Release of pyridoxal 5'-phosphate upon unfolding of mitochondrial aspartate aminotransferase.

Dimeric mitochondrial aspartate aminotransferase (mAAT) contains a molecule of pyridoxal 5'-phosphate (PLP) tightly attached to each of its two identical active sites. The presence of this natural reporter allows us to study separately local perturbations in the architecture of this critical region of the molecule during unfolding. Upon unfolding of the enzyme with guanidine hydrochloride (GdnHCl), the coenzyme is completely released from the active site. The transition midpoint for the dissociation of PLP is 1.4+/-0.02 M when determined by size-exclusion chromatography (SEC) and 1.6+/-0.02 M when the protein-bound PLP is estimated by electrospray mass spectrometry (ESI-MS). In both cases the transition midpoint is higher than that of inactivation (1.3+/-0.01 M). On the other hand, the midpoint of the unfolding transition obtained by monitoring changes in ellipticity at 356 nm, which reflects the asymmetric environment of the PLP cofactor at the active site, is 1.19+/-0.011 M guanidine. These results indicate that the unfolding of mAAT is a multi-step process which includes an intermediate containing bound PLP but lacking catalytic activity.

Retinal abnormalities associated with the G90D mutation in opsin.

Several mutations in the opsin gene have been associated with congenital stationary night blindness, considered to be a relatively nonprogressive disorder. In the present study, we examined the structural and functional changes induced by one of these mutations, i.e., substitution of aspartic acid for glycine at position 90 (G90D). Transgenic mice were created in which the ratio of transgenic opsin transcript to endogenous was 0.5:1, 1.7:1, or 2.5:1 and were studied via light and electron microscopy, immunocytochemistry, electroretinography (ERG), and spectrophotometry. Retinas with transgenic opsin levels equivalent to one endogenous allele (G0.5) appeared normal for a period of about 3-4 months, but at later ages there were disorganized, shortened rod outer segments (ROS), and a loss of photoreceptor nuclei. Higher levels of G90D opsin expression produced earlier signs of retinal degeneration and more severe disruption of photoreceptor morphology. Despite these adverse effects, the mutation had a positive effect on the retinas of rhodopsin knockout (R-/-) mice, whose visual cells fail to form ROS and rapidly degenerate. Incorporation of the transgene in the null background (G+/-/R-/- or G+/+/R-/-) led to the development of ROS containing G90D opsin and prolonged survival of photoreceptors. Absorbance spectra measured both in vitro and in situ showed a significant reduction of more than 90% in the amount of light-sensitive pigment in the retinas of G+/+/R-/- mice, and ERG recordings revealed a >1 log unit loss in sensitivity. However, the histological appearances of the retinas of these mice show no significant loss of photoreceptors and little change in the lengths of their outer segments. These findings suggest that much of the ERG sensitivity loss derives from the reduced quantal absorption that results from a failure of G90D opsin to bind to its chromophore and form a normal complement of light-sensitive visual pigment.

[Activation of NF-kappa B in the mouse retina after ischemia reperfusion].

OBJECTIVE: To investigate the expression of NF-kappa B following retinal ischemia and reperfusion injury in mice. METHODS: Retinal ischemia was induced by elevation of intraocular pressure. Retinal degeneration and atrophy were quantified by an image analysis system. Immunohistochemistry using p65 monoclonal antibody was performed on the retina and co-related with TUNEL labeling. RESULTS: Inner retinal thickness was increased in the initial 24 hours following retinal ischemia and was ascribed to tissue edema, but was significantly decreased by 168 hours after reperfusion. Six hours after retinal ischemia, p65 immunoreactivity was increased in the ganglion cell and the inner nuclear layers, reached a peak at 24 hours, and was parallel to TUNEL labeling. Double labeling with p65 and TUNEL showed partial co-localization of p65 and TUNEL labeling, predominantly in the inner nuclear layer. CONCLUSIONS: Activation of NF-kappa B appears to play an important role in retinal degeneration following retinal ischemia and reperfusion injury. The pro- and anti-apoptotic effects of NF-kappa B after retinal ischemia are being further investigated.