Trypsin and calcium ions elicit changes of the membrane potential in pig blood platelets.

The addition of trypsin or thrombin or of Ca²⁺ ions to pig blood platelets was followed by a K⁺- dependent change of the membrane potential similar to that produced by the ionophore valinomycin. The effect of trypsin and of Ca²⁺, but not of valinomycin, was prevented by La³⁺ and by EGTA. It is proposed that upon the modification of the platelet surface by trypsin (and by thrombin under physiological conditions) membrane Ca²⁺ move from the external to the internal side of the platelet surface membrane and open the gates of K⁺ - specific channels.     

Ca2+-activated, phospholipid-dependent protein kinase catalyzes the phosphorylation of actin-binding proteins

Chicken gizzard vinculin and filamin were found to be phosphorylated by Ca2+-activated, phospholipid-dependent protein kinase (protein kinase C). These two actin-binding proteins serve as substrates for protein kinase C specifically in the free form, whereas they are little phosphorylated by protein kinase C in the presence of F-actin. In contrast, alpha-actinin from chicken gizzard is less susceptible to phosphorylation by protein kinase C, either in the presence or in the absence of F-actin. In light of these data, the possibility that Ca2+ and phospholipid-dependent phosphorylation by protein kinase C may modulate the function of actin-binding proteins has to be considered.     

Ca2+-dependent K+ permeability of heart sarcolemmal vesicles. Modulation by cAMP-dependent protein kinase activity and by calmodulin

The Ca2+-dependent K+ permeability of heart sarcolemma vesicles was measured by following the transmembrane movement of the charge compensating tetraphenylborate anion. The increase in vesicles permeability induced by Ca2+ is lost when membrane proteins are dephosphorylated by an endogenous protein phosphatase and is restored by a phosphorylation process catalysed by a cAMP-dependent protein kinase. The calmodulin antagonist R 24571 lowers the Ca2+-dependent K+ permeability by decreasing the Ca2+ affinity of the K+ transporting system.     

Adrenocortical cytochrome P-450 responsible for cholesterol side chain cleavage (P-450scc) is phosphorylated by the calcium-activated, phospholipid-sensitive protein kinase (protein kinase C)

Purified bovine adrenocortical cytochrome P-450scc (specific for cholesterol side chain cleavage in the inner mitochondrial membrane) was selectively phosphorylated in vitro by a Ca2+-activated, phospholipid-sensitive protein kinase (protein kinase C) preparation, whereas cyclic AMP dependent and two cyclic nucleotide independent kinases were ineffective. Cytochrome P-450scc incorporated a maximum of 4 mol of phosphate in the presence of protein kinase C within 15 min at 30 degrees C, with apparent Km and Vmax of 0.14 mumol and 0.76 pmol/min, respectively. Serine and threonine were the two target aminoacids phosphorylated in a ratio of about 1:1. In the presence of 1 microM Ca2+, a mixture of phosphatidylserine and diolein (or a potent tumor promoter phorbol ester) was required for optimal cytochrome P-450scc phosphorylation. In addition, purified inner mitochondrial membrane preparations from adrenocortical mitochondria were found to contain protein kinase C activity. These findings, together with the previous demonstration that activators of protein kinase C such as a potent phorbol ester activates steroidogenesis of intact adrenocortical cells, suggest that phosphorylation of P-450scc should be examined for its possible role in the regulation of adrenocortical functions.     

Conformational changes of F-actin-ε-ADP in thin filaments in myosin-free muscle fibers induced by Ca2+

Polarized fluorescence from F-actin-ε-ADP in thin filaments reconstituted in a myosin-free single muscle fiber was measured at various concentrations of Ca²⁺. Four components of polarized fluorescence changed with increasing Ca²⁺ concentration at pCa values of around 7 to 6, concomitant with a change of the tension generated by the fiber irrigated with myosin in the presence of Mg-ATP. From analysis of observed values of the four components, it was found that the flexibility of the thin filament increased, or the elastic modulus for bending decreased from 5.7 × 10^?17 dyn cm² to 4.7 × 10^?17 dyn cm², when the pCa value decreased from 7 to 6. In the same range of pCa values, the angles of absorption and emission dipoles of ε-ADP changed, suggesting a small rotation of the base-plane of ε-ADP around an axis perpendicular to the F-actin axis.     

