Structural and dynamic states of actin in the erythrocyte.

Analysis of the nucleotide tightly associated with isolated erythrocyte cytoskeletons show it to be ADP, rather then ATP. This confirms that at least a major part of the erythrocyte actin is in the F-form. A re-evaluation of the stoichiometry of spectrin and actin in the erythrocyte (taking account of a gross difference between the color responses of the two proteins on staining of electrophoretic gels) leads to values of 1x10(5) and 5x10(5) for the number of molecules of spectrin tetramer and actin respectively per cell. It has been found possible to perform spectrophotometric DNAase I assays fro actin on lysed whole cells. The concentration of monomeric actin at 0 degrees C is approximately 16 mug/ml packed cells. After washing the lysed cells the monomer pool is not re-established, indicating that only a small proportion of the actin subunits are free to dissociate. The actin monomer concentration in the cytosol remains unchanged after equilibration of the cells with cytochalasin E. The ability of actin-containing complexes in the membrane to nucleate the polymerization of added G-actin was measured fluorimetrically; it was found that membranes incubated with cytochalasin E were completely inert with respect to nucleating activity under conditions that favor appreciable growth at the slowly-growing ("pointed") ends of free actin filaments. This suggests that these ends of the actin "protofilaments" in the red cell are blocked or sterically obstructed. After treatment of the membranes with guanidine hydrochloride under conditions that dissociate F-actin, the measured concentration of actin monomer rises to approximately 180 mug/ml of packed cells, which is nearly 70 percent of the total actin content. On treatment with trypsin in the presence of DNAase, the spectrin and 4.1 are extensively degraded, but the actin remains undamaged. This treatment, followed by exposure to guanidine hydrochloride, causes a further rise in the concentration of actin responsive to the DNAase assay to 250 mug/ml of cells, compared with 270 mug/ml estimated by densitometry of stained gels. The oligomeric complex, consisting of actin, spectrin, and 4.1, that is extracted from the membrane at low ionic strength, generates no detectable actin monomer after the same treatment. From literature data on the number of cytochalasin binding sites per cell and our value for the total actin content, we obtain a number-average degree of polymerization for actin in the membrane of 12-17. The results lead to a model for the structure of the cytoskeletal network and suggest some consequences of metabolic depletion.

Gel electrophoretic analysis of poly(riboadenylic acid).

It is shown that molecular weights and molecular-weight distributions of poly(rA), and by implication other single-stranded polynucleotides, and synthetic and natural polyelectrolytes in general, can be determined by electrophoresis in polyacrylamide gels. It is shown that fractions of very narrow molecular-weight distribution can be obtained by preparative electrophoresis of polydisperse samples. Molecular-weight calibrations based on sedimentation coefficients of such fractions are given, and in aqueous systems do not coincide with calibrations for partially base-paired RNA species. Poly(rU) fractions fall on the same calibration as poly(rA). Relations between mobilities, relative to standard markers, and molecular weight for poly(rA) over a wide range of molecular weights are given, which allow rapid molecular-weight determination on poly(rA) samples, such as the segments found in many types of messenger RNA.

Study of actin filament ends in the human red cell membrane.

There is conflicting evidence concerning the state of the actin protofilaments in the membrane cytoskeleton of the human red cell. To resolve this uncertainty, we have analysed their characteristics with respect to nucleation of G-actin polymerization. The effects of cytochalasin E on the rate of elongation of the protofilaments have been measured in a medium containing 0.1 M-sodium chloride and 5 mM-magnesium chloride, using pyrene-labelled G-actin. At an initial monomer concentration far above the critical concentration for the negative ("pointed") end of F-actin, high concentrations of cytochalasin reduce the elongation rate of free F-actin by about 70%. The residual rate is presumed to correspond to the elongation rate at the negative ends. By contrast, the elongation rate on red cell ghosts or cytoskeletons falls to zero, allowing for the background of self-nucleated polymerization of the G-actin. The critical concentration of the actin in the red cell membrane has been measured after elongation of the filaments by added pyrenyl-G-actin in the same solvent. It was found to be 0.07 microM, compared with 0.11 microM under the same conditions for actin alone. This is consistent with prediction for the case of blocked negative ends on the red cell actin. The rate of elongation of actin filaments, free and in the red cell membrane cytoskeleton, has been measured as a function of the concentration of an added actin-capping protein, plasma gelsolin, with a high affinity for the positive ends. The elongation rate falls linearly with increasing gelsolin concentration until it approaches a minimum when the gelsolin has bound to all positive filament ends. The elongation rate at this point corresponds to the activity of the negative ends, and its ratio to the unperturbed polymerization rate (in the absence of capping proteins) is indistinguishable from zero in the case of ghosts, but about 1 : 4 in the case of F-actin. When ATP is replaced in the system by ADP, so that the critical concentrations at the two filament ends are equalized, the difference is equally well-marked: for F-actin, the rate at the equivalence point is about 40% of that in the absence of capping protein, whereas for ghosts the nucleated polymerization rate at the equivalence point is again zero, indicating that under these conditions the negative ends contribute little or not at all to the rate of elongation.(ABSTRACT TRUNCATED AT 400 WORDS)

