PubMed Health⌕ Search

Biomedical subjects

W B Gratzer

Publications and source records attributed to W B Gratzer.

At least 91 records · Page 5Linked to original sources
PubMed1978

Isolation of protein subunits by coupling to an insoluble matrix: analysis of interactions and application to G-actin.

A criterion has been devised for the assessment of intermolecular contacts between protein subunits coupled to a gel matrix. After reversible coupling by way of disulfide groups to Sepharose 4B, the system is exposed to a bifunctional reagent. The protein is then released by reduction, and the proportion of dimers and higher oligomers formed by cross-linking is determined by gel electrophoresis, followed by densitometry of the stained gels. Experiments were performed with G-actin coupled to dithio-2-dipyridyl Sepharose 4B. At low concentration of coupled protein, no species other than the monomer were obtained; under these conditions any intermolecularly cross-linked species would represent pre-existing associated states coupled to the matrix. At higher protein concentrations dimers and higher species progressively appear. Their proportion is much higher than predicted on the basis of a random statistical distribution of coupled molecules throughout the accessible internal volume of the matrix. It follows that protein-protein contacts can occur either because of high flexibility of the polysaccharide chains of the matrix, or because the protein is partly (but not wholly) present as a shell on the surface of the beads. With Affi-gel 10, which has N-hydroxysuccinimide ester coupling groups, similar experiments were performed, taking advantage of the degradation of the matrix under relatively mild acid conditions. In this case the degree of cross-linking was much lower than in the Sepharose 4B system at the same protein concentration. However, this medium proved unsatisfactory for the measurement of interactions of the bound actin with other muscle proteins present in the mobile phase. The results with Sepharose 4B support the validity of previous studies on interaction of monomeric actin with other muscle proteins.

Actins↗
PubMed1978

Binding of protoporphyrin and haemin to human spectrin.

Haemin and protoporphyrin IX, but not bilirubin, are extensively bound by human spectrin. The absorption spectrum of the bound haemin is indicative of coordination of the iron by nitrogenous ligands in the protein. The protoporphyrin IX generates difference spectra on binding, which change with ligand:protein ratio, showing the existence of at least two structurally distinct types of site. The binding of both ligands is complex, and may be cooperative. Binding isotherms, based on spectrophotometric titrations, are given. Haemin and protoporphyrin IX also bind strongly to erythrocyte ghosts. At ionic strengths near physiological we can find no evidence of binding of haemoglobin to spectrin, as judged by sedimentation velocity, and it appears that reported interactions of this nature represent only non-specific binding at low ionic strength.

Bilirubin↗
PubMed1977

Structural study of spectrin from human erythrocyte membranes.

Human erythrocyte spectrin prepared from fresh blood is a mixture of different association states. Depending on the manner of preparation, the two-chain dimer or the tetramer predominates. These forms are not in rapid thermodynamic equilibrium. The molecular weight of the dimer by sedimentation and diffusion and by light scattering is about 5 X 10(5). The frictional properties indicate a low or moderate asymmetry (axial ratio in the range 2-10), and from the angular dependence of light scattering intensity an upper limit of about 80 A can be set for the radius of gyration. The tetramer similarly has a moderate asymmetry. Electron microscopy reveals that the dimer is a compact, slightly elongated molecule, and that the tetramer probably consists of two parallel dimers. On increasing the concentration of solutions containing spectrin dimers, oligomers are formed, which are not rapidly dissociated on dilution. At very low protein concentrations (below about 0.05 mg/mL) there is evidence of the onset of a rapid dissociation equilibrium between dimers and single chains. Other physical properties of the spectrin have been measured. The size and shape of the spectrin molecule would seem to rule out any major physical resemblance to myosin.

Erythrocyte Membrane↗
PubMed1977

An investigation of heavy meromyosin-ADP binding equilibria by proton release measurements.

The interaction of magnesium-ADP with skeletal muscle heavy meromyosin has been studied by measuring the accompanying release of protons. Total pH changes of the order of 0.03 were involved, and measurements were performed with a discrimination of some ten-thousandths of a pH unit. At pH 8.0 and 25 degrees C about 0.5 mol of protons per mol of heavy meromyosin is released at saturation. A stoichiometry of binding close to 2 mol of ADP per mol of protein was found, with a binding constant, obtained from the proton release titration curve (pH 8.0, 25 degrees C), of 2 X 10(5) M-1. At 5 degrees C the release of protons per mole is slightly greater, and the binding constant is somewhat increased, reflecting a negative enthalpy of binding. Similar proton release behavior is observed in the presence of manganous ions in place of magnesium. The liberation of protons is thus unrelated to the temperature-dependent isomerization of myosin in the presence of substrate. Alkylation of a reactive thiol group (SH1) does not change the proton liberation at pH 8.0. From the pH dependence of proton release, the association constant of heavy meromyosin with magnesium-ADP at other pH values can be inferred and shows an appreciable rise as the pH increases. The pH-proton release profile also allows the pK of the ionizing groups perturbed by the ligand to be deduced. At least two groups ionizing above pH 7 and one below are involved. Their pK's in the unperturbed state are assigned as 8.5, 9.3, and about 6.6, respectively; they are displaced in the complex to about 8.0, 9.1, and 6.3. A relation to the pH-activity profile of myosin ATPase is indicated. The pH-proton release profile is somewhat changed when the SH1 group is alkylated. Measurements with potassium-ADP, in the absence of magnesium, show that at pH 8.0 there is no proton release but rather a sizeable proton absorption (about 0.5 mol of protons per mol of heavy meromyosin). The association constant derived from the titration curves (pH 8.0, 25 degrees C) is 3 X 10(4) M-1.

