Tryptic digestion of spectrin in variants of hereditary elliptocytosis.

Spectrin, either in the form of unfractionated low ionic strength extracts of erythrocyte membranes or purified by chromatography on Sepharose (CL)4B, was subjected to tryptic digestion at 0 degrees C. Four patients, each with a different variant of hereditary elliptocytosis, were studied. In one patient, whose erythrocytes showed significant fragmentation on heating on 45 degrees C, such preparations generated a remarkably different pattern of polypeptide fragments on tryptic digestion at low ionic strength. In this patient 32P was released at a slower rate on tryptic digestion of labeled band 2, and an unusual 32P-labeled peptide fragment was also generated, in contrast to control preparations in which such a peptide could not be easily distinguished. There was increased susceptibility of this patient's spectrin to tryptic digestion at physiological ionic strength, but the qualitative pattern of polypeptide fragments was normal. Phosphorylation of spectrin by membrane protein kinase was markedly impaired in this patient, whereas phosphorylation of casein ws unimpaired. However, the phosphorylation of spectrin in her intact erythrocytes was normal. Our findings suggest an abnormality of spectrin structure which we postulate is causally related to the predisposition to hemolysis in this patient, but do not distinguish whether this is a primary abnormality or a post-translational modification of the spectrin molecule. The other three patients showed normal tryptic digestion of spectrin.

Erythrocyte membrane proteins in hereditary glucosephosphate isomerase deficiency.

Erythrocytes (approximately equal to 50% reticulocytes) obtained from a splenectomized patient with a thermolabile variant of glucosephosphate isomerase (GPI) deficiency showed a striking degree of crenation and decreased filterability through 3-micrometer Nuclepore filters (Nuclepore Corp., Pleasanton, Calif.). Membranes prepared by hypotonic lysis of such erythrocytes were found to contain a high molecular weight aggregate which was probably disulphide-bonded. The 10% most dense erythrocyte fraction showed an accentuation of aggregate formation while aggregates could not be detected in the 10% least dense erythrocyte fraction. The aggregate consisted mainly of spectrin (band 1) and a protein with the mobility of 4.2. "Extractability" of spectrin from these membranes was also markedly diminished. Incubation of the erythrocytes for 24 h in substrate-free medium caused more pronounced spectrin aggregation than in low or high reticulocyte controls. Incubation of low or high reticulocyte controls for 24 h in medium that contained glucose completely prevented the formation of the high molecular weight aggregate. GPI-deficient erythrocytes incubated with glucose in the medium showed an accentuation of membrane protein aggregate formation; however, this was almost completely reversed by the addition of adenine and inosine to the incubation medium or by the use of fructose, the intermediate just distal to the "block" in glycolysis, as the sole substrate. ATP and reduced glutathione levels in the GPI-deficient erythrocytes incubated with glucose were similar to that found in the low and high reticulocyte controls. Our findings suggest that only a proportion of erythrocytes (the older, more dense population of cells) are susceptible to the formation of disulphide-bonded aggregates, and that this is directly related to an impairment of substrate flow through the glycolytic sequence. The exact mechanism of aggregate formation in these erythrocytes remains to be elucidated.

Defective binding of spectrin to ankyrin in a kindred with recessively inherited hereditary elliptocytosis.

The interaction of spectrin with spectrin-depleted inside-out membrane vesicles was studied in a kindred with an atypical variant of hereditary elliptocytosis inherited in a recessive manner. The probands are characterized by prominent elliptocytosis, decreased erythrocyte thermal stability, an altered limited tryptic peptide pattern of spectrin digested at low ionic strength, and defective spectrin dimer-dimer association. The parents are normal. The spectrin/band 3 ratio determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of isolated membranes of the probands was decreased to approximately 70% of control values, and total erythrocyte spectrin content in one proband was also decreased on SDS-PAGE. When a monospecific antispectrin antibody was used, a faintly labeled fragment of molecular weight approximately 28,000 was detected on immunoblots of whole cell lysates of one proband and a control, but could not account for the decreased erythrocyte spectrin content of the proband on SDS-PAGE. Binding and competitive inhibition studies revealed an alteration in the spectrin-ankyrin interaction due to an abnormality of spectrin in the probands. No defect was found in the mother; the father's spectrin showed decreased binding affinity, although it was not so severe as in the probands. Separation of bound and unbound spectrin dimers from one proband and subsequent conversion to tetramers showed that the self-association defect was detectable only on the bound subpopulation of her spectrin. These findings demonstrate a hitherto undescribed functional abnormality of spectrin in this kindred which could result in decreased stability of the membrane skeleton and contribute to the elliptocytic shape of these erythrocytes.

