John F. Brandts

A relationship between anion transport and a structural transition of the human erythrocyte membrane

Scanning microcalorimetry was employed as an aid in examining some structural features of the anion transport system in red blood cell vesicles. Two structural transitions were previously shown to be sensitive to several covalent and non-covalent inhibitors of anion transport in red cells. In this study, these transitions were selectively removed, either thermally or enzymatically, and the subsequent effect on 35SO2- 4 efflux in red cell vesicles was determined. It is shown that removal of one of these transitions (B2) has a negligible inhibitory effect on anion transport. Cytoplasmic, intermolecular disulfide linkages between band 3 dimers are known to form during the B2 transition. The integrity of the 4,4-diisothiocyanostilbene-2,2-disulfonate-sensitive C transition, on the other hand, is shown to be a requirement for anion transport. The localized region of the membrane giving rise to this transition contains the transmembrane segment of band 3, as well as membrane phospholipids. The calorimetric results suggest a structure of band 3 which involves independent structural domains, and are consistent with the transmembrane segment playing a direct role in the transport process.

Substitution of a proline for alanine 183 in the hinge region of phosphoglycerate kinase: effects on catalysis, activation by sulfate, and thermal stability.

A “hinge-bending” domain movement has been postulated as an important part of the catalytic mechanism of phosphoglycerate kinase (PGK) (Bankset al., 1979). In order to test the role of the flexibility of a putative interdomain hinge in the substrate- and sulfate-induced conformational transitions, alanine-183 was replaced by proline using site-directed mutagenesis. The maximal velocity of the Ala 183→Pro mutant, measured at saturating concentrations of ATP and phosphoglycerate (5 mM and 10 mM, respectively) and in the absence of sulfate ions, is increased approximately 21% in comparison to the wild type PGK. TheKm values for both substrates are essentially unchanged. The effect of sulfate on the specific activity of the Ala 183→Pro mutant and the wild type PGK was measured in the presence of 1 mM ATP and 2 mM 3-phosphoglycerate (3-PG). A maximum activation of 70% was observed at 20 mM sulfate for the mutant enzyme, as compared to 130% activation at 30 mM sulfate for the wild type PGK. These results demonstrate that the increased rigidity of the putative hinge, introduced by the Ala→Pro mutation, does not impair catalytic efficiency of phosphoglycerate kinase, while it appears to decrease the sulfate-dependent activation. The differential scanning calorimetry (DSC) studies demonstrate an increased susceptibility of the Ala 183 → Pro mutant to thermal denaturation. In contrast to one asymmetric transition observed in the DSC scan for the wild type PGK, withTm near 54°C, two transitions are evident for the mutant enzyme withTm values of about 45 and 54°C. Using a thermodynamic model for two interacting domains, a decrease in the free energy of domain-domain interactions of about 2 kcal was estimated from the DSC data.

The interaction of adenine nucleotides with the red cell membrane: A calorimetric study

Abstract The interaction of two adenine nucleotides with the red cell membrane was investigated using highly sensitive differential scanning calorimetry. It was found that ADP and AMP-PNP (an ATP analogue) preferentially modify the A transition, which has been shown to involve the unfolding of a portion of spectrin, an erythrocyte membrane protein complex. The interaction of ADP with spectrin was shown to be reversible and facilitated by the usual cofactor, Mg2+. The ADP-induced modification, however, is only observed for membrane associated spectrin; ADP has no effect on extracted spectrin. The results presented are consistent with an ADP-induced conformational change in the spectrin complex which leads to a change in the spectrin-membrane interaction. ADP, but not AMP-PNP, is shown to modify an additional calorimetric transition (B2) associated with a structural change in the transmembrane protein band 3. This behavior is characteristic of inhibitors of anion transport in the red cell. ADP is also found to be an inhibitor of anion transport in red cells.

A calorimetric comparison of structural transitions of erythrocyte ghosts from normal individuals and from patients with muscular dystrophy

Abstract Using a highly sensitive scanning calorimeter, the thermally induced structural transitions of erythrocyte ghosts from normal individuals and from patients with Duchenne muscular dystrophy (DMD) were carefully examined. No differences were observed under a variety of conditions. This finding is consistent with the idea that the composition, structure, and organization of membrane proteins and lipids in DMD erythrocyte membranes is very similar to normal erythrocyte membranes, in contrast to many other reports in the literature which utilized different techniques.

Binding of plasma membrane glycoproteins to the cytoskeleton during patching and capping is consistent with an entropy-enhancement model

Concentrations of concanavalin A that induced patching and capping of cell surface receptors on Dictyostelium discoideum also induce binding of the receptors to the cortical cytoskeleton, which was isolated by density-gradient centrifugation. The receptors were solubilized by deoxycholate, purified by affinity chromatography, and used to determine whether the receptors bound directly to the cytoskeletal protein, actin. As the concentration of actin was increased, many of the receptors became bound to purified filamentous rabbit muscle actin, even in the absence of concanavalin A. As in the ligation-induced binding of receptors to the cortical cytoskeleton in cells, concanavalin A induced much stronger binding of the purified receptors to filamentous actin. The results were consistent with a previously stated hypothesis that induction of receptor binding to the cytoskeleton during their patching and capping is driven by clustering the receptors, which reduces their translational entropy and by doing so enhances their avidity for the cytoskeleton.