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R Donato

Publications and source records attributed to R Donato.

At least 91 records · Page 5Linked to original sources
PubMed1988

S-100b protein regulates the activity of skeletal muscle adenylate cyclase in vitro.

We have investigated the effect of the b isoform of S-100 proteins on adenylate cyclase activity of rat skeletal muscle. S-100b inhibits the adenylate cyclase activity in the presence of Mg2+ (5.0-50 mM), while it activates the same enzyme in the presence of Ca2+ (0.1-1.0 mM) dose-dependently in both cases. S-100b counteracts the stimulatory effect of NaF on adenylate cyclase in the presence of Mg2+ and the inhibitory effect of RMI 12330 A in the presence of Ca2+.

Adenylyl Cyclase Inhibitors↗
PubMed1988

Calcium-independent, pH-regulated effects of S-100 proteins on assembly-disassembly of brain microtubule protein in vitro.

At alkaline pH, Ca2+ is no longer required for S-100 proteins to inhibit the assembly and to promote the disassembly of brain microtubules in vitro, though the presence of Ca2+ significantly favors the S-100 effects. These effects are inversely related to the microtubule protein concentration and directly related to the S-100 concentration and the pH. Ca2+-independent, pH-regulated inhibition of assembly of phosphocellulose-purified tubulin by S-100 is also described. The microtubule disassembling effect of S-100 is additive to that of alkali (used to raise the pH), and S-100 further disassembles microtubules after alkalinization. Thus the larger inhibitory effect of S-100 on microtubule assembly at alkaline versus acid pH depends on both a decrease in the assembly rate and an increase in the disassembly rate. Together with previous data on this topic, the present findings indicate that S-100 proteins act on microtubule protein in vitro primarily by binding to tubulin, this event being Ca2+-regulated at a given pH, and pH-regulated at a given free Ca2+ concentration.

Animals↗
PubMed1988

Ultracytochemical localization of adenylate cyclase and guanylate cyclase in crushed peripheral nerves.

Cellular and subcellular distribution of adenylate cyclase (AC) and guanylate cyclase (GC) activities in crushed peripheral nerves during regeneration were studied at the electron microscope level. In unlesioned nerves, no AC reaction product could be evidenced, whereas GC was detectable on the plasma membranes of Schwann cells, myelinated and nonmyelinated fibers, and within nonmyelinated axons. At 24 hours after the crush, AC reaction product was found within axonal segments proximal to the zone of the crush in association with mitochondria. At this stage, macrophage-like cells, which probably are transformed Schwann cells, polymorphonuclear leucocytes, and endothelial cells displaying an intense AC reaction product could be detected. On the other hand, at 24 hours after the crush, GC was no longer detectable, except on occasional unlesioned nerve fibers. At 48 hours after the lesion, AC reaction product was no longer detectable within axons, and all AC positivity was associated with plasma membranes of non-neuronal cells, including transformed Schwann cells, occasional macrophages, polymorphonuclear leucocytes, fibroblasts, and elongated cells. As to GC, images similar to those obtained at 24 hours were observed until 48 hours after the crush. From the 7th to the 28th postlesion day, AC activity was localized exclusively to the plasma membranes of fibroblasts and elongated cells. Transformed Schwann cells were no longer detectable, whereas normal Schwann cells and regenerating axons could be seen, and these showed no AC reaction product in analogy to the absence of AC reaction product of unlesioned nerves. During the same period, GC again was detectable on regenerating fibers with the same subcellular localization as that of unlesioned nerves. The present results strongly suggest that starting from the second postcrush day, cells invading the lesioned zone and transformed Schwann cells, all taking part in the formation of the new perineurial tissue, display a high AC activity, which should be taken into account when measuring cyclic adenosine monophosphate (cAMP) levels under these conditions. Also, our data suggest that GC is involved primarily in regeneration processes that occur in crushed peripheral nerves. Thus, the pattern of AC distribution in peripheral unlesioned and lesioned nerves appears to be exactly the opposite of the GC localization examined under similar experimental conditions insofar as nervous fibers are concerned.

Adenylyl Cyclases↗
PubMed1988

Interaction of S-100b protein with cardiolipin vesicles as monitored by electron spin resonance, pyrene fluorescence and circular dichroism.

