Madan G. Luthra

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 Murphys 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.

Studies on an activator of the (Ca2++Mg2+)-ATPAse of human erythrocyte membranes

Abstract 1. 1. An activator of the (Ca 2+ +Mg 2+ )-stimulated ATPase present in the human erythrocytes (membrane) has been isolated in soluble form from hemolysates of these cells. Partial purification has been achieved through use of carboxymethyl-Sephadex chromatography. The resulting activator fraction contained no hemoglobin and only 0.3% of the total adenylate kinase activity of the cell. 2. 2. Whereas the activator was released from erythrocytes subjected to hemolysis in 20 miosM buffer at pH 7.6 or at pH 5.8 only the membranes prepared at pH 7.6 were affected by it. 3. 3. When (Ca 2+ +Mg 2+ )-ATPase activity was measured by 32 P i release from [γ- 32 ]ATP, freeze-thawed erythrocytes, as well as membranes prepared at pH 5.8 and at pH 7.6, expressed lower values than noted by assay for total P i release. When ADP instead of ATP was used as substrate, significant amounts of P i were released by these erythrocyte preparations. Further study revealed (a) production of ATP and AMP from ADP with membranes and hemolysate alone, and (b) exchange of the γ- and β-position phosphate on [γ- 32 P]ATP in the presence of membranes plus hemolysates. These observations established the presence of adenylate kinase activity in the (membrane-free) hemolysates and in membranes. It further supports the conclusion that P i release from ADP by human erythrocytes (freeze-thawed) and by their isolated membranes is due to formation of ATP by adenylate kinase and hydrolysis of this generated ATP by (Ca 2+ +Mg + )-ATPase. 4. 4. The following points were also established: (a) absence of an ADPase in human erythrocytes: (b) the (Ca 2+ +Mg 2+ )-ATPase activator enhanced cleavage only of the γ-position of ATP and (c) the (Ca 2+ +Mg 2+ )-ATPase activator is neither adenylate kinase nor hemoglobin.

(Ca++ + Mg++)-ATPase of red cells in Duchenne and myotonic dystrophy: effect of soluble cytoplasmic activator.

Red cell (Ca++ + Mg++)-ATPase was investigated in Duchenne and myotonic dystrophy patients. Saponin-lysed red cells of patients with Duchenne dystrophy displayed slightly higher (Ca++ + Mg++)-ATPase activity (66.2 ± 2.6 ±moles Pi [inorganic phosphorus] per 2 hours per milliliter cells) than age-matched male controls (54.3 ± 2.4 ±moles Pi per 2 hours per milliliter cells). The activity of the enzyme in myotonic dystrophy patients was somewhat lower (59.6 ± 1.3 μnoles Pi per 2 hours per milliliter cells) than in controls (66.7 ± 1.6 ±moles Pi per 2 hours per milliliter cells). In all cell types, a cytoplasmic activator protein is present, which stimulated the (Ca++ + Mg++)-ATPase of hypotonically isolated membranes. In age-matched Duchenne controls, the membrane enzyme was maximally stimulated by the activator, increasing from 14.4 ± 0.5 ±moles Pi per 2 hours per milliliter cells to 28.5 ± 1.4 ±moles Pi per 2 hours per milliliter cells. Duchenne (Ca++ + Mg++)-ATPase was enhanced from 15.8 ± 0.7 ±moles Pi per 2 hours per milliliter cells in the membrane to 33.3 ± 1.8 ±moles Pi per 2 hours per milliliter cells by the activator. Similarly, myotonic (Ca++ + Mg++)-ATPase activity was augmented from 13.7 ± 0.5 ±moles Pi per 2 hours per milliliter cells to 29.4 ± 1.5 ±moles Pi per 2 hours per milliliter cells by the activator, whereas enzymes from age-matched controls displayed a stimulation from 15.0 ± 0.6 ±moles Pi per 2 hours per milliliter cells to 36.2 ± 1.7 ±moles Pi per 2 hours per milliliter cells in the presence of their own activator. The extent of the enzyme stimulation by the activator was independent of the source of its origin in all cases, suggesting that the differences observed in the (Ca++ + Mg++)-ATPase from patients and controls were more likely related to differences in membrane structure than to alteration in the activator protein.