Basis of unique red cell membrane properties in hereditary ovalocytosis.

Hereditary ovalocytes from a Mauritian subject are extremely rigid, with a shear elastic modulus about three times that of normal cells, and have increased resistance to invasion by the malaria parasite Plasmodium falciparum in vitro. The genetic anomaly resides in band 3; the protein gives rise to chymotryptic fragments with reduced mobility in SDS/polyacrylamide gel electrophoresis, but this is a result of anomalous binding of SDS and not a higher molecular weight. Analysis of the band 3 gene reveals (1) a point mutation (Lys56----Glu), which also occurs in a common asymptomatic band 3 (Memphis) variant and governs the electrophoretic properties, and (2) a deletion of nine amino acid residues, including a proline residue, encompassing the interface between the membrane-associated and the N-terminal cytoplasmic domains. The interaction of the mutant band 3 with ankyrin appears unperturbed. The fraction of band 3 capable of undergoing translation diffusion in the membrane is greatly reduced in the ovalocytes. Cells containing the asymptomatic band 3 variant were normal with respect to all the properties that we have studied. Possible mechanisms by which a structural change in band 3 at the membrane interface could regulate rigidity are examined.

An examination of the soluble oligomeric complexes extracted from the red cell membrane and their relation to the membrane cytoskeleton.

A part of the spectrin extracted from red cell membranes at low ionic strength occurs in the form of a high-molecular weight oligomeric complex with actin and proteins 4.1 and 4.9. When the extraction is performed at 35 degrees, the spectrin is present in this complex as the dimer, all higher forms being dissociated. We have been unable to establish any correlation between the fraction of the spectrin thus complexed and the metabolic state of the cell. At least a large part of the complex appears to be a defined monodisperse species, sedimenting at 31S. The actin is present as short protofilaments. The average number of spectrin molecules associated with each molecule of complex has been studied by cytochalasin binding and electron microscopy. The complexes present the appearance in the electron microscope of spiders, in which the legs are spectrin dimers, attached to a globular element, containing by inference, actin and proteins 4.1 and 4.9; they are active in nucleating the polymerization of G-actin. The complexes are extremely stable, being resistant to dissociation under the conditions of the deoxyribonuclease assay, even after treatment with trypsin to degrade the actin-associated proteins. It is suggested that the complexes represent intact junctions of the membrane cytoskeletal network. Relevant structural features of the network are revealed by electron microscopy. The results lead to inferences concerning the mechanism of dissociation of the network from the membrane.

Spectrin and protein 4.1 as an actin filament capping complex.

Spectrin and protein 4.1, when added to G- or F-actin, cause the formation of short filaments, as judged by the appearance of powerful nucleating activity for G-actin polymerisation. F-Actin filaments are rapidly fragmented under physiological solvent conditions. The effect of cytochalasin E on the polymerisation reaction and the extent of reduction in the critical monomer concentration of actin when spectrin and 4.1 are added suggest that these proteins form a capping system for the more slowly growing, or 'pointed' ends of actin filaments. The interaction is not affected by calcium or by 4.9, the remaining constituent of the purified red cell membrane cytoskeleton.

Investigation of the actin-deoxyribonuclease I interaction using a pyrene-conjugated actin derivative.