The interaction of S-100b protein with cardiolipin (CL) vesicles has been studied by electron spin resonance, pyrene fluorescence, and circular dichroism. Electron spin resonance and pyrene fluorescence data indicate that S-100b binds to the polar surface of vesicles Ca2+-independently. In the presence of Ca2+, S-100b potentiates the Ca2+-induced clustering of the polar headgroups of CL molecules and causes a further reduction in the Ca2+-dependent decrease in the lateral mobility of the pyrene inserted into the lipid bilayer, which points to an effect of the protein on the hydrophobic core of the lipid bilayer through a larger perturbation of its polar surface. Circular dichroism analyses indicate that CL vesicles cause a decrease in the alpha-helical content of S-100b, analogous to that produced by Ca2+ and that the effects of CL vesicles and of Ca2+ on the secondary structure of the protein are supra-additive. By this technique, we found that the affinity of Ca2+ for S-100b increases substantially in the presence of CL vesicles, even in the presence of physiologic concentrations of KCl, suggesting that once S-100b had interacted with CL vesicles it assumes a new conformation in which its Ca2+-binding properties are greatly enhanced. These results are discussed in relation to binding of S-100b proteins to natural membranes, and to a possible involvement of S-100b in the regulation of membrane structural organization.

Calcium↗
PubMed1987

Quantitative analysis of the interaction between S-100 proteins and brain tubulin.

S-100 was shown to regulate the in vitro assembly of brain microtubule proteins (MTPs) in a Ca2+-mediated way by acting on both the nucleation and the elongation of microtubules (MTs). Here data will be shown suggesting that S-100 binds to tubulin. The binding is time-, temperature-, Ca2+-, and pH-dependent, and saturable with respect to S-100. At pH 6.75, the saturation curve is biphasic, displaying a high affinity component (dissociation constant, Kd1, approximately 0.1 microM) and a low affinity component (Kd2 approximately 3.8 microM). At pH 6.75, as the free Ca2+ concentration raises from 0 to 100 microM, the overall binding capacity increases from 0.065 to 0.66 mol S-100/mol tubulin dimer. This finding, together with the observation that the S-100 effect on MTP assembly is Ca2+-dependent at that pH, suggests that the S-100-induced inhibition of MTP assembly depends on S-100 binding to the low affinity sites on the tubulin molecule. The S-100 binding to tubulin is pH-dependent; as the pH raises from 6.75 to 8.3, both binding components are affected, the major changes consisting of an increase in the binding capacity and a decrease in the overall affinity. Moreover, as the pH raises, Ca2+ is no longer required for S-100 to bind to tubulin. S-100 also interacts with a component of whole MTPs (probably tubulin, on the basis of the above results). No S-100 binding to microtubule-associated proteins (MAPs) could be evidenced by the techniques employed in this study. On the contrary, some competition between S-100 and MAPs for binding sites or tubulin seems to occur.

Ammonium Sulfate↗
PubMed1987

Characteristics of the effect of S-100 proteins on the assembly-disassembly of brain microtubule proteins at alkaline pH in vitro.

The ability of S-100 proteins to inhibit the assembly of brain microtubule proteins (MTPs) in the presence of microM levels of Ca2+ increases as a function of pH. This seems to be due to an increasingly larger inhibitory effect of S-100 on the nucleation and, probably, on the elongation of microtubules (MTs) as the pH raises. In the presence of microM Ca2+ levels, the ability of S-100 to disassemble MTs also increases linearly with the pH, suggesting that the larger inhibitory effect of S-100 on MTP assembly at alkaline than at acidic pH may depend on both a decrease in the assembly rate and an increase in the disassembly rate. Also, S-100 inhibits the assembly of phosphocellulose-purified tubulin to a larger and larger extent as the pH raises. S-100 brings about its effect on MT assembly-disassembly probably by sequestering soluble tubulin, though additional mechanisms cannot be excluded. The present data are briefly discussed in relation to the role attributed to changes in intracellular pH in the regulation of the state of assembly of cytoplasmic MTs.

Animals↗
PubMed1986

S-100 proteins.

S-100 is a group of closely related, small, acidic Ca2+-binding proteins (S-100a0, S-100a and S-100b, which are alpha alpha, alpha beta, and beta beta in composition, respectively). S-100 is structurally related to calmodulin and other Ca2+-binding proteins. S-100 is abundant in the brain and is contained in well defined cell types of both neuroectodermal and non-neuroectodermal origin, as well as in their neoplastic counterparts. In the mammalian brain, S-100a and S-100b are confined to glial cells, while S-100a0 is neuronal in localization. Single S-100 isoforms bind Ca2+ with nearly the same affinity. K+ antagonizes the binding of Ca2+ to high affinity sites on S-100. S-100 binds Zn2+ with high affinity. S-100 is found in a soluble and a membrane-bound form and has the ability to interact with artificial and natural membranes. S-100 has no enzymatic activity. S-100 has been involved in several activities including memory processes, regulation of diffusion of monovalent cations across membranes, modulation of the physical state of membranes, regulation of the phosphorylation of several proteins, control of the assembly-disassembly of microtubules. Some of these effects are strictly Ca2+-dependent, while other are not. S-100 is being secreted or released to the extracellular space. In some cases, this event is hormonally regulated. Several S-100 binding proteins are being described.

