Limited breakdown of cytoskeletal proteins by an endogenous protease controls Ca-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.     

Plasma membrane NADH dehydrogenase and Ca2+-dependent potassium transport in erythrocytes of several animal species

Ca²⁺-dependent K⁺ transport and plasma membrane NADH dehydrogenase activities have been studied in several ‘high-K⁺’ (human, rabbit and guinea pig) and ‘low-K⁺’ (dog, cat and sheep) erythrocytes. All the species except sheep showed Ca²⁺-dependent K⁺ transport. NADH-ferricyanide reductase was detected in all the species and showed positive correlation with the flavin contents of the membranes. NADH-cytochrome $\text{c}$ reductase was very low or absent in dog, sheep and guinea pig membranes. No correlation was found between NADH dehydrogenase and Ca²⁺-dependent K⁺ channel activities in the species studied. Nor were any of the above activities correlated with $\text{(}\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ activity.     

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 alpha-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.     

Interaction of Ca2+ with (Na+ + K+)-ATPase: Properties of the Ca2+-stimulated phosphatase activity

Ca²⁺ inhibited the Mg²⁺-dependent and K⁺-stimulated p-nitrophenylphosphatase activity of a highly purified preparation of dog kidney (Na⁺ + K⁺)-ATPase. In the absence of K⁺, however, a Mg²⁺-dependent and Ca²⁺-stimulated phosphatase was observed, the maximal velocity of which, at pH 7.2, was about 20% of that of the K⁺-stimulated phosphatase. The Ca²⁺-stimulated phosphatase, like the K⁺-stimulated activity, was inhibited by either ouabain or Na⁺ or ATP. Ouabain sensitivity was decreased with increase in Ca²⁺, but the K_0.5 values of the inhibitory effects of Na⁺ and ATP were independent of Ca²⁺ concentration. Optimal pH was 7.0 for Ca²⁺-stimulated activity, and 7.8–8.2 for the K⁺-stimulated activity. The ratio of the two activities was the same in several enzyme preparations in different states of purity. The data indicate that (a) Ca²⁺-stimulated phosphatase is catalyzed by (Na⁺ + K⁺)-ATPase; (b) there is a site of Ca²⁺ action different from the site at which Ca²⁺ inhibits in competition with Mg²⁺; and (c) Ca²⁺ stimulation can not be explained easily by the action of Ca²⁺ at either the Na⁺ site or the K⁺ site.     

Quinolinic acid, an endogenous metabolite with neurotoxic properties, alters the physical state of membrane proteins in human erythrocytes

Quinolinic acid (2,3-pyridinedicarboxylic acid), an endogenous, tryptophan metabolite, is neurotoxic when injected into rat striatum (1). To begin to investigate the molecular interactions of quinolinic acid with membranes, electron spin resonance studies of the effects of this neurotoxin on the physical state of lipids, proteins, and cell-surface sialic acid in human erythrocyte ghosts have been performed. Quinolinic acid induced a highly significant alteration in the physical state of membrane proteins (P less than 0.01) while that of sialic acid and membrane lipids was unaffected. These results are similar to those induced by ibotenic acid, an exogenous neurotoxin, and are discussed with reference to possible molecular characteristics of the interaction of these neurotoxins with membrane proteins.     

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.     

Stimulation of Ca2+ efflux by N-formyl chemotactic peptides in guinea-pig peritoneal macrophages

Effects of N-formyl chemotactic peptides on the Ca²⁺ influx and efflux were investigated in guinea-pig peritoneal macrophages using an isotope tracer. fMet-Leu-Phe did not enhance the influx of ⁴⁵Ca²⁺ into macrophages, whereas it stimulated the efflux of ⁴⁵Ca²⁺ from macrophages at concentrations ranging from 10^?10 M to 10^?7 M. fMet-Met-Met and fMet-Leu also stimulated the ⁴⁵Ca²⁺ efflux, albeit at much higher concentrations, while there was no stimulation with fMet. The mitochondrial inhibitors, oligomycin and NaN₃, did not modify the ⁴⁵Ca²⁺ efflux induced by the chemoattractants, yet they did induce the release of ⁴⁵Ca²⁺ from the mitochondria. On the other hand, higher concentrations of the calmodulin antagonists, chlorpromazine and trifluoperazine, induced the release of ⁴⁵Ca²⁺ from the NaN₃-insensitive Ca²⁺ store site and mimicked the enhancement of the ⁴⁵Ca²⁺ efflux by N-formyl chemotactic peptides. Thus, N-formyl chemotactic peptides appear to increase the levels of intracellular free Ca²⁺ in guinea-pig peritoneal macrophages, probably by inducing the release of Ca²⁺ from the NaN₃-insensitive Ca²⁺ store site.     