F-Actin Structure Destabilization and DNase I Binding Loop Fluctuations: Mutational Cross-Linking and Electron Microscopy Analysis of Loop States and Effects on F-Actin

The conformational dynamics of filamentous actin (F-actin) is essential for the regulation and functions of cellular actin networks. The main contribution to F-actin dynamics and its multiple conformational states arises from the mobility and flexibility of the DNase I binding loop (D-loop; residues 40-50) on subdomain 2. Therefore, we explored the structural constraints on D-loop plasticity at the F-actin interprotomer space by probing its dynamic interactions with the hydrophobic loop (H-loop), the C-terminus, and the W-loop via mutational disulfide cross-linking. To this end, residues of the D-loop were mutated to cysteines on yeast actin with a C374A background. These mutants showed no major changes in their polymerization and nucleotide exchange properties compared to wild-type actin. Copper-catalyzed disulfide cross-linking was investigated in equimolar copolymers of cysteine mutants from the D-loop with either wild-type (C374) actin or mutant S265C/C374A (on the H-loop) or mutant F169C/C374A (on the W-loop). Remarkably, all tested residues of the D-loop could be cross-linked to residues 374, 265, and 169 by disulfide bonds, demonstrating the plasticity of the interprotomer region. However, each cross-link resulted in different effects on the filament structure, as detected by electron microscopy and light-scattering measurements. Disulfide cross-linking in the longitudinal orientation produced mostly no visible changes in filament morphology, whereas the cross-linking of D-loop residues > 45 to the H-loop, in the lateral direction, resulted in filament disruption and the presence of amorphous aggregates on electron microscopy images. A similar aggregation was also observed upon cross-linking the residues of the D-loop (> 41) to residue 169. The effects of disulfide cross-links on F-actin stability were only partially accounted for by the simulations of current F-actin models. Thus, our results present evidence for the high level of conformational plasticity in the interprotomer space and document the link between D-loop interactions and F-actin stability.     

Effect of hemolysate on calcium inhibition of the (Na+ + K+)-ATPase of human red blood cells

The sensitivity of the (Na+ + K+)-ATPase in human red cell membranes to inhibition by Ca2+ is markedly increased by the addition of diluted cytoplasm from hemolyzed human red blood cells. The concentration of Ca2+ causing 50% inhibition of the (Na+ + K+)-ATPase is shifted from greater than 50 microM free Ca2+ in the absence of hemolysate to less than 10 microM free Ca2+ when hemolysate diluted 1:60 compared to in vivo concentrations is added to the assay mixture. Boiling the hemolysate destroys its ability to increase the sensitivity of the (Na+ + K+)-ATPase to Ca2+. Proteins extracted from the membrane in the presence of EDTA and concentrated on an Amicon PM 30 membrane increased the sensitivity of the (Na+ + K+)-ATPase to Ca2+ in a dose-dependent fashion, causing over 80% inhibition of the (Na+ + K+)-ATPase at 10 microM free Ca2+ at the highest concentration of the extract tested. The active factor in this membrane extract is Ca2+-dependent, because it had no effect on the (Na+ + K+)-ATPase in the absence of Ca2+. Trypsin digestion prior to the assay destroyed the ability of this protein extract to increase the sensitivity of the (Na+ + K+)-ATPase to Ca2+.     

Protein phosphorylation in pancreatic islets: evidence for separate Ca2+ and cAMP-enhanced phosphorylation of two 57,000 Mr proteins

There is a phosphopeptide that has an Mr of 53,000 to 60,000 in insulin-secreting tissues and there is general agreement that this peptide can be phosphorylated in a calcium-dependent manner. The present report shows that there are at least two phosphoproteins with Mr's near 57,000 in rat pancreatic islet cytosol. One peptide has an Mr of 57,000, a pl of 7.5 - 8 and is phosphorylated in a Ca2+-enhanced manner, and the other has an Mr of 54,000, a pl of 5 - 5.5 and is phosphorylated in a cAMP-enhanced manner, as judged by two-dimensional polyacrylamide gel electrophoresis. Sepharose 4B chromatography indicated that the former polypeptide resides in a native protein complex that has an Mr of about 500,000 and the latter in a complex that has an Mr of about 180,000. Tritiated azido cyclic AMP binds to an islet polypeptide that has an Mr of 54,000. The results suggest that Ca2+ and cAMP could regulate stimulus-secretion coupling in pancreatic islets via protein phosphorylation.     

Reconstitution of succinate-Q reductase.

Reconstitution of succinate-Q reductase is achieved by admixing soluble succinate dehydrogenase (SDH) and ubiquinone-protein-S (QP-S), a new protein isolated from the soluble cytochrome $\text{b-c}{}_1$ complex. The reconstituted reductase catalyzes reduction of Q by succinate. The reaction is fully sensitive to thenoyltrifluoroacetone. The reconstituted reductase (same as succinate-cytochrome $\text{c}$ reductase or submitochondrial particles) does not show “low concentration ferricyanide reductase activity” as soluble dehydrogenase does. In other words, this enzymic site on SDH is occupied by QP-S. When an artificial dye, such as phenazine methosulfate or Wurster's Blue, is used as electron acceptor the rate of oxidation of succinate by SDH is not significantly changed regardless of whether the dehydrogenase is in the free or in the reconstituted succinate-Q reductase forms.     

