Diminished spectrin extraction from ATP-depleted human erythrocytes. Evidence relating spectrin to changes in erythrocyte shape and deformability.

Abstract

A B S T R A C T We measured spectrin "extractal)ilitvy in erythrocytes which were metabolically depleted ly ineubatioin at 37°C in plasma or glucose-free buffers. Membranes were extracted with 1 mnl EDTA (pH 8, 40 h, 4°C) 12 September 1977. exposed to extremes of pH and temlperature. WVhen the pH in the ervthrocvte interior fell below 5.5, a pH where spectrini was aggregated and isoelectricallv precipitated, erythrocyte and ghost viscosity increased coincident with a marked decrease in spectrini extractability. Similarly above 49°C, a temperatture where spectrin was denatured and precipitated, ervthrocvte viscosity rose as inextractable spectrin a(-cumulated. These observations provide direct evidence of a chanige in the physical state of spectrini associated with a change in ervthrocvte shape and deformability. They sutpport the concept that erythrocyte shape and deformability are largely determiined by the shape aind deformal)ilitv of the spectrin-actiii protein meshwork which laminates the inner membrane surface.

Reconstitution of spectrin-deficient, spherocytic mouse erythrocyte membranes.

“…This suggestion is further supported by observations of close correlations between the in vitro conditions which cause changes in the extractability or aggregation state of spectrin, and conditions which induce sphering and loss of deformability oferythrocytes (22)(23)(24). Finally, the recent finding of marked spectrin deficiency in a strain of mice with severe hemolysis and hereditary spherocytosis again strongly suggests a role for spectrin in maintenance of normal cell shape (25).…”

Regulation of the interaction of purified human erythrocyte AMP deaminase and the human erythrocyte membrane.

“…Both AMP and ATP have beeni shown to be activators of AMP deaminase2 and it is possible that the dissociation of the enzymemembranie complex promoted by AMP is mediated through an isosteric site of AMP on the enzyme, distinct from the AMP substrate binding site. ATP, although an allosteric activator of the enzyme, also has pronounced effects on the erythrocyte membrane (18) whichi may mediate ATP promoted dissociation. ATP is not an allosteric effector of G3PD but effectively dissociates the G3PD membrane complex (3).…”

“…After separation ofthe membranes by centrifugation, the supernatant solution was dialyzed against 1,000 vol of 10 mM KCI, 10 Preparatiot oferythrocyte melemabr(ane fractionis. 5 Spectrin (bands 1 and 2) and actin (band 5) were solubilized from unsealed erythrocyte ghosts by incubation of washed hemoglobin free ghosts in 2 vol of 1 mM EDTA (pH 8.0) and dialysis against 1,000 vol of the diluent at 4°C, as described bv Lux et al (18).…”

Membrane protein lesions in erythrocytes with Heinz bodies.

“…Waugh and Low (12) and Low and colleagues (13) have focused on phenylhydrazine-induced hemoglobin oxidation to hemichrome, and its binding to and modulation ofthe organization of protein 3 (12,13), a feature that is also found in SS-RBCs (16 Preparation ofmembranes and membrane proteins. Venous blood was collected in citrate-phosphate-dextrose and stored at 40C for up to 5 d. Abnormal blood samples were stored for up to 48 h. Erythrocyte membranes were prepared by hypotonic hemolysis (24). Isotonic membrane shells were prepared from membranes in a modification of the method of preparing shells from whole cells by incubating them in a buffer of the following composition: Triton X-100 150 mg/ml, NaCl 150 mM, Hepes 24 mM, EGTA 1 mM, phenylmethylsulfonyl fluoride (PMSF) 10 ,ug/ml, leupeptin (Sigma Chemical Co., St. Louis, MO) 10 ;Lg/ml, pepstatin A (Sigma Chemical Co.) 10 ,ag/ml, pH 7.0, 5 min, 0C (25).…”