scholarly journals Diisopropylfluorophosphate Is Not a Specific Label for the Red Cell Membrane

Blood ◽  
1969 ◽  
Vol 34 (3) ◽  
pp. 376-379 ◽  
Author(s):  
DAVID A. SEARS ◽  
ROBERT I. WEED
Keyword(s):  
Cell Membrane ◽  
Red Cells ◽  
Red Cell ◽  
Red Cell Membrane ◽  
Membrane Enzyme ◽  
Specific Label

Abstract When red cells were labeled either in vitro or in vivo with DF32P, the label was attached primarily at intracellular, not membrane, sites. Thus, despite the fact that it binds to and inactivates the red cell membrane enzyme, acetylcholinesterate, DF32P is not a specific label for the red cell membrane.

scholarly journals The relationship between in vivo generated hemoglobin skeletal protein complex and increased red cell membrane rigidity

Blood ◽  
1988 ◽  
Vol 71 (5) ◽  
pp. 1427-1431 ◽  
Author(s):  
N Fortier ◽  
LM Snyder ◽  
F Garver ◽  
C Kiefer ◽  
J McKenney ◽  
...  
Keyword(s):  
Protein Complex ◽  
Sodium Dodecyl ◽  
Red Cells ◽  
Red Cell ◽  
Red Cell Membrane ◽  
Cellular Dehydration ◽  
Thalassemia Minor ◽  
Membrane Rigidity

Abstract In vitro induced oxidative damage to normal human RBCs has previously been shown to result in increased membrane rigidity as a consequence of the generation of a protein complex between hemoglobin and spectrin. In order to determine if in vivo generated hemoglobin-spectrin complexes may play a role in increased membrane rigidity of certain pathologic red cells, we measured both these parameters in membranes prepared from hereditary xerocytosis (Hx), sickle cell disease (Sc), and red cells from thalassemia minor (beta thal). Membranes were prepared from density-fractionated red cells, and membrane deformability was measured using an ektacytometer. Hemoglobin-spectrin complex was determined by sodium dodecyl sulfate (SDS)-polyacrylamide gel analysis, as well as by Western blot analysis using a monoclonal antibody against the beta- subunit of hemoglobin. For these three types of pathologic red cells, progressive cellular dehydration was associated with increased membrane rigidity and increased content of hemoglobin-spectrin complex. Moreover, the increase in membrane rigidity appeared to be directly related to the quantity of hemoglobin-spectrin complex associated with the membrane. Our findings imply that hemoglobin-spectrin complex is generated in vivo, and this in turn results in increased membrane rigidity of certain pathologic red cells. The data further suggest that oxidative crosslinking may play an important role in the pathophysiology of certain red cell disorders.

scholarly journals Partial spectrin deficiency in hereditary pyropoikilocytosis

Blood ◽  
1986 ◽  
Vol 67 (4) ◽  
pp. 919-924 ◽  
Author(s):  
TL Coetzer ◽  
J Palek
Keyword(s):  
Degradation Products ◽  
Membrane Skeleton ◽  
Red Cells ◽  
Proteolytic Degradation ◽  
Red Cell ◽  
Red Cell Membrane ◽  
Erythroid Precursor ◽  
Single Defect

Abstract Hereditary pyropoikilocytosis (HPP) is a severe hemolytic anemia in which an instability of the red cell membrane skeleton has been correlated with structural and functional defects of spectrin. We now report that 13 unrelated HPP subjects have approximately 30% less spectrin than normal as evidenced by a decreased spectrin/band 3 ratio. We also examine the role of spectrin degradation as an underlying cause of this partial spectrin deficiency. Our studies demonstrate that the reduced spectrin content of HPP red cells remains constant during in vivo aging of the cells in the peripheral blood, as well as during in vitro incubation. Furthermore, immunoblotting experiments using an affinity-purified antispectrin antibody indicate that there is no loss of spectrin during membrane preparation and also that neither whole HPP red cells nor ghosts nor cytosol contains any abnormal spectrin degradation products. These data suggest that spectrin is not degraded and that it is stable on the membrane of the circulating HPP red cell. In contrast, however, incubation of free spectrin with a lysate of nucleated erythroid precursor cells indicates that HPP alpha I/46 spectrin, but not HPP alpha I/74 spectrin, is more susceptible to proteolytic degradation than a control. These data imply that the decreased spectrin content of HPP is not due to a single defect but that a more complex mechanism is involved. In HPP Sp alpha I/46 subjects, an increased proteolytic degradation in bone marrow erythroid precursors of cytosolic spectrin, prior to its assembly on the membrane, could contribute toward the partial spectrin deficiency.