Inosine from liver as a possible energy source for pig red blood cells

Abstract Adult pig red blood cells are unable to metabolize glucose due to a membrane permeability barrier to glucose developed shortly after birth. In vitro , pig red cells incubated in their own plasma are unable to maintain normal ATP levels, thus the question has been raised as to the nature of the metabolic energy source. We have suggested that organs, such as the liver, might supply low levels of substrate to the red cells as they transit through the organ. In this paper, evidence is presented to show that perfused pig livers supply a metabolic substrate used by pig red cells. This substrate has been tentatively identified as inosine.

Observations on the (Ca2+−Mg2+)-ATPAse activator found in various mammalian erythrocytes

1. A soluble activator of membrane (Ca2+ plus Mg2)-ATPase is present in hemolysates of the newborn calf and cow, the new born and adult pig as well as human erythrocytes. 2. The activator is also found in reticulocytes of the adult pig. 3. The activator obtained from any of the above species is capable of stimulating the membrane (Ca2+ plus Mg2+)-ATPases of the other species, regardless of the age of the animals. 4. The results obtained from density fractionation of human erythrocytes revealed that the soluble factor has little simulatory effect on membranes of young erythrocytes from which it is derived but caused a marked stimulation on (Ca2+ plus Mg2+)-ATPase activity of the intermediate aged and old erythrocyte membranes. 5. The above observations support the following conclusions: (a) the extremely low levels of (Ca2+ plus Mg2+)-ATPase in cow erythrocytes is not due to the lack of a (Ca2+ plus Mg2+)-ATPase activator; (b) the distribution of (Ca2+ plus Mg2+)-atpase activator is not species specific and the differences in the level of membrane (Ca2+ plus Mg2+)-ATPase activity in various species of cells is an inherent property of that particular membrane (c) the (Ca2+ plus Mg2+)-ATPase activator is present at least from the time of reticulocyte formation and remain during tthe life span of the erythrocyte.

(Ca2+ + Mg2+)-ATPase of density-separated human red cells. Effects of calcium and a soluble cytoplasmic activator (Calmodulin)☆

The effect of calcium and a soluble cytoplasmic activator on (Ca2+ + Mg2+)-ATPase of density-separated human red cells was investigated. At all calcium concentrations tested, dense (old) lysed cells and their isolated membranes displayed lower activities as compared to the light (young) cells and their membranes. Isolated membranes from all density red cell fractions showed two distinct (Ca2+ + Mg2+)-ATPase activities; one at low calcium and another at moderate calcium concentrations. At high calcium concentration, (Ca2+ + Mg2+)-ATPase activity of isolated membranes was low in all cell fractions. In contrast to the isolated membranes, lysed cells from all density fractions had a maximum (ca2+ + Mg2+)-ATPase activity only at a low concentration of calcium, while moderate and high calcium concentrations produced low activity. Upon isolation of membranes, a substantial loss of (Ca2+ + Mg2+)-ATPase activity took place from all density cell fractions. Upon membrane isolation, the relative loss of (Ca2+ + Mg2+)-ATPase activity at low Ca2+ concentration was greater in older cells. The extent of stimulation of (Ca2+ + Mg2+)-ATPase by the activator at low calcium concentration was 3-4-fold greater in older cell membranes than in the young ones. These data suggest that the lower (Ca2+ + Mg2+)-ATPase activity in old cells could be accounted for by a selective loss of (Ca2+ + Mg2+)-ATPase activity at low Ca2+ concentration presumably due to reduced affinity of old cell membranes to activator protein.

Effects of calcium and soluble cytoplasmic activator protein (calmodulin) on various states of (Ca2+ + Mg2+)-ATPase activity in isolated membranes of human red cells.

