A single-pool model for intracellular calcium oscillations and waves in the Xenopus laevis oocyte.

We construct a minimal model of cytosolic free Ca2+ oscillations based on Ca2+ release via the inositol 1,4,5-trisphosphate (IP3) receptor/Ca2+ channel (IP3R) of a single intracellular Ca2+ pool. The model relies on experimental evidence that the cytosolic free calcium concentration ([Ca2+]c) modulates the IP3R in a biphasic manner, with Ca2+ release inhibited by low and high [Ca2+]c and facilitated by intermediate [Ca2+]c, and that channel inactivation occurs on a slower time scale than activation. The model produces [Ca2+]c oscillations at constant [IP3] and reproduces a number of crucial experiments. The two-dimensional spatial model with IP3 dynamics, cytosolic diffusion of IP3 (Dp = 300 microns 2 s-1), and cytosolic diffusion of Ca2+ (Dc = 20 microns 2 s-1) produces circular, planar, and spiral waves of Ca2+ with speeds of 7-15 microns.s-1, which annihilate upon collision. Increasing extracellular [Ca2+] influx increases wave speed and baseline [Ca2+]c. A [Ca2+]c-dependent Ca2+ diffusion coefficient does not alter the qualitative behavior of the model. An important model prediction is that channel inactivation must occur on a slower time scale than activation in order for waves to propagate. The model serves to capture the essential macroscopic mechanisms that are involved in the production of intracellular Ca2+ oscillations and traveling waves in the Xenopus laevis oocyte.     

Dopamine receptor-mediated Ca(2+) signaling in striatal medium spiny neurons

Inositol 1,4,5-trisphosphate (InsP(3)) and cAMP are the two second messengers that play an important role in neuronal signaling. Here, we investigated the interactions of InsP(3)- and cAMP-mediated signaling pathways activated by dopamine in striatal medium spiny neurons (MSN). We found that in approximately 40% of the MSN, application of dopamine elicited robust repetitive Ca(2+) transients (oscillations). In pharmacological experiments with specific agonists and antagonists, we found that the observed Ca(2+) oscillations were triggered by activation of D1 class dopamine receptors (DARs). We further demonstrated that activation of phospholipase C was required for induction of dopamine-induced Ca(2+) oscillations and that maintenance of dopamine-evoked Ca(2+) oscillations required both Ca(2+) influx and Ca(2+) mobilization from internal Ca(2+) stores. In "priming" experiments with a type 2 5-hydroxytryptamine receptor agonist, we have shown a likely role for calcyon in coupling D1 class DARs with Ca(2+) oscillations in MSN. In experiments with the DAR-specific agonist SKF83959, we discovered that phospholipase C activation alone could not account for dopamine-induced Ca(2+) oscillations. We further demonstrated that direct activation of protein kinase A by 8-bromo-cAMP or inhibition of protein phosphatase-1 (PP1) or calcineurin (PP2B) resulted in elevation of basal Ca(2+) levels in MSN, but not in Ca(2+) oscillations. In experiments with competitive peptides, we have shown an importance of type 1 InsP(3) receptor association with PP1alpha and with AKAP9.protein kinase A for dopamine-induced Ca(2+) oscillations. In experiments with MSN from DARPP-32 knock-out mice, we demonstrated a regulatory role of DARPP-32 in dopamine-induced Ca(2+) oscillations. Our results indicate that, following D1 class DAR activation, InsP(3) and cAMP signaling pathways converge on the type 1 InsP(3) receptor, resulting in Ca(2+) oscillations in MSN.     

Fertilization stimulates long-lasting oscillations of CaMKII activity in mouse eggs

