Dissociation and aggregation of calpain in the presence of calcium

Calpain is a heterodimeric Ca(2+)-dependent cysteine protease consisting of a large (80 kDa) catalytic subunit and a small (28 kDa) regulatory subunit. The effects of Ca(2+) on the enzyme include activation, aggregation, and autolysis. They may also include subunit dissociation, which has been the subject of some debate. Using the inactive C105S-80k/21k form of calpain to eliminate autolysis, we have studied its disassociation and aggregation in the presence of Ca(2+) and the inhibition of its aggregation by means of crystallization, light scattering, and sedimentation. Aggregation, as assessed by light scattering, depended on the ionic strength and pH of the buffer, on the Ca(2+) concentration, and on the presence or absence of calpastatin. At low ionic strength, calpain aggregated rapidly in the presence of Ca(2+), but this was fully reversible by EDTA. With Ca(2+) in 0.2 m NaCl, no aggregation was visible but ultracentrifugation showed that a mixture of soluble high molecular weight complexes was present. Calpastatin prevented aggregation, leading instead to the formation of a calpastatin-calpain complex. Crystallization in the presence of Ca(2+) gave rise to crystals mixed with an amorphous precipitate. The crystals contained only the small subunit, thereby demonstrating subunit dissociation, and the precipitate was highly enriched in the large subunit. Reversible dissociation in the presence of Ca(2+) was also unequivocally demonstrated by the exchange of slightly different small subunits between mu-calpain and m-calpain. We conclude that subunit dissociation is a dynamic process and is not complete in most buffer conditions unless driven by factors such as crystal formation or autolysis of active enzymes. Exposure of the hydrophobic dimerization surface following subunit dissociation may be the main factor responsible for Ca(2+)-induced aggregation of calpain. It is likely that dissociation serves as an early step in calpain activation by releasing the constraints upon protease domain I.     

A membrane glycoprotein-containing fraction which promotes all aggregation.

Glycoproteins have been extracted from 16C rat fibroblasts using acetone/lithium di-iodosalicylate/phenol purification. This fraction increases the aggregation of the fibroblasts $\text{in vitro}$.     

Investigation on the surface hydrophobicity and aggregation kinetics of human calprotectin in the presence of calcium

Calcium and zinc binding protein, calprotectin is a multifunctional protein with broad spectrum antimicrobial and antitumoural activity. It was purified from human neutrophil, using a two-step ion exchange chromatography. Since surface hydrophobicity of calprotectin may be important in membrane anchoring, membrane penetration, subunits oligomerization and some biological roles of protein, in this study attempted to explore the effect of calcium in physiological range on the calprotectin lipophilicity. Incubation of human calprotectin (50 microg/ml) with different calcium concentrations showed that 1-anilino-8-naphthalene sulfonic acid (ANS) fluorescence intensity of the protein significantly elevates with calcium in a dose dependent manner, suggesting an increase in calprotectin surface hydrophobicity upon calcium binding. Our study also indicates that calcium at higher concentrations (6, 8 and 10 mM) induces aggregation of human calprotectin. Our finding demonstrates that the starting time and the rate constant of calprotectin aggregation depend on the calcium concentration.     

Preparation of amphiphilic poly(L-lactide)-graft-chondroitin sulfate copolymer self-aggregates and its aggregation behavior

Novel polymeric amphiphilic copolymers were synthesized using chondroitin sulfate (CS) as a hydrophilic segment and poly(L-lactide) (PLLA) as a hydrophobic segment. Micelles of those copolymers were formed in an aqueous phase and were characterized by 1H NMR spectra, fluorescence techniques, dynamic light scattering (DLS), atomic force microscopy (AFM), and confocal microscopy. Their critical aggregation concentrations (CAC) are in the range of 0.0043-0.0091 mg/mL at 25 degrees C. The partition equilibrium constants, Kv, of the pyrene probe in the aqueous solution were from 3.65 x 10(5) to 1.41 x 10(6) at 25 degrees C. The mean diameters of the micelles were below 200 nm, and their sizes were narrowly distributed. The AFM images revealed that the self-aggregates were spherical. Additionally, the CSn-PLLA micelles can efficiently transport within the cells via endocytosis as observed from confocal microscopy.     

