Association between human erythrocyte calmodulin and the cytoplasmic surface of human erythrocyte membranes

This report describes Ca2+-dependent binding of 125I-labeled calmodulin (125I-CaM) to erythrocyte membranes and identification of two new CaM-binding proteins. Erythrocyte CaM labeled with 125I-Bolton Hunter reagent fully activated erythrocyte (Ca2+ + Mg2+)-ATPase. 125I-CaM bound to CaM depleted membranes in a Ca2+-dependent manner with a Ka of 6 x 10(-8) M Ca2+ and maximum binding at 4 x 10(-7) M Ca2+. Only the cytoplasmic surface of the membrane bound 125I-CaM. Binding was inhibited by unlabeled CaM and by trifluoperazine. Reduction of the free Ca2+ concentration or addition of trifluoperazine caused a slow reversal of binding. Nanomolar 125I-CaM required several hours to reach binding equilibrium, but the rate was much faster at higher concentrations. Scatchard plots of binding were curvilinear, and a class of high affinity sites was identified with a KD of 0.5 nM and estimated capacity of 400 sites per cell equivalent for inside-out vesicles (IOVs). The high affinity sites of IOVs most likely correspond to Ca2+ transporter since: (a) Ka of activation of (Ca2+ + Mg2+)-ATPase and KD for binding were nearly identical, and (b) partial digestion of IOVs with alpha-chymotrypsin produced activation of the (Ca2+ + Mg2+)-ATPase with loss of the high affinity sites. 125I-CaM bound in solution to a class of binding proteins (KD approximately 55 nM, 7.3 pmol per mg of ghost protein) which were extracted from ghosts by low ionic strength incubation. Soluble binding proteins were covalently cross-linked to 125I-CaM with Lomant's reagent, and 2 bands of 8,000 and 40,000 Mr (Mr of CaM subtracted) and spectrin dimer were observed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis autoradiography. The 8,000 and 40,000 Mr proteins represent a previously unrecognized class of CaM-binding sites which may mediate unexplained Ca2+-induced effects in the erythrocyte.     

Proteolytic activity associated with human erythrocyte membranes. Self-digestion of isolated human erythrocyte membranes

At least two kinds of enzymes are active in the proteolytic self-digestion of erythrocyte membranes. The specific activities of these enzymes do not decrease with repeated washings of purified stroma. The effects of a variety of inhibitors on the membrane preparation's capacity to digest ¹²⁵I-labelled casein, covalently linked to latex beads, have been examined.Pepstatin-inhibitable enzyme, active at low pH, digests the membrane extensively to small polypeptide fragments. Spectrin, located at the internal part of the membrane, is readily degraded. Diisopropylfluorophosphate-inhibitable enzyme, active at pH 8–9, has only limited digestive capacity. Some of the membrane components, such as the small molecular weight glycoproteins, are resistant to digestion. The restricted capacity of digestion is due to the membrane molecular arrangement; increased disaggregation removes the restriction and increases the activity. Spectrin is not digested unless the membrane topography is disrupted by NP-40 neutral detergent. These observations suggest that the enzymes active at basic pH are located external to the cell. Intact cells do possess a limited capacity to degrade ¹²⁵I-labelled casein when their surfaces are brought into contact with substrate-coated beads.     

Proteolytic activity associated with human erythrocyte membranes. Self-digestion of isolated human erythrocyte membranes.

At least two kinds of enzymes are active in the proteolytic self-digestion of erythrocyte membranes. The specific activities of these enzymes do not decrease with repeated washings of purified stroma. The effects of a variety of inhibitors on the membrane preparation's capacity to digest 125-I-labelled casein, covalently linked to latex beads, have been examined. Pepstatin-inhibitable enzyme, active at low pH, digests the membrane extensively to small polypeptide fragments. Spectrin, located at the internal part of the membrane, is readily degraded. Diisopropylfluorophosphate-inhibitable enzyme, active at pH 8-9, has only limited digestive capacity. Some of the membrane components, such as the small molecular weight glycoproteins, are resistant to digestion. The restricted capacity of digestion is due to the membrane molecular arrangement; increased disaggregation removes the restriction and increases the activity. Spectrin is not digested unless the membrane topography is disrupted by NP-40 neutral detergent. These observations suggest that the enzymes active at basic pH are located external to the cell. Intact cells do possess a limited capacity to degrade 125-I-labelled casein when their surfaces are brought into contact with substrate-coated beads.     