Elucidation of the biochemical mechanisms by which specific proteins transduce the all important intracellular calcium (Ca2+) signal at fertilization into events of egg activation will increase our understanding of the regulation of the onset of development and the extent to which these signals can be experimentally modified. Previously, we reported data supporting the hypothesis that mouse eggs have the capability to generate oscillations of the activity of Ca2+ and calmodulin-dependent kinase II (CaMKII), regulating the cell cycle and secretion. This study directly demonstrates transient increases of enzyme activity in relatively close synchrony with Ca2+ oscillations for the first hour of fertilization in single mouse eggs monitored for both Ca2+ and CaMKII activity. The extent of the enzyme activity increase was correlated with the level of intracellular Ca2+. After a rise in activity, the decrease in activity did not appear to be due to negative feedback from elevated Ca2+ or CaMKII activity over time, since enzyme activity persisted after 8 min of elevated Ca2+ from 7% ethanol activation. The contribution of CaMKII from a single sperm to the rise in CaMKII activity at fertilization appeared to be negligible. Also, long-term cell cycle inhibition was observed in fertilized eggs with the CaMKII antagonist myrAIP (50 microM), which did not inhibit the first large Ca2+ transient or subsequent early oscillations but did reduce the percentage of eggs fertilized. Thus, mammalian eggs appear to drive many activation events over time to completion with repeated short bursts of Ca2+ oscillation-dependent CaMKII activity, rather than by a steady-state, continuously elevated level of CaMKII activity that is maintained by periodic Ca2+ oscillations.     

Activation of the liver glycogen phosphorylase by Ca(2+)oscillations: a theoretical study

Cytosolic calcium plays a crucial role as a second messenger in cellular signalling. Various cell types, including hepatocytes, display Ca(2+)oscillations when stimulated by an extracellular signal. However, the biological relevance of this temporal organization remains unclear. In this paper, we investigate theoretically the effect of Ca(2+)oscillations on a particular example of cell regulation: the phosphorylation-dephosphorylation cycle controlling the activation of glycogen phosphorylase in hepatocytes. By modelling periodic sinusoidal variations in the intracellular Ca(2+)concentration, we show that Ca(2+)oscillations reduce the threshold for the activation of the enzyme. Furthermore, as the activation of a given enzyme depends on the kinetics of its phosphorylation-dephosphorylation cycle, specificity can be encoded by the oscillation frequency. Finally, using a model for signal-induced Ca(2+)oscillations based on Ca(2+)-induced Ca(2+)release, we show that realistic Ca(2+)oscillations can potentiate the response to a hormonal stimulation. These results indicate that Ca(2+)oscillations in hepatocytes could contribute to increase the efficiency and specificity of cellular signalling, as shown experimentally for gene expression in lymphocytes (Dolmetsch et al., 1998).     

Localization of a fibrin polymerization site

The formation of a fibrin clot is initiated after the proteolytic cleavage of fibrinogen by thrombin. The enzyme removes fibrinopeptides A and B and generates fibrin monomer which spontaneously polymerizes. Polymerization appears to occur though the interaction of complementary binding sites on the NH2-terminal and COOH-terminal (Fragment D) regions of the molecule. A peptide has been isolated from the gamma chain remnant of fibrinogen Fragment D1 which has the ability to bind to the NH2-terminal region of fibrinogen as well as to inhibit fibrin monomer polymerization. The peptide reduces the maximum rate and extent of the polymerization of thrombin or batroxobin fibrin monomer and increases the lag time. The D1 peptide does not interact with disulfide knot, fibrinogen, or Fragment D1, but it binds to thrombin-treated disulfide knot with a Kd of 1.45 X 10(-6) M at approximately two binding sites per molecule of disulfide knot. Fibrin monomer formed either by thrombin or batroxobin binds approximately two molecules of D1 peptide per molecule of fibrin monomer, indicating that the complementary site is revealed by the loss of fibrinopeptide A. The NH2-terminal sequence (Thr-Arg-Trp) and COOH-terminal sequence (Ala-Gly-Asp-Val) of the D1 peptide were determined. Therefore the gamma 373-410 region of fibrinogen contains a polymerization site which is complementary to the thrombin-activated site on the NH2-terminal region of fibrinogen.     

Fibrinogen binding to human platelet plasma membranes. Identification of two steps requiring divalent cations

