Radiation effects on erythrocyte membrane structure studied by the intrinsic fluorescence.

The changes in the intrinsic fluorescence, primarily from tryptophan residues, of sheep erythrocyte membranes following X-irradiation (0--4000 R) were investigated. The experiments showed that there was (1) a decrease in the intensity of fluorescence with increasing dose of X-rays, (2) a small shift of fluorescence emission to longer wavelengths, (3) a decrease in the fluorescence polarization, and that (4) treatment of membranes with a perturbing solvent, 2-chloroethanol, can eliminate the effects of X-rays. The amount of tryptophan in the membranes was not altered after X-irradiation. It was also shown that sulphydryl reagents, N-ethylmaleimide and 2,2'-dithiodipyridine, induced similar fluorescence changes. From these results it was concluded that the fluorescence changes could result from a change in the environment surrounding tryptophan residues, from being relatively non-polar to being more polar, implying that conformational changes of membrane proteins are brought about by low doses of X-rays.     

Human erythrocyte transglutaminase. Purification and properties

Transglutaminase has been isolated from human erythrocytes, and some of its molecular and catalytic properties have been determined. An enzyme preparation of about 15% purity is readily obtained in about 25% yield after DEAE-cellulose fractionation and gel filtration. In order to achieve this yield of enzyme it is essential to add to the buffers a dialyzable stabilizing factor which is present in the early enzyme fractions. This natural factor can be partly replaced by chelating compounds and totally replaced by ATP, and in practice, the purification of the enzyme is best carried out with ATP present in the buffers. The role of ATP in stabilizing the enzyme is unknown. Complete purification of the erythrocyte transglutaminase can be accomplished by preparative acrylamide gel electrophoresis. The pure enzyme has a molecular weight of 82 000 ± 5000, as established by gel filtration and SDS gel electrophoresis, and its catalytic properties are essentially identical to those of guinea pig liver transglutaminase. The guinea pig liver and the erythrocyte enzymes have also been compared as catalysts for protein modification reactions, and have been found to have quite similar specificity requirements for protein substrates. Both enzymes catalyzed significant incorporation of amines into 4 of 20 soluble proteins tested and into proteins 1, 2 and 3 of the red cell membrane. The partially purified erythrocyte enzyme has been found to be completely satisfactory for protein modification experiments, and the ready availability of outdated human blood and the simple purification procedure should make this enzyme a convenient protein-modifying or crosslinking reagent.     

Calcium effects on human erythrocyte membrane proteins

The effects of Ca²⁺ on human erythrocyte membrane proteins were examined by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis. Ca²⁺ had several effects on normal human erythrocyte membrane proteins. It affected the binding of cytoplasmic proteins to the membrane, produced a non-reversible aggregation of several membrane proteins and activated apparent proteolysis of membrane proteins. The Ca²⁺ effect could be obtained with isolated, washed membranes when the erythrocyte cytoplasm was added. These studies indicate that the Ca²⁺-induced membrane proteolysis and aggregation effects are not due simply to its presence at the time of hemolysis as previously suggested (Carraway, K.L., Triplett, R.B. and Anderson, D.R. (1975) Biochim. Biophys. Acta 379, 571–581), but are the result of more complex interactions between the erythrocyte membrane and cytoplasmic factors.     

Genomic structure of keratinocyte transglutaminase. Recruitment of new exon for modified function.

The gene for keratinocyte transglutaminase (TGK) spans 14 kilobase pairs and contains 15 exons. Many features of the TGK gene are very similar, if not identical, to those of the gene encoding the catalytic subunit of human clotting factor XIII: they have the same number of exons, corresponding introns always interrupt the coding region in the same phase of the codon, and most exons are of similar size (10 or 15 are exactly the same size). In these respects, the TGK and factor XIII catalytic subunit genes resemble each other more than either resembles the gene for erythrocyte band 4.2, a noncatalytic transglutaminase superfamily member. Exon II in both the TGK and factor XIII genes encodes an amino-terminal extension of nonhomologous sequence which in each protein confers a specialized function (membrane anchorage or activation of cross-linking, respectively). This suggests that the evolution of these genes included recruitment of a new exon to modify the enzyme action. Southern blots of genomic DNA reveal the presence of a TGK-like gene in birds, amphibians, and fish, but not in flies.     

Carotenoids as a Raman-active probes of erythrocyte membrane structure.

1. Erythrocyte ghosts exhibit resonance-enhanced Raman bands at 1530 cm(-1) and 1165 cm(-1) attributable to v(-C=C-) and v(=C-C=), respectively, of the conjugated polyene chains in carotenoids. In lipid extract of ghosts, these resonance-enhanced bands lie at 1527 and 1158 cm(-1). The spectra indicate the presence of membrane-bound beta-carotene. 2. The resonance-enhanced Raman spectrum of beta-carotene in lecithin liposomes is identical to that obtained with hexane or chloroform solutions. 3. Increasing proportions of cholesterol in cholesterol-lecithin liposomes up to a cholesterol: phospholipid molar ratio of 0.8-0.9 drastically decreases the intensity of both resonance-enhanced bands. 4. In ghosts the carotenoid bands respond to membrane perturbations. Trypsinization, lysolecithin treatment and reduction of pH increase the intensities of the 1530 and 1165 cm(-1) bands. In contrast, a decrease in the intensity of both bands follows equilibration of ghosts for 15 min at approx. 50 degrees C or addition of (0.1%) sodium dodecyl sulfate. 5. We suggest that perturbants known to change lipid-protein interactions in erythrocyte membranes modify the microenvironment and/or configuration of the membrane-bound carotenoid.     

