Erythrocyte spectrin. Purification in deoxycholate and preliminary characterization

Abstract

Erythrocyte spectrin, isolated by aqueous extraction of erythrocyte ghosts, may be freed from contaminating membrane lipids and small amounts of other proteins by gel chromatography in 5 or 10 mM deoxycholate. The purified protein, in deoxycholate, is a mixture of monomers and dimers, both highly asymmetric molecules. The hydrodynamic properties of the dimer closely resemble those of muscle myosin, and spectrin and myosin also have similar circular dichroism spectra. The proportion of dimer to monomer in the purified protein varies from one preparation to another, an observation for which there is no simple explanation. In the absence of deoxycholate, spectrin associated beyond the dimer stage, possibly by loose end-to-end aggregation involving hydrophobic forces.

Self-assembly of synthetic collagen triple helices

“…Nonsedimenting baselines of 0.03 OD for fragment 1 (32 M), and 0.09 and 0.14 OD for fragment 2 (20 and 32 M), which were based on high-speed data, were included in the data analysis. The final choice of model was made by analyzing each data set as a combination of species from monomer to hexamer (monomer molecular masses used were 6,721 and 7,105 Da for 1 and 2, respectively) (38). Although the data did not identify a single model uniquely, data for (1) n were described best by a model with a combination of dimers and trimers, and data for (2) n were described best by a model with a combination of dimers and tetramers (Fig.…”

Identification of functional domains of human erythrocyte spectrin.

“…The composition and organization of the erythrocyte cytoskeleton has become the object of intense investigative effort (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16). A major component of the cytoskeleton and the predominant protein associated with the cytoplasmic surface of the erythrocyte membrane is spectrin (6,7).…”

Peripheral proteins and smooth membrane from erythrocyte ghosts. Segregation of ATP-utilizing enzymes into smooth membrane.

“…The evidence presented here does not distinguish between these two possibilities. When this evidence is considered together with the body of evidence which exists concerning the nature of spectrin (11,25,27), it appears most likely that membrane movement occurs as a result of a cooperation between the smooth membrane, which has the ATP-splitting enzymes to supply the energy, and the spectrin-actin, which has structural properties similar to those which occur in filaments found in other contractile systems.…”

“…The peripheral proteins have been widely thought of in terms of a structural and/or motile system lying on the inside of the membrane; many workers have emphasized the resemblance of spectrin to myosin (25); the presence of actin together with spectrin and the weak immunological cross-reaction between spectrin and myosin (27) have strengthened this analogy. However, the absence of any significant level of ATP splitting in the peripheral protein fractions, combined with the presence of a number of ATPases and other ATP-splitting activities on the membrane proper, raises interesting possibilities concerning the way in which energy can be put into the spectrin-actin system in order for movement to occur.…”

“…Spectrin has been said to resemble, to some degree, the actin-binding protein of rabbit pulmonary macrophages (30), and it resembles even more closely the actin-associated proteins of echinoderm sperm (32). The unusually large monomer molecular weight of spectrin, together with its circular dichroism, is similar to that of myosin (25), but it can be isolated in a form which forms only monomers and dimers in solution (11), unlike myosin, which forms much larger aggregates.…”