W.B. Gratzer

Properties of brain spectrin (fodrin)

Fodrin, a protein from bovine brain, immunologically related to spectrin, is shown, unlike some other proteins of generally similar appearance in the electron microscope, to resemble spectrin closely in its most distinctive structural characteristic, the very high α‐helix content. Like spectrin, it is also insoluble below pH 5. One of the subunits only is phosphorylated by the cAMP‐independent red cell membrane kinase, that phosphorylates the smaller subunit of spectrin. Fodrin also forms a ternary complex with F‐actin and the third constituent of the red cell membranes cytoskeleton, protein 4.1. In the presence of 4.1 the interaction between fodrin and F‐actin is enhanced. It is surmised that fodrin plays an analogous functional role in neuronal cells to that of spectrin in the red cell.

Electrophoretic examination of native myosin.

Whereas it is firmly established that the light chains of myosins from different types of muscle within one species are different [l-3] , the evidence regarding the heavy chains is more tenuous. Examination of published amino acid compositions shows that the variation between data for different myosins is of the same order as those for the same material investigated in different laboratories. The only strong evidence for differences appears to be that of Huszar and Elzinga [4] , who have reported substitutions in the sequences of corresponding methylhistidine-containing peptides isolated from skeletal and cardiac muscle myosins. This approach is arduous and is of course limited to the detection of substitutions in small, highly localised parts of the chains, and gives little idea of the total extent of the overall differences, A related question is whether the myosin in a given muscle is homogeneous, and several, so far inconclusive, attempts have been made to determine whether heterogeneity exists, either between the two heavy chains of a single myosin molecule, or within the population of myosin molecules in one type of muscle (for discussion see Lowey [ 51). The problem has also now acquired a particular interest in regard to the biosynthesis of muscle proteins in embryos [6], and to the reported change in the nature of the myosin, so far only in terms of the light chains, after cross-innervation of fast and slow twitch muscles [7]. The simplest approach to the examination of heterogeneity is in general zone electrophoresis. In the case of myosin the electrophoretic studies so far described have involved either myosin fragments, such as heavy, meromyosin or subfragment-l [S] , or the denatured

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.

Binding of hydrophobic ligands to spectrin

Spectrin is the major protein of the red cell membrane cytoskeleton, a network, which covers the inner surface of the membrane and is responsible for maintaining the shape, stability and mechanical properties of the cell. The primary site of attachment of the cytoskeleton to the membrane is by way of an association between spectrin and a receptor protein, 2.1 or ankyrin [ 1,2], which is itself bound to the preponderant transmembrane protein, band 3 [3,4]. The mechanism by which the cytoskeleton acts to control the membrane properties is not clear; some evidence has been adduced in favour of a direct interaction between spectrin and the lipid bilayer, and it has been suggested [S ,6] that this interaction stabilises the membrane and preserves the asymmetric distribution of the phospholipids between the inner and outer leaflet [7]. No direct binding studies with dispersed phospholipids have been carried out, probably due to the technical problems that such measurements present, but there are several reports [8--131 of perturbations in the properties of phospholipid vesicles, (all, however, by crude spectrin, contaminated with other cytoskeletal proteins) and one [ 141 of a relatively high propensity of spectrin to penetrate phospholipid monolayers. The observed effects have been reported to be greatest with phosphatidylserine [6,14], which is a component present exclusively in the inner leaflet of the membrane [7]. Spectrin, in terms of its amino acid composition and indeed its solubility properties, is by no means a particularly hydrophobic protein. For example the ‘hydrophobicity index’, based on the free energies of transfer of the amino acid side chains from water to an organic solvent [ 151, is 880, which is similar to that of myosin and considerably lower than for

Proteolytic digestion patterns of spectrin subunits

Spectrin, which is the most abundant protein associated with the erythrocyte membrane, and is evidently involved in maintaining the shape of the cell [I], contains two kinds of subunit, tightly associated with each other [2], in equimolar proportions. These chains have approx. mol. wt 240 000 and 220 000. It has been suggested [3-61 that spectrin may resemble myosin, though the only direct argument for such a view is its reported immunological cross-reaction with the myosin of a smooth muscle [6]. In chemical terms, little is known about the relationship between the two spectrin subunits, and one possibility which must be considered is that one is derived by post-synthetic proteolysis of the other. To establish whether two species are derived from the same gene, originate from genes of common ancestry, or are totally unrelated, presents problems for proteins of such high molecular weight. Conventional peptide maps in particular will be expected to contain too many spots to permit of quantitative comparisons. Papain can be used to accomplish partial degradation of proteins into fragments in a desired size range, in the presence of the dissociating agent, sodium dodecyl sulphate (SDS) [7]. A useful variant of this procedure consists in performing the digestion on an electrophoretic zone cut from an acrylamide gel. This ensures that the starting material is pure, and obviates problems of isolation. The gel slice is then reintroduced as the sample on an acrylamide gel, and electrophoresis is performed as before. Breakdown patterns thus displayed can be compared with great precision. We describe here the results of such a comparison between the two spectrin subunits. We have also compared the patterns of peptides generated by cyanogen bromide cleavage [8]. The results show at once that the sequence of the two chains are t different, but there is clear evidence that they are nevertheless related, and therefore presumably share an ancestral gene. A comparison with myosin and with the cytoplasmic high molecular weight filamentous protein, filamin [9], is also shown.