Menadione-induced cytotoxicity is associated with protein thiol oxidation and alteration in intracellular Ca2+ homeostasis

The toxicological implications of alterations in intracellular thiol homeostasis during menadione metabolism have been investigated using freshly isolated rat hepatocytes. A strict correlation between depletion of protein sulfhydryl groups and loss of cell viability was observed. Loss of protein thiols preceded cell death, and occurred more rapidly in cells with decreased levels of reduced glutathione. Depletion of protein thiols was also associated with inhibition of Ca²⁺ efflux from the cells and perturbation of intracellular Ca²⁺ homeostasis. It is proposed that the oxidative stress induced by menadione metabolism in isolated hepatocytes results in the depletion of both soluble and protein thiols, and that the latter effect is critically associated with a perturbation of Ca²⁺ homeostasis and loss of cell viability.     

Ca2+-dependent regulation of calmodulin binding and adenylate cyclase activation in bovine cerebellar membranes

The binding parameters of ¹²⁵I-labeled calmodulin to bovine cerebellar membranes have been determined and correlted with the activation of adenylate cyclase by calmodulin. In the presence of saturating levels of free Ca²⁺, calmodulin binds to a finite number of specific membrane sites with a dissociation constant (K_d) of 1.2 nM. Furthermore, Scatchard analysis reveals a second population of binding sites with a 100-fold lower affinity for calmodulin. The Ca²⁺-dependence of calmodulin binding and of adenylate cyclase activation varies with the amount of calmodulin present, as can be infered from the model of sequential equilibrium reactions which describes the activation of calmodulin-dependent enzymes. On the basis of this model, a quantitative analysis of the effect of free Ca²⁺ and of free calmodulin concentration on both binding and activation of adenylate cyclase was carried out. This analysis shows that both processes take place only when calmodulin is complexed with at least three Ca²⁺ atoms. The concentration of the active calmodulin ·Ca²⁺ species required for half-maximal activation of adenylate cyclase is very similar to the K_d of the high affinity binding sites on brain membranes. A Hill coefficient of approx. 1 was found for both processes indicating an absence of cooperativity. Phenothiazines and thioxanthenes antipsychotic agents inhibit calmodulin binding to membranes and calmodulin-dependent activation of adenylate cyclase with a similar order of potency. These results suggest that the Ca²⁺-dependent binding of calmodulin to specific high affinity sites on brain membranes regulates the activation of adenylate cyclase by calmodulin.     

Two cytosolic,Ca2+-dependent,neutral proteinases from rabbit liver: Purification and properties of the proenzymes

Two Ca²⁺-requiring proteinases have been purified from rabbit liver cytosol and shown to be present in isolated hepatocytes. They differ in relative molecular mass, with the major and minor forms, M_r = 150,000 and M_r = 200, 000, accounting for 75 and 18% of the total cytosolic neutral proteinase activity, respectively. Both are recovered as inactive proenzymes that can be converted to the active, low-Ca²⁺-requiring proteinases by incubation with Ca²⁺ and substrate [S. Pontremoli, E. Melloni, F. Salamino, B. Sparatore, M. Michetti, and B. L. Horecker (1984) Proc. Natl. Acad. Sci. USA81, 53–56. Each proenzyme is composed of two subunits, with molecular masses of 80 and 100 kDa, respectively. Activation of the proenzymes was found to correlate with their dissociation into subunits. The optimum pH for conversion of the proenzymes to the active proteinases in the presence of 5 mm Ca²⁺ and 2 mg/ml of denatured globin was approximately 7.5, and the same pH optimum was observed for the digestion of denatured globin by the activated proteinases. Following activation, each proteinase was observed to undergo autolytic inactivation at rates that were dependent on the concentration of both Ca²⁺ and the digestible substrate. A model is proposed for the activation of the proenzymes and the subsequent inactivation of the active proteinases.     

Regulation of the Ca2+-dependent neutral proteinases from rabbit liver by an endogenous inhibitor

