Hsp60 is targeted to a cryptic mitochondrion-derived organelle ("crypton") in the microaerophilic protozoan parasite Entamoeba histolytica.
Entamoeba histolytica is a microaerophilic protozoan parasite in which neither mitochondria nor mitochondrion-derived organelles have been previously observed. Recently, a segment of an E. histolytica gene was identified that encoded a protein similar to the mitochondrial 60-kDa heat shock protein (Hsp60 or chaperonin 60), which refolds nuclear-encoded proteins after passage through organellar membranes. The possible function and localization of the amebic Hsp60 were explored here. Like Hsp60 of mitochondria, amebic Hsp60 RNA and protein were both strongly induced by incubating parasites at 42 degreesC. 5' and 3' rapid amplifications of cDNA ends were used to obtain the entire E. histolytica hsp60 coding region, which predicted a 536-amino-acid Hsp60. The E. histolytica hsp60 gene protected from heat shock Escherichia coli groEL mutants, demonstrating the chaperonin function of the amebic Hsp60. The E. histolytica Hsp60, which lacked characteristic carboxy-terminal Gly-Met repeats, had a 21-amino-acid amino-terminal, organelle-targeting presequence that was cleaved in vivo. This presequence was necessary to target Hsp60 to one (and occasionally two or three) short, cylindrical organelle(s). In contrast, amebic alcohol dehydrogenase 1 and ferredoxin, which are bacteria-like enzymes, were diffusely distributed throughout the cytosol. We suggest that the Hsp60-associated, mitochondrion-derived organelle identified here be named "crypton," as its structure was previously hidden and its function is still cryptic. (+info)
UV irradiation of polycyclic aromatic hydrocarbons in ices: production of alcohols, quinones, and ethers.
Polycyclic aromatic hydrocarbons (PAHs) in water ice were exposed to ultraviolet (UV) radiation under astrophysical conditions, and the products were analyzed by infrared spectroscopy and mass spectrometry. Peripheral carbon atoms were oxidized, producing aromatic alcohols, ketones, and ethers, and reduced, producing partially hydrogenated aromatic hydrocarbons, molecules that account for the interstellar 3.4-micrometer emission feature. These classes of compounds are all present in carbonaceous meteorites. Hydrogen and deuterium atoms exchange readily between the PAHs and the ice, which may explain the deuterium enrichments found in certain meteoritic molecules. This work has important implications for extraterrestrial organics in biogenesis. (+info)
Solid-state NMR and hydrogen-deuterium exchange in a bilayer-solubilized peptide: structural and mechanistic implications.
Hydrogen-deuterium exchange has been monitored by solid-state NMR to investigate the structure of gramicidin M in a lipid bilayer and to investigate the mechanisms for polypeptide insertion into a lipid bilayer. Through exchange it is possible to observe 15N-2H dipolar interactions in oriented samples that yield precise structural constraints. In separate experiments the pulse sequence SFAM was used to measure dipolar distances in this structure, showing that the dimer is antiparallel. The combined use of orientational and distance constraints is shown to be a powerful structural approach. By monitoring the hydrogen-deuterium exchange at different stages in the insertion of peptides into a bilayer environment it is shown that dimeric gramicidin is inserted into the bilayer intact, i.e., without separating into monomer units. The exchange mechanism is investigated for various sites and support for a relayed imidic acid mechanism is presented. Both acid and base catalyzed mechanisms may be operable. The nonexchangeable sites clearly define a central core to which water is inaccessible or hydroxide or hydronium ion is not even momentarily stable. This provides strong evidence that this is a nonconducting state. (+info)
Anaerobic degradation of phthalate isomers by methanogenic consortia.
Three methanogenic enrichment cultures, grown on ortho-phthalate, iso-phthalate, or terephthalate were obtained from digested sewage sludge or methanogenic granular sludge. Cultures grown on one of the phthalate isomers were not capable of degrading the other phthalate isomers. All three cultures had the ability to degrade benzoate. Maximum specific growth rates (microseconds max) and biomass yields (YXtotS) of the mixed cultures were determined by using both the phthalate isomers and benzoate as substrates. Comparable values for these parameters were found for all three cultures. Values for microseconds max and YXtotS were higher for growth on benzoate compared to the phthalate isomers. Based on measured and estimated values for the microbial yield of the methanogens in the mixed culture, specific yields for the phthalate and benzoate fermenting organisms were calculated. A kinetic model, involving three microbial species, was developed to predict intermediate acetate and hydrogen accumulation and the final production of methane. Values for the ratio of the concentrations of methanogenic organisms, versus the phthalate isomer and benzoate fermenting organisms, and apparent half-saturation constants (KS) for the methanogens were calculated. By using this combination of measured and estimated parameter values, a reasonable description of intermediate accumulation and methane formation was obtained, with the initial concentration of phthalate fermenting organisms being the only variable. The energetic efficiency for growth of the fermenting organisms on the phthalate isomers was calculated to be significantly smaller than for growth on benzoate. (+info)
Incubation of OKP cells in low-K+ media increases NHE3 activity after early decrease in intracellular pH.