Vitamin A acid-induced activation of Ca2+-activated, phospholipid-dependent protein kinase from rabbit retina

Ca2+-activated, phospholipid-dependent protein kinase from rabbit retina was partially purified. Vitamin A acid (retinoic acid) stimulated this protein kinase in the presence of Ca2+, while other metabolites of vitamin A such as retinol or retinal were less effective. The order of the extent of phosphorylation of the various substrate proteins by this protein kinase was identical in the presence of vitamin A acid or phosphatidylserine. The major spots of the 32P labeled peptide from histone H1 phosphorylated in the presence of vitamin A acid by this protein kinase did not differ from those obtained from histone H1 phosphorylated in the presence of phosphatidylserine. Retinol caused a further enhancement of the enzymatic activity, whereas the addition of retinal inhibited the activation by vitamin A acid. Thus, vitamin A and its metabolites may play an important role in the regulation of Ca2+-activated, phospholipid-dependent protein kinase activity in the retina.     

Specific binding of the calcium-dependent regulator protein to brain membranes from the guinea pig

The interaction of a calcium-dependent regulator protein (CDR) of brain adenylate cyclase (EC 4.6.1.1) with synaptic membranes from guinea pig brain was examined using ¹²⁵I-CDR as a tracer molecule. ¹²⁵I-CDR binding was reversible, saturable, and temperature sensitive. The same Ca²⁺ and Mg²⁺ dependence was observed for ¹²⁵I-CDR binding and for brain adenylate cyclase activation by CDR.     

Partial characterization of protein kinase-catalyzed phosphorylation of low molecular weight proteins in purified preparations of pigeon heart sarcolemma and sarcoplasmic reticulum

Pigeon heart microsomes contain three minor size protein kinase substrates of minimal molecular weights of 22 000, 15 000, and 11 500, as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. When the microsomes were partially loaded with calcium oxalate and subjected to rate zonal and isopynic centrifugations in sucrose density gradient columns, the 22 000 and the 15 000 dalton proteins settled in the heaviest fraction, which was composed mainly of vesicles of sarcoplasmic reticular membranes; the 11 500 dalton protein was concentrated in the lightest fractions, which consisted chiefly of vesicles of sarcolemmal origin. During incubation of the membrane fractions with Mg[γ-³²P]ATP significant amounts of ³²P were incorporated into all these proteins. Incorporation of ³²P into the 15 000 dalton protein was moderately and ³²P incorporation into the 22 000 dalton protein was markedly enhanced in the presence of exogenous soluble cyclic AMP-dependent protein kinase and cyclic AMP. The phosphorylation of the three proteins was virtually unaffected by CA²⁺ concentrations up to 0.1 mM and by ethyleneglycol-bis(β-aminoethylether)-N,N′-tetraacetic acid in the absence of added Ca²⁺.Phosphorylation of the 22 000 and the 11 500 dalton proteins occurred mainly at serine residues. In the 15 000 dalton protein threonine residues were the main site of endogenous phosphorylation. Nearly equal amounts of [³²P]-phosphate were incorporated into threonine and serine residues of this protein when phosphorylation was supported by exogenous cyclic AMP-dependent protein kinase and cyclic AMP.The 15 000 dalton protein could be removed from its membrane attachment by extraction with an acidic chloroform/methanol mixture. This step opens the way for the purification of this membrane-bound protein kinase substrate.     

Structure-activity study of cytotoxicity and microtubule assembly in vitro by taxol and related taxanes

Fractionation of bovine brain cytosol by DEAE cellulose chromatography revealed the presence of a calcium-dependent protein kinase. This soluble neuronal protein kinase selectively phosphorylated several endogenous substrates. The most prominent substrate was a polypeptide with an apparent M_r of 45,000 which was stimulated 20-fold by addition of both calcium and calmodulin. Activation was dose-dependent, with half-maximal phosphorylation occurring at 0.9 μM free Ca²⁺ and 60nM calmodulin. The effect of calmodulin was competitively inhibited by a variety of calmodulin inhibitors, in a manner characteristic of most calmodulin-dependent enzymes. This calcium- and calmodulin-dependent protein kinase is distinct from any previously described protein kinase.     

Detection of sequences with Z-DNA forming potential in higher plants

Sequences of alternating purine-pyrimidine residues with Z-DNA forming potential have been detected in the nuclear DNA of two higher plant species: wheat and radish. Poly (dG-dT) and poly (dG-dC) stretches have been detected by hybridization of the corresponding nick-translated probes to Southern blots. These stretches are scattered throughout the genome and some of them belong to moderately repeated sequence families interspersed with other DNA sequences.     

Effect of visible light on the mitochondrial inner membrane.