An endogenous inhibitor of neutral Ca2+-dependent proteinases has been isolated from rabbit liver cytosol. The inhibitor is a heat-stable, 240-kDa, tetrameric protein. It is dissociated into its 60-kDa subunits by high concentrations of Ca2+ (0.1-1 mM), but not by lower concentrations in the physiological range. Inhibition of the 150-kDa proteinase of rabbit liver [Melloni, E., Pontremoli, S., Salamino, F., Sparatore, B., Michetti, M. and Horecker, B.L. (1984) Arch. Biochem. Biophys. 232, 505-512] requires the monomeric form of the inhibitor, and occurs only at the high concentrations of Ca2+ which also cause dissociation of the dimeric 150-kDa proteinase into its 80-kDa subunits. The molecular weight of the inactive proteinase-inhibitor complex was estimated by the equilibrium gel penetration method to be 140 kDa, suggesting that it contains one subunit of proteinase and one of inhibitor. The mechanism of interaction of the inhibitor with the 200-kDa proteinase at high concentrations of Ca2+ is identical to that observed for the 150-kDa proteinase, namely dissociation of both proteinase and inhibitor into subunits and formation of an inactive 160-kDa proteinase-inhibitor complex. However, unlike the 150-kDa proteinase, which does not interact with the inhibitor at low Ca2+ concentrations, the 200-kDa proteinase is also inhibited at low concentrations of Ca2+. Under these conditions, the high-molecular-weight complex (greater than 400 kDa) formed between the tetrameric inhibitor and the dimeric proteinase prevents conversion of the 200-kDa proenzyme to the active, low-Ca2+-requiring form.     

Active Ca2+ transport by vesicles reconstituted from triton X-100-solubilized pigeon erythrocyte membrane

Pigeon erythrocyte membrane was solubilized partially, but relatively unselectively by Triton X-100. Vesicles were reconstituted from mixtures of Triton-solubilized membrane and lipid (phosphatidylcholine plus phosphatidylethanolamine plus cholesterol) by addition of bovine high-density lipoprotein. This efficiently removed the Triton X-100. Sodium dodecyl sulfate-polyacrylamide gel electropherograms of reconstituted vesicles showed band patterns resembling those of the original membrane. The reconstituted vesicles showed ATP-dependent active accumulation of ⁴⁵Ca²⁺. ATP-dependent ⁴⁵Ca²⁺ uptake by the reconstituted vesicles resembled the corresponding activity of the original membrane vesicles; in both preparations the Ca²⁺ uptake rate depended on the square of the Ca²⁺ concentration and had similar $\text{[Ca}{}^{\mathrm{2+}}\text{]}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ values, 0.16 μM and 0.18 μM, respectively.     

Regulation of neutral protease activity through the life cycle of Tetrahymena pyriformis

Substantial fluctuations in the intracellular specific activity of neutral proteases, as assayed at pH 8 with azocasein as substrate, occur during the life cycle of the protozoan Tetrahymena pyriformis. Specific activity increases during growth in 2% proteose peptone, despite slow secretion into the medium. The most rapid increase occurs during late stationary phase and appears to be a response to one or more low molecular weight (less than 10 000), heatstable, trypsin-insensitive, polar molecules secreted into the medium. In contrast, intracellular specific activity drops by a factor of 2–5 within the first 2–3 h after transfer to non-nutritive medium. The decrease in activity under these conditions results from an enhanced rate of secretion and the cessation of net synthesis. Its kinetics are unaffected by cycloheximide and concanavalin A.     

The degradation of endogenous and exogenous proteins in cultured smooth muscle cells

The pathways of degradation followed by endogenous proteins in cultured smooth muscle cells were compared with the well-characterized lysosomal pathway involved in the degradation of apolipoprotein B of endocytosed LDL. Under conditions in which lysosomal activity towards ¹²⁵I-labeled LDL was almost completely inhibited by chloroquine and/or ammonium chloride, the degradation of short-lived and abnormal proteins, assessed by the release of [³H]phenylalanine, was reduced by only 10–17%. The basal rate of degradation of long-lived proteins was reduced by about 30% by the same inhibitors while the accelerated proteolysis found under nutrient-poor conditions could be completely accounted for by the lysosomal system as defined by these lysosomotrophic agents. Temperature studies indicated differences between the mechanisms involved in the degradation of long-lived proteins (E_a = 18 kcal/mol) and short-lived proteins (E_a = 10 kcal/mol). Arrhenius plots for the degradation of endogenous proteins showed no transitions between 15 and 37°C in contrast to the breakdown of LDL which ceased below 20°C. The results indicate that the degradation of rapid-turnover proteins is largely extralysosomal and that a significant breakdown of long-lived proteins occurs also outside lysosomes.     

The use of phospholipase C to detect structural changes in the membranes of human erythrocytes aged by storage

Human blood was stored under blood transfusion conditions for up to 10 weeks. At various times samples were removed, erythrocytes isolated and the susceptibility of the erythrocyte membrane lipids to non-lytic concentrations of phospholipase C from either Bacillus cereus or Clostridium perfringens tested. The morphology of the cells at various times and the release of microvesicles from the erythrocytes were also assessed. Initially the cells were attacked very little by the phospholipases at the concentrations chosen, but their susceptibility increased markedly after about 2 weeks, stabilised until 5 weeks, and then increased again to approach a nearly stable value after 8–10 weeks. The first rise accompanied the conversion of most of the cells to crenated and echinocytic configurations and was reversed if cells were incubated in a ‘rejuvenating’ medium designed to restore their energy supplies. The second rise occurred during the period when the cells underwent extensive microvesiculation and eventually became spherocytes: this phase involved, in particular, an increase in availability of phosphatidylethanolamine for hydrolysis by phospholipase C and was not reversed by attempts at ‘rejuvenation’. When microvesicles released from the cells were harvested and their phospholipase susceptibility compared with that of the residual cells it was found that the microvesicles were the more susceptible. These changes in phospholipase susceptibility presumably reflect subtle changes in membrane organization that occur during storage and vesiculation of erythrocytes; the possible nature of such changes is discussed.     