Chronic hypokalemia increases the activity of proximal tubule apical membrane Na+/H+ antiporter NHE3. The present study examined the effect of the incubation of OKP cells (an opossum kidney, clone P cell line) in control medium (K+ concn ([K+]) = 5.4 mM) or low-K+ medium ([K+] = 2.7 mM) on NHE3. The activity of an ethylisopropyl amiloride-resistant Na+/H+ antiporter, whose characteristics were consistent with those of NHE3, was increased in low-K+ cells beginning at 8 h. NHE3 mRNA and NHE3 protein abundance were increased 2.2-fold and 62%, respectively, at 24 h but not at 8 h. After incubation in low-K+ medium, intracellular pH (pHi) decreased by 0.27 pH units (maximum at 27 min) and then recovered to the control level. Intracellular acidosis induced by 5 mM sodium propionate increased Na+/H+ antiporter activity at 8 and 24 h. Herbimycin A, a tyrosine kinase inhibitor, blocked low-K+- and sodium propionate-induced activation of the Na+/H+ antiporter at 8 and 24 h. Our results demonstrate that low-K+ medium causes an early decrease in pHi, which leads to an increase in NHE3 activity via a tyrosine kinase pathway. (+info)
Ontogeny of intestinal safety factors: lactase capacities and lactose loads.
We measured intestinal safety factors (ratio of a physiological capacity to the load on it) for lactose digestion in developing rat pups. Specifically, we assessed the quantitative relationships between lactose load and the series capacities of lactase and the Na+-glucose cotransporter (SGLT-1). Both capacities increased significantly with age in suckling pups as a result of increasing intestinal mass and maintenance of mass-specific activities. The youngest pups examined (5 days) had surprisingly high safety factors of 8-13 for both lactase and SGLT-1, possibly because milk contains lactase substrates other than lactose; it also, however, suggests that their intestinal capacities were being prepared to meet future demands rather than just current ones. By day 10 (and also at day 15), increased lactose loads resulted in lower safety factors of 4-6, values more typical of adult intestines. The safety factor of SGLT-1 in day 30 (weanling) and day 100 (adult) rats was only approximately 1.0. This was initially unexpected, because most adult intestines maintain a modest reserve capacity beyond nutrient load values, but postweaning rats appear to use hindgut fermentation, assessed by gut morphology and hydrogen production assays, as a built-in reserve capacity. The series capacities of lactase and SGLT-1 varied in concert with each other over ontogeny and as lactose load was manipulated by experimental variation in litter size. (+info)
Pre-steady-state kinetics of the reactions of [NiFe]-hydrogenase from Chromatium vinosum with H2 and CO.
Results are presented of the first rapid-mixing/rapid-freezing studies with a [NiFe]-hydrogenase. The enzyme from Chromatium vinosum was used. In particular the reactions of active enzyme with H2 and CO were monitored. The conversion from fully reduced, active hydrogenase (Nia-SR state) to the Nia-C* state was completed in less than 8 ms, a rate consistent with the H2-evolution activity of the enzyme. The reaction of CO with fully reduced enzyme was followed from 8 to 200 ms. The Nia-SR state did not react with CO. It was discovered, contrary to expectations, that the Nia-C* state did not react with CO when reactions were performed in the dark. When H2 was replaced by CO, a Nia-C* EPR signal appeared within 11 ms; this was also the case when H2 was replaced by Ar. With CO, however, the Nia-C* state decayed within 40 ms, due to the generation of the Nia-S.CO state (the EPR-silent state of the enzyme with bound CO). The Nia-C* state, induced after 11 ms by replacing H2 by CO in the dark, could be converted, in the frozen enzyme, into the EPR-detectable state with CO bound to nickel (Nia*.CO) by illumination at 30 K (evoking the Nia-L* state), followed by dark adaptation at 200 K. This can be explained by assuming that the Nia-C* state represents a formally trivalent state of nickel, which is unable to bind CO, whereas nickel in the Nia-L* and the Nia*.CO states is formally monovalent. (+info)
Denatured states of human carbonic anhydrase II: an NMR study of hydrogen/deuterium exchange at tryptophan-indole-H(N) sites.
Hydrogen/deuterium (H/D) exchange measurements in low and moderate concentrations of GuHCI were conducted on the side chain H(N) atoms of the seven tryptophans of pseudo wild-type human carbonic anhydrase II. Tryptophans 5, 16 and 245, situated in or close to the N-terminal domain were found to have little protection against exchange. The H/D exchange results for Trp-123, Trp-192 and Trp-209 showed that a previously identified molten globule and the native state gave a similar protection against exchange. Global unfolding of the protein is necessary for the efficient exchange at Trp-97, which is located in the central part of the beta-sheet. (+info)