Subject(s)
Aorta/abnormalities , Lameness, Animal , Receptors, Nerve Growth Factor/genetics , Receptors, Nerve Growth Factor/physiology , Alternative Splicing/genetics , Animals , Aorta/embryology , Cell Count , Embryonic and Fetal Development , Humans , Mice , Mice, Inbred C57BL , Mice, Transgenic , Mutation , Neurons, Afferent/physiology , Protein Isoforms , Receptor, Nerve Growth Factor , Schwann Cells/chemistry , Schwann Cells/physiologySubject(s)
Nerve Growth Factors/metabolism , Neurons/physiology , Signal Transduction/physiology , Animals , Cell Differentiation , Cell Division , Cell Size , Cell Survival , Humans , Nerve Growth Factors/physiology , Nervous System/cytology , Nervous System/metabolism , Neurons/cytology , Neurons/metabolism , Synaptic Transmission/physiology , VertebratesABSTRACT
The gene for the alpha-glucosidase AglA of the hyperthermophilic bacterium Thermotoga maritima MSB8, which was identified by phenotypic screening of a T. maritima gene library, is located within a cluster of genes involved in the hydrolysis of starch and maltodextrins and the uptake of maltooligosaccharides. According to its primary structure as deduced from the nucleotide sequence of the gene, AglA belongs to family 4 of glycosyl hydrolases. The enzyme was recombinantly expressed in Escherichia coli, purified, and characterized. The T. maritima alpha-glucosidase has the unusual property of requiring NAD+ and Mn2+ for activity. Co2+ and Ni2+ also activated AglA, albeit less efficiently than Mn2+. T. maritima AglA represents the first example of a maltodextrin-degrading alpha-glucosidase with NAD+ and Mn2+ requirement. In addition, AglA activity depended on reducing conditions. This third requirement was met by the addition of dithiothreitol (DTT) or beta-mercaptoethanol to the assay. Using gel permeation chromatography, T. maritima AglA behaved as a dimer (two identical 55-kDa subunits), irrespective of metal depletion or metal addition, and irrespective of the presence or absence of NAD+ or DTT. The enzyme hydrolyzes maltose and other small maltooligosaccharides but is inactive against the polymeric substrate starch. AglA is not specific with respect to the configuration at the C-4 position of its substrates because glycosidic derivatives of D-galactose are also hydrolyzed. In the presence of all cofactors, maximum activity was recorded at pH 7.5 and 90 degrees C (4-min assay). AglA is the most thermoactive and the most thermostable member of glycosyl hydrolase family 4. When incubated at 50 degrees C and 70 degrees C, the recombinant enzyme suffered partial inactivation during the first hours of incubation, but thereafter the residual activity did not drop below about 50% and 20% of the initial value, respectively, within a period of 48 h.
Subject(s)
Manganese/pharmacology , NAD/pharmacology , Sulfhydryl Compounds/pharmacology , Thermotoga maritima/enzymology , alpha-Glucosidases/isolation & purification , alpha-Glucosidases/metabolism , Cations, Divalent/metabolism , Cations, Divalent/pharmacology , Dithiothreitol/pharmacology , Enzyme Stability/drug effects , Escherichia coli , Genes, Bacterial , Hydrogen-Ion Concentration , Kinetics , Manganese/metabolism , Molecular Sequence Data , Multigene Family , NAD/metabolism , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Sequence Analysis, DNA , Substrate Specificity , Sulfhydryl Compounds/metabolism , Temperature , Thermotoga maritima/genetics , alpha-Glucosidases/geneticsABSTRACT
Neurotrophins bind to two structurally unrelated receptors, the trk tyrosine kinases and the neurotrophin receptor p75(NTR). Ligand activation of these two types of receptor can lead to opposite actions, in particular the prevention or activation of programmed cell death. Many cells co-express trk receptors and p75(NTR), and we found that p75(NTR) was co-precipitated with trkA, trkB and trkC in cells transfected with both receptor types. Co-precipitation of p75(NTR) was not observed with the epidermal growth factor receptor. Experiments with deletion constructs of trkB (the most abundant trk receptor in the brain) and p75(NTR) revealed that both the extracellular and intracellular domains of trkB and p75(NTR) contribute to the interaction. Blocking autophosphorylation of trkB substantially reduced the interactions between p75(NTR) and trkB constructs containing the intracellular, but not the extracellular, domains. We also found that co-expression of p75(NTR) with trkB resulted in a clear increase in the specificity of trkB activation by brain-derived neurotrophic factor, compared with neurotrophin-3 and neurotrophin-4/5. These results indicate a close proximity of the two neurotrophin receptors within cell membranes, and suggest that the signalling pathways they initiate may interact soon after their activation.
Subject(s)
Receptor Protein-Tyrosine Kinases/chemistry , Receptor Protein-Tyrosine Kinases/metabolism , Receptors, Nerve Growth Factor/chemistry , Receptors, Nerve Growth Factor/metabolism , Animals , Base Sequence , Binding Sites , CHO Cells , Cell Line , Cricetinae , DNA Primers/genetics , Ligands , Mice , Phosphorylation , Rats , Receptor Protein-Tyrosine Kinases/genetics , Receptor, Ciliary Neurotrophic Factor , Receptor, trkA/chemistry , Receptor, trkA/genetics , Receptor, trkA/metabolism , Receptor, trkC , Receptors, Nerve Growth Factor/genetics , Sequence DeletionABSTRACT
In addition to the previously identified 4-alpha-glucanotransferase gene mgtA and the alpha-amylase gene amyA of Thermotoga maritima strain MSB8 we have now isolated three further genes encoding amylolytic enzymes from a gene library of this ancestral bacterium. The genes code for the extremely thermostable enzymes pullulanase (pulA), maltodextrin phosphorylase (agpA) and alpha-glucosidase (aglA) and have the potential to encode polypeptides with calculated molecular masses of 96.3 kDa, 96.1 kDa and 52.5 kDa, respectively. Comparative amino acid sequence analysis revealed that PulA and AgpA are clearly related to other known enzymes with similar function. AglA, on the other hand, was not related to other alpha-glucosidases but appears to belong to an enzyme family containing alpha-galactosidases and 6-phospho-beta-glucosidases. Enzyme properties are reported which demonstrate the extreme thermostability of these T. maritima enzymes.