Electrophoretic analysis of the major polypeptides of human erythrocyte membranes prepared by low and high osmolarity haemolysis.

Human erythrocyte membranes were prepared in three ways: washing in hypotonic Tris buffer, pH 7.6, by lysis in isotonic Tris buffer pH 7.6 after incubation at 37 degrees C for 2 hours and by ultrasonication in an isotonic medium, pH 7.6. Analysis of the major polypeptides of the erythrocyte membranes by sodium dodecylsulphate polyacrylamide gel electrophoresis revealed a selective depletion of a major polypeptide representing glyceraldehyde-3-phosphate dehydrogenase in the membranes prepared by high osmolarity lysis. The pattern of seperation of the remaining polypeptides was identical in the 3 different membrane preparations.

Lactate dehydrogenase isoenzymes of human erythrocyte membranes.

Human erythrocyte membranes and cytosol were fractionated in detergent-containing polyacrylamide gels and stained for lactate dehydrogenase activity. A highly significant difference in the distribution of three isoenzymes of lactate dehydrogenase (LD-1, LD-2 and LD-3) between membranes and cytosol was found, a striking feature being the poor binding of LD-3 to the membrane fraction.

Polyacrylamide gel electrophoresis of human erythrocyte membrane enzymes solubilized with triton X-100.

Human erythrocyte ghosts were solubilized in a low ionic strength medium containing 1% Triton X-100 and subjected to electrophoresis in polyacrylamide gels containing Triton X-100. Five major bands were stained with Coomassie Blue, all except one band being heterogenous when re-electrophoresed in gels containing sodium dodecyl sulphate. It was possible to detect acetylcholinesterase, non-specific esterase, ATPase, alkaline phosphatase, 5'-nucleotidase, glyceraldehyde-3-phosphate dehydrogenase, lactate dehydrogenase, and aldolase activities on the Triton-containing polyacrylamide gels. Two of the enzymes, ATPase and 5'-nucleotidase, showed substantial inhibition by Triton X-100 in quantitative studies. This appears to be a useful method for studying membrane enzymes in normal and pathological red cells.

Cross-linking of membrane proteins of metabolically-depleted and calcium-loaded erythrocytes.

The membranes of erythrocytes undergoing metabolic depletion or an influx of calcium undergo several changes in structure and function. In erythrocytes incubated without substrate we find extensive cross-linking of membrane proteins by disulphide bonding occurring after 24--48 h, involving all major membrane proteins as well as haemoglobin. Aggregates of mol wt 40 x 10(6) or greater are formed. These changes are partially reversible by repletion with adenosine. Rapid introduction of calcium (intracellular concentrations approximately 0.6 mM) into metabolically replete erythrocytes with the ionophore A23187 results in transglutaminase-dependent cross-linking of membrane proteins. Cellular calcium concentrations of approximately 0.3 mM have no cross-linking effect. Cells undergoing metabolic depletion show a progressive loss of transglutaminase activity to undetectable levels at 12 h, so that influx of calcium into such cells cannot cause cross-linking by a transglutaminase-mediated reaction. These studies suggest that the metabolic state of the cell and the rate and degree of calcium influx into erythrocytes are critical factors in determining the type of membrane protein cross-linkage.

The use of glycosylated haemoglobin measurements in the control of the diabetic patient.

Glycosylated haemoglobin (HbA1c) has recently been used as an indicator of long-term diabetic control. This study compares the efficacy of HbA1c measurements and postprandial blood glucose estimations in assessing diabetic control in 51 diabetic patients. It was found that the HbA1c levels reflected overall diabetic control significantly better than did a single postprandial blood glucose estimation. HbA1c measurements give considerable aid in the assessment of the longitudinal blood sugar control in the diabetic, and may be a useful indicator of the efficacy of diabetic treatment.