Effect of temperature on membrane fluidity and calcium conductance of the excitable ciliary membrane from Paramecium

Fluorescence anisotropy and average fluorescence lifetime of diphenylhexatriene were measured in artificial lipid membrane vesicles. Within the temperature range investigated (15–52°C) both parameters correlate and can be used interchangeably to measure membrane fluidity. Fluorescence anisotropy of DPH in membrane vesicles of cilia from the protozoan Paramecium tetraurelia decreased slightly from 5 to 37°C, yet, no phase transition was observed. An estimated flow activation energy of approx. 2 kcal/mol indicated that the ciliary membrane is very rigid and not readily susceptible to environmental stimuli. The ciliary membrane contains two domains of different membrane fluidity as indicated by two distinct fluorescence lifetimes of diphenylhexatriene of 7.9 and 12.4 ns, respectively. Ca²⁺ flux into ciliary membrane vesicles of Paramecium as measured with the Ca²⁺ indicator dye arsenazo III showed a nonlinear temperature dependency from 5 to 35°C with a minimum around 15°C and increasing flux rates at higher and lower temperatures. The fraction of vesicles permeable for Ca²⁺ remained unaffected by temperature. The differences in temperature dependency of Ca²⁺ conductance and membrane fluidity indicate that the Ca²⁺ permeability of the ciliary membrane is a membrane property which is not directly affected by the fluidity of its lipid environment.     

A study of protein phosphorylation in shape change and Ca++-dependent serotonin release by blood platelets

Upon treatment with agents such as thrombin, collagen or concanavalin A, blood platelets change shape, secrete serotonin and phosphorylate two proteins having molecular weights of approximately 20,000 and 40,000. We have analyzed the relationship of this protein phosphorylation to shape change and release aided by the fact that while shape change occurs independently of extracellular calcium, release of serotonin displays a rather strict calcium requirement. Under limited calcium conditions, where virtually no serotonin release occurs, (Con A)-stimulated phosphorylation is uninhibited. Divalent cations (Mg⁺⁺, Co⁺⁺ and Zn⁺⁺) also inhibit release but not phosphorylation. The microtubule effectors colchicine and D₂O show concomitant effects on release and phosphorylation, indicating a microtubule involvement prior to phosphorylation. Papaverine inhibits release and phosphorylation while not strongly influencing shape change, suggesting that shape change does not require phosphorylation. We therefore conclude that phosphorylation of these proteins takes place after shape change but prior to release, and although it may be required for secretion to occur, the two processes are easily separated. Thus phosphorylation of these proteins is not likely to be an integral component of the release mechanism.     

Elevation of [3H]cimetidine binding by CuCl2 in brain membranes of rats

Influences of dithiothreitol (DTT), p-chloromercuriphenyl sulfonate (PCMPS) and ascorbate on CuCl₂-induced elevation of [³H]cimetidine binding were investigated in brain membranes of rats. CuCl₂ (10–500 μM) elevated specific [³H]cimetidine binding in a concentration-dependent manner. There were two types of [³H]cimetidine binding in the presence of 50 μM CuCl₂: high affinity binding with K_d = 1.97 nM and low affinity with K_d = 21.6 nM. PCMPS (10 and 100 μM) reduced the binding in both media with and without CuCl₂. DTT (1–30 μM) or ascorbate (0.1 and 1.0 mM) markedly elevated the binding in the presence of CuCl₂ but showed no effect and ascorbate rather inhibited the binding in the absence of CuCl₂. DTT (0.1 mM) diminished the binding in the presence and absence of CuCl₂. CuCl₂ (50 μM) significantly (P < 0.01) increased the IC₅₀ of histamine for [³H]cimetidine binding and the effect was greater than that from 100 μM GTP. It is suggested that sulfhydryl groups sensitive to PCMPS could interact with Cu²⁺ and thus be involved in an elevation of cimetidine binding. Cu²⁺ seems to regulate affinity of agonist binding for cimetidine binding sites presumably by acting on cimetidine binding sites and/or GTP binding regulatory proteins.