monensin and antimycin

Crithidia deanei, a monoxenic trypanosomatid, presents an endosymbiotic bacterium in its cytoplasm. Both the protozoan and the bacterium maintain intensive metabolic exchange, resulting in an interesting model to study the coevolution of metabolisms. The relevance of l-proline for the growth of C. deanei and its transport into these cells was studied. Both the endosymbiont-containing (wild) and the endosymbiont-free protozoa (aposymbiont or cured) strains, when grown in medium supplemented with l-proline, reached higher cell densities than those grown in unsupplemented media. We biochemically characterized the uptake of l-proline in both the wild (K(m)=0.153+/-0.022 mM, V(max)=0.239+/-0.011 nmol min(-1) per 4 x 10(7) cells) and the aposymbiont strains (K(m)=0.177+/-0.049 mM, V(max)=0.132+/-0.012 nmol min(-1) per 4 x 10(7) cells). These data suggest a single type of proline transporter whose activity is upregulated by the presence of the symbiotic bacterium. Proline transport was further characterized and was found to be insensitive to the extracellular concentration of Na+, but sensitive to K+ and pH. The abolition of proline uptake by respiratory chain inhibitors and valinomycin indicates that the proline transport in C. deanei is dependent on the plasma membrane K+ gradient.

Topics: Animals; Antimycin A; Bacteria; Crithidia; Culture Media; Depression, Chemical; DNA, Bacterial; Hydrogen-Ion Concentration; Monensin; Potassium; Proline; RNA, Ribosomal, 16S; Rotenone; Sodium; Symbiosis; Temperature; Time Factors; Up-Regulation; Valinomycin

Relationships between the Na+ dependent amino acid uptake displayed by fertilized sea urchin eggs and the electrochemical gradient of Na+ was investigated. The time course of Na+ content and valine or alanine uptake was simultaneously monitored in Na+ loaded eggs [by fertilization in K+-free artificial sea water (OK-ASW), or by using monensin, antimycin, cyanide, or ciguatoxin]. Our results demonstrate that the uphill amino acid uptake follows the "Na+ gradient hypothesis." Subsequent fertilization of eggs Na+ depleted by ammonia for 40 min stimulates to a great extent the development of amino acid uptake as compared with controls eggs. By using simultaneous change of external and intracellular Na+ concentration, we studied the specific role of this ion. An increase in internal Na+ inhibits the uptake through trans inhibitory action while an increase in external Na+ stimulates the efficiency of the uptake system. In eggs fertilized since 30 min, hyperpolarization obtained in K+-free ASW stimulates amino acid uptake while depolarization (transfer from K+ free ASW to ASW) inhibits it. This potential-dependent effect developed after fertilization with a time course similar to that the establishment of K+ conductance described by R. A. Steinhardt, L. Lundin, and D. Mazia (1971, Proc. Natl. Acad. Sci. USA 68, 2426-2430). In conclusion, our results point out that slight modulations in the activity of the Na+ pump can widely affect the amino acid uptake, suggesting that activation of Na+/K+ ATPase has a key role in the stimulation of amino acid transport.

Topics: Amino Acids; Ammonium Chloride; Animals; Antimycin A; Biological Transport; Ciguatoxins; Fertilization; Hydrogen-Ion Concentration; Ion Channels; Membrane Potentials; Monensin; Ovum; Potassium; Sea Urchins; Sodium; Sodium-Potassium-Exchanging ATPase