Calcium binding proteins in the brain

It is astonishing how a single ion, Ca++, can give rise to so many intracellular effects. The current explanation is that various combinations and permutations of a repertoire of calcium channels, calcium binding proteins and calcium pumps make up the individual answer of each neuronal type to the calcium signal. This review describes the major calcium binding proteins of the brain, which represent intracellular tools to decipher and transform the quantitative Ca+(+)-signal in qualitatively different cellular responses.     

Calmodulin-binding proteins in brain

It is now widely accepted that actions of intracellular Ca²⁺ are mediated by a four-domain Ca²⁺-binding protein, calmodulin. Brain is especially rich in calmodulin, containing about 400 mg (24 μmol) of EGTA-extractable calmodulin per kg of brain. However, only a fraction of the above amount is required for the calmodulin-activated enzymes and most of the rest may be assigned to calmodulin-binding proteins, proteins which are apparently devoid of enzyme activities but undergo Ca²⁺-dependent associations with calmodulin. Several of such proteins have been recently discovered in brain. These include a heat-labile 80 K phosphodiesterase inhibitor protein (calcineurin), a heat-stable 70 K phosphodiesterase inhibitor protein, a 50 K protein, myelin basic protein, tubulin, microtubule τ (tau) factor, a spectrin-like doublet protein (240 plus 235 K) (calspectin; fodrin) and a particle-associated 155 K protein.Functions of these calmodulin-binding proteins have not been fully elucidated yet. Some proteins may be calmodulin-regulated enzymes catalyzing yet unknown biochemical reactions, e.g. a protein phosphatase activity was found for calcineurin. Some proteins may interact with contractile elements or cytoskeleton of the cell, e.g. τ factor and calspectin interacted with tubulin and F-actin, respectively and tubulin itself is a calmodulin-binding protein. So, interesting possibilities are the regulation of the functions of cytoskeleton by calmodulin through these calmodulin-binding proteins. Regulation of microtubule assembly by Ca²⁺-dependent binding of calmodulin to tubulin and/or τ factor and possible involvement of calspectin in the mechanism regulating axonal transport of neuronal proteins have been suggested. Thus, the exploration of the regulating functions of Ca²⁺/calmodulin in brain depends largely upon the further study of the properties of these calmodulin-binding proteins.     

Specific calcium antagonist binding sites in brain

The binding of the calcium antagonist [³H] nitrendipine ([³H] NDP) to brain and heart is described and the brain site is characterized. The binding is saturable, specific and of very high affinity with K_D values of 0.16 nM in brain and 0.21 nM in heart. Our kinetic results are similar to those recently reported by two other groups (1,2), indicating a saturable, high affinity binding site in brain. In brain the binding sites are enriched in crude nuclear and synaptosomal fractions. The highest levels of binding are seen in the hippocampus, caudate and cerebral cortex with much lower levels in the cerebellum and pons. Calcium has a marked stimulatory effect on [³H] NDP binding at 10^?4 M. Addition of 0.5 mM CaCl₂ to EDTA treated membranes nearly doubles the number of binding sites. Of the many drugs and neurotransmitters tested only other calcium antagonists, i.e., verapamil, inhibit binding (IC₅₀ = 250 nM). The inhibition of [³H] NDP binding by verapamil is apparently non-competitive and not complete, suggesting that [³H] NDP binds to several sites, only some of which are inhibited by verapamil. The [³H] NDP binding site is probably a protein since it is very sensitive to trypsin, heat and sulfhydryl reagents.     

Specific interactions in proteins due to proton fluctuations

A theory of site–site interaction due to proton/charge fluctuations in protein molecules has been developed. It is shown that, with the proper geometric configuration of identical ionizable groups on matching sites, a specific attraction may be established. This attractive force has a bell-shaped pH dependence and is maximal close to the pK of the groups involved. Various types of protein interactions are examined in the light of this theory.     