Animals↗
PubMed1986

Identity between cytoplasmic and membrane-bound S-100 proteins purified from bovine and rat brain.

Cytoplasmic and membrane-bound S-100 proteins were purified to homogeneity from bovine and rat brain. Cytoplasmic and membrane-bound S-100 from single species are identical by immunological, electrophoretic, spectrophotometric, and functional criteria. Cytoplasmic and membrane-bound S-100 from bovine brain consists of nearly equal amounts of S-100a and S-100b, whereas cytoplasmic and membrane-bound S-100 from rat brain consists mostly of S-100b. The functional role of membrane-bound S-100 remains to be elucidated.

Animals↗
PubMed1986

Effects of S-100 proteins on assembly of brain microtubule proteins: correlation between kinetic and ultrastructural data.

Microtubules formed in vitro in the presence of S-100 proteins and micromolar Ca2+ concentrations are fewer in number and longer than those formed in the presence of Ca2+ alone. Moreover, microtubules growing after addition of microtubule fragments to a microtubule protein solution in the presence of S-100 are shorter than those growing in its absence. These data lend support to previous results of kinetic studies indicating that S-100 interferes with both the nucleation and the elongation of microtubules in vitro.

Animals↗
PubMed1985

S-100 proteins and microtubules: analysis of the effects of rat brain S-100 (S-100b) and ox brain S-100a0, S-100a and S-100b on microtubule assembly-disassembly.

Rat brain S-100 (S-100b) and ox brain S-100a0, S-100a and S-100b have been tested for their ability to control the assembly and disassembly of brain microtubule proteins in the presence of either Ca2+ or Zn2+, in vitro. In the presence of Ca2+, single S-100 isoforms have similar, if not identical, effects, i.e. they inhibit assembly and promote disassembly. In the presence of Zn2+ from 0.1 to 1 mM (free concentration), rat S-100 and ox S-100a and S-100b inhibit assembly, while S-100a0 is without effect. These data are briefly discussed in relation to the cellular localization of single S-100 isoforms in the brain.

Animals↗
PubMed1985

S-100 protein-induced changes in the physical state of synaptosomal particulate fractions as monitored by spin labels.

This report documents changes in the physical state of synaptosomal particulate fractions (SYN) upon binding of S-100 protein, as monitored by spin labels. Studies were conducted on SYN labeled with either 5-doxylstearic acid or 16-doxylstearic acid, which probe the polar region and the hydrophobic core of the lipid bilayer, respectively. S-100 perturbs to some extent both the polar surface and the hydrophobic core of SYN in a time- and temperature-dependent manner. Ca2+ is essential for S-100 to perturb the membranes. K+ almost completely inhibits the S-100 perturbing effect if present in the incubation medium, but fails to reverse the S-100-induced changes if added after S-100 has interacted with SYN. At room temperature and below, the overall S-100 effect registered after about 30 min of association of the protein with SYN is an increase in the fluidity of both the surface and the interior of the membranes. Spectra registered at intervals at room temperature indicate that the S-100 perturbing effect on the membrane surface is practically monophasic, consisting of an increase in fluidity, while that on the membrane interior is multiphasic, consisting of a decrease in fluidity during the first 10 min of association, followed by an increase in fluidity during the subsequent 20 min and a return to starting values during the second 30 min of association. Around 37 degrees C, on the contrary, a decrease in fluidity is registered in both regions. The data suggest that S-100 induces a spatial rearrangement of membrane components (proteins) involved in the specific binding and/or partially penetrates into the lipid bilayer.

Animals↗
PubMed1985

Calcium-sensitivity of brain microtubule proteins in the presence of S-100 proteins.

