G.B. Ralston

Identification by 1H NMR spectroscopy of flexible C-terminal extensions in bovine lens α-crystallin

Two‐dimensional 1H NMR spectroscopy of bovine eye lens α‐crystallin and its isolated αA and αB subunits reveals that these aggregates have short and very flexible C‐terminal extensions of eight (αA) and ten (αB) amino acids which adopt little preferred conformation in solution. Total α‐crystallin forms a tighter aggregate than the isolated αA and αB subunit aggregates. Our results are consistent with a micelle model for α‐crystallin quaternary structure. The presence of terminal extensions is a general feature of those crystallins, α and β, which form aggregates.

A Retinal Ganglion Cell Neurotrophic Factor Purified from the Superior Colliculus

Abstract: Dissociated neonatal rat retinal ganglion cells can be maintained by the addition of an extract from the neonatal superior colliculus. This extract can support 95% of ganglion cells over 24 h in culture; in addition it promotes the expression of neurites from these cells. This report describes the purification of a neurotrophic factor from the superior colliculus which supports the survival of 80% of retinal ganglion cells over 24 h in vitro. The purification procedure involves a combination of dye‐ligand, anion‐exchange, and molecular sieve chromatography. The purified neurotrophic factor has a Stokes radius of approximately 200 Å using molecular sieve chromatography in the presence of a chaotropic agent. Sodium dodecyl sulfate‐polyacrylamide gel electrophoresis of the purified factor indicates that it is a glycoprotein that migrates with a molecular mass >400 kDa. Further characterization of this high‐molecular‐mass glycoprotein by enzymatic digestion demonstrated that it is a chondroitin sulfate pro‐teoglycan. This factor is clearly distinguishable from other neurotrophic factors that have an effect on retinal ganglion cells such as brain‐derived neurotrophic factor and fibroblast growth factor. The chondroitin sulfate proteoglycan from the neonatal superior colliculus is the first proteoglycan to be identified as a neurotrophic factor.

The temperature-dependent dissociation of spectrin

Spectrin, isolated from both human and bovine erythrocyte membranes, was found to exist predominantly as a tetramer of 960 000 daltons when prepared at 4 degrees C, but as a dimer of 480 000 daltons when prepared at 37 degrees C. The purified tetramer from both species could be dissociated to dimers by low ionic strength incubation at 37 degrees C.

Physico-chemical characterization of the spectrin tetramer from bovine erythrocyte membranes

The tetramer of bovine spectrin has been purified and characterized in terms of its hydrodynamic and optical properties. (1) The molecular weight, from both sedimentation equilibrium and sedimentation velocity/diffusion measurements, is close to one million. (2) The hydrodynamic properties suggest a highly expanded but basically symmetrical molecule of Stokes radius 200 A. (3) Optical rotatory dispersion measurements indicate a high degree of order in the tertiary structure of the molecule. These results are not consistent with the assumption that is often made, that the spectrin molecule is a long fibrous rod.

Macromolecular crowding: effects on actin polymerisation

Dextran has been found to enhance the polymerisation of actin. This enhancement increases exponentially with increasing mass concentrations of dextran, in a manner that is consistent with excluded volume theory. Mathematical prediction of experimental results is difficult due to the fact that all participating species, namely F-actin, G-actin and dextran are best represented by differently shaped hard particles. Modelling dextran as a sphere of radius defined by an effective thermodynamic radius (Reff), we have predicted our experimental results to an acceptable degree, given the relative crudity of the model. The results imply that the highly crowded cellular environment may help to stabilise the filamentous actin network in vivo.

Effects of dextran on the self-association of human spectrin.

The self-association of human spectrin is enhanced in the presence of dextran. The equilibrium constant for association of two heterodimers to form a tetramer is increased by an order of magnitude in the presence of 20% dextran. The rate constant for association is also enhanced, while the rate constant for dissociation is almost independent of dextran concentration. The degree of enhancement of association is dependent only on the mass concentration of dextran; for a given mass concentration of dextran the effect is independent of dextran molecular weight. These effects are believed to be due to excluded volume phenomena, and a model is presented that realistically accounts for the effects. These results imply that the association of spectrin within the erythrocyte will be enhanced by the presence of hemoglobin.

The action of organic mercurials on the erythrocyte membrane

The solubilisation of proteins from erythrocyte membranes by treatment with organic mercurials has been studied with different species. The marked solubilisation previously reported for human membranes does not seem to be a general phenomenon. All of the other species examined showed less than 50% of the solubilisation shown by human membranes. The protein-solubilising effect seems to be dependent on hydrophobic mercury derivatives carrying a net negative charge. Uncharged compounds like phenylmercuric acetate blocked the effect, although N-ethylmaleimide and iodoacetamide did not. With the aid of radioactively labelled compounds, and of atomic absorption spectrophotometry, the proteins reactive towards the mercurials were identified. The major integral protein, band 3, was the major protein capable of binding the mercurial. Reaction with the mercurial appears to disrupt interaction of band 3 with bands 2.1 and 4.2, allowing dissociation of the cytoskeleton from the membrane. In addition, band 4.9 was also found to react with the mercurials, possibly resulting in disruption of the cytoskeleton.

Salt and temperature-dependent conformation changes in spectrin from human erythrocyte membranes

ORD and CD measurements of spectrin, in both the dimer and tetramer association state, indicate a high proportion of alpha-helix in this protein. At temperatures below 27 degrees C and in 0.1 M NaCl, the tetramer has an apparent helix content of 73% and the dimer, 68%. The conformation of both states is dependent on salt concentration and temperature. Low ionic strength solutions of spectrin display lowered sedimentation coefficients and a decreased apparent helix content, indicating perhaps a slight refolding and expansion of the molecule. In addition, spectrin in low ionic strength solutions undergoes a broad temperature-dependent transition spread from 20 to 50 degrees C, while in the presence of salt the transition is sharp and centered on 49 degrees C. The temperature-dependent changes in low ionic strength solutions appear to parallel the dissociation of tetramer to dimer.

The influence of salt on the aggregation state of spectrin from bovine erythrocyte membranes

Sedimentation velocity and gel filtration experiments have been performed with bovine spectrin over a wide range of neutral salt concentrations. Increasing salt concentrations tend to increase both the sedimentation coefficient of spectrin and the elution volume of the protein from 4% agarose columns. No conformation change can be detected by means of optical rotation measurements as the salt concentration is raised. The results are incompatible with the hypothesis that salt causes the aggregation of spectrin, but are consistent with the existence of marked charge effects operative at low salt concentrations. In support of the charge effect hypothesis, acidic groups have been detected on the agarose gels, and ion-exclusion behaviour on the column has been observed with other proteins of similar size.

A water-soluble Mg2+-ATPase from erythrocyte membranes

A ouabain-insensitive ATPase activity associated with the water-soluble proteins of the human and bovine erythrocyte membrane is demonstrated by means of activity-staining in polyacrylamide gels. The ATPase activity from both sources had an absolute requirement for Mg2+, activity becoming easily detectable at 0.2 mM Mg2+. At low Mg2+ concentrations added Ca2+ appeared to decrease the intensity of the ATPase stain. The activity is unaffected by monovalent cations, does not hydrolyse p-nitrophenyl phosphate and is not inhibited by 2 : 4 dinitrophenol. The ATPase has an apparent molecular weight of approximately 100 000 as determined by electrophoresis in acrylamide gels containing dodecyl sulphate.