Background: Acute intermittent porphyria (AIP) is an autosomal dominant disorder that results from the partial deficiency of porphobilinogen deaminase (PBGD) in the heme biosynthetic pathway. Patients with AIP can experience acute attacks consisting of abdominal pain and various neuropsychiatric symptoms. Although molecular biological studies on the porphobilinogen deaminase (PBGD) gene have revealed several mutations responsible for AIP, the properties of mutant PBGD in eukaryotic expression systems have not been studied previously. Materials and Methods: Seven mutations were analyzed using transient expression of the mutated polypeptides in COS-1 cells. The properties of mutated polypeptides were studied by enzyme activity measurement, Western blot analysis, pulse-chase experiments, and immunofluorescence staining. Results: Of the mutants studied, R26C, R167W, R173W, R173Q, and R225X resulted in a decreased enzyme activity (0-5%), but R225G and 1073delA (elongated protein) displayed a significant residual activity of 16% and 50%, respectively. In Western blot analysis, the polyclonal PBGD antibody detected all mutant polypeptides except R225X, which was predicted to result in a truncated protein. In the pulse-chase experiment, the mutant polypeptides were as stable as the wild-type enzyme. In the immunofluorescence staining both wild-type and mutant polypeptides were diffusely dispersed in the cytoplasm and, thus, no accumulation of mutated proteins in the cellular compartments could be observed. Conclusions: The results confirm the causality of mutations for the half normal enzyme activity measured in the patients' erythrocytes. In contrast to the decreased enzyme activity, the majority of the mutations produced a detectable polypeptide, and the stability and the intracellular processing of the mutated polypeptides were both comparable to that of the wild-type PBGD and independent of the cross-reacting immunological material (CRIM) class.
Nonequilibrium molecular dynamics simulations have been performed on model fluids representing eicosane isomers in order to investigate the effect of branching and side chain position on fluid rheology. A heterogeneous, united-atom model with 20 Lennard-Jones interaction sites located at carbon centers was used to model the fluids. Vibrations and bond rotations were frozen, but torsional rotation was included. It was found that viscosity increases significantly from the n-alkane structure to a branch on carbon 2, but the movement of the branch along the carbon backbone has a smaller increasing than decreasing effect. The size of the group in a branched position has a more substantial effect upon the viscosity.
Nonequilibrium molecular dynamics simulations of viscosity were
performed using various molecular
representations of 3-methylhexane in order to study the influence of
potential models on simulated viscosity.
The models investigated were united atom models with fixed bond
lengths and bond angles. The effect of
intermolecular potential was examined by comparing results from a
homogeneous (in which all −CH
x
groups
are equivalent) model and two heterogeneous models. The effect of
intramolecular potential was investigated
by comparing results from three different torsional potential models.
The simulations were carried out at
three different densities to investigate the sensitivity of the
contributions from the various models to the
viscosity at different conditions. Large changes in viscosity were
produced by relatively small changes in
the intermolecular potential parameters of the branched methyl group.
The viscosity was found to be less
sensitive to the intermolecular potential parameters of the chain
methyl groups and the torsional potential.
Our results suggest that an accurate representation of the
molecular structure and size as governed by
intermolecular interactions is more important in accurate viscosity
predictions than careful modeling of the
intramolecular potential.
Molecular dynamics simulations were carried out to study the
conformational properties of branched alkanes
(C40H82). The populations of different
isomeric conformers, cooperative transitions, and transition rates
by
analyzing a trajectory at 450 K in the gas phase were calculated.
The results extracted from the molecular
dynamics trajectory were mostly in very good agreement with the results
for previously simulated n-alkanes.
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