Membrane potential and human erythrocyte shape.

Altered external pH transforms human erythrocytes from discocytes to stomatocytes (low pH) or echinocytes (high pH). The process is fast and reversible at room temperature, so it seems to involve shifts in weak inter- or intramolecular bonds. This shape change has been reported to depend on changes in membrane potential, but control experiments excluding roles for other simultaneously varying cell properties (cell pH, cell water, and cell chloride concentration) were not reported. The present study examined the effect of independent variation of membrane potential on red cell shape. Red cells were equilibrated in a set of solutions with graduated chloride concentrations, producing in them a wide range of membrane potentials at normal cell pH and cell water. By using assays that were rapid and accurate, cell pH, cell water, cell chloride, and membrane potential were measured in each sample. Cells remained discoid over the entire range of membrane potentials examined (-45 to +45 mV). It was concluded that membrane potential has no independent effect on red cell shape and does not mediate the membrane curvature changes known to occur in red cells equilibrated at altered pH.     

Thermotropic behavior of intact human erythrocyte membranes detected by measuring their surface conductivity

A relative surface conductivity Hz/Hm of intact human erythrocytes was investigated by scanning the temperature interval 5-55 degrees C. The form of thus obtained thermogram implies a relation between Hz/Hm and thermotropic behavior of the erythrocyte membrane in vivo. Conditions for the best manifestation of this relation were studied.     

Calcium binding by human erythrocyte membranes

1. The characteristics of Ca²⁺ binding to haemoglobin-free human erythrocyte membranes were investigated by using ⁴⁵Ca and centrifugation partition of `ghosts' from their external incubation medium. Equilibrium of `ghosts' with external Ca²⁺ required less than 15min. 2. The binding did not vary with temperature in the range 0–37°C. 3. At pH7.4 `ghosts' bound a maximum of 283μmol of Ca<sup>2+</sup>/g of `ghost' protein, equivalent to 6.85×10⁷ Ca²⁺ ions per cell. 4. Increasing the ionic strength from 0.01 to 0.46 diminished Ca²⁺ binding, as did ATP in concentrations ranging from 0 to 15mm in the incubation medium. 5. An increase of the pH from 3.0 to 9.3 caused a marked increase in the amount of Ca²⁺ bound. 6. Extraction of ⁴⁵Ca-labelled `ghosts' with chloroform–methanol showed that the distribution of Ca²⁺ was: 79% protein-bound, 16% lipid-bound, 5% in the aqueous phase, presumably non-bound. Most of the lipid-bound Ca²⁺ (about 80%) was associated with a phospholipid fraction containing phosphatidylserine, phosphoinositides and phosphatidylethanolamine, giving a molar Ca²⁺: phosphorus ratio of about 1:2.     

Voltage-induced conductance in human erythrocyte membranes

Isotonic suspensions of erythrocytes were exposed to intense electric fields for a duration in microseconds. Time-dependent increase in the conductivity of the suspension was observed under fields greater than a threshold of about 1.5 kV/cm. The threshold was independent of the ionic strength of the medium, and changed little with temperature or with the rise time of the applied field. Under fields greater than 3 kV/cm, the time course of the conductivity increase consisted of a rapid (approx. 1 μs) and a slow (approx. 100 μs) phases. The increase is attributed primarily to large membrane conductance induced by the applied field. The membrane conductance is in the order of $\text{10 Ω}{}^{\mathrm{?1}}\text{/}\text{cm}{}^2$ in the rapid phase and $\text{10}{}^2\text{Ω}{}^{\mathrm{?1}}\text{/}\text{cm}{}^2$ in the slow phase. Comparison with previous results indicates that this induced membrane conductance corresponds to the formation of aqueous pores in the cell membrane. After the applied field was removed, the conductivity of the suspension returned nearly to its initial value, indicating that the induced membrane conductance is strongly dependent on the membrane potential. The conductivity then increased again in the time range of 10 s. This is attributed to the diffusional efflux of intracellular ions through the voltage-induced pores. From the rate of the efflux, number of the pores/cell is estimated to be in the order of 10². Final stage of the conductivity change was a slow decrease, corresponding to the colloid osmotic swelling of the perforated cells.     

