Jennifer C. Pinder

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.

Conservation of the structure of ribosomal RNA during evolution

Abstract Limited digestion of ribosomal RNA with T 1 ribonuclease results in breakage of the polynucleotide chain at specific loci. The fragments are separated according to size with good resolution by polyacrylamide gel electrophoresis. The electrophoretic pattern is characteristic of the type of RNA and provides an index of the similarity or differences between closely related species. In this work we have examined the ribosomal RNA from several mammalian and microbial species. The results warrant the following general conclusions: (1) the structure of ribosomal RNA has differentiated appreciably in the course of evolution; (2) the over-all structure as opposed to the nucleotide sequence tends to be conserved during evolution; (3) there is no evidence of heterogeneity in ribosomal RNA population from a given species.

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.

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

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.

Integrity of polypeptide chains of spectrin from human erythrocytes

Abstract The suggestion that the high molecular weight erythrocyte membrane protein, spectrin, consists of subunits resistant to dissociation by both sodium dodecyl sulfate and 6 m guanidine hydrochloride has been reevaluated. By gel electrophoresis in dodecyl sulfate and thin-layer gel filtration in 6 m guanidine hydrochloride as well as in the much more powerful denaturant guanidine thiocyanate, and by sedimentation velocity in 6 m guanidine hydrochloride, the molecular weight emerges in the range 2–2.5 × 105. Denaturation profiles as a function of guanidine hydrochloride concentration, observed by circular dichroism, reveal that the spectrin conformation is unusually labile, with a mid-point for the unfolding process at a denaturant concentration near 1 m . Complete acylation with succinic anhydride, as well as reaction with citraconic anhydride, leaves the molecular weight unchanged even in 6 m guanidine hydrochloride. The possibility of measuring molecular weights of proteins by viscosity determination in trifluoroacetic acid was explored. A calibration with a series of proteins gave a Mark-Houwink plot with high scatter, which did not result from low precision of viscosity determination or protein degradation. Evidence is adduced from infrared spectra that the scatter is due to a variable degree of protonation of the polypeptide backbone in the acid, leading to altered hydrodynamic characteristics. Within the semiquantitive limits of the method, spectrin is not further disaggregated in trifluoroacetic acid. The presence of refractory noncovalent interactions and of covalent cross-links has been variously invoked to explain an apparent microheterogeneity in spectrin preparations. The results here described appear to render the former explanation untenable.

Separation of proteins by two-dimensional sedimentation and gel electrophoresis

Abstract A method is described for the two-dimensional separation of protein mixtures by sucrose gradient sedimentation and polyacrylamide gel electrophoresis. This technique gives high resolution of components, and makes possible the determination of approximate sedimentation coefficients for all the components in a mixture in a single experiment with very small amounts of material.