The interaction of deoxyribonuclease I with muscle actin was studied with the aid of a pyrenyl derivative of the actin [Kouyama, T., & Mihashi, K. (1981) Eur. J. Biochem. 114, 33-38] that increases its quantum yield by an order of magnitude on polymerization. It is shown that this derivative copolymerizes with unlabeled G-actin in a random manner and will also bind to deoxyribonuclease with inhibition of enzymic activity. The derivative affords a highly sensitive means of following nucleated polymerization. Preincubation of F-actin with deoxyribonuclease at a concentration of 5% or less of that of total subunits causes inhibition of polymerization of additional G-actin onto the filaments. In red cell membranes that contain stabilized short filaments of actin such that the concentration of filament ends is large relative to monomers, complete inhibition of nucleated polymerization of G-actin is achieved by preincubation with deoxyribonuclease. The results indicate that binding of DNase occurs at the "plus" ends of the actin filaments. Competition with cytochalasin E, which is known to have a high affinity for the plus or preferentially growing ends of F-actin, can be observed. Whereas the activity of deoxyribonuclease in the 1:1 complex with G-actin is inhibited, the enzyme attached to the ends of filaments appears to be fully active. This causes a reduction in the inhibition of enzymic activity with increasing F-actin concentration, presumably by reason of a change in the partition of the enzyme between monomers and filament ends. The degree of inhibition increases with time, however, as the actin depolymerizes. Implications for measurements of actin monomer concentrations by the deoxyribonuclease assay procedure are considered.

Interaction of protein 4.1 with the red cell membrane: effects of phosphorylation by protein kinase C.

Phosphorylation with endogenous protein kinase C causes the membrane skeletal protein, band 4.1, to lose its capacity to attach to one of two classes of high-affinity binding sites on the red cell membrane. These sites are the ones eliminated by proteolysis in situ of glycophorin C; the surviving type of site is located in a C-terminal peptide of the glycophorin C, retained on the membrane after proteolysis, which is also the site of attachment of p55. A synthetic peptide, comprising the 28 C-terminal residues of glycophorin C, also binds protein 4.1. Phosphorylation of the intact cells, stimulated by phorbol ester, approximately halves the retention of glycophorin C in the membrane cytoskeletons prepared from these cells and reduces the affinity of extracellular glycophorin C epitopes for their antibody.

Membrane attachment sites for the membrane cytoskeletal protein 4.1 of the red blood cell.

The identity of the membrane binding sites for the membrane cytoskeletal protein 4.1 of the human red blood cell has been investigated. Exhaustive proteolysis of the membrane with a range of proteases led to the elimination of only some 60% of all binding sites. The predominant integral membrane protein, band 3, as well as glycophorin A, was totally digested at levels of proteolysis that were essentially without effect on the number of 4.1 binding sites. Proteolysis caused scission of the polypeptide chain of glycophorin C (together with the minor product, glycophorin D, of the same gene), but left a fragment from the region of the C-terminus still attached to the membrane. We have found a low-molecular weight protein, possessing an epitope (recognized by an antibody directed against the cytoplasmic domain of glycophorin C) in common with this proteolytic fragment, in cells of a Leach phenotype, which are characterized by lack of extracellular epitopes of glycophorin C. When these membranes were extracted at low ionic strength to dissociate the membrane cytoskeleton, approximately half the content of 4.1 was liberated, compared with only some 25% from normal membranes. Cells of a different variant of the Leach phenotype, which are totally devoid of glycophorin C, lost close to 70% of their 4.1 under these circumstances. The Rh(D) transmembrane protein, which interacts with the membrane cytoskeleton, is also resistant to proteolysis of the cytoplasmic membrane surface, but Rhnull cells, devoid of this protein, showed no decreased retention of 4.1. The results suggest that glycophorin C (with D) may contain two types of binding site for 4.1, which would be sufficient in number to account for all the strong binding of 4.1 on normal membranes; modulation of binding at one of the sites by another protein or by lipid is not excluded. A possible site for reinitiation of translation overlapping the premature stop codon in the mutant expressing the truncated glycophorin C can be discerned.

Composition of the ternary protein complex of the red cell membrane cytoskeleton.