scholarly journals Partial spectrin deficiency in hereditary pyropoikilocytosis

Blood ◽  
1986 ◽  
Vol 67 (4) ◽  
pp. 919-924
Author(s):  
TL Coetzer ◽  
J Palek
Keyword(s):  
Degradation Products ◽  
Membrane Skeleton ◽  
Red Cells ◽  
Proteolytic Degradation ◽  
Red Cell ◽  
Red Cell Membrane ◽  
Erythroid Precursor ◽  
Single Defect

Hereditary pyropoikilocytosis (HPP) is a severe hemolytic anemia in which an instability of the red cell membrane skeleton has been correlated with structural and functional defects of spectrin. We now report that 13 unrelated HPP subjects have approximately 30% less spectrin than normal as evidenced by a decreased spectrin/band 3 ratio. We also examine the role of spectrin degradation as an underlying cause of this partial spectrin deficiency. Our studies demonstrate that the reduced spectrin content of HPP red cells remains constant during in vivo aging of the cells in the peripheral blood, as well as during in vitro incubation. Furthermore, immunoblotting experiments using an affinity-purified antispectrin antibody indicate that there is no loss of spectrin during membrane preparation and also that neither whole HPP red cells nor ghosts nor cytosol contains any abnormal spectrin degradation products. These data suggest that spectrin is not degraded and that it is stable on the membrane of the circulating HPP red cell. In contrast, however, incubation of free spectrin with a lysate of nucleated erythroid precursor cells indicates that HPP alpha I/46 spectrin, but not HPP alpha I/74 spectrin, is more susceptible to proteolytic degradation than a control. These data imply that the decreased spectrin content of HPP is not due to a single defect but that a more complex mechanism is involved. In HPP Sp alpha I/46 subjects, an increased proteolytic degradation in bone marrow erythroid precursors of cytosolic spectrin, prior to its assembly on the membrane, could contribute toward the partial spectrin deficiency.

scholarly journals The relationship between in vivo generated hemoglobin skeletal protein complex and increased red cell membrane rigidity

Blood ◽  
1988 ◽  
Vol 71 (5) ◽  
pp. 1427-1431 ◽  
Author(s):  
N Fortier ◽  
LM Snyder ◽  
F Garver ◽  
C Kiefer ◽  
J McKenney ◽  
...  
Keyword(s):  
Protein Complex ◽  
Sodium Dodecyl ◽  
Red Cells ◽  
Red Cell ◽  
Red Cell Membrane ◽  
Cellular Dehydration ◽  
Thalassemia Minor ◽  
Membrane Rigidity

In vitro induced oxidative damage to normal human RBCs has previously been shown to result in increased membrane rigidity as a consequence of the generation of a protein complex between hemoglobin and spectrin. In order to determine if in vivo generated hemoglobin-spectrin complexes may play a role in increased membrane rigidity of certain pathologic red cells, we measured both these parameters in membranes prepared from hereditary xerocytosis (Hx), sickle cell disease (Sc), and red cells from thalassemia minor (beta thal). Membranes were prepared from density-fractionated red cells, and membrane deformability was measured using an ektacytometer. Hemoglobin-spectrin complex was determined by sodium dodecyl sulfate (SDS)-polyacrylamide gel analysis, as well as by Western blot analysis using a monoclonal antibody against the beta- subunit of hemoglobin. For these three types of pathologic red cells, progressive cellular dehydration was associated with increased membrane rigidity and increased content of hemoglobin-spectrin complex. Moreover, the increase in membrane rigidity appeared to be directly related to the quantity of hemoglobin-spectrin complex associated with the membrane. Our findings imply that hemoglobin-spectrin complex is generated in vivo, and this in turn results in increased membrane rigidity of certain pathologic red cells. The data further suggest that oxidative crosslinking may play an important role in the pathophysiology of certain red cell disorders.

