7 research outputs found
Aptamer-mediated inhibition of mycobacterium tuberculosis polyphosphate kinase 2
Inorganic polyphosphate (polyP) plays a number of critical roles in bacterial persistence, stress, and virulence. PolyP intracellular metabolism is regulated by the polyphosphate kinase (PPK) protein families, and inhibition of PPK activity is a potential approach to disrupting polyP-dependent processes in pathogenic organisms. Here, we biochemically characterized Mycobacterium tuberculosis (MTB) PPK2 and developed DNA-based aptamers that inhibit the enzymes catalytic activities. MTB PPK2 catalyzed polyP-dependent phosphorylation of ADP to ATP at a rate 838 times higher than the rate of polyP synthesis. Gel filtration chromatography suggested MTB PPK2 to be an octamer. DNA aptamers were isolated against MTB PPK2. Circular dichroism revealed that aptamers grouped into two distinct classes of secondary structure; G-quadruplex and non-G-quadruplex. A selected G-quadruplex aptamer was highly selective for binding to MTB PPK2 with a dissociation constant of 870 nM as determined by isothermal titration calorimetry. The binding between MTB PPK2 and the aptamer was exothermic yet primarily driven by entropy. This G-quadruplex aptamer inhibited MTB PPK2 with an IC 50 of 40 nM and exhibited noncompetitive inhibition kinetics. Mutational mechanistic analysis revealed an aptamer G-quadruplex motif is critical for enzyme inhibition. The aptamer was also tested against Vibrio cholerae PPK2, where it showed an IC 50 of 105 nM and insignificant inhibition against more distantly related Laribacter hongkongensis PPK2. © 2011 American Chemical Society.link_to_subscribed_fulltex
A case of hyperzincemia with functional zinc depletion: a new disorder?
We report the case of an 11-y-old boy with a plasma Zn concentration greater than 200 μmol/L, but with symptoms consistent with Zn deficiency. He has had hepatosplenomegaly, rashes, stunted growth (<3rd centile), anemia, and impaired immune function since infancy. He also has vasculitis and osteoporosis. A plasma Zn-binding protein has been separated and characterized by a combination of size exclusion and ion exchange chromatography and electrophoretic studies and by immunologic methods. Antibodies to the partially purified protein have been raised in rabbits. Size exclusion chromatography shows that Zn is bound to a protein with a mass 110 000-300 000 kD. Electrophoretic and mass spectrometry studies suggest that the protein may be composed of several subunits. One component of the isolated protein reacts with antiserum to α2-macroglobulin; immunoprecipitation studies confirm that the protein is not α2-macroglobulin or a histidine-rich glycoprotein. Kinetic studies of zinc metabolism in the patient and his mother with stable Zn isotopes show the presence of increased exchangeable Zn, with a rapid flux from plasma to a stable pool. Liver and muscle Zn and Cu concentrations are raised, but with no abnormal liver histology. Immunoreactive metallothionein in the liver is increased. We suggest that this boy may suffer from a previously unrecognized inborn error of Zn metabolism causing symptomatic zinc deficiency