Michal Bental

Polyphosphate metabolism in the alga Dunaliella salina studied by 31P-NMR

Polyphosphate synthesis and the state of the intracellular polyphosphates in the unicellular green alga Dunaliella salina were studied using in vivo 31P-NMR spectroscopy. By perfusing phosphate-depleted algal cells trapped inside agarose beads with orthophosphate (Pi) containing medium, we were able to follow the process of polyphosphate synthesis in whole, living cells. The results suggest that, in Dunaliella, low molecular weight, probably cyclic, polyphosphate intermediates are synthesized from Pi, and are then condensed to high molecular weight polymers. Studies of the intracellular organization of the polyphosphates by electron microscopy and solid-state NMR techniques indicate that most of these polymers are stored in the cell in a soluble form, and not in solid-like structures.

Ca2+-independent, protein-mediated fusion of chromaffin granule ghosts with liposomes

We have investigated the interaction between isolated membrane vesicles from chromaffin granules and large unilamellar phospholipid vesicles (liposomes). Mixing of membrane lipids has been monitored continuously, utilizing the fluorescence resonance energy transfer assay described by Struck et al. ((1982) Biochemistry 20, 4093-4099). To demonstrate coalescence of the internal vesicle volumes the transfer of colloidal gold from the liposomes to the interior of the granule membrane vesicles has been examined. Efficient fusion of the liposomes with the granule membranes was observed. Significant fusion occurred in the absence of Ca2+, although the extent of interaction was enhanced in its presence. The sensitivity of the interaction to pretreatment of the granule membranes with trypsin showed the fusion reaction to be a protein-mediated process.

An NMR View of Primary T-Lymphocyte Activation

Publisher Summary This chapter discusses the nuclear magnetic resonance (NMR) view of primary T-lymphocyte activation. It focuses on studies of primary cells, specifically lymphocytes isolated from human blood. In culture, quiescent lymphocytes can be activated by lectins, a combination of phorbol esters and calcium ionophores, lymphocyte growth factors, or antibodies to surface antigens, thus providing a model for proliferation of nontransformed cells. Lymphocyte activation is dependent on cellular metabolism, because adenosine triphosphate (ATP) and anabolic precursors are needed to support modification of proteins, synthesis of nucleic acids and proteins, and membrane turnover. Magnetic resonance spectroscopy (MRS) is well suited for the study of mitogenesis in situ. The chapter explains the characterization by MRS techniques of quiescent and stimulated lymphocytes and describes the changes observed following mitogen stimulation. It also discusses studies of lymphocytes that speculate on innovative uses of immune cells in magnetic resonance imaging and MRS applications.

The H+-Export Capacity of Dunaliella acidophila and the Permeability of the Plasma Membrane for H+ and Weak Acids

The acid resistant green alga Dunaliella acidophila tolerates H+ concentrations in the medium up to 0.3 M (1, 2) without any significant change of the cytoplasmic pH, which is kept close to pH 7 (3–6). Factors contributing to this extreme acid resistance are a positive membrane potential, a positive surface charge, a low permeability coefficient (P value) of the plasma membrane (PM) for H+ (4, 5), and an efficient PM-H+s -ATPase (Sekler, unpublished). In order to get some information regarding the H+ transport capacity of intact cells, we exposed the algae to internal H+ stress by incubation with weak acids. When incubated at pH 1.0 the protonated species of weak acids (HAc) is expected to diffuse a-cross the PM into the cells along the chemical gradient. Inside the cells HAc dissociates into the anion (Ac−) and H+. Since the cytoplasmic pH of D. acidophila is close to 7 and that of the medium is 1.0, internal accumulations of Ac− and H+ are expected. To maintain cytoplasmic pH, the protons are expected to be reexported by ATPases of the PM. This will depend on the ATP-ase capacity in vivo and the extent of internal H+ stress. Monitoring the uptake of weak acids and simultaneously cytoplasmic pH by means of NMR techniques can provide an estimate for the minimal proton transport capacitity, which is a measure for the acid resistance of intact cells.