Pyridoxal phosphate. An anionic probe for protein amino groups exposed on the outer and inner surfaces of intact human red blood cells.

Pyridoxal phosphate is a potent probe for exploring the "sidedness" of proteins in the membrane of the intact red blood cell. It reacts with amino groups with a high degree of specificity, forming a Schiff's base that can be fixed as an irreversible bond upon reduction with NaBH4; its binding site can be identified by use of [3-H]pyridoxal phosphate or NaB3-H4; it can be used as a surface probe under conditions of minimal penetration, or it can be used as a probe for proteins on the inside of the membrane under conditions of substantial uptake. Pyridoxal phosphate uptake involves a rapid and a slow component. The former represents the binding to the outer surface of the membrane; it is not substantially affected by pH and temperature, but is reduced considerably by pretreatment of cells by 4,4-diisothiocyano-2,2-stilbenedisulfonic acid, a specific inhibitor of anion transport. The slow component represents penetration into the cell; it is blocked by high pH, low temperature, or pretreatment with the disulfonic stilbene. Pyridoxal phosphate itself is also an effective and specific inhibitor of the permeation of other anions. Under conditions of minimal uptake, the only labeled proteins are three glycoproteins and a protein of apparent molecular weight 95,000. Under conditions of substantial uptake into the cell, the other major protein bands seen by staining on acrylamide gels after electrophoresis are labeled. It is concluded that virtually all of the major membrane proteins interact with pyridoxal phosphate from one side of the membrane or the other. The differences in labeling under conditions of minimal or maximal uptake can, therefore, be attributed to the sidedness in the distribution of the membrane proteins rather than to differences in their reactivity.     

Functional characterization of anion transport system isolated from human erythrocyte membranes.

The anion transport system of human red blood cells was isolated in vesicles containing the original lipids of the membrane and predominantly the 95,000-dalton polypeptides (Band 3) associated with intralipid particles. The vesicles display various characteristic properties of anion permeation closely resembling those of the native system. The properties include energy of activation, pH dependence, anion sleectivity, sensitivity to specific inhibitors, and exchange and net rates of sulfate transport. Based on these and other criteria, the functional properties of isolated vesicles could be equated with those of the intact cell system. Direct support for the involvement of 95,000-dalton polypeptides in permeation functions is provided.     

Characterization of mitochondrial iron uptake in HepG2 cells

There is increasing evidence that accumulation of redox-active iron in mitochondria leads to oxidative damage and contributes to various neurodegenerative diseases, such as Friedreich's ataxia and Parkinsons disease. In this work, we examined the existence of regulatory mechanisms for mitochondrial iron uptake and storage. To that end, we used rhodamine B-[(1,10-phenanthrolin-5-yl)amino carbonyl] benzyl ester, a new fluorescent iron-sensitive probe that is targeted specifically to the mitochondrion. We found that extracellular iron was incorporated readily into mitochondria in an apparently saturable process. Moreover, the rate of iron incorporation responded to the Fe status of the cell, an indication that the mitochondrion actively regulates its iron content.     

Characterization of Arabidopsis NEET reveals an ancient role for NEET proteins in iron metabolism

The NEET family is a newly discovered group of proteins involved in a diverse array of biological processes, including autophagy, apoptosis, aging, diabetes, and reactive oxygen homeostasis. They form a novel structure, the NEET fold, in which two protomers intertwine to form a two-domain motif, a cap, and a unique redox-active labile 2Fe-2S cluster binding domain. To accelerate the functional study of NEET proteins, as well as to examine whether they have an evolutionarily conserved role, we identified and characterized a plant NEET protein. Here, we show that the Arabidopsis thaliana At5g51720 protein (At-NEET) displays biochemical, structural, and biophysical characteristics of a NEET protein. Phenotypic characterization of At-NEET revealed a key role for this protein in plant development, senescence, reactive oxygen homeostasis, and Fe metabolism. A role in Fe metabolism was further supported by biochemical and cell biology studies of At-NEET in plant and mammalian cells, as well as mutational analysis of its cluster binding domain. Our findings support the hypothesis that NEET proteins have an ancient role in cells associated with Fe metabolism.     

Localization and characterization of transport-related elements in the plasma membrane of turtle bladder epithelial cells.

