UBC Theses and Dissertations
Membrane perturbational effects of inorganic cations Garnett, Maureen Elizabeth
The membrane perturbational effects of divalent and monovalent cations were examined by studying kinetic and chemical characteristics of trinitrobenzenesulfonic acid (TNBS) incorporation into the amino groups of human erythrocyte membranes. Both monovalent and divalent cations stimulated TNBS incorporation. The monovalent cation-stimulated TNBS incorporation, which was not size dependent, was attributed to non-specific ionic strength effects while divalent cation-stimulated TNBS incorporation, which was size dependent, was explained in terms of membrane structural perturbations which indirectly affected amino group reactivity. A parallel was noted between the effects of divalent cations on the activation energy for TNBS incorporation and the first ionization potential of the cations. It was concluded that the activation energy term is determined primarily by electrostatic properties affecting probe permeation. Labelling studies indicated an important role of membrane proteins in mediating these effects. Similarly, parallels were also noted between cation-induced rate enhancement of TNBS incorporation, associated with a corresponding increase in phospholipid labelling by TNBS, and the second ionization potential for the cation. This was thought to indicate that the rate of TNBS incorporation was dependent primarily on phospholipids and that divalent cations exerted their effects on rate by altering the hydration of phospho- lipid polar head groups, leading to a change in configuration of the phospholipids and hence a change in the accessibility of membrane amino groups. Differences were found between the relative extent and the characteristics of protein and phospholipid labelling by TNBS in the presence of various cations. Based on the ratios of phospholipid to protein labelling, the divalent cations could be divided into two groups, a homogeneous group consisting of the alkaline earth cations Mg²⁺, Ca²⁺, Sr²⁺, and Ba²⁺, and a second heterogeneous group including Co²⁺, Ni2²⁺, and Mn²⁺. A similar grouping of these ions can be made on the basis of their known effects on the functional properties of certain excitable tissues. Studies of divalent cation effects on membrane-associated enzymes indicated that divalent cations can interact with both inner and outer membrane surfaces. The influence of divalent and monovalent cations on ultrasonic disruption of membranes was examined in another series of experiments. The results obtained closely paralleled those found with TNBS incorporation, indicating that cation effects analyzed using TNBS as a probe are a result of cation-membrane interactions, with minimal contribution from the probe itself. Furthermore, in experiments involving a hypotonic challenge of intact erythrocytes in the presence of divalent or monovalent cations, the anti-hemolytic effects of these ions closely followed their ability to stimulate TNBS incorporation into membranes. Finally, preliminary experiments with bovine synaptic vesicles and rat liver microsomal membranes gave results qualitatively similar to those obtained with erythrocytes, suggesting that information on the molecular mechanisms by which divalent cations perturb erythrocyte membranes may be applicable to other more complicated membrane systems.
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