ABSTRACT
Nerve growth factor (NGF) is involved in the development and maintenance of the nervous system. NGF binds with high affinity to the extracellular region of the tyrosine kinase receptor TrkA. This domain comprises leucine and cysteine rich motifs, followed by two immunoglobulin like (Ig-like) domains. We describe the expression and purification of recombinant Ig-like domains. Fluorescence and circular dichroism spectroscopy show that the protein is folded into a compact globular structure and contains mainly beta-sheet secondary structure. Recombinant protein binds to NGF and can inhibit NGF bioactivity both in vitro and in vivo.
Subject(s)
Immunoglobulins/genetics , Immunoglobulins/metabolism , Nerve Growth Factors/metabolism , Proto-Oncogene Proteins/genetics , Proto-Oncogene Proteins/metabolism , Receptor Protein-Tyrosine Kinases/genetics , Receptor Protein-Tyrosine Kinases/metabolism , Receptors, Nerve Growth Factor/genetics , Receptors, Nerve Growth Factor/metabolism , Amino Acid Sequence , Animals , Binding Sites/genetics , Binding, Competitive , Biotechnology , Circular Dichroism , Humans , Immunoglobulins/chemistry , In Vitro Techniques , Male , Molecular Sequence Data , Nerve Growth Factors/antagonists & inhibitors , PC12 Cells , Protein Folding , Protein Structure, Secondary , Proto-Oncogene Proteins/chemistry , Rats , Rats, Wistar , Receptor Protein-Tyrosine Kinases/chemistry , Receptor, trkA , Receptors, Nerve Growth Factor/chemistry , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Spectrometry, FluorescenceABSTRACT
The crystal structure of despentapeptide insulin, a monomeric insulin, has been refined at 1.3 A spacing and subsequently used to predict and model the organization in the insulin fibril. The model makes use of the contacts in the densely packed despentapeptide insulin crystal, and takes into account other experimental evidence, including binding studies with Congo red. The dimensions of this model fibril correspond well with those measured experimentally, and the monomer-monomer contacts within the fibril are in accordance with the known physical chemistry of insulin fibrils. Using this model, it may be possible to predict mutations in insulin that might alleviate problems associated with fibril formation during insulin therapy.
Subject(s)
Insulin/analogs & derivatives , Insulin/chemistry , Protein Structure, Secondary , Protein Structure, Tertiary , Computer Simulation , Crystallography, X-Ray , Models, Molecular , Water/analysisABSTRACT
Apolipoprotein E (ApoE) is a potent risk factor for Alzheimer's disease. Since the loss of cholinergic function in Alzheimer's disease is known to occur at an early stage in the disease we have examined this function in normal subjects with an Apoepsilon4 allele to see if the deficit occurs in the absence of Alzheimer pathology or symptoms. We report that brain tissue obtained post-mortem from normal subjects and Alzheimer patients with an Apoepsilon4 allele has a lower cholinergic activity than tissue from those subjects without this allele. This has important significance for the interpretation of the cholinergic deficits found in Alzheimer's disease.
