Michelle Comte

Distribution pattern of three neural calcium-binding proteins (NCS-1, VILIP and recoverin) in chicken, bovine and rat retina

SummaryNeural Ca2+-binding proteins (NCaPs) constitute a subfamily of 4-EF-hand proteins, and display a histological and structural dichotomy: the A-type NCaPs are selectively expressed by the retina and pineal organ and display two canonical EF-hands, whereas the B-type NCaPs are found in the entire brain and present three regular EF-hands. In this study, antisera were raised against the A-type NCaP recoverin (26 kDa) and the B-type NCaPs VILIP and NCS-1 (22 kDa). Since the sequence identity among NCaPs is high, specific polyclonal antibodies were purified by double cross-immunoaffinity chromatography; both ELISA and immunoblot analyses determined that the resulting antibodies showed selectivity ratios inferior to 1/363 for the two other related NCaPs. Besides, the anti-VILIP antibodies displayed some affinity toward neurocalcin δ, and the antirecoverin antibodies recognized a 24 kDa protein, which is most likely visinin. Thus, immunohistochemical studies on the chicken, rat and cow retina revealed that anti-recoverin antibodies recognized the vertebrate photoreceptors and a small number of mammalian bipolar cells. Anti-VILIP antibodies exclusively labelled the inner Retina, I.e. the amacrine and ganglion cells. NCS-1 was mainly present in the photoreceptor inner segments, the inner plexiform layer and the ganglion cells. NCS-1 showed the highest species disparity. The retinal localization of NCS-1 and VILIP offered an important morphological basis for the understanding of their function. Furthermore, specific antibodies against the NCaPs may enable the identification of cell populations in more complex neural tissues, such as the brain.

Evidence for four capital and six auxiliary cation-binding sites on calmodulin: Divalent cation interactions monitored by direct binding and microcalorimetry

Recently, Mills and Johnson [7] and our group [9] provided evidence that calmodulin contains, in addition to the four Ca2+-binding sites (capital sites), which are essential for drug- and enzyme-binding, a number of divalent cation-binding sites of different ion selectivity (auxiliary sites), which modulate drug-binding as well as the affinity of Ca2+ for the capital sites. In the present study, the number of auxiliary sites and their relationship to the capital sites were determined by equilibrium gel filtration and by flow microcalorimetry with Zn2+ and Mn2+ as selective probes for the auxiliary sites and with Cd2+ as a probe for both types of sites. In the absence of other divalent cations, 6 mol of Zn2+ bind to calmodulin with an identical affinity constant of 2,850 M-1 and a delta H0 of 106 kJ/mol calmodulin. In the presence of millimolar free Ca2+ calmodulin binds, in addition to four Ca2+, six Zn2+ with an affinity constant of 1,200 M-1 and a delta H0 of 47 kJ/mol calmodulin. The Zn2+-Ca2+ antagonism is governed by negative free energy coupling between the capital and auxiliary sites. In contrast, the Zn2+-Mg2+ antagonism follows the rule of straight competition at all six auxiliary sites. Mn2+ also binds exclusively to the auxiliary sites with affinity constants of 800 or 280 M-1 and delta H0 of 45 or 46 kJ/mol calmodulin in the absence and presence of saturating [Ca2+], respectively. Cd2+ binds to the capital sites with an affinity constant of 3.4 10(4) M-1 (delta H = 35 kJ/mol calmodulin) and to the auxiliary sites with ca. 100-fold lower affinity. The Zn2+ much greater than Mn2+ greater than or equal to Cd2+ greater than Mg2+ selectivity of the auxiliary sites corroborates the potencies of these cations in modulating drug binding. The auxiliary site-specific cations are unable to promote high-affinity complex formation between calmodulin and melittin.

A new solid-phase chelator with high affinity and selectivity for calcium: Parvalbumin-polyacrylamide

A solid-phase chelator for calcium was prepared by linking parvalbumin (a muscle calcium-binding protein of remarkable stability) to the polyacrylamide matrix of Bio-Gel P-60. The immobilized parvalbumin can be used repeatedly, and, due to its remarkable affinity for calcium, it is capable of lowering calcium concentration to ≤10−10m at neutrality. The affinity for calcium remains relatively high even at pHs as low as 4–5, at which complexants such as EDTA, EGTA, or Chelex would be quite inefficient. As immobilized parvalbumin binds Mg2+ with an apparent Kdiss 3.5 orders of magnitude higher than that of Ca2+, it can be used to control calcium concentrations even in the presence of magnesium. The affinity for calcium of any strong complexant can be determined by measuring Ca2+ distribution between this complexant and the solid-phase parvalbumin. Due to its outstanding affinity and selectivity for calcium, immobilized parvalbumin could prove to be a powerful tool in investigating the role of calcium in the regulation of metabolic processes.

Cation Binding to Calmodulin and Relation to Function

Calmodulin (CaM) is intimately involved in the stimulus-response coupling in eukaryotic cells since it is the prime sensor of transient increases of the free intracellular [Ca2+] and conveys the signal to multiple target enzymes, especially protein kinases and phosphatases. For a general survey of the properties of CaM, the reader is referred to Cheung (1980), Klee and Vanaman (1982), Manalan and Klee (1984) and Wang et al. (1985) (see also Cheung this Vol.). This paper deals more specifically with the mode of action of CaM. Although in the last decade our understanding of its action has considerably increased, some enigmas remain and, unfortunately, some basic and long-lasting controversies on its mode of action have not been solved yet. In this paper, two of these are examined: (a) cation binding to CaM, and (b) the thermodynamics of the interaction of CaM with its targets.

Immunolocalization of calcium vector protein and its target protein in amphioxus

Three proteins, sarcoplasmic CA2+-binding protein (SCP), Ca2+ vector protein (CaVP) and its target protein (CaVPT), are found abundantly in the higher invertebrate amphioxus. Whereas the function of SCP is likely to be related to Ca2+ and Mg2+ buffering, that of the latter two proteins, apparently linked together, is still not clear. In this study, affinity-purified polyclonal antibodies to these three proteins were used to study the extractability under physiological ionic conditions, the distribution in different tissues and the immunocytochemical localization in striated muscle. Our data show that SCP is essentially cytosolic whereas CaVP and CaVPT are partially associated with non-soluble components in amphioxus tissues. The tissue distribution, studied in transverse sections, shows that SCP is merely confined to striated muscle, whereas CaVP and CaVPT are also abundant in other tissues such as the spinal chord and the gonads. Thus the protein pair CaVP/CaVPT is likely to serve a general role in many tissues; however, no strict correlation was found in the distribution of the latter two proteins, suggesting that they may function independently. The detailed cytochemical localization of the three proteins in longitudinal sections of striated muscle revealed a discrete striation pattern in addition to a diffuse background. For SCP these striations are coincident with the Z line. The immunostaining for CaVP shows intense striations at the level of the Z lines alternating with weak striations at the M lines. For CaVPT the striations at the Z and M line are more or less of equal intensity, leading to a pattern with a 1μm periodicity. The data lead to the conclusion that CaVP and CaVPT can form dynamic complexes with structural components of the sarcomere.