Adenosine Diphosphate↗
PubMed1976

The interaction of actin monomers with myosin heads and other muscle proteins.

The simplest interacting unit of actomyosin, viz., single myosin heads (subfragment 1) with actin monomers, has been studied at physiological ionic strength, by isolating the actin molecules from each other on a solid support. The interaction is characterized by a binding constant of 10(5) to 10(6) M-1 in the temperature range 4-30degrees C. It is endothermic with a standard enthalpy of 24 +/- 10 kcal mol-1, and a standard entropy of 110 +/- 40 eu. It is thus, like many protein-protein association processes, entropy-driven. Despite the high affinity of the association, which is comparable in its binding constant to that of subfragment 1 with F-actin, there is only very small activation of myosin ATPase. The ionic-strength dependence of the interaction shows unusual features. Binding of the proteins of the relaxing system to the monomeric actin was also examined: troponin binds both in the presence and absence of calcium ions, but neither tropomyosin nor the tropomyosin-troponin complex was found to bind significantly. Monomeric actin has also been examined as a function of ionic strength by spectroscopic methods; it appears that conformational differences between the G and the F state are the consequence of polymerization, and not of the change in ionic strength required to being the conversion about.

Actins↗
PubMed1975

Effects of specific chemical modification of actin.

G-actin has been nitrated with tetranitromethane in conditions that lead to the modification of one tyrosine residue. The reactive residue was found by earlier workers to be Tyr-69. The nitrated actin is conformationally similar to native G-actin, as judged by sedimentation velocity and circular dichroism analysis. A small proportion only is in the form of covalently linked dimers and trimers. The nitrated G-actin will polymerise to form filaments, indistinguishable in the electron microscope from those of native F-actin, but the polymerisation process is slower. Reduction of the nitrophenol group to the corresponding aminophenol leaves the properties of the protein in respect of polymerisation unchanged. When a dansyl group is introduced at the same point, however, the ability of the actin to polymerise is lost. The nitrated actin and its reduced counterpart will also bind heavy meromyosin, and the characteristic arrowhead formation of the bound molecules along the filaments can be seen in the electron microscope. Neither of the modified F-actins, however, significantly activates or inhibits the myosin ATPase activity. The fluorescence of nitrated actin is strongly quenched through the presence of the nitrophenol chromophore. In soluble complexes with heavy meromyosin the fluorescence is indistinguishable from the sum of the separate contributions of the two protein components. There is thus no measurable excitation transfer between any tryptophan residues on the myosin heads, such as that inferred to be present in the ATPase site, and the nitrotyrosine in position 69 of the actin sequence. Implications of this observation are considered in relation to the different interaction sites in myosin and in actin. The activation of heavy meromyosin ATPase by copolymers containing actin and nitroactin in different proportions has been measured, and is not proportional to the fraction of native actin. The results are consistent with the view that the function of actomyosin depends on the interaction of the myosin heads with more than one actin subunit.

Actins↗
PubMed1975

Properties of the high-molecular-weight protein (spectrin) from human-erythrocyte membranes.

The high-molecular-weight protein component from human erythrocytes has been isolated and its solubility properties studied. In physiological solvent conditions the spectrin is not aggregated and is unaffected, both in hydrodynamic properties and conformation, as judged by circular dichroism and intrinsic fluorescence, by the addition of calcium ions. When the pH is decreased an opalescence first sets in, which corresponds to an associated fibrous state of the protein, and when a critical pH is reached precipitation ensues. The precipitation profile is characterised by extreme sharpness, of the kind observed in the phase separation of polyacid-polybase mixtures or of polyampholytes. The addition of calcium ions displaces this precipitation edge towards higher pH. Sodium ions have a similar but smaller effect. The position of the profile is significantly displaced in aged spectrin preparations, or those from frozen erythrocyte ghosts. Fresh preparations of spectrin consist predominantly of a component sedimenting at 9.7 S, with a minor component at 4.4 S (and traces of higher aggregates). The pattern is independent of the ionic strength, or of the presence or absence of calcium ions. The proportion of the small component increases with time, and in spectrin preparations from frozen ghosts it invariably predominates. At low concentrations of guanidine hydrochloride the larger component gives place progressively to the smaller, and vanishes completely when the concentration of the denaturant reaches 1 M. The two components coexist at concentrations below this, and are not in rapid interconversion equilibrium. On recovery of the protein from the guanidine hydrochloride by dialysis, the original pattern of two components is regained. On the other hand the larger component is not found in the material recovered from guanidine hydrochloride solutions of preparations that contain only the small component at the outset. The recovered spectrin is similar to the starting material in its circular dichroism, in its pH-precipitation profiles, and the manner in which the latter is modified by calcium ions. Molecular weight determination by sedimentation equilibrium shows that the 4.4-S species has a molecular weight of some 230 000, which is also the value derived from the extrapolated sedimentation coeffiecient in 6 M guanidine hydrochloride, and thus corresponds to single chains (of which two or more species are resolved in acrylamide gel electrophoresis in the presence of sodium dodecylsulphate); the 9.7-S species, which characterises what is evidently the native state of the extracted spectrin, is found to be a dimer. The frictional coefficients of the monomer and dimer are appreciably different. That of the dimer is compatible with a somewhat asymmetric structure, but by no means to the extent expected for a myosin-like or paramyosin-like molecule.

Blood Proteins↗