Fatty acid composition of erythrocytes in hereditary spherocytosis.

The fatty acid distribution of the four major phospholipids has been determined in the erythrocytes of five patients with hereditary spherocytosis and in five control subjects. In contrast to a report by other workers, we are unable to confirm their findings of a defect in fatty acid chain lengthening activity leading to disappearance of long chain fatty acid conjugates of more than 20 carbon atoms in some of the phospholipid fractions.

Serum human placental lactogen levels in intra-uterine fetal growth retardation.

Serum human placental lactogen (HPL) levels were measured in the last trimester of pregnancy in 16 mothers who delivered small-for-gestational-age babies. Only 3 patients had levels which were below the normal range, while 4 others had levels close to the lower limit of the normal range. The finding of a normal serum HPL level therefore does not exclude the possibility of intra-uterine fetal growth retardation. No correlation was found between serum HPL levels at 37-39 weeks and infant or placental weights in full-term normal deliveries.

The topology of red cell membrane lipids in hereditary spherocytosis.

The topology of membrane phospholipids in the red cells of patients with hereditary spherocytosis has been studied with the non-penetrating probe 2,3,5-trinitrobenzenesulphonate. There was no significant difference in the assymetric distribution of the aminophospholipids phosphatidylserine and phosphatidylethanolamine across the two halves of the membrane bilayer in red cells of five patients with hereditary spherocytosis as compared to seven normal controls. These studies indicated that the complex processes responsible for membrane lipid assymetry are intact in hereditary spherocytosis.

Membrane proteins of incubated erythrocytes: effect of sulphydryl inhibition.

Incubation of erythrocytes with various sulphydryl inhibitors for 24 h leads to an accumulation in isolated membranes of globin as well as an unidentified polypeptide of apparent molecular weight 24 000 on polyacrylamide gel electrophoresis. Accumulation of both these components does not appear to be directly related to erythrocyte adenosine triphosphate and reduced glutathione levels. The 24 000 molecular weight polypeptide is probably derived from the erythrocyte cytosol, as determined by polyacrylamide gel electrophoresis of the non-haemoglobin proteins of the cytosol. Together with globin it forms an integral part of the 'spectrin-actin lattice' of sulphydryl-inhibited erythrocytes, as both these components can be detected, together with spectrin and actin, after extraction of such membranes by Triton X-100. We postulate that this leads to inherent damage and distortion to the membranes of such erythrocytes.

Studies on calcium transport and calcium-dependent adenosine triphosphatase activity of erythrocyte membranes in hereditary spherocytosis.

Evidence has been recently presented of a relative deficiency of Ca2+ - dependent adenosine triphosphatase activity of erythrocyte membranes obtained from patients with hereditary spherocytosis. We have sought to confirm these findings by measuring calcium efflux from intact erythrocytes of patients with hereditary spherocytosis, as well as erythrocyte calcium concentrations, but find both these parameters to be normal. Ca2+-dependent adenosine triphosphatase activity, as well as Ca2+ -dependent membrane phosphorylation was also not found to be deficient in erythrocyte membranes from subjects with hereditary spherocytosis. These studies do not support the postulate that an accumulation of calcium affects the deformability of erythrocytes and their subsequent destruction in the spleen.

The effect of vitamin E on haemolysis in paroxysmal nocturnal haemoglobinuria: in vitro and in vivo studies.

The administration of vitamin E, a natural antioxidant, to a patient with paroxysmal nocturnal haemoglobinuria (PNH) failed to diminish the urinary excretion of 59-Fe as monitored by 59-Fe whole body counting and urinary loss of isotope. However, in vitro vitamin E corrected the increased sensitivity of PNH red cells to haemolysis by hydrogen peroxide. These results support the concept that the susceptibility of PNH red cells to lipid peroxidation is an in vitro phenomenon bearing little relation to the mechanism of in vivo haemolysis.