The basement membrane glycoprotein entactin promotes cell attachment and binds calcium ions

Mouse entactin derived from the extracellular matrix of M1536-B3 cells and from insect cells infected with a recombinant virus containing entactin sequences were shown to promote the attachment of mouse mammary tumor, human melanoma, and other cells. The cell attachment was inhibited by antibodies against mouse entactin but not by anti-fibronectin or anti-laminin antibodies. On a weight basis entactin was as effective as laminin in promoting the attachment of mouse mammary tumor cells. The attachment of cells to entactin was in part mediated by the integrin recognition RGD peptide sequence. This was demonstrated by the cell attachment properties of peptides derived from entactin which contained this sequence. Furthermore, the peptide RGDS could inhibit the attachment of mouse mammary tumor cells to entactin to approximately 60% of control. It is suggested that additional cell recognition sequences may be present in entactin. The direct binding of calcium ions to entactin was observed. It is probable that the binding sites reside in peptide sequences located toward the NH2 terminus region of entactin. This conclusion was supported by the demonstration that synthetic peptides, containing potential calcium binding sequences derived from entactin, bound calcium. In addition, a recombinant peptide containing the amino-terminal 330 amino acids of entactin also bound calcium ions. The significance of these properties of entactin is discussed.     

Self-oligomerization and protein aggregation of alpha-synuclein in the presence of Coomassie Brilliant Blue.

alpha-Synuclein has been implicated in various neurodegenerative disorders, including Parkinson's and Alzheimer's diseases, by its participation in abnormal protein depositions. As the protein has been suggested to play a significant role in the formation of the deposits which might be responsible for neurodegeneration, there is a strong demand to screen for alpha-synuclein-interactive small molecules. In this report, Coomassie Brilliant Blue (CBB) interaction of alpha-synuclein has been investigated with respect to induction of protein self-oligomerization in the presence of the chemical coupling reagent N-(ethoxycarbonyl)-2-ethoxy-1,2-dihydroquinoline. Both CBB-G and CBB-R, which differ by only two methyl groups, induced the self-oligomerization of alpha-synuclein in a biphasic manner with optimal dye concentrations of 250 microM and 150 microM, respectively. The protein aggregates of alpha-synuclein induced by the dyes in the absence of the coupling reagent were analysed by electron microscopy. Whereas CBB-G induced formation of protein aggregates with a worm-like structure, CBB-R induced clear fibrilization of alpha-synuclein on a background of granular structures. CBB-R interacted with alpha-synuclein approximately twice as effectively as CBB-G (dissociation constants 0.63 microM and 1.37 microM, respectively). These dye interactions were independent from the acidic C-terminus of alpha-synuclein, which was reminiscent of the Alphabeta25-35 interaction of alpha-synuclein. However, the metal-catalysed oxidative self-oligomerization of alpha-synuclein in the presence of Cu2+/H2O2, which was augmented synergistically by Alphabeta25-35, was not affected by the dyes. This indicates that the dye binding site is also distinctive from the Alphabeta25-35 interaction site on alpha-synuclein. These biochemically specific interactions between alpha-synuclein and the dyes indicate that alpha-synuclein-interactive small molecules could provide a tool with which to approach development of diagnostic, preventive, or therapeutic strategies for various alpha-synuclein-related neurodegenerative disorders.     

The aggregation of isolated human platelets in the presence of lipoproteins and prostacyclin.