Structure and function of human erythrocyte membrane skeletons

On the basis of extensive studies of the literature and of own results the present knowledge about the structure of the membrane skeleton of human erythrocytes is summarized and functional and clinical aspects are described. The spectrins are the centre of interest. Their interconnections, spatial arrangement and association with other components of the membrane are explained in greater detail. With regard to the membrane skeleton questions of erythrocyte shape, membrane integrity, phospholipid asymmetry, distribution of transmembrane proteins and cell deformation are discussed.     

Cellular transglutaminase. Lung matrix-associated transglutaminase: characterization and activation with sulfhydryls

Transglutaminase in the rat lung is tightly associated with the insoluble matrix which is not extractable with detergent, 0.5 M NaCl, and 40% glycerol solutions. The insoluble matrix was found to be rich in heparin sulfate and poor in collagen, elastin, and DNA. The lung transglutaminase was found to be distinct from tissue transglutaminase (identifiable with the well-characterized guinea pig liver transglutaminase) in its retention volume in DEAE-Sephacel columns and its Kd value in gel-filtration columns. The enzyme was activated 6-8-fold with the sulfhydryl reagent dithiothreitol. This activation was accompanied with the dissociation of enzyme from the tightly bound insoluble matrix and resulted in changes of the molecular properties of the enzyme--increase in affinity for anion-exchanger and decrease in Stokes radius. Addition of 50 mM KSCN induced a 2-fold increase in SH-dependent activation of transglutaminase activity. These results suggest that sulfhydryl agents may play a role in the activation and compartmental translocation of the transglutaminase in the lung.     

Effect of acute intestinal obstruction on erythrocyte membrane function

The amino acid composition of red blood cell membrane proteins had been studied in different stages of acute intestinal obstruction. Hydrophobic amino acids were revealed to increase and glutamate was found to decrease during the early period of acute intestinal obstruction. Later neutral amino acids and some of the main amino acids were stated to decrease. Shifts in the ratio of protein fractions seen in red blood cell membrane of rats with acute intestinal obstruction could be explained by changes followed in the amino acid composition. The data accumulated had demonstrated that such a significant modification of protein component of the red blood cell membrane could be one of the reasons of the erythrocyte membrane penetrability violation and could play the pathogenetic role in the occurrence of irreversibility changes in cases of the intestinal obstruction. All that was mentioned above had shown the necessity to use membrane protectors and antienzyme drugs in the postoperative period.     

Temperature effects on osmotic fragility, and the erythrocyte membrane

Results are reported on the temperature-dependence of intact-cell surface area, isotonic volume, hemolytic volume, and ghost steady-state surface area and volume, using several techniques of resistive pulse spectroscopy. Temperature was found not to alter the intact cell surface area permanently: the area remains constant at 130 +/- 1 micron 2, at temperatures ranging from 0 to 40 degrees C. Temperature does alter the steady-state volume of the cells, with a colder temperature inducing swelling by about 0.29 micron 3/deg. C. Such a temperature-induced volume change is sufficient to explain only approximately half of the fragility differences which result from temperature changes. The remainder was found to result from higher temperatures enabling a substantial transient increase in surface area of intact cells (up to at least 14% of 40 degrees C), with a corresponding increase in the cell's hemolytic volume (up to 21%). The hemolytic volume apparently increases linearly with temperature, since steady-state ghost volumes are found to increase linearly with the temperature at which the ghosts were produced. In the steady state (at high temperature), the membranes of electrically-impermeable resealed ghosts can remain extended by more than 10%, compared with membranes of the corresponding unhemolyzed, intact red cells.     

Microsomal membrane structure and function subsequent to calcium activation of an endogenous phospholipase

An endogenous system in the membranes of rat liver endoplasmic reticulum is capable upon Ca²⁺ activation of considerable disruption of normal structure and function. Phosphatidylethanolamine (PE) and to a lesser extent phosphatidylcholine (PC) are degraded to hydrophilic products. This lipid loss is greater at an alkaline pH, preferentially utilizes millimolar Ca²⁺ rather than Mg²⁺ ions, and is inhibited by KCl. Diethyl ether has no effect on the rate of loss of PE or PC, and the Ca²⁺ ionophore A23187 does not lower the Ca²⁺ requirement. Phospholipids are most likely lost from the membranes in a two-step process. Lysophospholipids generated in the first, Ca²⁺-dependent step are removed by an endogenous lysophospholipase demonstrated by the hydrolysis of either added lyso PE or lysophospholipids generated from endogenous substrates by Naja naja phospholipase A₂. The depletion of microsomal membrane phospholipid is accompanied by a loss of glucose 6-phosphatase and of cytochrome P-450. The latter is not associated with any change in total heme content. Polyacrylamide gel electrophoresis showed no difference between the pattern or relative amounts of solubilized membrane proteins before or after depletion of membrane phospholipid. It is concluded that activation of an endogenous phospholipase by Ca²⁺ can result in significant depletion of PE and PC that is accompanied by considerable disruption of membrane function. The significance of this system with respect to the maintenance of cell integrity and its possible role in cell injury are discussed.