(Ca2+ + Mg2+)-ATPase activity of red cells and their isolated membranes was investigated in the presence of various Ca2+ concentrations and cytoplasmic activator protein. Red cell ATPase activity was high at low Ca2+ concentrations, and low at moderate and high concentrations of Ca2+. In the case of isolated membranes, both low and moderate ca2+ concentrations produced higher (Ca2+ + Mg2+)-ATPase activity than high Ca2+ concentration. Membrane-free hemolysate containing soluble activator of (Ca2+ + Mg2+)-ATPase produced a significant increase in (Ca2+ + Mg2+)-ATPase activity only at low ca2+ concentration. Regardless of Ca2+ and activator concentrations, the enzyme activity in the membrane was lower than lysed red cells. The low level of (Ca2+ + Mg2+)-ATPase activity seen at high Ca2+ concentration can be augmented by lowering the Ca2+ concentration of EGTA in the assay medium. However, once the membrane was exposed to a high Ca2+ concentration, the activator could no longer exert it maximum stimulation at the low Ca2+ concentration brought about by addition of EGTA. This loss of activation was not attributable to the Ca2+-induced denaturation of activator protein but rather related to the alteration of (Ca2+ + Mg2+)-ATPase states in the membrane. On the basis of these data, it is suggested that only a small portion of (Ca2+ + Mg2+)-ATPase activity of isolated membranes can be stimulated by the soluble activator and that (ca2+ + Mg2+)ATPase most likely exists in various states depending upon ca2+ concentration and the presence of activator. The enzyme state exhibiting the high degree of stimulation by activator may undergo irreversible damage in the presence of high Ca2+ concentrations.

Effects of tris and histidine on human erythrocytes and conditions influencing their mode of action

1. The incubation of human erythrocytes in 0.172 M Tris - HCl, pH 7.6 buffer at 37 degrees leads to (1) a pronounced cellular volume increase, (2) a preferential release of Na+, and (3) if continued sufficiently long, hemolysis. These effects are pH dependent and also are influenced to a considerable degree by such diverse reagents as NaC glucose, and histidine. In each instance, increasing levels of the latter compounds in a Tris - HCl incubation mixture led to diminished cellular volume increase and prolonged time of onset of hemolysis. 2. Histidine solutions of 0.31 M, pH 7.5 caused a rapid and dramatic decrease in cellular volume of human erythrocytes and a concomitant rapid exit of cations. However, in a prolonged incubation, human erythrocytes slowly regained their cell volume as a result of histidine entry into the cell. Of considerable interest: Tris swollen cells undergo immediate shrinkage to far below the initial cell volume when incubated in histidine at 37 degrees C. Through repetition of this process two additional times, as much as 90-95% of the total cellular Na+ and K+ was removed without hemolysis. 3. Human erythrocytes washed in 0.12 M MgCl2 and then suspended in 0.31 M histidine, pH 7.5, lost upwards of 60% of their total Na+ and 30% of their total K+ after a 40 min incubation at 37 degrees C. However, when increasing amounts of 0.172 M Tris - HCl, pH 7.6 were added to the histidine suspension of cells, the release of K+ was reduced to 5% but the release of Na+ decreased only to 40% of the total cellular level. On the basis of these observations, it is evident that Tris exerts a preferential activity towards the efflux of Na+ from the human erythrocyte, whereas histidine results in high efflux of K+ and Na+ from the cell.

Factors influencing osmotic fragility of red blood cells in Duchenne muscular dystrophy

Factors affecting osmotic fragility were studied in red blood cells of patients with Duchenne muscular dystrophy. The mean osmotic fragility (MOF), operationally defined as the NaCl concentration for 50% hemolysis, was found to be higher by 3.63 ± 0.51 mM in Duchenne cells than in normal cells having an MOF of 60.1 ± 0.5 mM NaCl buffered with 10 mM sodium phosphate at pH 7.0. However, about 20% of Duchenne patients had red cells indistinguishable from their age- and sex-matched controls. Temperature, pH, preincubation in plasma, and proteolytic digestion all affected Duchenne and normal cells to the same extent. However, after salt loss, induced either by preincubation in isotonic nonelectrolyte solutions or by exposure to ionophore A23187, Duchenne cells showed a greater change in MOF. Osmotic fragility of Duchenne cells was increased even in younger blood cells, suggesting that the membrane was abnormal in the early stages of red cell maturation.

Calmodulin an activator of human erythrocyte (Ca2+ + Mg2+)-ATPase phosphorylation

The effect of purified calmodulin on the calcium-dependent phosphorylation of human erythrocyte membranes was studied. Under the conditions employed, only one major peak of phosphorylation was observed when solubilized membrane proteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The molecular weight of this phosphorylated protein band was estimated to be 130000 and in the presence of purified red blood cell calmodulin, the rate of phosphorylation of this band was increased. These data suggest that calmodulin activation of (Ca2+ + Mg2+)-ATPase could be a partial reflection of an increased rate of phosphorylation of the (Ca2+ + Mg2+)-ATPase of human erythrocyte membranes.