Tyrosyl and phosphatidylinositol kinases of human erythrocyte membranes

The tyrosyl kinase and phosphatidylinositol (PI) kinase activities of human red cells have been partially purified and characterized. Although the PI kinase required detergent for solubilization, the major tyrosyl kinase of the red cell could be extracted by high salt. A very small residual activity remained associated with the membranes, however, that was solubilized with the PI kinase and copurified through an ammonium sulfate precipitation and diethylaminoethyl (DEAE) ion-exchange step gradient elution. However, the two activities were found to differ with respect to their apparent KmS for ATP and Mg2+; they showed different half-lives for temperature inactivation, possessed different relative activities in the presence of Mn2+ and Ca2+, and were separable by elution from a DEAE-Trisacryl ion exchange column using a linear NaCl gradient. The kinetic parameters of the membrane-associated tyrosyl kinase differed from those of the salt-extracted enzyme. PI kinase was not activated by pretreatment with the tyrosyl kinase p68v-ros or by addition of the phosphotyrosyl phosphatase inhibitor, vanadate, to intact membranes, and was not competitively inhibited by the tyrosyl kinase substrate poly(Glu4, Tyr). We conclude that the human red cell phosphatidylinositol and tyrosyl kinases are distinct and separate activities, and that at least two separable tyrosyl kinases are present in human erythrocytes.     

Blood-group-Ii-active gangliosides of human erythrocyte membranes.

More than ten new types of gangliosides, in addition to haematoside and sialosylparagloboside, were isolated from human erythrocyte membranes. These were separated by successive chromatographies on DEAE-Sephadex, on porous silica-gel columns and on thin-layer silica gel as acetylated compounds. Highly potent blood-group-Ii and moderate blood-group-H activities were demonstrated in some of the ganglioside fractions. The gangliosides incorporated into cholesterol/phosphatidylcholine liposomes stoicheiometrically inhibited binding of anti-(blood-group I and i) antibodies to a radioiodinated blood-group-Ii-active glycoprotein. The fraction with the highest blood-group-I-activity, I(g) fraction, behaved like sialosyl-deca- to -dodeca-glycosylceramides on t.l.c. Certain blood-group-I and most of the -i determinants were in partially or completely cryptic form and could be unmasked by sialidase treatment. Thus the I and i antigens, which are known to occur on internal structures of blood-group-ABH-active glycoproteins in secretions, also occur in the interior of the carbohydrate chains of erythrocyte gangliosides.     

Norepinephrine and epinephrine in human erythrocyte plasma membranes

Acetone extract prepared from the plasma membranes of human erythrocytes contained substances which induced the contraction of the thoracic aortic strip of rabbit in vitro and caused blood pressure elevation in rat upon intravenous injection. The contractile response was inhibited by the alpha 1-adrenergic antagonist prazosin. By HPLC/electrochemical detection as well as radioenzymatic assay, large amounts of norepinephrine (NE) (14 +/- 4 [SE] ng/ml packed cells) and epinephrine (E) (16 +/- 2 ng/ml packed cells) were found in the extract. Using the same amounts as in the extract, we were able to demonstrate additive effect between NE and E. The possibility that erythrocyte membranes may play a role in the regulation of NE and E in circulation is suggested.     

Guanosine triphosphatase activity in human erythrocyte membranes

Human red cell membranes have the capacity to hydrolyze enzymatically GTD to GDP. The reaction requires magnesium, is not appreciably affected by sodium, potassium or calcium, and is not inhibited by ouabain. Kinetic analysis suggests that there are two separate enzymes in membranes which cleave GTP, a 'high Km' GTPase and a 'low Km' GTPase. Both enzymes are also ATPases, with an approximately equal affinity for GTP and ATP. GTPase activity did not extract from the membrane with spectrin and was not inactivated by antispectrin antibody. Activity was partially destroyed by 0.5% Triton X-100. It seems probable that the low Km GTPase is the sodium- and potassium-independent ATPase of red cell membranes. The identity of the high Km enzyme is not clear.