Endocytosis in adenosine triphosphate-depleted erythrocytes.

The extent of membrane invagination or endocytosis in intact erythrocytes was quantified by measuring the loss of acetylcholinesterase activity. Primaquine-induced endocytosis was completely inhibited in ATP-depleted cells. However, chlorpromazine and vinblastine were capable of inducing membrane invagination in depleted cells. With both drugs, the loss of enzyme activity was less than that measured in fresh cells. We conclude that drug-induced endocytosis is not necessarily an energy-dependent process.     

Actin-activated ATPase from human erythrocytes.

A fibrillar protein complex, possessing ouabain-insensitive Ca2+-ATPase activity was isolated from human erythrocyte membranes by using a low ionic strength extraction procedure. Mg2+-ATPase activity was revealed upon addition of rabbit skeletal muscle actin, thus demonstrating the presence of a myosin-like protein in the crude extract of the erythrocyte membrane. Upon sodium dodecylsulfate gel electrophoresis, the extract showed mainly the doublet of subunit molecular weight bands of 230 000 and 210 000, and more than 10 faster moving bands. Gel filtration of the erythrocyte membrane extract on Sepharose 4B furnished 4 fractions. Fraction I, containing the doublet and 80 000, 60 000 and 46 000 subunit molecular weight bands was 5-fold purified with respect to Ca2+-ATPase activity, but was devoid of actin-activated Mg2+-ATPase activity. Fraction II, containing only the doublet, was devoid of Ca2+ and actin-activated Mg2+-ATPase activity. The 210 000 subunit molecular weight protein could be phosphorylated in the presence of Mg2+ in the crude extract and Fraction I but not in Fraction II.     

Perfluoro compounds as artificial erythrocytes.

Some liquid perfluoro compounds dissolve relatively large amounts of oxygen and can be used in dispersed form as substitutes for erythrocytes. The commonly used perfluoro compounds contain about the same amount of oxygen as do equal volumes of erythrocytes when equilibrated with 100% oxygen. However, when equilibrated with alveolar air, the perfluoro compounds contain much less oxygen than erythrocytes. The dispersed fluorochemicals are adequate substitutes for perfusion of isolated preparations of mammalian brain, heart kidney, lung and liver. However, when put into the circulation of the intact animal, the dispersed fluorochemicals tends to produce lesions of the lungs, dilation of the right heart, and ultimately fatal hypoxia. It is suggested that the course of events following intravenous injection of dispersed fluorochemical is initiated by an interaction of the perfluoro particles with blood platelets or blood clotting factors. The ensuing intravascular clotting could then cause the changes in the lungs which lead to a marked increase in pulmonary artery pressure and dilation of the right heart. These events would terminate in fatal hypoxia due to pulmonary pathology and heart failure.     

Amyloid insulin interaction with erythrocytes.

Erythrocyte membrane interactions with insulin fibrils (amyloid) have been investigated using centrifugation, fluorescence spectroscopy, light scattering, and flow cytometric techniques. The results indicate that insulin fibrils are having moderate affinity to erythrocyte membrane. However, analysis of the apparent dissociation constants of human erythrocyte membranes (leaky and resealed vesicles) with amyloid insulin reveal that the insulin binding is drastically reduced on attaining the fibrillar state compared with native insulin. To understand the role of insulin receptors on erythrocytes binding to amyloid, we have studied the interaction of biotinylated forms of denatured and amyloidic insulin with erythrocytes. FITC-streptavidin was used as a counter staining in flow cytometry measurements. We found that insulin fibrils bind 10 times more with erythrocyte membranes than with amylin and denatured insulin.     

Phosphoglycolate phosphatase in human erythrocytes.

Human erythrocytes were found to contain an enzyme capable of dephosphorylating phosphoglycolate. The rates of hydrolysis of 14 other metabolites by the enzyme preparation were less than 6% of the rate with phosphoglycolate. The pH optimum is in the range of 6 to 7 and the Km for phosphoglycolate is 0.76 mM. The molecular weight appears to be about 79,000. Such an activity has previously been reported only for plant cells.     

Iron-mediated oxidative stress in erythrocytes.

Erythrocytes subjected extracellularly to iron-mediated oxidant stress undergo haemoglobin oxidation and membrane damage, which can be modulated by maintaining the energy requirements of the cells. The results presented here suggest that a balance exists between the oxidation state of the haemoglobin and the oxidative deterioration of the membrane lipids, which is dependent on the metabolic state of the erythrocytes. These findings have important implications for thalassaemic erythrocytes that may be exposed to excess plasma iron levels, in which excessive membrane-bound iron in the form of haemichromes is a characteristic feature and in which cellular ATP levels are lowered.     

Membrane proteins in senescent erythrocytes.

The examination of erythrocyte senescence has been facilitated by recent advances in techniques for the isolation of aged red cells. One of these methods, which uses biotinylated rabbit erythrocytes, has been used to examine the state of membrane proteins in effete cells. These aged red cells were found to have normal ratios of alpha-spectrin and beta-spectrin as well as normal levels of ankyrin. The observation concerning ankyrin is particularly important due to the sensitivity of this protein to proteolysis and the postulated action of proteinases in the aging process. The senescent erythrocytes were also found to have an altered ratio of bands 4.1a and 4.1b without any apparent change in the total level of 4.1. In addition, the analysis of the aged cell membranes did not show any large-molecular-mass aggregated protein at the origin of the SDS/polyacrylamide gels, indicating a lack of transglutaminase activity in the senescence process for rabbit erythrocytes. These results indicate that aging of the rabbit erythrocyte is not accompanied by gross proteolytic degradation or transglutaminase-catalysed cross-linking of membrane components.     

