Chicken parvalbumin. Comparison with parvalbumin-like protein and three other components (Mr = 8,000 to 13,000).

Procedures for a rapid isolation and purification of parvalbumin (Mr = 12,600), parvalbumin-like protein (Mr = 12,800), and three other polypeptides with molecular weights of 12,400 (Component 1), 11,700 (Component 2), and 8,000, respectively, from chicken leg muscle, are described. A direct comparison of parvalbumin with these other proteins showed distinct differences in the amino acid compositions, charge, and immunological behavior. Parvalbumin has two high affinity sites for Ca2+ with a KDiss less than or equal to 10(-6) M (Blum, H. E., Lehky, P., Kohler, L., Stein, E.A., and Fischer, E. H. (1977) J. Biol. Chem. 252, 2834-2838), in contrast to parvalbumin-like protein. Components 1 and 2, and the Mr = 8,000 protein, where only low affinity sites for Ca2+ could be detected (KDiss greater than 10(-3) M). From our results it is concluded that the co-extracted proteins do not constitute isoproteins of parvalbumin. The very low affinity for Ca2+ suggests that these proteins are not involved in processes of Ca2+ transport or Ca2+ regulation as proposed for parvalbumin. Parvalbumin could not be localized within isolated myofibrils and also did not accumulate in primary myogenic cell cultures together with proteins forming the myofibrillar structure. Parvalbumin was not even detected in myotubes in which myofibrils and sarcoplasmatic reticulum were already assembled and functioning. Parvalbumin (or cross-reacting material) was detected in leg muscle and brain 1 day after hatching of the chick. Possible roles for parvalbumin are discussed.     

Implications on zinc binding to S100A2

Human S100A2 is an EF-hand calcium-binding S100 protein that is localized mainly in the nucleus and functions as tumor suppressor. In addition to Ca2+ S100A2 binds Zn2+ with a high affinity. Studies have been carried out to investigate whether Zn2+ acts as a regulatory ion for S100A2, as in the case of Ca2+. Using the method of competition with the Zn2+ chelator 4-(2-pyridylazo)-resorcinol, an apparent Kd of 25 nM has been determined for Zn2+ binding to S100A2. The affinity lies close to the range of intracellular free Zn2+ concentrations, suggesting that S100A2 is able to bind Zn2+ in the nucleus. Two Zn2+-binding sites have been identified using site directed mutagenesis and several spectroscopic techniques with Cd2+ and Co2+ as probes. In site 1 Zn2+ is bound by Cys21 and most likely by His 17. The binding of Zn2+ in site 2 induces the formation of a tetramer, whereby the Zn(2+) is coordinated by Cys2 from each subunit. Remarkably, only binding of Zn2+ to site 2 substantially weakens the affinity of S100A2 for Ca2+. Analysis of the individual Ca2+-binding constants revealed that the Ca2+ affinity of one EF-hand is decreased about 3-fold, whereas the other EF-hand exhibits a 300-fold decrease in affinity. These findings imply that S100A2 is regulated by both Zn2+ and Ca2+, and suggest that Zn2+ might deactivate S100A2 by inhibiting response to intracellular Ca2+ signals.     

S100 and S100 fused-type protein families in epidermal maturation with special focus on S100A3 in mammalian hair cuticles

Epithelial Ca²⁺-regulation, which governs cornified envelope formation in the skin epidermis and hair follicles, closely coincides with the expression of S100A3, filaggrin and trichohyalin, and the post-translational modification of these proteins by Ca²⁺-dependent peptidylarginine deiminases. This review summarizes the current nomenclature and evolutional aspects of S100 Ca²⁺-binding proteins and S100 fused-type proteins (SFTPs) classified as a separate protein family with special reference to the molecular structure and function of S100A3 dominantly expressed in hair cuticular cells. Both S100 and SFTP family members are identified by two distinct types of Ca²⁺-binding loops in an N-terminal pseudo EF-hand motif followed by a canonical EF-hand motif. Seventeen members of the S100 protein family including S100A3 are clustered with seven related genes encoding SFTPs on human chromosome 1q21, implicating their association with epidermal maturation and diseases. Human S100A3 is characterized by two disulphide bridges and a preformed Zn²⁺-pocket, and may transfer Ca²⁺ ions to peptidylarginine deiminases after its citrullination-mediated tetramerization. Phylogenetic analysis utilizing current genome databases suggests that divergence of the S100A3 gene coincided with the emergence of hair, a defining feature of mammals, and that the involvement of S100A3 in epithelial Ca²⁺-cycling occurred as a result of a skin adaptation in terrestrial mammals.     

The clues to species specificity of pollination among Brassicaceae

The Brassica S product of the pollen-signalling S-gene (SP11 for Specificity Pollen, or SCR for S locus Cysteine Rich) has recently been identified as a member of the family of PCPs (Pollen Coat Proteins), a group of small basic proteins. In female plants SRK (S Receptor Kinase) has been demonstrated, among several stigma-expressed S genes, to exclusively control self-pollen rejection. Genes and gene products that are highly related to the sporophytic SI system appear to take part in the control of several stages of pollen-stigma interactions in the Brassicaceae. These genes ensure, in particular, the species specificity of late pollen adhesion. Pollen capture itself is not species specific, since it depends essentially on lipophilic affinities between pollen coatings and waxy pellicles on the stigma. These interactions and their control are reviewed here. Received: 2 August 2000 / Revision accepted: 11 August 2000     