In the presence of the usual 0.1 M Mes buffer, pH 6.7, mM free Ca2+ levels are required for half-maximal decrease in the rate and extent of brain microtubule protein (MTP) assembly in the absence of ox brain S-100, while microM free Ca2+ levels are sufficient in the presence of S-100. At the same pH 6.7, but in the presence of 0.12 M KCl, as low as 1.5 microM free Ca2+ is sufficient for S-100 to produce half-maximal reduction in the rate of assembly, while as high as 0.5 mM free Ca2+ is required in the absence of S-100. Similar results are obtained with rat brain S-100 (S-100b), indicating that single S-100 iso forms are equipotent in affecting the MTP assembly. At pH 7.5, MTPs are remarkably resistant to Ca2+ in the absence of S-100. In the presence of S-100, not only is the free Ca2+ concentration required for complete inhibition of assembly at least one order of magnitude smaller than that required in the absence of S-100, but significant S-100-dependent inhibition of assembly occurs in the absence of Ca2+. Under the two conditions where S-100 is particularly effective in inhibiting the assembly, i.e. at pH 6.7 in the presence of KCl and at pH 7.5, S-100 increases the disassembly rate even in the presence of microM Ca2+ levels. Our results suggest that the free Ca2+ concentration regulates the way S-100 disassembles microtubules (MTs): at microM Ca2+ levels, S-100 sequesters tubulin with concomitant increase in the disassembly rate; at mM Ca2+ levels, the S-100-Ca2+ complex probably interacts with MTs producing endwise disassembly.

Animals↗
PubMed1984

Mechanism of action of S-100 protein(s) on brain microtubule protein assembly.

The inhibitory effect of S-100 on microtubule protein assembly is inversely related to the microtubule protein concentration and/or the temperature of assembly. Moreover, the S-100-induced decrease in the rate and extent of assembly is positively correlated with the length of the lag of assembly. When microtubule fragments are added to the microtubule protein solution, the S-100 effect is reduced but not abolished. These data suggest that S-100 interferes with both the nucleation and the elongation of microtubules. Since S-100 also inhibits the assembly of purified tubulin, S-100 is suggested to affect the microtubule assembly by interacting with and sequestering tubulin.

Animals↗
PubMed1984

Chlorpromazine inhibits the calcium-mediated effects of S-100 protein(s) on assembled brain microtubule proteins, but not those on microtubule protein assembly.

We have examined the S-100-chlorpromazine interplay at the level of brain microtubule proteins in vitro. The results indicate that in the presence of 0.12 M KCl and 10 microM free Ca2+ the inhibitory effect of S-100 on microtubule assembly is additive to that of chlorpromazine, but S-100 fails to potentiate the disassembling effect of 0.1 mM Ca2+ if added to assembled microtubule proteins after chlorpromazine and Ca2+, probably because of inhibition of S-100 by the phenothiazine. Chlorpromazine does not compete with S-100 for binding to purified tubulin.

Animals↗
PubMed1984

Binding of chlorpromazine to S-100 protein.

Chlorpromazine (CPZ) induces in S-100 conformational changes resulting in the exposure of titratable SH groups of the protein to the solvent. This effect is even greater in the presence of Mg2+ +/- Ca2+. S-100 possesses binding sites for CPZ. The binding of CPZ to 3 microM S-100 is half-saturated by 0.18 microM CPZ in the presence of Mg2+ plus Ca2+ and by 0.24 microM CPZ in the presence of Mg2+ plus EGTA. The extent of the binding is greater in the presence of Ca2+ than in the presence of EGTA, especially at low CPZ concentrations.

Animals↗
PubMed1983

Effect of S-100 protein on assembly of brain microtubule proteins in vitro.

S-100 protein inhibits the assembly of brain microtubule proteins in vitro in the presence of 10 microM free Ca2+. The S-100 effect is generally greater on the rate than on the extent of assembly, and even greater as the microtubule protein concentration decreases and the time of preincubation between S-100 and microtubule proteins before GTP addition increases, at a given S-100/tubulin dimer molar ratio. The S-100 effect is greatly enhanced in the presence of physiological concentrations of K+ and is completely reversed by EGTA.

Animals↗
PubMed1983

Biochemical and physicochemical properties of the solubilized S-100 protein binding activity of synaptosomal particulate fractions.

The 125I-labeled S-100 specific binding to a Triton X-100 (TX-100) extract of synaptosomal particulate fractions (SYN) was investigated. The results indicate that (a) S-100 binding to the TX-100 extract is partially irreversible after a critical association time at 37 degrees C, while it is fully reversible after any association time at 4 degrees C; (b) trypsin and phospholipase C partially reverse the S-100 binding, while phospholipase D enhances the interaction to some extent, in a dose-dependent way; (c) EDTA and high concentrations of NaCl or KCl are more efficient as inhibitors of the S-100 binding to the TX-100 extract than as 125I-labeled S-100 dissociating agents, in analogy with previous observations with SYN; and (d) two main populations of solubilized S-100 binding sites can be evidenced by gel filtration and sucrose gradient centrifugation when low amounts of the TX-100 extract are processed and/or low S-100 concentrations are used, while two additional molecular species are separated when greater amounts of either factors are tested. These results suggest the possibility that S-100 may be involved in the regulation of some membrane activities.

Animals↗