A mixed membrane preparation obtained from turtle bladder epithelial cells contains (Na+ + K+)-ATPase, adenylate cyclase and protein kinase, which interact with ouabain, norepinephrine and cyclic AMP, respectively. When such a preparation is obtained from bladders which had been preexposed to serosal fluids containing the tritiated form of 4,4'-diisothiocyano-2,2'-disulfonic stilbene, the subsequently isolated membrane proteins are enriched in tritium as well as in the afore-mentioned enzymes, none of which is inhibited. Free-flow electrophoresis separates the mixed membrane preparation into two distinguishable groups: one, construed as apical membranes, is enriched in norepinephrine-sensitive adenylate cyclase and cyclic AMP-sensitive protein kinase; the other, construed as basal-lateral membranes, is enriched in ouabain-sensitive ATPase and 4,4'-diisothiocyano-2,2'-disulfonic stilbene-binding proteins. The physiological counterparts of these enzymatically defined membrane markers are the mucosal sidedness of the transport effects of norepinephrine and cyclic AMP derivatives and the serosal sidedness of the transport effects of ouabain and disulfonic stilbenes in the intact turtle bladder. The discreteness and ion selectivity of each membrane-bound, transport-related element are discussed in relation to the corresponding characteristics of each transport process in vivo; the possibility of regulation of anion transport by adenylate cyclase-protein kinase system is also discussed.     

Intracellular labile iron

Cells maintain organellar pools of "labile iron" (LI), despite its propensity for catalyzing the formation of reactive oxygen species. These pools are identifiable by iron-chelating probes and accessible to pharmacological agents. Cytosolic LI has been assumed to have a dual function: providing a rapidly adjustable source of iron for immediate metabolic utilization, and for sensing by iron-regulatory proteins (IRPs) that regulate iron uptake and compartmentalization via transferrin receptors and ferritin. However, it now appears that IRPs may respond both to fluctuations in LI per se and to secondary signals associated with redox-active species. Recent information also indicates that iron can be delivered to mitochondria via pathways that circumvent cytosolic LI, suggesting possible alternative mechanisms of cell iron mobilization and trafficking. We discuss the changing views of intracellular LI pools in relation to iron homeostasis and cellular distribution in physiological and pathological states.     

Characterization of permeation pathways in the plasma membrane of human erythrocytes infected with early stages of Plasmodium falciparum: association with parasite development

Human intraerythrocytic malarial parasites (Plasmodium falciparum) induce permeability changes in the membrane of their host cells. The differential permeability of infected erythrocytes at various stages of parasite growth, in combination with density gradient centrifugation, was used to fractionate parasitized cells according to their developmental stage. By this method it was possible to obtain cell fractions consisting essentially of erythrocytes infected with the youngest parasite stage (i.e., rings). These preparations were used for the measurement of transport of various solutes. It is shown that permeabilization of host erythrocyte membrane appears as early as 6 h after parasite invasion of the erythrocyte and increases gradually with parasite maturation. Since the selectivity for several different solutes and the enthalpy of activation of transport remain unaltered with maturation-related increase of permeability, it is concluded that the number of transport agencies in the host cell membrane increases with parasite maturation. Evidence is presented to indicate the need for parasite protein synthesis as an essential factor for the generation of the new permeability pathways.     

Transport domain of the erythrocyte anion exchange protein

Summary The anion transport domain of the anion exchange protein (AEP) of human erythrocyte membranes (band 3, 95 kD mol wt) was probed with the substrate and affinity label pyridoxal-5-phosphate (PLP). Acting from outside, this probe labels two chymotryptic fragments of 65 and 35 kD of AEP but only the 35-kD fragment is protected from labeling by reversibly acting disulfonic stilbenes (DS). It is shown here by functional studies and by immunoblotting with anti-PLP antibodies that transmembrane gradients of anions determine the availability of a 35-kD fragmentlys residue to surface labeling by PLP, in analogy with their effects on labeling of 65-kD fragment by DS. On this basis, it is suggested that both fragments contribute to the formation of the transport domain. However, unlike DS, PLP blocks transport when reacted from within resealed membranes, indicating that the 35-kD fragment might contain components of the mobile unit of the AEP. Using impermeant fluorescence quenchers of PLP of both complexation type (anti-PLP antibodies) or collisional type (acrylamide) as topological probes for PLP-labeled sites, it is deduced that the 65-kD PLP-labeled and the 35-kD PLP-labeledlys groups are inaccessible to macromolecules from either surface, but the 65-kD PLP-lys is accessible to low molecular weight molecules from without while the 35-kD PLP-labeledlys shows accessibility primarily from within the cell surface. The studies indicate that the accommodation of a wide class of anions by AEP might be associated with the flexibility of the transport domain of the protein and its capacity to undergo transport-related conformational changes.