Addition of prostacyclin (PGI2) temporarily inhibits platelet aggregation and permits the isolation of platelets free from plasma proteins, which have the same sensitivity as those in plasma [Moncada, Radomski & Vargas (1982) Br. J. Pharmacol. 75, 165P]. By using a modification of this technique we have established that platelets isolated from normal subjects aggregate more readily in response to ADP and adrenaline when physiological concentrations of low-density lipoproteins (LDL) are present. At high LDL concentrations spontaneous aggregation occurs. High-density lipoproteins (HDL) and very-low-density lipoproteins (VLDL) had no effect on agonist-induced platelet aggregation at normal concentrations, but HDL sensitized at higher concentrations. These effects by lipoproteins are not accompanied by changes in platelet lipid content. Cyclohexanedione treatment of LDL to modify apolipoproteins appeared to abolish the sensitization effect, indicating that binding to receptors was essential for the effects of LDL. LDL, but not HDL, overcame the inhibitory effect of PGI2 on platelet aggregation, except at very high concentrations of PGI2. PGI2 raised the cyclic AMP content of isolated platelets, but LDL only partially prevented this rise. These results suggest that LDL may have a greater role in platelet aggregation than previously recognized and may also regulate effects of PGI2. These findings may be of relevance to an understanding of cardiovascular diseases.     

The interaction of beta-adrenoceptor blocking drugs with platelet aggregation, calcium displacement and fluidization of the membrane

beta-Adrenoceptor blocking drugs interfere with adenosine diphosphate-stimulated platelet aggregation. Alprenolol, exaprolol, K? 1124 and propranolol inhibited the aggregation, metipranolol decreased the extent and rate of aggregation significantly. Atenolol potentiated the aggregation measured by amplitude significantly. The interaction of beta-adrenoceptor blocking drugs with aggregation correlated with the displacement of calcium ions from binding sites in isolated platelets and the fluidization of the whole platelets and isolated platelet membrane as measured with electron spin resonance of the spin probe. The most potent were highly liposoluble drugs alprenolol, exaprolol, metipranolol and propranolol which increased the calcium displacement and membrane fluidity, the least active was atenolol decreasing these phenomena. The inhibition by beta-adrenoceptor blocking drugs of stimulated platelet aggregation is rather a result of unspecific than specific receptor interaction.     

Streamless aggregation of Dictyostelium in the presence of isopropylidenadenosin

Starving cells of Dictyostelium discoideum undergo a developmental cycle where cAMP is autocatalytically produced and relayed from cell to cell, resulting in the propagation of excitation waves over a spatially extended population. Later on the homogeneous cell layer transforms into a pattern of cell streams directed perpendicular to the cAMP waves. Here we chemically influence aggregation competent cells by isopropylidenadenosin (IPA), an adenosine derivative. It can be assumed, that IPA acts via specific adenosine binding sites localized in the cellular membrane. We find, however, that pattern formation and cellular aggregation under the influence of IPA differ considerably compared to experiments with adenosine. In particular, our observations point towards an inhibitory effect on adenylate cyclase (ACA), the key enzyme in the autocatalytic production process of cAMP inside the cell. Our results suggest the existence of a direct coupling (via intracellular affection) or indirect coupling (via inhibition of cAMP binding) of the specific adenosine receptors to the regulatory circuit that controls cyclic intra- and extracellular cAMP concentration.     

Effects of dihydropyridines and inorganic calcium blockers on aggregation and on intracellular free calcium in platelets.