Fibrinogen binding to platelet plasma membranes, which is a prerequisite for platelet aggregation, was determined by incubating 125I-labeled fibrinogen with isolated membranes and measuring the amount of radioactivity sedimenting with the membranes through 15% sucrose. Fibrinogen binding was optimal at 10(-3) M Ca2+. Scatchard analyses of the fibrinogen binding showed that the membrane capacity for fibrinogen was 1.6 X 10(-12) mol/mg of membrane protein, with a dissociation constant (Kd) = 1.2 X 10(-8) M. When Ca2+ levels were manipulated by the addition of varying amounts of EGTA at a fixed Mg2+ concentration of 3 X 10(-3) M, specific binding of fibrinogen to platelet membranes occurred only at Ca2+ concentrations greater than or equal to 10(-6) M. Membranes isolated from platelets of an individual with Glanzmann's thrombasthenia bound only 12% as much fibrinogen as control platelets. The data in the present study suggest that there are two divalent cation binding sites that must be occupied for fibrinogen to bind: one site is specific for calcium and is saturated at 10(-6) M Ca2+; the other site is less specific and is saturated at a 10(-3) M concentration of either Ca2+ or Mg2+. Fibrinogen binding to intact platelets and, consequently, platelet aggregation only required 10(-3) M extracellular divalent cation and was not specific for Ca2+. These data indicate that the cytoplasm is a potential source for the requirement of 10(-6) M Ca2+, and that changes in the intracellular concentration of Ca2+ may cause the expression of fibrinogen receptors during ADP-induced platelet activation.     

Effects of calcium binding and of EDTA and CaEDTA on the clotting of bovine fibrinogen by thrombin

Studies were carried out at pH 7.0 and gamma/2 0.15 before addition of CaCl2 or EDTA. Clotting time, tau, at 3.03 microM fibrinogen and 0.91 u/ml thrombin was determined for equilibrium systems. With added Ca2+, tau decreases, from tau 0 at 0 added Ca2+ (mean, 29.7 +/- 3 s), by approximately 3 s at 5 mM added Ca2+. With added EDTA, tau increases sigmoidally from tau 0 at 0 EDTA to a maximum (mean tau m = 142 +/- 23 s) at approximately 200 microM EDTA. tau then decreases slightly to a minimum at approximately 1.3 mM and finally increases to infinity at approximately 10 mM EDTA. Between 0 and 1.3 mM EDTA, effects on clotting time are completely reversed by adding Ca2+ and, after equilibration at 400 microM EDTA, tau is independent of EDTA concentration. Thus, up to 400 microM EDTA, effects on clotting time are attributed to decreasing fibrinogen bound Ca2+. Between 5 mM Ca2+ and 200 microM EDTA it is assumed that an equilibrium distribution of fibrinogen species having 3, 2, 1, or 0 bound calcium ions is established and that a clotting time is determined by the sum of products of species fractional abundance and pure species clotting time. Analysis indicates that pure species clotting times increase proportionately with decreasing Ca2+ binding, binding sites are nearly independent, and the microscopic association constant for the first bound Ca2+ is approximately 4.9 X 10(6) M-1. Effects of adding Ca2+ at times t1 after thrombin addition to systems initially equilibrated at 200 microM EDTA were determined. Analysis of the relation between tau and t1 indicates that as Ca2+ binding decreases, rate constants for release of B peptides decrease less than those for release of A peptides. As EDTA concentration is increased above 1.3 mM, inhibitory effects of EDTA and CaEDTA progressively increase.     

Localization of a fibrinogen calcium binding site between gamma-subunit positions 311 and 336 by terbium fluorescence

Calcium is required for effective fibrin polymerization. The high affinity Ca2+ binding capacity of fibrinogen was directly localized to the gamma-chain by autoradiography of nitrocellulose membrane blots of fibrinogen subunits incubated with 45Ca2+. Terbium (Tb3+) competitively inhibited 45Ca2+ binding to fibrinogen during equilibrium dialysis, accelerated fibrin polymerization, and limited fibrinogen fragment D digestion by plasmin. The intrinsic fluorescence of Ca2+-depleted fibrinogen was maximally enhanced by Ca2+ and Tb3+, but not by Mg2+, at about 3 mol of cation/mol of fibrinogen. Protein-bound Tb3+ fluorescence at 545 nm was maximally enhanced by resonance energy transfer from tryptophan (excitation at 290 nm) at about 2 mol of Tb3+mol of fibrinogen and about 1 mol of Tb3+/mol of plasmic fragment D94 (Mr 94,000). Fibrinogen fragments D78 (Mr 78,000) and E did not show effective enhancement of Tb3+ fluorescence, suggesting that the Ca2+ site is located within gamma 303 to gamma 411, the peptide which is absent in fragment D78 but present in D94. When CNBr fragments of the carboxyamidated gamma-subunit were assayed for enhancement of Tb3+ fluorescence, peptide CBi (gamma 311-336) bound 1 mol of Tb3+/mol of CBi. Thus, the Ca2+ site is located within this peptide. The sequence between gamma 315 and gamma 329 is homologous to the calmodulin and parvalbumin Ca2+ binding sites.     

