Divalent cation chelators citrate and EDTA unmask an intrinsic uncoupling pathway in isolated mitochondria

We demonstrate a suppression of ROS production and uncoupling of mitochondria by exogenous citrate in Mg²⁺ free medium. Exogenous citrate suppressed H₂O₂ emission and depolarized mitochondria. The depolarization was paralleled by the stimulation of respiration of mitochondria. The uncoupling action of citrate was independent of the presence of sodium, potassium, or chlorine ions, and it was not mediated by the changes in permeability of the inner mitochondrial membrane to solutes. The citrate transporter was not involved in the citrate effect. Inhibitory analysis data indicated that several well described mitochondria carriers and channels (ATPase, IMAC, ADP/ATP translocase, mPTP, mKATP) were not involved in citrate’s effect. Exogenous MgCl₂ strongly inhibited citrate-induced depolarization. The uncoupling effect of citrate was demonstrated in rat brain, mouse brain, mouse liver, and human melanoma cells mitochondria. We interpreted the data as an evidence to the existence of a hitherto undescribed putative inner mitochondrial membrane channel that is regulated by extramitochondrial Mg²⁺ or other divalent cations.     

Simulation of Migration of Human Epidermal Keratinocytes over Three-Dimensional Collagen Gel

We developed a novel model for studying migration of keratinocyte colonies over 3D collagen gel. It was shown that keratinocytes previously cultured on plastic and then seeded on the surface of collagen gel proliferate more actively than the primary cells. The gel density could significantly affect the morphology and migration of keratinocyte colonies. Modification of collagen gel by fibronectin-like protein or poly-L-lysine altered the morphometric parameters of colonies without significant changes in the velocity of migration.     

Fibroblasts Stimulate Epithelization of Collagen Gel

We studied the influence of human embryonic fibroblasts on epithelization of collagen gel. Introduction of the fibroblasts into the gel stimulates proliferation of keratinocytes plated on the gel surface. The presence of fibroblasts in the gel also affects the pattern of keratinocyte migration on the gel and induces pronounced polarization of the migrating colonies.     

EGFP as a fusion partner for the expression and organic extraction of small polypeptides

Green fluorescent protein (GFP) is widely used as an excellent reporter module of the fusion proteins. The unique structure of GFP allows isolation of the active fluorescent protein directly from the crude cellular sources by extraction with organic solvents. We demonstrated the stable expression of four short polypeptides fused to GFP in Escherichia coli cells, including antimicrobial cationic peptides, which normally kill bacteria. EGFP module protected fusion partners from the intracellular degradation and allowed the purification of the chimerical proteins by organic extraction. The nature of the polypeptide fused to GFP, as opposed to the order of GFP and the polypeptide modules in the fusion protein, influenced the efficiency of the described purification technique.     

Effect of zinc and temperature on the conformation of the gamma subunit of retinal phosphodiesterase: a natively unfolded protein

The cyclic GMP phosphodiesterase gamma-subunit (PDEgamma) was shown to belong to the family of natively unfolded proteins. Increasing temperature transforms the protein into a more ordered (but still relatively disordered) conformation. The C-terminal part of PDEgamma has a high-affinity zinc-binding site (Kd approximately 1 microM), with His75 and His79 being directly involved into the coordination of Zn2+. Zinc-loaded protein remains effectively unfolded. Possible implications of these findings to the functioning of PDEgamma are discussed.     

Activation of the cytochrome cd1 nitrite reductase from Paracoccus pantotrophus. Reaction of oxidized enzyme with substrate drives a ligand switch at heme c

Cytochromes cd(1) are dimeric bacterial nitrite reductases, which contain two hemes per monomer. On reduction of both hemes, the distal ligand of heme d(1) dissociates, creating a vacant coordination site accessible to substrate. Heme c, which transfers electrons from donor proteins into the active site, has histidine/methionine ligands except in the oxidized enzyme from Paracoccus pantotrophus where both ligands are histidine. During reduction of this enzyme, Tyr(25) dissociates from the distal side of heme d(1), and one heme c ligand is replaced by methionine. Activity is associated with histidine/methionine coordination at heme c, and it is believed that P. pantotrophus cytochrome cd(1) is unreactive toward substrate without reductive activation. However, we report here that the oxidized enzyme will react with nitrite to yield a novel species in which heme d(1) is EPR-silent. Magnetic circular dichroism studies indicate that heme d(1) is low-spin Fe(III) but EPR-silent as a result of spin coupling to a radical species formed during the reaction with nitrite. This reaction drives the switch to histidine/methionine ligation at Fe(III) heme c. Thus the enzyme is activated by exposure to its physiological substrate without the necessity of passing through the reduced state. This reactivity toward nitrite is also observed for oxidized cytochrome cd(1) from Pseudomonas stutzeri suggesting a more general involvement of the EPR-silent Fe(III) heme d(1) species in nitrite reduction.