[Ca2+]i increase is necessary in physiological platelet activity, particularly aggregation and release. The increase of [Ca2+]i observed during platelet activation depends in part on Ca2+ influx from the extracellular medium. The participation of voltage-operated Ca2+ channels as a pathway for Ca2+ entry is controversial. In the present study we have attempted to reinvestigate this problem by measuring aggregation and [Ca2+]i changes in platelets activated by ADP or thrombin and incubated with organic or inorganic blockers of calcium channels. The main findings of the present paper can be summarized as follows: (i) Ni2+, Co2+ and Mn2+, well known inorganic blockers of Ca2+ channels, inhibited platelet aggregation induced by ADP or thrombin in a dose-dependent manner, Ni2+ being the most effective agent. (ii) Thrombin induced a rise in free [Ca2+]i in platelets incubated both in 1 mmol/l Ca(2+)-containing medium and in nominally Ca(2+)-free medium; the rise of free [Ca2+]i was in the first case up to 370 +/- 31 nmol/l and in the second case up to 242 +/- 26 nmol/l, indicating that this observed difference was due to Ca2+ entry from the extracellular medium. Co2+ and Ni2+ abolished that difference by inhibiting Ca2+ influx. (iii) Nisoldipine, nitrendipine and nimodipine (10-50 nmol/l) inhibited in a dose-dependent manner platelet aggregation induced by either ADP or thrombin in platelets incubated in normal-Ca2+ normal-K+ medium, also, aggregation was inhibited to a similar extent in platelets incubated in normal-Ca2+ high-K+ medium. (iv) Nisoldipine--the most effective dihydropyridine to inhibit platelet aggregation--also inhibited Ca2+ influx in platelets incubated in normal-Ca2+ medium, either in normal-K+ or high-K+ media. Our data support the existence of voltage-operated, dihydropyridine-sensitive calcium channels (L-type) and a physiological role for them in platelet function.     

Handling time promotes the coevolution of aggregation in predator-prey systems

Predators often have type II functional responses and live in environments where their life history traits as well as those of their prey vary from patch to patch. To understand how spatial heterogeneity and predator handling times influence the coevolution of patch preferences and ecological stability, we perform an ecological and evolutionary analysis of a Nicholson-Bailey type model. We prove that coevolutionarily stable prey and searching predators prefer patches that in isolation support higher prey and searching predator densities, respectively. Using this fact, we determine how environmental variation and predator handling times influence the spatial patterns of patch preferences, population abundances and per-capita predation rates. In particular, long predator handling times are shown to result in the coevolution of predator and prey aggregation. An analytic expression characterizing ecological stability of the coevolved populations is derived. This expression implies that contrary to traditional theoretical expectations, predator handling time can stabilize predator-prey interactions through its coevolutionary influence on patch preferences. These results are shown to have important implications for classical biological control.     

Effect of cholesterol on Ca2+-induced aggregation of liposomes and calcium diphosphatidate membrane traversal

Sonicated cholesterol-phosphatidylcholine (PC) liposomes containing 4 mol % phosphatidic acid (PA) aggregate in 10 mM Ca2+, slowly at low molar fractions of cholesterol (up to 30%) and 15 times faster at higher concentrations; the inflection point is at ca. 35 mol % bilayer cholesterol. O-[[(Methoxyethoxy)ethoxy]ethyl]cholesterol (OH-blocked cholesterol) does not give this rate enhancement. If PC is replaced by diether PC (CO groups abolished), cholesterol does not accelerate aggregation at concentrations in the bilayer below 50 mol %. No change in Ca2+-induced aggregation rates was observed if the ester CO groups of the bridge-forming PA only were replaced by CH2 (diether PA) in liposomes containing PC and cholesterol. PA-mediated Ca2+ membrane traversal seems to be accelerated by the addition of cholesterol to the PC-PA membrane, but analysis shows that the effect is due to the bilayer condensation effect of cholesterol resulting in an increase in the surface concentration of PA and that membrane cholesterol in fact slightly reduces the rate of Ca(PA)2 traversal; OH-blocked cholesterol, however, increases this rate 3-fold. It appears that lipid OH and CO groups interact, directly or with the mediation of water, in establishing the structure of the membrane "hydrogen belts", i.e., the strata containing those hydrogen-bond donors and acceptors. Cholesterol hydroxyl above 33 mol % (saturation of a 2:1 PC/cholesterol complex?) causes a restructuring of the hydrogen belts that facilitates membrane-water-membrane dehydration, the prerequisite for liposome aggregation by trans-Ca(PA)2 formation. On the other hand, the formation of the dehydrated cis-Ca(PA)2 complex that precedes Ca2+ membrane traversal is not accelerated by presence of the cholesterol hydroxyl group.     