Ca2+ oscillations induced by histamine H1 receptor stimulation in HeLa cells: Fura-2 and patch clamp analysis

The response of HeLa cells to histamine H1 receptor stimulation is characterized by periodic increases in cytosolic free Ca2+ concentration. The mechanisms underlying this oscillatory behaviour are not well understood. Fura-2 and patch clamp experiments carried out on HeLa cells have previously shown: (a) that Ca2+ oscillations are not initially dependent on the presence of external Ca2+, that external Ca2+ is required to maintain the oscillatory activity; (b) that a depolarization of the cell membrane leads to an inhibition of Ca2+ oscillations during the external Ca2+ dependent phase of the process; and (c) that Ca2+ oscillations can be abolished during this latter phase by the exogenous addition of Ca2+ channel blocking agents, such as Co2+ or La3+. The contribution of the inositol phosphate pathway to Ca2+ oscillations was more recently investigated in whole cell experiments performed with patch pipettes containing IP3 or the non-hydrolysable GTP analogue GTP-gamma S. Clear periodic current fluctuations were recorded using both patch pipette solutions. Assuming that the intracellular IP3 level remained constant under these conditions, these findings provide direct evidence that the Ca2+ oscillations in HeLa cells do not arise from a periodic production of IP3. The effect of the internal and external cell pH on the oscillatory process was also investigated in Fura-2 and patch clamp experiments. It was found that an increase in intracellular pH from 7.4 to 7.7 during the external Ca2+ dependent phase of the histamine stimulation abolishes the appearance of Ca2+ spikes whereas, a cellular acidification to pH 7.2 maintains or stimulates the Ca2+ oscillatory activity. The former effect was observed in the absence of Ca2+ in the bathing medium, indicating that the inhibitory action of alkaline pH was not related to a reduced Ca2+ entry. An increase in extracellular pH from 7.3 to 9.0 in contrast elicited an intracellular Ca2+ accumulation which resulted in most cases in an inhibition of the oscillatory process. This effect was dependent on external Ca2+ and was observed in alkaline internal pH conditions (pH 7.7). These observations suggest: (a) that the net Ca2+ influx in HeLa cells is strongly dependent on the cell internal and external pH; and (b) that the magnitude of this Ca2+ influx controls to a large extent the oscillation frequency. Finally, an inhibition of the histamine induced Ca2+ oscillatory activity was observed following the addition of the Ca(2+)-induced Ca(2+)-release (CICR) inhibitor adenine to the external medium.(ABSTRACT TRUNCATED AT 250 WORDS)     

Temporal order in mammalian cells. I. The periodic synthesis of lactate dehydrogenase in the cell cycle

Chinese hamster cells were synchronized by the Colcemid-selection system. In cells with a division cycle time of 11.5–12 hr, the activity of the enzyme lactate dehydrogenase (LDH) underwent marked oscillations with a 3.5-hr period. Precipitation of labeled LDH enzyme with specific antibody indicated that the enzyme activity changes were the result of intermittent enzyme synthesis and relatively constant degradation. Inhibition of normal DNA replication with 4 mM of thymidine, while reducing the amount of new enzyme synthesized, did not prevent oscillations from occurring. Similarly, actinomycin D (AcD) added at the time of synchronization allowed some new enzyme synthesis to proceed in an oscillatory manner. LDH synthesis went on at nearly normal rates when AcD was added in the middle of S phase. However, addition of cycloheximide to cultures at any time in the cycle caused an immediate drop in levels of activity and in enzyme protein. The half-life of LDH, calculated either from loss of enzyme activity or precipitable radioactivity in cycloheximide-treated cultures, was between 2 and 2.5 hr.     