The red cell membrane skeletal network is constructed from actin, spectrin and protein 4.1 in a molar ratio of actin subunits/spectrin heterodimer/protein 4.1 of 2:1:1. This represents saturation of the actin filaments, since incubation with extraneous spectrin and protein 4.1 leads to no binding of additional spectrin, either to the inner surface of ghost membranes or to lipid-free membrane cytoskeletons. Partial extraction of spectrin from the membrane is accompanied by release of actin under all conditions. Regardless of the proportion of spectrin extracted, the molar ratio of spectrin dimers/actin subunits is constant at 1:2. This is not the result of release or cooperative breakdown of whole lattice junctions from the network, for the number of actin filaments, judged by capacity to nucleate polymerisation of added G-actin, remains unchanged even when as much as 60% of the total spectrin has been lost. A similar 1:2:1 stoichiometry characterises the complex formed when G-actin is allowed to polymerise in the presence of varying amounts of spectrin and protein 4.1. When this complex is treated with the depolymerising agent, 1 M guanidine hydrochloride, it breaks down into smaller units of the same stoichiometry. After cross-linking these can be recovered from a gel-filtration column. Complexes prepared starting from G-actin appear to be much more stable than those formed when spectrin and protein 4.1 are bound to F-actin.

Fluorescence quenching of spectrin and other red cell membrane cytoskeletal proteins. Relation to hydrophobic binding sites.

The intrinsic fluorescence of spectrin is strongly quenched by low concentrations of 2-bromostearate. This results from binding at a series of hydrophobic sites. Analysis of dynamic fluorescence quenching by acrylamide, iodide and caesium ions, separately and in conjunction with 2-bromostearate, leads to the conclusion that most of the tryptophan side-chains are exposed to solvent. The sites at which the fatty-acid-quenched tryptophans are located apparently interact with the lipid bilayer in the cell, as judged by quenching by bromostearate dissolved in the lipid phase. A minor proportion of the side-chains in native spectrin give rise to sharp proton magnetic resonance signals, indicative of segmental mobility; these chain elements contain some tryptophan residues, as revealed by weak downfield signals from the heterocyclic ring protons. These signals are not appreciably perturbed by stearic acid or by phosphatidylserine liposomes, suggesting that the hydrophobic binding sites are not in mobile chain elements. By contrast with a series of globular proteins which, with the exception of serum albumins, show little or no quenching by 2-bromostearate, the peripheral red cell membrane skeletal proteins ankyrin (and its spectrin-binding domain), protein 4.1 and (to a lesser extent) actin show evidence of a high affinity for the hydrophobic ligand and may, like spectrin, interact directly with the bilayer in situ.

Myosins of Babesia bovis: molecular characterisation, erythrocyte invasion, and phylogeny.

Using degenerate primers, three putative myosin sequences were amplified from Australian isolates of Babesa bovis and confirmed as myosins (termed Bbmyo-A, Bbmyo-B, and Bbmyo-C) from in vitro cultures of the W strain of B. bovis. Comprehensive analysis of 15 apicomplexan myosins suggests that members of Class XIV be defined as those with greater than 35% myosin head sequence identity and that these be further subclassed into groups bearing above 50-60% identity. Bbmyo-A protein bears a strong similarity with other apicomplexan myosin-A type proteins (subclass XIVa), the Bbmyo-B myosin head protein sequence exhibits low identity (35-39%) with all members of Class XIV, and 5'-sequence of Bbmyo-C shows strong identity (60%) with P. falciparum myosin-C protein. Domain analysis revealed five divergent IQ domains within the neck of Pfmyo-C, and a myosin-N terminal domain as well as a classical IQ sequence unusually located within the head converter domain of Bbmyo-B. A cross-reacting antibody directed against P. falciparum myosin-A (Pfmyo-A) revealed a zone of approximately 85 kDa in immunoblots prepared with B. bovis total protein, and immunofluorescence inferred stage-specific myosin-A expression since only 25% of infected erythrocytes with mostly paired B. bovis were immuno-positive. Multiplication of B. bovis in in vitro culture was inhibited by myosin- and actin-binding drugs at concentrations lower than those that inhibit P. falciparum. This study identifies and classifies three myosin genes and an actin gene in B. bovis, and provides the first evidence for the participation of an actomyosin-based motor in erythrocyte invasion in this species of apicomplexan parasite.

Preparation and properties of human red-cell ankyrin.