Specific protein-protein binding in many-component mixtures of proteins

Proteins must bind to specific other proteins in vivo in order to function. The proteins are required to bind to only one or a few other proteins of the few thousand proteins typically present in vivo. To quantify this requirement we introduce a property of proteins called the capability. The capability is the maximum number of specific-binding interactions possible in a mixture, or in other words the size of largest sustainable interactome. This calculation of the maximum number possible is closely analogous to the work of Shannon and others on the maximum rate of communication through noisy channels. Using a simple model of proteins, we find specific binding to be a demanding function in the sense that it demands that the binding sites of the proteins be encoded by long sequences of elements, and the requirement for specific binding then strongly constrains these sequences.     

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.     

ADP-ribosylation of nuclear proteins in the rat brain

The distribution of (ADP-ribose)n synthesized from [14C]NAD labeled at the adenyl ring in several protein fractions of isolated rat brain nuclei was studied. Preferential ADP-ribosylation of nonhistone nuclear proteins was shown to occur. It was demonstrated that pol (ADP-ribose)polymerase and DNA-topoisomerase II are located spatially close to each other. A correlation between ADP-ribosylation and the activity of nuclear matrix DNA-topoisomerase II was established.     

Specific G proteins mediate endothelin induced contraction

Endothelin is a potent vasoconstrictor peptide which has recently been localized in the gastrointestinal tract. We have investigated the transmembrane signaling properties of endothelin in isolated smooth muscle cells of the rabbit rectosigmoid. Endothelin induced a dose dependent contraction of smooth muscle cells in a range of 10⁻¹⁰ to 10⁻⁶M. In normal buffer, contraction peaked at 30 sec and was sustained for up to 8 min. Incubation in 0Ca/2mM EGTA abolished the sustained contraction induced by endothelin, but had no effect on the initial transient contraction. Preincubation of saponin treated cells with G protein antisera had no effect on control cell length. Preincubation of saponin treated isolated smooth muscle cells with specific G protein antisera (rabbit antisera) for Go or Gs for 60 minutes did not inhibit contraction induced by endothelin. Preincubation with an antiserum to Gi₃ inhibited the initial transient contraction induced by endothelin and preincubation with an antiserum to Gi₁₋₂ inhibited the sustained phase of the endothelin induced contraction. Our data indicate that: 1) Endothelin induces a direct sustained contraction of smooth cells from the rectosigmoid; 2) The transmembrane signalling of endothelin is through two specific GTP binding components that are Gi, one for the initial transient contraction, and the other for the sustained phase of the contraction.     

Specific proteins in stem meristems during transition to flowering

Immunodiffusion tests were used for studying protein composition of apical buds ofRudbeckia bicolor andPerilla nankinensis during their transition from vegetative to reproductive state under inductive photoperiodic conditions or GA₃ treatment. In both species the induced buds differ from the vegetative ones in the presence of specific proteins (P): P1, P2, P3 appear inRudbeckia apical buds 2, 8, 16 d after the start of inductive treatment; P4 appears inPerilla apical buds 6 d after inductive treatment. P1, P2, P4 are revealed in induced buds in the early period of apex development when morphogenetic changes are not yet present. The similarity between antigenic spectra of induced buds and of those treated by GA₃ appears only inRudbeckia. These observations support the hypothesis of a change in gene expression at floral evocation. Presented at the International Symposium “Plant Growth Regulators” held on June 18–22, 1984 at Liblice, Czechoslovakia.     

Specific for apurine-apyrimidine DNA endodeoxyribonuclease from the rat brain

Endodeoxyribonuclease was detected in rat neocortex chromatin. The partly purified enzyme was found to influence the superhelical apurine-apyrimidine DNA 50 times as effectively as compared to the native substrate. The enzyme hydrolyzes the phosphodiester bond with the formation of 3'-OH- and 5'-phosphate terminal groups. The enzyme-hydrolyzed DNA is an effective primer for DNA-polymerase I from E. coli. It was assumed that DNAase from rat brain chromatin is an apurine-apyrimidine endonuclease II.