LACK OF EFFECT OF CORTICOSTEROIDS ON THE IN VIVO DISAPPEARANCE OF SULFHYDRYL-INHIBITED AND ANTIBODY-COATED ERYTHROCYTES

1964 ◽  
Vol 47 (3_Suppl) ◽  
pp. S28-S36
Author(s):  
Kailash N. Agarwal
Keyword(s):  
Red Cells ◽  
List Type ◽  
Red Cell

ABSTRACT Red cells were incubated in vitro with sulfhydryl inhibitors and Rhantibody with and without prior incubation with prednisolone-hemisuccinate. These erythrocytes were labelled with Cr51 and P32 and their disappearance in vivo after autotransfusion was measured. Prior incubation with prednisolone-hemisuccinate had no effect on the rate of red cell disappearance. The disappearance of the cells was shown to take place without appreciable intravascular destruction.

scholarly journals Red Cell Membrane Permeability Deduced from Bulk Diffusion Coefficients

1974 ◽  
Vol 64 (6) ◽  
pp. 706-729 ◽  
Author(s):  
W. R. Redwood ◽  
E. Rall ◽  
W. Perl
Keyword(s):  
Cell Membrane ◽  
Membrane Permeability ◽  
Diffusion Coefficients ◽  
Bulk Diffusion ◽  
Red Cells ◽  
Cell Membrane Permeability ◽  
Red Cell ◽  
Cell Column ◽  
Red Cell Membrane ◽  
One Dimensional

The permeability coefficients of dog red cell membrane to tritiated water and to a series of[14C]amides have been deduced from bulk diffusion measurements through a "tissue" composed of packed red cells. Red cells were packed by centrifugation inside polyethylene tubing. The red cell column was pulsed at one end with radiolabeled solute and diffusion was allowed to proceed for several hours. The distribution of radioactivity along the red cell column was measured by sequential slicing and counting, and the diffusion coefficient was determined by a simple plotting technique, assuming a one-dimensional diffusional model. In order to derive the red cell membrane permeability coefficient from the bulk diffusion coefficient, the red cells were assumed to be packed in a regular manner approximating closely spaced parallelopipeds. The local steady-state diffusional flux was idealized as a one-dimensional intracellular pathway in parallel with a one-dimensional extracellular pathway with solute exchange occurring within the series pathway and between the pathways. The diffusion coefficients in the intracellular and extracellular pathways were estimated from bulk diffusion measurements through concentrated hemoglobin solutions and plasma, respectively; while the volume of the extracellular pathway was determined using radiolabeled sucrose. The membrane permeability coefficients were in satisfactory agreement with the data of Sha'afi, R. I., C. M. Gary-Bobo, and A. K. Solomon (1971. J. Gen. Physiol. 58:238) obtained by a rapid-reaction technique. The method is simple and particularly well suited for rapidly permeating solutes.

scholarly journals Effect of oxalate and malonate on red cell metabolism

Blood ◽  
1987 ◽  
Vol 70 (5) ◽  
pp. 1389-1393
Author(s):  
E Beutler ◽  
L Forman ◽  
C West
Keyword(s):  
Pyruvate Kinase ◽  
Cell Metabolism ◽  
Inhibitory Effect ◽  
Red Cells ◽  
Red Cell ◽  
Acid Analogue ◽  
2,3 Dpg

The addition of oxalate to blood stored in Citrate-phosphate-dextrose (CPD) produces a marked improvement in 2,3-diphosphoglycerate (2,3-DPG) preservation; an increase in 2,3-DPG levels can also be documented in short-term incubation studies. Oxalate is a potent in vitro inhibitor of red cell lactate dehydrogenase, monophosphoglycerate mutase, and pyruvate kinase (PK). In the presence of fructose 1,6-diphosphate the latter inhibitory effect is competitive with phospho(enol)pyruvate (PEP). Determination of the levels of intermediate compounds in red cells incubated with oxalate suggest the presence of inhibition at the PK step, indicating that this is the site of oxalate action. Apparent inhibition at the glyceraldehyde phosphate dehydrogenase step is apparently due to an increase in the NADH/NAD ratio. Oxalate had no effect on the in vivo viability of rabbit red cells stored in CPD preservatives for 21 days. Greater understanding of the toxicity of oxalate is required before it can be considered suitable as a component of preservative media, but appreciation of the mechanism by which it affects 2,3-DPG levels may be important in design of other blood additives. Malonate, the 3-carbon dicarboxylic acid analogue of oxalate late did not inhibit pyruvate kinase nor affect 2,3-DPG levels.

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