Regulation of cAMP dynamics by Ca2+ and G protein-coupled receptors in the pancreatic beta-cell: a computational approach

In this report we describe a mathematical model for the regulation of cAMP dynamics in pancreatic beta-cells. Incretin hormones such as glucagon-like peptide 1 (GLP-1) increase cAMP and augment insulin secretion in pancreatic beta-cells. Imaging experiments performed in MIN6 insulinoma cells expressing a genetically encoded cAMP biosensor and loaded with fura-2, a calcium indicator, showed that cAMP oscillations are differentially regulated by periodic changes in membrane potential and GLP-1. We modeled the interplay of intracellular calcium (Ca(2+)) and its interaction with calmodulin, G protein-coupled receptor activation, adenylyl cyclases (AC), and phosphodiesterases (PDE). Simulations with the model demonstrate that cAMP oscillations are coupled to cytoplasmic Ca(2+) oscillations in the beta-cell. Slow Ca(2+) oscillations (<1 min(-1)) produce low-frequency cAMP oscillations, and faster Ca(2+) oscillations (>3-4 min(-1)) entrain high-frequency, low-amplitude cAMP oscillations. The model predicts that GLP-1 receptor agonists induce cAMP oscillations in phase with cytoplasmic Ca(2+) oscillations. In contrast, observed antiphasic Ca(2+) and cAMP oscillations can be simulated following combined glucose and tetraethylammonium-induced changes in membrane potential. The model provides additional evidence for a pivotal role for Ca(2+)-dependent AC and PDE activation in coupling of Ca(2+) and cAMP signals. Our results reveal important differences in the effects of glucose/TEA and GLP-1 on cAMP dynamics in MIN6 beta-cells.     

Characterization of the kinetic pathway for liberation of fibrinopeptides during assembly of fibrin

The time dependence of the release of fibrinopeptides from fibrinogen was studied as a function of the concentration of fibrinogen, thrombin, and Gly-Pro-Arg-Pro, an inhibitor of fibrin polymerization. The release of fibrinopeptides during fibrin assembly was shown to be a highly ordered process. Rate constants for individual steps in the formation of fibrin were evaluated at pH 7.4, 37 degrees C, gamma/2 = 0.15. The initial event, thrombin-catalyzed proteolysis at Arg-A alpha 16 to release fibrinopeptide A (kcat/Km = 1.09 X 10(7) M-1s-1) was followed by association of the resulting fibrin I monomers. Association of fibrin I was found to be a reversible process with rate constants of 1 X 10(6) M-1s-1 and 0.064 s-1 for association and dissociation, respectively. Assuming random polymerization of fibrin I monomer, the equilibrium constant for fibrin I association (1.56 X 10(7) M-1) indicates that greater than 80% of the fibrin I protofibrils should contain more than 10 monomeric units at 37 degrees C, pH 7.4, when the fibrin I concentration is 1.0 mg/ml. Association of fibrin I monomers was shown to result in a 6.5-fold increase in the susceptibility of Arg-B beta 14 to thrombin-mediated proteolysis. The 6.5-fold increase in the observed specificity constant from 6.5 X 10(5) M-1s-1 to 4.2 X 10(6) M-1s-1 upon association of fibrin I monomers and the rate constant for fibrin association indicates that most of the fibrinopeptide B is released after association of fibrin I monomers. The interaction between a pair of polymerization sites in fibrin I dimer was found to be weaker than the interaction of fibrin I with Gly-Pro-Arg-Pro and weaker than the interaction of fibrin I with fibrinogen.     

The course and prerequisites of Lys-plasminogen formation during fibrinolysis

Plasmin-catalyzed modification of the native plasma zymogen Glu1-plasminogen to its more reactive Lys78 form has been shown to be enhanced in the presence of fibrin. The aim of the present work has been to characterize the influence of fibrinopeptide release, fibrin polymerization, and plasmin cleavage of fibrin on the rate of Lys78-plasminogen formation. 125I-Labeled Glu1- to Lys78-plasminogen conversion was catalyzed by performed Lys78-plasmin, or by plasmin generated during plasminogen activation with tissue plasminogen activator or urokinase. The two forms of plasminogen were quantitated following separation by polyacrylamide gel electrophoresis in acetic acid/urea. Plasmin generated by plasminogen activator was monitored by a fixed-time amidolytic assay. The rate of Lys78-plasminogen formation was correlated, in separate experiments, to the simultaneous, plasmin-catalyzed cleavage of 125I-labeled fibrinogen or fibrin to fragments X, Y, and D. The radiolabeled components were quantitated after separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The results show that the formation of both bathroxobin-catalyzed des-A-fibrin and thrombin-catalyzed des-AB-fibrin leads to marked stimulation of Lys78-plasminogen formation, whereas inhibition of fibrin polymerization, with Gly-Pro-Arg-Pro, abolishes the stimulatory effect. The rate of Lys78-plasminogen formation varies markedly in the course of fibrinolysis. The apparent second-order rate constant of the reaction undergoes a transient increase upon transformation of fibrin to des-A(B) fragment X polymer and decreases about 10-fold to the level observed during fibrinogenolysis upon further degradation to soluble fragments Y and D.(ABSTRACT TRUNCATED AT 250 WORDS)     