We describe a procedure for the preparation of ankyrin from human red cells with a yield of 2-3 mg of protein from 30 ml of packed cells. This represents an improvement of an order of magnitude over the usual earlier procedure. Moreover, the product is, in our hands, much more stable against adsorption and proteolysis, and can in general be stored for at least 2 months at 4 degrees C without significant decrease in concentration and binding activity. The preparation depends on the release of the ankyrin-band-3 complex from the membrane cytoskeleton when intact cells are lysed in a medium containing concentrated Triton X-100. The complex is dissociated at high ionic strength, and the final purification is achieved by gel filtration in a medium containing 2 M-Tris or 0.6 M-NaBr. The ankyrin contains all the progression of components present in the intact membrane. All react with affinity-purified polyclonal anti-ankyrin antibodies, and all give widely similar patterns of peptides in partial proteolytic digests. The ankyrin is fully active, as judged by its capacity to bind to band-3-containing membrane vesicles and to Sepharose-coupled spectrin. All components bind to the membrane vesicles. Purified components 2.1 and 2.2, as well as the calmodulin-binding cytoskeletal constituent adducin, can be isolated in pure form by a single anion-exchange column step.

Actin polymerisation regulates integrin-mediated adhesion as well as rigidity of neutrophils.

Activation of adherent neutrophils causes them to convert from selectin-mediated rolling to integrin-mediated immobilisation and migration. Migration is known to depend on formation and redistribution of filamentous (F) actin, but although immobilisation in seconds parallels early cortical actin polymerisation, no link has been proven. We tested the effect of the actin-polymerising agent jasplakinolide (10 microM) on adhesive and mechanical properties of neutrophils. Pretreated cells were able to adhere and roll on immobilised platelets in a flow-based adhesion assay, but whereas untreated rolling cells became immobilised in seconds when chemotactic formyl peptide (fMLP, 0.1 microM) was superfused over them, the cells treated with jasplakinolide continued rolling. Pretreatment with jasplakinolide also blocked de novo expression of integrin CD11b and shape change which otherwise occurred in minutes after treatment with fMLP. Jasplakinolide directly caused actin polymerisation within neutrophils, evidenced by a marked increase in rigidity (resistance to aspiration into a 5 microm micropipette) and increase in association of actin with the Triton-insoluble cytoskeleton. These results indicate that rearrangement of the actin cytoskeleton regulates integrin-mediated adhesion of activated neutrophils, as well as their migration and mechanical properties.

Association state of human red blood cell band 3 and its interaction with ankyrin.

We have studied the association state of band 3, the anion channel and predominant transmembrane protein of the human red blood cell, and the anomalous stoichiometry and dynamics of its interaction with ankyrin, which acts as a link to the spectrin of the membrane skeletal network. Band 3 exists in benign nonionic detergent solutions as a dimer. Tetramer is formed irreversibly in the course of manipulations, particularly in ion-exchange chromatography. The dimer in solution binds ankyrin without self-associating. In ankyrin-free inside-out membrane vesicles and when incorporated into phosphatidylcholine liposomes, only some 10% to 15% of band 3 chains bind ankyrin at saturation. Moreover, in liposomes this was independent of protein:lipid ratio between 1:2 and 1:40. The bound fraction of band 3 remains with the detergent-extracted membrane cytoskeleton, but is released if the ankyrin has been cleaved with chymotrypsin before detergent treatment; thus, the attachment to the membrane cytoskeleton is entirely through ankyrin and not through other constituents such as protein 4.1. The ratio of band 3 to ankyrin in this complex implies that it consists of two chains of band 3 and one chain of ankyrin, at least after detergent extraction. The bound and free populations of band 3 exchange freely in the membrane. In the artificial liposome membrane binding of ankyrin to band 3 dimers cause association of the band 3 into higher aggregates, as seen in freeze-fracture electron microscopy. Successive manipulations of the red blood cell membrane, which are involved in the preparation of ghosts, of inside-out vesicles, and of inside-out vesicles stripped of peripheral proteins are accompanied by progressive aggregation of intramembrane particles, as judged by freeze-fracture electron microscopy. Thus the intramembrane particles are evidently stabilized in the intact cell by the peripheral protein network and the cytosolic milieu. Aggregation may be expected to limit the number of functional ankyrin binding sites. However, although extraneous ankyrin binds to the unoccupied binding site on the spectrin tetramers in intact ghost membranes, little or no ankyrin can bind to the unoccupied band 3 dimers in situ, perhaps by reason of occlusion of binding sites by the membrane skeletal network.