Exposure of mitochondria to visible light in the presence of riboflavin resulted in the initial release of respiratory control, followed by inhibition of electron transport and dissolution of structural integrity. Under these conditions, however, cytochrome c oxidase activity remained unchanged. ATPase activity was stimulated initially and remained in this activitated state even under continued illumination. In submitochondrial preparations, both electron transport and ATPase declined as a function of illumination time; cytochrome c oxidase was not sensitive to light. Enzyme inactivation also occurred to a lesser extent in the absence of riboflavin.     

Increase in polymerized liver tubulin during stimulation of hepatic plasma protein secretion in the rat

Involvement of hepatic microtubules in plasma protein secretion by the liver was investigated by stimulating protein secretion in rat liver and then measuring the different forms of tubulin. Total and free tubulin were estimated in liver supernatants by the [³H] colchicine-binding assay. Polymerized tubulin, assumed to reflect the presence of microtubules, was calculated from the difference between total and free tubulin. To enhance liver plasma protein secretion, an acute inflammatory reaction was induced in one group of rats and a nephrotic syndrome in another. In both cases, total liver tubulin increased significantly compared to normal animals, but free tubulin was unchanged. Accordingly, polymerized tubulin rose by 50% during the inflammatory reaction and by 90% during the nephrotic syndrome. These results support the hypothesis that hepatic microtubules are involved in plasma protein secretion by the liver and also suggest that enhanced secretion requires additional microtubules.     

Properties of an Ezrin Mutant Defective in F-actin Binding

Ezrin, radixin and moesin are a family of proteins that provide a link between the plasma membrane and the cortical actin cytoskeleton. The regulated targeting of ezrin to the plasma membrane and its association with cortical F-actin are more than likely functions necessary for a number of cellular processes, such as cell adhesion, motility, morphogenesis and cell signalling. The interaction with F-actin was originally mapped to the last 34 residues of ezrin, which correspond to the last three helices (αB, αC and αD) of the C-terminal tail. We set out to identify and mutate the ezrin/F-actin binding site in order to pinpoint the role of F-actin interaction in morphological processes as well as signal transduction. We report here the generation of an ezrin mutant defective in F-actin binding. We identified four actin-binding residues, T576, K577, R579 and I580, that form a contiguous patch on the surface of the last helix, αD. Interestingly, mutagenesis of R579 also eliminated the interaction of band four-point one, ezrin, radixin, moesin homology domains (FERM) and the C-terminal tail domain, identifying a hotspot of the FERM/tail interaction. In vivo expression of the ezrin mutant defective in F-actin binding and FERM/tail interaction (R579A) altered the normal cell surface structure dramatically and inhibited cell migration. Further, we showed that ezrin/F-actin binding is required for the receptor tyrosine kinase signal transfer to the Ras/MAP kinase signalling pathway. Taken together, these observations highlight the importance of ezrin/F-actin function in the development of dynamic membrane/actin structures critical for cell shape and motility, as well as signal transduction.     

A three-dimensional FRET analysis to construct an atomic model of the actin-tropomyosin complex on a reconstituted thin filament

Fluorescence resonance energy transfer (FRET) was used to construct an atomic model of the actin–tropomyosin (Tm) complex on a reconstituted thin filament. We generated five single-cysteine mutants in the 146–174 region of rabbit skeletal muscle α-Tm. An energy donor probe was attached to a single-cysteine Tm residue, while an energy acceptor probe was located in actin Gln41, actin Cys374, or the actin nucleotide binding site. From these donor–acceptor pairs, FRET efficiencies were determined with and without Ca²⁺. Using the atomic coordinates for F-actin and Tm, we searched all possible arrangements for Tm segment 146–174 on F-actin to calculate the FRET efficiency for each donor–acceptor pair in each arrangement. By minimizing the squared sum of deviations for the calculated FRET efficiencies from the observed FRET efficiencies, we determined the location of the Tm segment on the F-actin filament. Furthermore, we generated a set of five single-cysteine mutants in each of the four Tm regions 41–69, 83–111, 216–244, and 252–279. Using the same procedures, we determined each segment's location on the F-actin filament. In the best-fit model, Tm runs along actin residues 217–236, which were reported to compose the Tm binding site. Electrostatic, hydrogen-bonding, and hydrophobic interactions are involved in actin and Tm binding. The C-terminal region of Tm was observed to contact actin more closely than did the N-terminal region. Tm contacts more residues on actin without Ca²⁺ than with it. Ca²⁺-induced changes on the actin–Tm contact surface strongly affect the F-actin structure, which is important for muscle regulation.     

Disassembly of microtubules by the action of calmodulin-dependent protein kinase (Kinase II) which occurs only in the brain tissues

Microtubules assembled by the incubation of GTP at 37 °C were disassembled by the action of calmodulin-dependent protein kinase (Kinase II) which occrs only in the brain tissues. This disassembly required the presence of ATP and physiological concentrations of Ca²⁺ and calmodulin.