A congenitally abnormal fibrinogen (Vlissingen) with a 6-base deletion in the gamma-chain gene, causing defective calcium binding and impaired fibrin polymerization

A congenitally abnormal fibrinogen (Vlissingen) was isolated from the blood of a young woman suffering from massive pulmonary embolism. Fibrinogen Vlissingen showed an abnormal clotting time with both thrombin and Reptilase. The release of the fibrino-peptides A and B by thrombin was normal, but fibrin polymerization was impaired both in the presence and absence of Ca2+ ions. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis performed according to Laemmli the gamma-chain of fibrinogen Vlissingen showed two bands, one normal and one having an apparently lower molecular mass of about 1,500 daltons. The previously described protective effect of Ca2+ ions on plasmin degradation of the carboxyl terminus of the gamma-chain of normal fibrinogen was only partially detectable in fibrinogen Vlissingen. In addition the binding of Ca2+ ions was decreased. Fibrinogen Vlissingen bound 2.4 Ca2+ ions per fibrinogen molecule at pH 7.4, whereas normal fibrinogen bound 3.1 Ca2+ ions. At pH 5.8 fibrinogen Vlissingen bound 1.1 Ca2+ ions, whereas normal fibrinogen bound 2.0 Ca2+ ions per molecule fibrinogen in the D-domains, again indicating a structural change in the carboxyl terminus of fibrinogen. The structural defect was determined by sequence analysis of DNA amplified by use of the polymerase chain reaction. Exons VIII, IX, and X of the gamma-chain gene were amplified and the DNA sequence of the amplified fragments was determined. A 6-base deletion was found in 50% of the fragments corresponding to exon VIII, indicating that the patient was heterozygous for the mutation. This deletion codes for amino acids Asn-319 and Asp-320 in the normal fibrinogen gamma-chain. The data indicate that Asn-319 and Asp-320 are crucial for maintaining the integrity of the carboxyl-terminal polymerization sites, the protective effect of Ca2+ ions on plasmin degradation of the carboxyl terminus of the gamma-chain, and the calcium binding domain at the carboxyl terminus of fibrinogen.     

Diffusion of a fluorescent probe in egg lecithin multilayers and the effect of a permanent magnetic field

A method is developed for determining the value of transverse diffusion coefficient through lipid multilayers. Using the absorption kinetics parameters of 1-aniline-8-sulphonate naphthalene (ANS) the value of diffusion coefficient was estimated as Dt = (3 +/- 1). X 10(-12) cm2 s-1 at (22 +/- 0.5) degrees C. The value of Dt was shown to increase under the action of the magnetic field parallel to the multilayer surface; a relative increase of Dt at field 1.3 T is +(40 +/- 15)%.     

The incipient stage in thrombin-induced fibrin polymerization detected by FCS at the single molecule level.

We used fluorescence correlation spectroscopy (FCS) to study the activation of fibrinogen by thrombin and the subsequent aggregation of fibrin monomers into fibrin polymers at a very low and at physiological fibrinogen concentrations. In the labeling procedure used the fibrinogen was randomly labeled and the label was bound to the fibrinopeptide A and/or to the part of fibrinogen which after activation takes part in fibrin formation. We measured a diffusion coefficient for fibrinogen of 2.48 x 10(-7) +/- 0.10 x 10(-7) cm2/s. After activation with thrombin both fibrinopeptide A and fibrin polymerization products could be demonstrated. From our findings we suggest a model for the formation of a three-dimensional network as two parallel processes, elongation and branching and that fibrin oligomers are not only intermediates in the polymerization process but also are substrates for branching.     

