Limited Tryptic Proteolysis of the Benzodiazepine Binding Proteins in Different Species Reveals Structural Homologies

Peptide mapping can be used to elucidate further the structural similarities of the benzodiazepine binding proteins in different vertebrate species. Crude synaptic membrane preparations were photoaffinity-labeled with [3H]flunitrazepam and subsequently degraded with various concentrations of trypsin. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by fluorography allowed a comparison of the molecular weights of photolabeled peptides in different species. Tryptic degradation led to a common peptide of 40K in all species investigated, a finding indicating that the benzodiazepine binding proteins are structurally homologous in higher bony fishes and tetrapods.     

Embryonic cholinesterase activity during morphogenesis of the mouse genital tract

Summary In the genital tract of male and female mouse embryos cholinesterase activity is described that is independent from innervation. The enzyme activity is localized in the mesenchyme at the junction of Wolffian and Müllerian ducts with the urogenital sinus. During male development prostate buds and vesicular glands grow out into the cholinesterase-active mesenchyme. During female development the active mesenchyme participates in the downgrowth of the vaginal anlage. Ultrastructurally the cholinesterase activity is localized in the perinuclear cisterna and in smooth endoplasmic reticulum of the mesenchymal cells. The enzyme activity disappears with definitive differentiation of the tissue. The embryonic cholinesterase is a component of a primitive muscarinic system. Its relation to the morphogenetic action of testosterone and its possible general functions are discussed.     

Structural organization and mutational analysis of the human uncoupling protein-2 (hUCP2) gene

Uncoupling proteins (UCPs) are mitochondrial membrane transporters which are involved in dissipating the proton electrochemical gradient thereby releasing stored energy as heat. This implies a major role of UCPs in energy metabolism and thermogenesis which when deregulated are key risk factors for the development of obesity and other eating disorders. From the three different human UCPs identified so far by gene cloning both UCP2 and UCP3 were mapped in close proximity (75-150 kb) to regions of human chromosome 11 (11q13) that have been linked to obesity and hyperinsulinaemia. At the amino acid level hUCP2 has about 55% identity to hUCP1 while hUCP3 is 71% identical to hUCP2. In this study we have deduced the genomic structure of the human UCP2 gene by PCR and direct sequence analysis. The hUCP2 gene spans over 8.7 kb distributed on 8 exons. The localization of the exon/intron boundaries within the coding region matches precisely that of the hUCP1 gene and is almost conserved in the recently discovered hUCP3 gene as well. The high degree of homology at the nucleotide level and the conservation of the exon /intron boundaries among the three UCP genes suggests that they may have evolved from a common ancestor or are the result from gene duplication events. Mutational analysis of the hUCP2 gene in a cohort of 172 children (aged 7 - 13) of Caucasian origin revealed a polymorphism in exon 4 (C to T transition at position 164 of the cDNA resulting in the substitution of an alanine by a valine at codon 55) and an insertion polymorphism in exon 8. The insertion polymorphism consists of a 45 bp repeat located 150 bp downstream of the stop codon in the 3'-UTR. The allele frequencies were 0.63 and 0.37 for the alanine and valine encoded alleles, respectively, and 0.71 versus 0.29 for the insertion polymorphism. The allele frequencies of both polymorphisms were not significantly elevated in a subgroup of 25 children characterized by low Resting Metabolic Rates (RMR). So far a direct correlation of the observed genotype with (RMR) and Body Mass Index (BMI) was not evident. Expression studies of the wild type and mutant forms of UCP2 should clarify the functional consequences these polymorphisms may have on energy metabolism and body weight regulation.     

Molecular Characterization of the Na+/H+-Antiporter NhaA from Salmonella Typhimurium

Na⁺/H⁺ antiporters are integral membrane proteins that are present in almost every cell and in every kingdom of life. They are essential for the regulation of intracellular pH-value, Na⁺-concentration and cell volume. These secondary active transporters exchange sodium ions against protons via an alternating access mechanism, which is not understood in full detail. Na⁺/H⁺ antiporters show distinct species-specific transport characteristics and regulatory properties that correlate with respective physiological functions. Here we present the characterization of the Na⁺/H⁺ antiporter NhaA from Salmonella enterica serovar Thyphimurium LT2, the causing agent of food-born human gastroenteritis and typhoid like infections. The recombinant antiporter was functional in vivo and in vitro. Expression of its gene complemented the Na⁺-sensitive phenotype of an E. coli strain that lacks the main Na⁺/H⁺ antiporters. Purified to homogeneity, the antiporter was a dimer in solution as accurately determined by size-exclusion chromatography combined with multi-angle laser-light scattering and refractive index monitoring. The purified antiporter was fully capable of electrogenic Na⁺(Li⁺)/H⁺-antiport when reconstituted in proteoliposomes and assayed by solid-supported membrane-based electrophysiological measurements. Transport activity was inhibited by 2-aminoperimidine. The recorded negative currents were in agreement with a 1Na⁺(Li⁺)/2H⁺ stoichiometry. Transport activity was low at pH 7 and up-regulation above this pH value was accompanied by a nearly 10-fold decrease of K_m ^Na (16 mM at pH 8.5) supporting a competitive substrate binding mechanism. K⁺ does not affect Na⁺ affinity or transport of substrate cations, indicating that selectivity of the antiport arises from the substrate binding step. In contrast to homologous E. coli NhaA, transport activity remains high at pH values above 8.5. The antiporter from S. Typhimurium is a promising candidate for combined structural and functional studies to contribute to the elucidation of the mechanism of pH-dependent Na⁺/H⁺ antiporters and to provide insights in the molecular basis of species-specific growth and survival strategies.