The phaseolin vacuolar sorting signal promotes transient, strong membrane association and aggregation of the bean storage protein in transgenic tobacco

Vacuolar storage proteins of the 7S class are co-translationally introduced into the endoplasmic reticulum and reach storage vacuoles via the Golgi complex and dense vesicles. The signal for vacuolar sorting of one of these proteins, phaseolin of Phaseolus vulgaris, consists of a four-amino acid hydrophobic propeptide at the C-terminus. When this sequence is deleted, phaseolin is secreted instead of being sorted to vacuoles. It is shown here that in transgenic tobacco plants newly-synthesized phaseolin has unusual affinity to membranes and forms SDS-resistant aggregates, but mutated phaseolin polypeptides that are either secreted or defective in assembly do not have these characteristics. Association to membranes and aggregation are transient events: phaseolin accumulated in vacuoles is soluble in the absence of detergents and is not aggregated. Association to membranes starts before the phaseolin glycan acquires a complex structure and therefore before the protein reaches the medial or trans-cisternae of the Golgi complex. These results support the hypothesis of a relationship between aggregation and vacuolar sorting of phaseolin and indicate that sorting may start in early compartments of the secretory pathway.     

Changes in membrane potential during calcium ion influx and efflux across the mitochondrial membrane.

1. A depolarisation of the membrane of rat liver mitochondria, as measured with the safranine method, is seen during Ca2+ uptake. The depolarisation is followed by a slow repolarisation, the rate of which can be increased by the addition of EGTA or phosphate. 2. Plots relating the initial rate of calcium ion (Ca2+) uptake and the decrease in membrane potential (delta psi) to the Ca2+ concentration show a half-maximal change at less than 10 micron Ca2+ and a saturation above 20 micron Ca2+. 3. Plots relating the initial rate of Ca2+ uptake to delta psi are linear. 4. Addition of Ca2+ chelators, nitriloacetate or EGTA, to deenergized mitochondria equilibrated with Ca2+ causes a polarisation of the mitochondrial membrane due to a diffusion potential created by electrogenic Ca2+ efflux. 5. If the extent of the response induced by different nitriloacetate concentrations is plotted against the expected membrane potential a linear plot is obtained up to 70 mV with a slope corresponding to two-times the extent of the response induced by valinomycin in the presence of different potassium ion gradients. This suggests that the Ca2+ ion is transferred across the membrane with one net positive charge in present conditions.     

Membrane depolarization inhibits thrombin-induced calcium influx and aggregation in human platelets.

The relationship between thrombin-evoked changes in intracellular calcium concentration [( Ca2+]i) and aggregation was examined in Indo-1-loaded human platelets. The stimulus-induced intracellular calcium release and external calcium influx, as well as platelet aggregation, were studied in the same cell preparation. A close correlation between the sustained high [Ca2+]i level, depending on calcium entry, and the aggregation response was found. Gramicidin, at a concentration high enough to induce membrane depolarization, strongly inhibited the calcium influx and aggregation, but did not influence the thrombin-induced intracellular calcium release. We conclude that calcium influx through depolarization-inhibited calcium channels is a prerequisite of thrombin-induced platelet aggregation.     

Current-voltage studies on the thylakoid membrane in the presence of ionophores.

The reversibility of the binding of ionophores to the thylakoid membrane is studied. While gramicidin binds practically irreversibly, valinomycin and nonactin bind reversibly, however, only a small fraction (about 1%) of the membrane-bound valinomycin or nonactin is active in ion transport. The current-voltage relationship is evaluated under these circumstances. We have found that it is practically linear. This together with the relationship between current and ion concentration agrees qualitatively with the results reported for bimolecular lipid membranes, which contain a large fraction of negatively charged lipids. For the ionophores, valinomycin and nonactin, the binding equilibria (K approximately equal to 10-4) and the turnover numbers (approximately equal to 3-10-4/s) are evaluated for their action on the thylakoid membrane. Possible reasons for the inactivity of the majority of membrane-bound ionophore molecules are discussed.