Kinetics Of Nucleotide Hydrolysis Research Articles

Disease Variants of the Human Mitochondrial DNA Helicase Encoded by C10orf2 Differentially Alter Protein Stability, Nucleotide Hydrolysis, and Helicase Activity

Missense mutations in the human C10orf2 gene, encoding the mitochondrial DNA (mtDNA) helicase, co-segregate with mitochondrial diseases such as adult-onset progressive external ophthalmoplegia, hepatocerebral syndrome with mtDNA depletion syndrome, and infantile-onset spinocerebellar ataxia. To understand the biochemical consequences of C10orf2 mutations, we overproduced wild type and 20 mutant forms of human mtDNA helicase in Escherichia coli and developed novel schemes to purify the recombinant enzymes to near homogeneity. A combination of molecular crowding, non-ionic detergents, Mg(2+) ions, and elevated ionic strength was required to combat insolubility and intrinsic instability of certain mutant variants. A systematic biochemical assessment of the enzymes included analysis of DNA binding affinity, DNA helicase activity, the kinetics of nucleotide hydrolysis, and estimates of thermal stability. In contrast to other studies, we found that all 20 mutant variants retain helicase function under optimized in vitro conditions despite partial reductions in DNA binding affinity, nucleotide hydrolysis, or thermal stability for some mutants. Such partial defects are consistent with the delayed presentation of mitochondrial diseases associated with mutation of C10orf2.

CGMP- and phosphodiesterase-dependent light-scattering changes in rod disk membrane vesicles: relationship to disk vesicle-disk vesicle aggregation.

Visible light activates a large guanosine cyclic 3',5'-phosphate (cGMP)- and phosphodiesterase (PDE)-dependent infrared light-scattering change in suspensions of photoreceptor disk membranes. Reconstitution experiments show that this signal requires bleached rhodopsin, G protein (three polypeptide subunits of Mr 39 000, 37 000, and 6000 which comprise the GTPase), phosphodiesterase, cGMP, and GTP. The lowest light intensity which elicits the light-scattering signal bleaches 0.002% rhodopsin. cGMP and GTP hydrolysis occurs more slowly than the initial phase of the scattering signal, and the kinetics of nucleotide hydrolysis do not correlate with any phase of the signal. Hydrolysis-resistant analogues of cGMP and GTP support the initial decreasing phase of the signal. Thus, the signal apparently depends upon nucleotide binding rather than hydrolysis. Microscopic observations made under the same conditions as light-scattering experiments show that vesicle-vesicle aggregation and disaggregation occur. The data suggest that light and nucleotide activations of the cyclic nucleotide cascade enzymes are responsible for the vesicle aggregation process and nucleotide hydrolysis for vesicle disaggregation. The vesicle aggregation-disaggregation phenomenon appears likely to be the physical basis of the cGMP- and PDE-dependent changes in infrared transmission.