The role of complementary E2 and D2 centers in the reaction between fibrin and fibrinogen

The effect of fibrinogen on the two steps of polymerization of two fibrin forms differing in the set of polymerization sites (fibrin-desAA and fibrin-desAABB) was studied. It was shown that fibrinogen inhibited the protofibril growth and fibril formation at the stage of lateral aggregation more effectively with fibrin-desAABB than with fibrin desAA. When the fibrinogen D2-site was blocked by tetrapeptide Gly-His-Arg-Pro, the key structure of the E2-site, the inhibitory activity of fibrinogen diminished. A conclusion is drawn that the high susceptibility of fibrin-desAABB to fibrinogen is due to the interaction of the E2-active site with the D2-site of the fibrinogen molecule. The concentration dependence of the tetrapeptide Gly-His-Arg-Pro-induced inactivation of fibrinogen and the effects of temperature and Ca2+ on the tetrapeptide interaction with fibrinogen were investigated.     

Reversible loss of cooperative calcium ion binding by sarcoplasmic reticulum calcium adenosinetriphosphatase

Incubation of rabbit skeletal muscle sarcoplasmic reticulum vesicles in solutions of very low [Ca2+] caused Ca2+ to bind noncooperatively, as determined by the dependence of the intrinsic tryptophan fluorescence intensity on added increments of Ca2+. Cooperative Ca2+ binding was obtained if the ATPase was incubated in [Ca2+] high enough (25 microM) to saturate the two high-affinity Ca2+ binding sites and then titrated with [ethylenebis(oxyethylenenitrilo)]tetraacetic acid. The cooperative binding had an apparent association constant of 6.3 X 10(6) M-1 and a Hill coefficient of 2.6; these constants for the noncooperative binding case were 5.0 X 10(5) M-1 and 1.2, respectively. The transitions from the noncooperative to the cooperative Ca2+ binding forms of the enzyme were slow compared to the time required for Ca2+ binding to reach equilibrium. Thus, it appears that sarcoplasmic reticulum CaATPase is a hysteretic enzyme. Intrinsic association constants for Ca2+ binding and equilibrium constants for the transitions between the two forms in low and high [Ca2+] were estimated from analyses of a general scheme for cooperative and noncooperative binding.     

Oscillations and waves of cytosolic calcium: insights from theoretical models.

Oscillations in cytosolic Ca2+ occur in a wide variety of cells, either spontaneously or as a result of external stimulation. This process is often accompanied by intracellular Ca2+ waves. A number of theoretical models have been proposed to account for the periodic generation and spatial propagation of Ca2+ signals. These models are reviewed and their predictions compared with experimental observations. Models for Ca2+ oscillations can be distinguished according to whether or not they rely on the concomitant, periodic variation in inositol 1,4,5-trisphosphate. Such a variation, however, is not required in models based on Ca(2+)-induced Ca2+ release. When Ca2+ diffusion is incorporated into these models, propagating waves of cytosolic Ca2+ arise, with profiles and rates comparable to those seen in the experiments.     

Regulation by calcium and calmodulin of adenylate cyclase from rabbit intestinal epithelium

The effect of calcium on adenylate cyclase from rabbit small intestine has been studied using a particulate preparation obtained from isolated epithelial cells. Both basal and vasoactive intestinal peptide-stimulated activities were inhibited by calcium concentrations in the micromolar range. In the presence of calmodulin, a biphasic response was obtained. At low calcium concentration (4 X 10(-9)-6 X 10(-8) M) the enzyme was activated up to 50%. As the Ca2+ concentration was increased, the enzyme was concomitantly inhibited. Half-maximal inhibition of calmodulin-dependent activity was obtained at 1 microM free Ca2+. The activation of the enzyme was also dependent on the concentration of Mg2+. At less than 1 microM Ca2+, the enzyme exhibited a biphasic response, being activated at below 3 mM Mg2+ and inhibited at higher concentrations. At Ca2+ concentrations that were inhibitory, the enzyme did not show the biphasic response to Mg2+. At concentrations above 3 mM, the maximal rate (Vmax) remained constant. Vmax was inversely proportional to the concentration of Ca2+ present. Calmodulin altered Vmax when acting on vasoactive intestinal peptide-stimulated enzyme. Calmodulin had no effect on the Km for hormone activation. The calmodulin-dependent activity was inhibited by incubation with trifluoperazine.