Metabolism of acetaldehyde by human erythrocytes.

Acetaldehyde metabolism in human erythrocytes was studied using head-space gas chromatographic determination methods, and it was found that acetaldehyde is metabolized in erythrocytes by NAD dependent cytosolic enzyme having an apparent Km value for acetaldehyde approximately 0.7 mM at pH 7.4, and more than 50% of this activity was reduced by 1 μM disulfiram. So, it is suggested that erythrocytes may have an enzyme system similar to the high Km isozyme of the liver aldehyde dehydrogenase.     

The osmometric behavior of human erythrocytes.

A reexamination of the data of Farrant and Woolgar (Cryobiology, 9, 9–15 (1972)) demonstrates that human erythrocytes exhibit osmometric behavior as predicted from the Boyle-Van't Hoff law when suspended in sodium chloride solutions as high as 3.5 osmolal. A Boyle-Van't Hoff plot is linear with r = 0.998. However, 24% of the water in isotonic red blood cells appears to be osmotically-inactive, although nearly 75% of this apparent non-osmotic water can be accounted for by the water of hydration of hemoglobin. These facts have direct significance to hypotheses concerning hemolysis as well as to the modeling of osmometric events which occur at sub-zero temperatures.     

Biochemical characterization of density-separated human erythrocytes.

A simple, reproducible method for the separation of human erythrocytes, described recently (Murphy, J. R. (1973) J. Lab. Clin. Med. 82, 334-341) has been utilized for the purpose of obtaining a wide range of biochemical data on these cells. Using phthalate ester density centrifugation of the fractions obtained by Murphy's method, we established that the cells were separated exclusively on the basis of their densities. Data on a wide range of biochemical and hematological parameters, when compared with previously reported density separation procedures showed that this simple technique can be used to fractionate the cells according to their densities (age) in their own plasma. Cells of increasing density consistently and reproducibly exhibited an increase in hemoglobin concentration, a moderate elevation in Na+ and a decrease in the following: K+, acetylcholinesterase, sialic acid, membrane protein, 2,3-diphosphoglycerate, ATP, cholesterol, phospholipid, mean corpuscular volume and critical hemolytic volume, However, no change in mean corpuscular hemoglobin was evident. The observed differences were not artifacts of the centrifugation process. This was determined in recentrifuged top fractions from which new top and bottom cells were obtained. The latter cells resembled the top fraction from which they were obtained, rather than the original bottom fraction. Whereas the parameters mentioned above exhibited consistency and reproducibility, such was not the case with the ATPase values. Depending on the cell density group examined and/or buffer as well as other conditions, significant variability in the activity levels of the ouabain sensitive, as well as the Ca2+ -stimulated ATPase, was observed. Use of these enzyme activities as indicators of cell age must be viewed with caution.     

Immunochemistry of Ii-active glycosphingolipids of erythrocytes.

Fractions of complex glycosphingolipids were prepared from adult, cord, and i phenotype erythrocytes by the method elaborated for the isolation of poly(glycosyl)ceramides. In contrast to poly(glycosyl)ceramides which comprise on the average 30 glycosyl units and about 5 branching points, i.e. 3,6-di-O-substituted galactopyranosyl residues, per mole of glucose, complex glycosphingolipids from cord and i erythrocytes comprise 6 and 15 glycosyl units respectively and only 0.7 branching points. The latter substances exhibited also a high i activity which was not detected in poly(glycosyl)ceramides. Erythrocyte membranes were labeled with radioactive N-acetylgalactosamine (GalNAc) from UDP-GalNAc using a purified A-blood-group gene-specified transfered of GalNAc. It was found that electrophoretic mobilities in dodecylsulfate-gel electrophoresis of all glycoconjugates which accepted GalNAc were increased in i as compared to I membranes. We conclude that the absence of highly branched glycosphingolipids in cord and i erythrocytes as well as the reduction of apparent molecular weights of the glycoconjugates, which are substrates for A-gene-specified transferase of GalNAc, result from a single cause, that is an inadequacy of the biosynthetic process which is responsible for the formation of GlcNAc1 leads to 6Gal structures.     

Electro-orientation of ellipsoidal erythrocytes. Theory and experiment.

The frequency-dependent orientation of human and llama erythrocytes suspended in isotonic solutions and subjected to linearly polarized electric fields is examined. Human erythrocytes may be represented as oblate spheroids (3.9:3.9:1.1 microns) with two distinguishable orientations, while the llama cells are approximated as ellipsoids with three distinct axes (4.0:2.0:1.1 microns). Under appropriate experimental conditions, both orientations of the human cells and all three orientations of the llama cells are observed. A theoretical cell model which accounts for the membrane as a thin confocal layer of ideal capacitance is used to predict the orientational spectra. The predicted spectra compare favorably in frequency range and orientational sequence with experimental data. Estimates for cell internal conductivity and permittivity are obtained by adjusting the values of these important parameters to achieve the closet fit of the theoretical curves to the data. By the use of this method, the internal conductivity of llama erythrocytes is estimated to be 0.26 S/m (+/- 20%), while the effective internal dielectric constant and conductivity of Euglena gracilis are estimated to be 120 (+/- 10%) and 0.43 S/m (+/- 20%), respectively.     

Glucokinase and hexokinase in pig erythrocytes.

We have shown different enzymatic activities responsible for the phosphorylation of glucose in pig erythrocytes. These activities were observed after partial purification from hemolyzed red cells. One of the enzymes involved is the hexokinase which is present in all tissues; the other is similar to hepatic glucokinase. We have determined the kinetic properties of these activities in hemolysates and in partially purified preparations. Their electrophoretic-migration characteristics were studied too.