Bioactivity of folding intermediates studied by the recovery of enzymatic activity during refolding

The peptide bond preceding proline residues realizes a cis/trans conformational switch with high switching resistance in native proteins and folding intermediates. Therefore, individual isomers have the potential to differ in bioactivity. However, information about isomer-specific bioactivities is difficult to obtain because of the risk of affecting isomeric distribution by bioactivity assay components.Here we present an approach that allows for the measurement of the recovery of enzymatic activities of wild-type RNase T₁ and RNase T₁ variants during refolding under conditions where the population of enzyme-substrate or enzyme-product complexes is negligible. Recovery of enzymatic activity was continuously monitored within the visible range of the spectrum by addition of a fluorescence-labeled nucleotide substrate to the refolding sample. We found that a nonnative trans conformation at Pro39 renders the RNase T₁ almost completely inactive. A folding intermediate having a nonnative trans conformation at Pro55 shows about 46% of the enzymatic activity referred to the native state. Pro55, in contrast to the active site located Pro39, is situated in a solvent-exposed loop region remote from active-site residues. In both cases, peptidyl prolyl cis/trans isomerases accelerate the regain of nucleolytic activity. Our findings show that even if there is a considerable distance between the site of isomerization and the active site, conformational control of the bioactivity of proteins is likely to occur, and that the surface location of prolyl bonds suffices for the control of buried active sites mediated by peptidyl prolyl cis/trans isomerases.     

UBE1L2, a novel E1 enzyme specific for ubiquitin

UBE1 is known as the human ubiquitin-activating enzyme (E1), which activates ubiquitin in an ATP-dependent manner. Here, we identified a novel human ubiquitin-activating enzyme referred to as UBE1L2, which also shows specificity for ubiquitin. The UBE1L2 sequence displays a 40% identity to UBE1 and also contains an ATP-binding domain and an active site cysteine conserved among E1 family proteins. UBE1L2 forms a covalent link with ubiquitin in vitro and in vivo, which is sensitive to reducing conditions. In an in vitro polyubiquitylation assay, recombinant UBE1L2 could activate ubiquitin and transfer it onto the ubiquitin-conjugating enzyme UbcH5b. Ubiquitin activated by UBE1L2 could be used for ubiquitylation of p53 by MDM2 and supported the autoubiquitylation of the E3 ubiquitin ligases HectH9 and E6-AP. The UBE1L2 mRNA is most abundantly expressed in the testis, suggesting an organ-specific regulation of ubiquitin activation.     

The Na(+)-independent Ca2+ efflux mechanism of liver mitochondria is not a passive Ca2+/2H+ exchanger

Whether the Na(2+)-independent Ca2+ efflux mechanism of liver mitochondria is a Ca2+/2H+ exchanger and whether this exchanger is a passive mechanism have been controversial since shortly after the discovery of this mechanism. Here, a new approach to determining if the mechanism is passive is developed based on the energy available to a passive Ca2+/2H+ exchanger. Conditions are identified in which the Na(+)-independent Ca2+ efflux mechanism transports Ca2+ out of mitochondria against a Ca2+ gradient many times greater than that possible for a passive Ca2+/2H+ exchanger, thus ruling this out as a possible mechanism.     

Long-Term GABA Administration Induces Alpha Cell-Mediated Beta-like Cell Neogenesis

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Characterisation of inosine monophosphate dehydrogenase expression during retinal development: differences between variants and isoforms

In mammals there are two ubiquitous, catalytically indistinguishable isoforms of inosine monophosphate dehydrogenase and mutations in the type I isoform, but not type II, cause retina-specific disorders. We have characterised the spatio-temporal expression of these proteins during development of the rat retina and performed functional investigations of the recently described retinal type I variants. Inosine monophosphate dehydrogenase was present in all immature cells throughout the retina during embryonic and neonatal development. Following eye opening and cell differentiation its distribution was restricted to the photoreceptors and bipolar cells, becoming prominent in Müller cells with aging. Type II was present in early, developing retinae whilst type I was undetectable. An isoform switch occurred around P10, after which the type I variants, type Ialpha and type Igamma, were the major forms. Functional investigations indicate type Igamma has greater catalytic activity compared with other variants and isoforms. Finally, all forms of type I show an increased propensity to form intracellular macrostructures compared to type II and these structures appear to be regulated in response to changing intracellular GTP levels. Collectively these data demonstrate that (i) type I does not play a role in early retinal development, (ii) type Igamma has greater activity and (iii) there are differences between type I and type II isoforms. These observations are consistent with the aetiology of retinitis pigmentosa and raise the possibility that programmed expression of specific inosine monophosphate dehydrogenase proteins may have arisen to meet the requirements of the cellular environment.     

Relationships between four novel ceratomyxid parasites from the gall bladders of labrid fishes from Heron Island, Queensland, Australia

Ceratomyxid parasites from the gall bladder of fishes from the family Labridae were examined for their taxonomic identity and their relatedness to other species of myxozoans. This paper describes four novel species Ceratomyxa choerodonae n. sp. from Choerodon cyanodus; Ceratomyxa cheilinae n. sp. from Cheilinus chlorourus; Ceratomyxa oxycheilinae n. sp. from Oxycheilinus digramma; and Ceratomyxa thalassomae n. sp. from Thalassoma lunare. Each species is characterised morphologically and small subunit (18 S) rDNA sequences were used in molecular phylogenetic analyses. Comparison of the small subunit rDNA sequences revealed four distinct species even though morphological differences indicated the presence of only two morphologically distinctive spore types. Phylogenetic analyses revealed that the ceratomyxids from labrid fishes did not form a clade to the exclusion of all other myxosporeans and provides evidence that host relatedness is not a good character to discriminate between species within the genus Ceratomyxa.