The interaction of 1-fluoro-2,4-dinitrobenzene with amino-phospholipids in membranes of intact erythrocytes,modified erythrocytes,and erythrocytes ghosts

Summary 1-Fluoro-2,4-dinitrobenzene (FDNB) has been used to study the availability of amino-containing phospholipids in erythrocyte membranes and ghosts in an aqueous, isotonic medium. It was found that the addition of bovine serum albumin (BSA) to the medium protects the cells from cation leak and protects some of the amino-phospholipids from reacting with the probe. In isotonic medium without BSA, 46% of the phosphatidylethanolamine and 12% of the phosphatidylserine of erythrocytes and 73% and 21% of these respective lipids of ghosts react with the probe. In the presence of 70 m BSA, 31% of phosphatidylethanolamine and 6.5% of phosphatidylserine of erythrocytes and 59% and 16% of these respective lipids of ghosts react with the probe. The labeling of these lipids does not change under conditions of varying tonicity, or after treatment of erythrocytes with pronase or lysolecithin. The data suggest that 46% of phosphatidylethanolamine and 12% of phosphatidylserine of the erythrocyte membrane are free in a lipid bilayer; 27% and 9% of these respective lipids are loosely bound to proteins which are lost during the preparation of ghosts and 27% of the phosphatidylethanolamine and 79% of the phosphatidylserine are tightly bound to core proteins which remain part of the erythrocyte membrane even after hemolysis.     

Glycolipids of fetal, newborn, and adult erythrocytes: glycolipid pattern and structural study of H3-glycolipid from newborn erythrocytes

The glycolipids of blood group type O adult, newborn, and fetal erythrocytes were compared. The total amount of glycolipids was indistinguishable between adult and newborn erythrocytes. However, glycolipids with long and neutral carbohydrates and the H determinant were greatly reduced in newborn cells. On the other hand, the amount of sialylated glycolipids (gangliosides) was significantly higher in newborn cells, suggesting that during erythropoiesis sialyltransferases are more active in fetuses than in adults. The amount of each core structure, lacto-N-tetraosyl, linear lacto-N-hexaosyl, and branched lacto-N-octaosyl, was compared between adult and newborn erythrocytes. It was found that branched lacto-series glycolipids were reduced in newborn cells compared with adult cells. Thus, development from fetal to adult human erythrocytes is associated with an increase of branching and a decrease of sialylation of N-acetyllactosaminyl carbohydrate chains. The study indicates that glycolipids are quantitatively different between adult and newborn or fetus.     

Cholesterol exchange in platelets, erythrocytes and megakaryocytes

Cholesterol exchange between plasma and human platelets and erythrocytes and guinea pig platelets, erythrocytes and megakaryocytes was studied. The characteristics of exchange of cholesterol between [3H]cholesterol-labeled plasma and human platelets and erythrocytes were similar: exchange per cell was independent of cell concentration in whole plasma, decreased only 2-fold over a wide range of cell concentrations in low concentrations of plasma and approached a plateau at 1/3 normal plasma cholesterol concentration, and there was no net change in the cholesterol content of either cell. The activation energy for exchange for both cells was 47 kJ/mol. In all experiments, erythrocyte cholesterol was labeled to approximately twice the specific activity of platelet cholesterol. Guinea pig megakaryocyte cholesterol exchanged at 25-33% of the rate of guinea pig platelet cholesterol in vitro. Similarly, when guinea pigs were fed [3H]cholesterol, erythrocyte cholesterol specific activity after 24 h was 90%, platelet 50-65%, and megakaryocyte 20-26% that of plasma. Guinea pig platelets incubated with plasma radiolabeled in free and esterified cholesterol incorporated radioactivity from free but not esterified cholesterol. The similarity of free cholesterol exchange in platelets and erythrocytes in vitro and in vivo and the apparent inability of platelets to take up cholesterol esters from lipoproteins suggest that the interaction between normal platelets and normocholesterolemic plasma is limited to cholesterol exchange.     

Phosphofructokinase from porcine heart, liver and erythrocytes

1. Phosphofructokinase from porcine heart, liver and erythrocytes were purified by affinity chromatographies on Cibacron Blue Sepharose and N6-ATP agarose. 2. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate revealed that the heart and liver enzymes consist of only one kind of subunit, namely, M and L type subunits, respectively, whereas the erythrocyte enzyme comprises of three kinds of subunits, M, L and C types. 3. Some kinetic and regulatory properties of the enzymes were also measured.     

Adenosine uptake, transport, and metabolism in human erythrocytes

Using rapid kinetic techniques, we have determined the kinetics of zero-trans influx and equilibrium exchange of adenosine, and its uptake and in situ phosphorylation at 25 degrees C in human erythrocytes which were pretreated with 2'-deoxycoformycin to inhibit deamination of adenosine. Both the Km and Vmax for adenosine transport were about 300 times higher than those for the in situ phosphorylation of adenosine (Km about 0.2 microM), so that the first order rate constants for both processes were about the same. In contrast, the first order rate constant for adenosine deamination by untreated, intact cells was about 20% of that of adenosine transport or phosphorylation. These kinetic properties of the various steps, in combination with substrate inhibition of adenosine phosphorylation above 1 microM adenosine, assure that, at extracellular concentrations of physiological relevance (less than 1 microM), adenosine is very rapidly and efficiently salvaged by the erythrocytes and converted to ATP, whereas at extracellular concentrations of 10 microM or higher, practically all adenosine transported into the cells is deaminated. When the concentration of adenosine was 0.1 microM, a 10% (v/v) suspension of erythrocytes depleted the extracellular fluid of adenosine within 1 min of incubation at 25 degrees C.     

Thermohemolysis of erythrocytes

Thermohemolysis kinetic curves of erythrocytes in the temperature interval of 37-75 degrees C in different media and in the course of blood storage were studied. Mechanism of thermohemolysis is proposed. The rate constants of hemolysis stages are introduced. It is shown that these parameters are sensitive to the changes of structural state of erythrocytes.     

Microencapsulation of erythrocytes

Human erythrocytes have been encapsulated in a polyacrylate membrane by a simple precipitation process. The encapsulated cells appeared to remain functional after encapsulation: the consumption of glucose and the ability to reversibly bind oxygen was unimpaired. Furthermore, storage at 4°C for almost 6 months had no effect on the P₅₀ and n₅₀ values. This is the first time to our knowledge that live mammalian cells have been encapsulated in a polymer other than alginate.