Determination of the catechol-O-methyltransferase inhibitor tolcapone and three of its metabolites in animal and human plasma and urine by reversed-phase high-performance liquid chromatography with ultraviolet detection

Reversed-phase HPLC procedures were developed for the determination of tolcapone (Ro 40-7592) and its metabolites Ro 40-7591, Ro 61-1448, and Ro 47-1669 in plasma and in urine samples. One of the procedures for plasma involved the determination of tolcapone and its metabolite Ro 40-7591 and the other, the determination of the two other metabolites. The urine assay enabled the simultaneous determination of tolcapone and all metabolites in one run. Sample preparation in plasma involved protein precipitation with acetonitrile. Urine was simply diluted. The compounds of interest were monitored in the UV at 270 nm. The limits of quantification were 0.05 μg/ml for each compound (plasma assay) and 0.2 μg/ml for the urine assay. The mean inter-assay precisions (C.V.) were ≤6% (plasma assay) and ≤8% (urine assay). The procedures were successfully applied to the sample analysis of animal pharmacokinetic (rat, dog, mouse, rabbit and cynomolgus monkey) and clinical pharmacology studies.     

Calcyclin is a calcium and zinc binding protein

Calcyclin, a cell cycle regulated protein, was recently purified from Ehrlich ascites tumour (EAT) cells and shown to be a calcium binding protein. Here we show that calcyclin monomer and dimer also bind zinc ions. Zinc binding sites seem to be different from calcium binding sites since: preincubation with Ca2+ lacks effect on the binding of Zn2+, and Ca2+ (but not Zn2+) increases tyrosine fluorescence intensity. Binding of Zn2+ reduces the extent of the conformational changes induced by Ca2+, and seems to affect Ca2(+)-binding. The data suggest that Ca2+ and Zn2+ might trigger the biological activity of calcyclin.     

Expression level of the SLG gene is not correlated with the self-incompatibility phenotype in the class II S haplotypes of Brassica oleracea

In Brassica, the S-locus glycoprotein (SLG) gene has been strongly implicated in the self-incompatibility reaction. Several alleles of this locus have been sequenced, and accordingly grouped as class I (corresponding to dominant S-alleles) and class II (recessive). We recently showed that a self-compatible (Sc) line of Brassica oleracea expressed a class II-like SLG (SLG-Sc) gene. Here, we report that the SLG-Sc glycoprotein is electrophoretically and immunochemically very similar to the recessive SLG-S15 glycoprotein, and is similarly expressed in stigmatic papillae. Moreover, by seed yield analysis, we observe that both alleles are associated with a self-compatibility response, in contrast with the other known recessive S haplotypes (S2 and S5). By genomic DNA blot analysis, we show the existence of molecular homologies between the Sc and S15 haplotypes, but demonstrate that they are not identical. On the other hand, we also report that the S2 haplotype expresses very low amounts of SLG glycoproteins, although it exhibits a self-incompatible phenotype. These results strongly question the precise role of the SLG gene in the molecular mechanisms that control the self-incompatibility reaction of Brassica.     

Appearance of parvalbumin-specific immunoreactivity in the cerebral cortex and hippocampus of the developing rat and gerbil brain

Developmental changes in the distribution of parvalbumin-specific immunoreactivity in the brain, in particular in the cerebral cortex and hippocampus, were followed immunohistochemically in two different species, the rat and the Mongolian gerbil (Meriones unguiculatus) using an antibody raised against for rat parvalbumin. The gerbil is known to develop its auditory and visual capacity later than rat. In both the rat and gerbil, parvalbumin-specific immunoreactivity appeared after birth in both the cerebral cortex and hippocampus. The timing of the development of expression of parvalbumin varied among different parts of the cerebral cortex. The parietal cortex showed evidence of the earliest expression of parvalbumin whilst the occipital and temporal cortices expressed parvalbumin at a later stage of a development. This feature was common to both the rat and gerbil but occurred at a relatively later stage in the gerbil. The profile of the distribution of parvalbumin in the brain of the developing and adult gerbil was similar to that of the rat, but there were some differences. The frequency of bead-like structures on the dendrites of the parvalbumin-positive cells in the CA1 region of the hippocampus was markedly lower in the gerbil; instead, straight non-beaded fibers which ran vertically into the pyramidal layer were stained. Parvalbumin-positive fibers were also found in the cerebral cortex of the gerbil.     

Parvalbumin in non-muscle tissues of the rat. Quantitation and immunohistochemical localization

Parvalbumin, a high affinity Ca2+-binding protein, is known to be expressed only in muscles and brain in the rat. We have investigated its distribution and characteristics in other rat tissues by several biochemical and immunohistochemical methods. Evidence for the presence of parvalbumin in teeth, bone, skin, prostate, seminal vesicles, testes, and ovary is given by two-dimensional polyacrylamide gel electrophoresis, immunoblotting ("Western technique") of one-dimensional gels, and its concentration measured by reverse phase high performance liquid chromatography. The distribution within several parvalbumin-positive organs was monitored by the immunohistochemical peroxidase-antiperoxidase method. In teeth, only ameloblasts reacted with anti-rat parvalbumin serum and in bone the calcified extracellular cartilage was the target of the immunoreaction. The panniculus carnosus was the exclusive site of parvalbumin in the skin. Besides the already known parvalbumin distribution in the brain, parvalbumin is also expressed in distinct cell types of the peripheral nervous system. Leydig cells were found to be the only parvalbumin location in testes. These observations lead us to conclude that parvalbumin in contrast to the multifunctional and constitutive calmodulin must function in Ca2+-dependent processes related to specific cell types.