Light and electron microscopical demonstration of the ouabain-sensitive, potassium-dependent p-nitrophenylphosphatase activity (K-NPPase) using a Ce-Mg-double capture technique.

The cerium-based method of Kobayashi et al. for the histochemical demonstration of K-NPPase activity was improved. Besides Ce3+ additionally Mg2+ ions as orthophosphate capture were employed (double capture technique). For light microscopical purposes the Mg-phosphate was converted into Ce-phosphate by treatment of the sections with Ce-citrate yielding higher quantity of reaction product. Unspecific background staining was eliminated by EGTA. In the electron microscope this technique brought about fine granular reaction products without diffusion artefacts.

Polarization-optical investigation (topo-optical analysis) of the structure of the human erythrocyte glycocalyx. Influence of pH, ionic strength and diamide-induced spectrin cross linking.

The value of membrane anisotropy after fixation and topo-optical analysis of erythrocytes stained with toluidine blue is a measure for the degree of spatial order of the dyestuff-binding acidic residues of the glycocalyx and thus a parameter for the characterization of the glycocalyx structure. Lowering the pH value and/or the ionic strength of the staining medium results in a decrease of membrane anisotropy indicating a lower order of the anionic residues. In agreement with the findings of other authors this phenomenon seems to be connected with an expansion of the glycocalyx. Diamide-induced oxidative crosslinking of spectrin before fixation and staining with toluidine blue at physiological pH and ionic strength also results in a decreased anisotropy. This indirect influence on the glycocalyx structure may be caused by an increase of the charge density within the glycocalyx due to a diamide-induced rearrangement of the membrane skeleton and of the transmembrane proteins bound to it.

Topo-optical studies of gradually disintegrated erythrocyte membrane derivates: different kinds of ghosts.

Several kinds of ghosts from human erythrocytes (blood-group A1D) were investigated by the topo-optical Toluidine-Blue (TB) reaction. In comparison to intact cells, all ghosts demonstrated a decreased TB-anisotropy. These results reflect an altered glycocalyx structure of ghosts, especially conformational changes of the TB-binding N-terminal extracellular segments of the glycophorines. It was assumed that this structural glycocalyx alteration was caused by substantial losses of membrane skeleton components during the ghost preparation. Moreover, disturbed molecular interactions between the membrane skeleton and the glycocalyx may contribute to this effect. Therefore, the glycocalyx and the membrane structure of ghosts in general are significantly different from the membrane of the intact erythrocyte. The experiments show that the effects of the reconstitution procedures for ghost membranes are restricted to a reconstitution of a defined membrane function (i.e. dynamic barrier for monovalent cations) in a widely disturbed membrane structure.

Topo-optical investigations of the human erythrocyte glycocalyx-age related changes.

The conformational state of the glycocalyx of the intact and altered erythrocyte membrane was studied by means of the topo-optical toluidine blue reaction, i.e. induced membrane birefringence. High membrane anisotropy represents the normal glycocalyx structure and its decline represents their perturbation. The results show that the glycocalyx structure is changed during ageing of the erythrocytes in vivo as well as in vitro. During fluid preservation, in vitro ageing and vesiculation of cells in vitro, a subpopulation of cells showed a decline of membrane anisotropy, but other cells demonstrated abnormally high values. In the latter cases, there is usually a correlation to spherocytes. From this point of view, it is to be assumed that spherogenesis during cell ageing is induced by cell vesiculation. This leads to a remodelling of an intact plasmalemma. In contrast, the cell fractions which are probably non-vesiculating seem to be more or less damaged by membrane and/or plasmic hydrolases. This can be mimicked by neuraminidase and protease treatment of erythrocytes in vitro. Membrane lesions caused by freeze preservation of red blood cells are rare. The topo-optical results are interpreted according to the assumptions of the theory of membrane anisotropy, i.e. the formation of dye-stuff micelles at distinct, clustered, sialylated carbohydrate chains of the glycophorin A.