca²⁺ pump were inhibited indicating the localization of a portion of this activity in the sarcolemma. Sarcolemma purified by a recent method (Morcos, N.C. and Drummond, G.I. (1980) Biochim. Biophys. Acta 598, 27–39) from N-ethylmaleimide-perfused hearts showed loss of approx. 85% of its (Ca²⁺ + Mg²⁺-ATPase and Ca²⁺ pump compared to control hearts. (Ca²⁺ + Mg²⁺)-ATPase and Ca²⁺ pump activities showed two classes of sensitivity to vanadate ion inhibition. The high vanadate affinity class ($\text{K}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ for inhibition approx. 1.5 μM) may be localized in the sarcolemma and represented approx. 20% of the total inhibitable activity in agreement with estimates from N-ethylmaleimide studies. Sucrose density fractionation indicated that only a small portion of Mg²⁺-ATPase and Ca²⁺-ATPase may be associated with the sarcolemma. The major portion of these activities seems to be associated with high density particles.     

Control of activity states of heart mitochondrial ATPase. Role of the proton-motive force and Ca2+

The ATPase complex of submitochondrial particles exhibits activity transitions that are controlled by the natural ATPase inhibitor (Gómez-Puyou, A., Tuena de Gómez-Puyou, M. and Ernster, L. (1979) Biochim. Biophys. Acta 547, 252–257). The ATPase of intact heart mitochondria also shows reversible activity transitions; the activation reaction is induced by the establishment of electrochemical gradients, whilst the inactivation reaction is driven by collapse of the gradient. In addition it has been observed that the influx of Ca²⁺ into the mitochondria induces a rapid inactivation of the ATPase; this could be due to the transient collapse of the membrane potential in addition to a favorable effect of Ca²⁺-ATP on the association of the ATPase inhibitor peptide to F₁-ATPase. This action of Ca²⁺ may explain why mitochondria utilize respiratory energy for the transport of Ca²⁺ in preference to phosphorylation. It is concluded that the mitochondrial ATPase inhibitor protein may exert a fundamental regulatory function in the utilization of electrochemical gradients.     

Inhibition by Sr2+ of specific mitochondrial Ca2+-efflux pathways

The effect of Sr²⁺ on the set point for external Ca²⁺ was studied in rat heart and liver mitochondria with the aid of a Ca²⁺-sensitive electrode. In respiring mitochondria the set point is determined by the rates of Ca²⁺ influx on the Ca²⁺ uniporter and efflux by various mechanisms. We studied the Ca²⁺-Na⁺ exchange pathway in heart mitochondria and the Δψ-modulated efflux pathway in liver mitochondria. Prior accumulation of Sr²⁺ was found to shift the set points towards lower external Ca²⁺ both in heart mitochondria under conditions of Ca²⁺-Na⁺ exchange and in liver mitochondria under conditions that should promote opening of the Δψ-modulated pathway. The effect on the set point was found to be due to inhibition of Ca²⁺ efflux by Sr²⁺ taken up by the mitochondria, while Sr²⁺ efflux was too slow to be measurable.     

Na+-driven Ca2+ transport in alkalophilic Bacillus

Ca²⁺ transport was studied in membrane vesicles of alkalophilic Bacillus. When Na⁺-loaded membrane vesicles were suspended in KHCO₃/KOH buffer (pH 10) containing Ca²⁺, rapid uptake of Ca²⁺ was observed. The apparent $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ value for Ca²⁺ measured at pH 10 was about 7 μM, and the $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ value shifted to 24 μM when measured at pH 7.4. The efflux of Ca²⁺ was studied with Ca²⁺-loaded vesicles. Ca²⁺ was released when Ca²⁺-loaded vesicles were suspended in medium containing 0.4 M Na⁺.Ca²⁺ was also transported in membrane vesicles driven by an artificial pH gradient and by a membrane potential generated by K⁺-valinomycin in the presence of Na⁺.These results indicate the presence of Ca²⁺/Na⁺ and H⁺/Na⁺ antiporters in the alkalophilic Bacillus A-007.     

Regulation of the reconstituted chloroplast phosphate translocator by an H+ gradient

This report describes the influence of ΔpH on the transport of phosphate, triose phosphate and 3-phosphoglycerate catalyzed by the phosphate translocator in a reconstituted system. The H⁺ gradient across the liposome membrane is adjusted by the addition of external buffer solution and maintained for several minutes. The following results are obtained: (1) An inward directed H⁺ gradient leads to an increase of 3-phosphoglycerate transport and to a decrease of phosphate and triose phosphate transport. (2) An H⁺ gradient in the opposite direction results in a restriction of 3-phosphoglycerate influx whereas the influx of phosphate and triose phosphate is enhanced. (3) The magnitude of the pH effect depends on the internal substrate. Compared to the homoexchange mode, the effect of applied ΔpH is more pronounced in the heteroexchange mode. (4) Transport of phosphate and 3-phosphoglycerate is influenced by ΔpH in a different manner. In the case of phosphate and triose phosphate transport the observed effects are associated with changes in the apparent K_m values whereas in the case of 3-phosphoglycerate transport the application of a pH gradient is linked to a change of V_max. (5) In competition experiments with both substrates in the external medium, ΔpH influences the effect of phosphate as a competitive inhibitor of 3-phosphoglycerate transport whereas the effect of 3-phosphoglycerate on phosphate transport is not affected by a pH gradient. (6) The measured apparent K_m and V_max values under the influence of ΔpH can be used for the calculation of substrate fluxes across the envelope during illumination. It can be demonstrated that the increase of stromal pH in the light gives rise to a considerable change in the ratio of the substrates transported. Under conditions without pH gradient, the species transported out is mainly 3-phosphoglycerate and the species transported in is mainly triose phosphate. These fluxes are reversed when a pH gradient is applied (light conditions).     

Limited breakdown of cytoskeletal proteins by an endogenous protease controls Ca2+-induced membrane fusion events in chicken erythrocytes

The profound morphological changes which follow the treatment of chicken erythrocytes with the ionophore A23187 and Ca²⁺ are associated with a concomitant breakdown of certain membrane-associated proteins including α-spectrin, goblin and microtubule-associated proteins (MAPS) which undergo a limited proteolysis to give large, well-defined fragments. The Ca²⁺-sensitive protease responsible for these changes appears to be present in the soluble fraction of the cells. Treatment with TLCK or iodoacetamide inhibits both the major morphological changes and the proteolytic events but these agents do not prevent the dissociation of microtubules or the activation of endogenous sphingomyelinase which occur in cells with raised levels of intracellular Ca²⁺. It is suggested that the sphingomyelinase is activated as a consequence of a Ca²⁺-induced loss of phospholipid asymmetry in the plasma membrane.