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)

Polymerisation of G-actin by spectrin preparations: identification of the active constituent

The cytoskeleton of the mammalian erythrocyte consists primarily of spectrin, with smaller quantities of actin and two other proteins. There is evidence to indicate that the shape and other characteristics of the cell are controlled by interactions amongst these proteins, probably in response to phosphorylation [l]. Work by Tilney and Detmers [2], as well as earlier results from this laboratory [3,4], indicated that interactions can occur between muscle actin and spectrin preparations, obtained by extracting the erythrocyte membranes at low salt concentration. Our experiments led to the conclusion that addition of spectrin brought about polymerisation of the G-actin in a medium of relatively low ionic strength. The mechanism of this interaction presumably involves the ‘seeding’ of the actin, and is a kinetic, rather than a thermodynamic effect. It has long been known [5] that spectrin preparations resulting from the standard, low-ionic strength extraction procedure [6,7] are heterogeneous, and on gel filtration give a series of components, viz spectrin dimer and tetramer, in a ratio depending on the method of preparation [8,9], an oligomer, containing spectrin together with endogenous actin and two other proteins, one of them identifiable as protein 4.1 (defined in terms of the indexing system of Fairbanks et al. [ 1 l] ), and contaminants of lower molecular weight, such as monomeric, apparently denatured, actin and traces of haemoglobin. The

Spectrin in primitive erythrocytes

The high-molecular weight protein, spectrin, comprises about one quarter of the total protein complement of the human erythrocyte membrane. It is a complex of two types of chain with molecular weights of about 2.2 and 2.4 X lo’, which are present in equimolar proportions [ 11. The function of this protein is still uncertain, but there is considerable evidence [2] to link it to a contractile role, essential for the maintaince of cell shape and flexibility. The question then arises whether spectrin or a close counterpart exerts a similar control over the properties of other cell types. High-molecular weight proteins have certainly been found in many other cells [3,4], and electrophoretic components similar in appearance to the spectrin doublet have been observed (and tentatively identified with spectrin) in the periacrosomal material of Thyone sperm [S]. On the other hand, Hiller and Weber [6] have reported that spectrin is absent from a range of tissueculture cells. To date therefore the only unequivocal identification of spectrin is confined to mammalian erythrocytes. By gel electrophoresis in the presence of sodium dodecyl sulphate (SDS), the total erythrocyte membrane protein pattern is strongly conserved between the mammalian species so far examined [7-91, and the spectrin doublet is indistinguishably present in all of them, including the camel [lo], the cells of which are not discoid in shape. The immunological similarity of spectrins from several species has also been reported [8]. In attempting a further assessment of the degree

Interaction of myosin with monomeric matrix-bound actin

Li,tde is Yuown abo~ lhe haleraet]on between m--)nomea5~ art,in ,(G-a~t-,~) wiL~ ~lyo~d.Xl; ~G-selkxl WaS sa~d [ 1 ] no~ t,o neONate myosSn ATPas~, bu~ n~or,e re eenlly I2] a small activation ,has been reported. When ~ e 5onCe strength o f an ac~in--myosin mixture is ~n,c~,eased. the ATPase acti~,,5ly risen aharply as the a t / in polymerJses. Possible reasons are (5) thai ~ae Sal~ i~self induces a confo,rra~lion change in the acI}n, whi,eh in¢ieases both i~ ability to bind ~o myosin, and ~to ~sec,iate t,o the F-form; (ii) tha~ peaymeaisa~ion leads I o a new cen~olmationM ~tat,e. i.~e, traps the actin ;m a eonfors~:.a~ion, wMch imeraets more efficiently wJlh .nayo~m, or ,(i~5) lhat effective rny,~sin-birafdng ~ e s ,havotve more ~han one aeIN monomer . A ¢onforma~ionM d~fference between a¢~fin in the G~n,d F-allies is su:ggeared by a number ,of spectroscopic changes th.~* accompany ~ae po]ymer]salian [3--6]. To prepare the p-u~alive rno~ome~ a,c~ha in tlae F-conformation, we have .coupled G-a~iin to a Seph,~iose naalfi.x, so lhai when ,the ioz_ic strength is increased any ,consequent c, onfonnafional ,change cannol pr,ov,okz potym.er~sat~on. We de , eftbe here now ~onomeric sclin can be so ¢0upied io a column wilho~;t xhe loss ,of its functional .chame~erimie~ in p~t icu lar its ab]lily t,o bind its nuelemide ]~gand, and w,e show ~h~t the monomers are capable 0f bimting myosin o~ s nbfmgmenl-] {3-1) in a specific mariner, lhai the complex is dissociated by ATP, and that acfiva~i,on ~f nayosin ATPase is small or absen t

STRUCTURAL BASIS FOR THE HIGH ACTIVATION-ENERGY OF SPECTRIN SELF-ASSOCIATION

The association of spectrin hetero‐dimer (αβ) to the tetramer (α2β2, which predominates in the cell) is marked by an exceptionally high activation energy, so that the reaction does not proceed measurably in the cold. We have tested the hypothesis that this is due to intra‐dimer association between the α‐ and β‐chain ends, which must be broken before tetramers can form. Two mutant univalent spectrins with association defects at the α and β ends, respectively, and incapable therefore of intra‐dimer bonding, were found to associate rapidly with one another at 4°C. The bimolecular rate constant is greater than for the association of normal dimers by 6 orders of magnitude.