P. Johnson

Molecular weight and symmetry of the pyruvate dehydrogenase multienzyme complex of Escherichia coli

The molecular weight and polypeptide chain stoichiometry of the native pyruvate dehydrogenase multienzyme complex from Escherichia coli were determined by independent techniques. The translational diffusion coefficient (Do20,w) of the complex was measured by laser light intensity fluctuation spectroscopy and found to be 0.90 (±0.02) × 10−11m2/s. When this was combined in the Svedberg equation with the measured sedimentation coefficient (so20,w = 60.2 (±0.4) S) and partial specific volume (v = 0.735 (±0.01) ml/g), the molecular weight of the intact native complex was calculated to be 6.1 (±0.3) × 106. The polypeptide chain stoichiometry (pyruvate decarboxylase: lipoate acetyltransferase: lipoamide dehydrogenase) of the same sample of pyruvate dehydrogenase complex was measured by the radioamidination technique of Bates et al. (1975) and found to be 1.56:1.0:0.78. From this stoichiometry and the published polypeptide chain molecular weights estimated by sodium dodecyl sulphate/polyacrylamide gel electrophoresis, a minimum chemical molecular weight of 283,000 was calculated. This structure must therefore be repeated approximately 22 times to make up the native complex, a number which is in good agreement with the expected repeat of 24 times if the lipoate acetyltransferase core component has octahedral symmetry. It is consistent with what appears in the electron microscope to be trimer-clustering of the lipoate acetyltransferase chains at the corners of a cube. It rules out any structure based on 16 lipoate acetyltransferase chains comprising the enzyme core. The preparation of pyruvate dehydrogenase complex was polydisperse: in addition to the major component, two minor components with sedimentation coefficients (so20,w) of 90.3 (±0.9) S and 19.8 (±0.3) S were observed. Together they comprised about 17% of the total protein in the enzyme sample. Both were in slowly reversible equilibrium with the major 60.2 S component but appeared to be enzymically active in the whole complex reaction. The faster-sedimenting species is probably a dimer of the complex, whereas the slower-sedimenting species has the properties of an incomplete aggregate of the component enzymes of the complex based on a trimer of the lipoate acetyltransferase chain.

The spontaneous transformation reactions of myosin

Abstract 1. 1. The changes in sedimentation pattern, sedimentation pattern, sedimentation rates, partial specific volume, solubility, light-scattering, viscosity and actin-binding capacity which occur spontaneously in myosin solutions have been followed at 20° or 25° by comparison with control solutions at 0–4°. 2. 2. From an analysis of sedimentation diagrams it is shown that the appearance of components sedimenting faster than native myosin obeys second-order kinetics with respect to unchanged myosin up to 78% transformation. The sedimentation and light-scattering evidence confirm a suggestion by Holtzer that aggregation occurs through an initial side-to-side dimerisation. 3. 3. The solubility of the protein shows rapid, approximately first-order changes during transformation; a large salt-soluble and a smaller water-soluble fraction (approx. 10%) being produced. In addition, the specific viscosity is shown to increase and the sedimentation coefficients of the various components to decrease; the partial specific volume also changes during transformation. These effects are interpreted in terms of a progressive first-order opening up of the tertiary structure of the molecule simultaneous with the different stages of aggregation. 4. 4. The relation of the fractions found in spontaneously transformed samples to sub-units of myosin found by other means is discussed. There is shown to be a general agreement between the various methods, and it is postulated that the salt-soluble and water-soluble fractions contain genuine sub-units of the myosin molecule. In the former case, at least, these are liberated in a denatured form. Actin-combining activity is largely confined to the water-soluble fraction. 5. 5. It is shown that these sub-units are not directly related to the meromyosins. A possible structural relationship within the myosin molecule is suggested.

Sedimentation studies of fumarase

The effect of thiocyanate on the sedimentation of fumarase has been studied over a range of experimental conditions. It was found that the action of this anion occurred in two distinct phases, an immediate lowering of the sedimentation constant from 9.28 to 8.6 S, followed by a slower dissociation into two similar subunits. Under certain conditions aggregation also accompanied dissociation. These physical changes are discussed in the light of fluorescence polarization studies already reported and of changes in the catalytic properties of the enzyme under similar conditions.

An electrophoretic and ultracentrifugal study of boar seminal plasma

Abstract 1. 1. Two main protein components (or groups of components) have been shown to occur in boar seminal plasma and separation by salt fractionation (Na2SO4) has been described. 2. 2. Component A possesses a sedimentation coefficient of 2.3 S and an isoelectric point of 8.2. It is the more soluble, heat-coagulable, and contains an excess of basic amino acids as well as carbohydrate. 3. 3. Component B, which is well defined electrophoretically (isoelectic point 5.1), sediments as several distinguishable peaks with sedimentation coefficients up to 40 S. It is less soluble, not heat-coagulable, and also contains carbohydrate. 4. 4. It seems likely that the main protein components of the seminal plasma of the boar have their origin in the vesicular secretion.

The disaggregation of Tamm-Horsfall mucroprotein by acetic acid

Abstract 1. 1. With a view to elucidating further its substructure, Tamm-Horsfall mucoprotein has been examined physicochemically in 50% acetic acid solution, in which the sialic acid content was not significantly lowered after several hours. 2. 2. Such material gave a well-defined single boundary in the analytical ultracentrifuge with s 0 20, w = 3.7S, though on removal of the acetic acid, reaggregation occurred rapidly. In free-boundary electrophoresis in 50% acetic acid, single well-defined boundaries were observed in both limbs, and in starch-gel electrophoresis in urea-Tris-citrate buffer, material freeze-dried from acetic acid gave a single band only. Immunodiffusion of a solution in acetic acid gave a single precipitin line, apparently identical with that for the normal mucoprotein. 3. 3. The molecular weight as determined by the Archibald procedure was 94 000, as compared with 136 000 indicated for the minimal value from amino acid content and tryptic fingerprinting studies. 4. 4. Viral hemagglutination inhibitory activity in acetic acid was considerably reduced as compared with that in a normal aqueous solvent.

Sedimentation studies on polymerised actin solutions

Abstract 1. 1. Polymerised actin solutions contain two components (F 1 - and F 2 -actin) giving solution boundaries and a gel component (gel-actin). The proportion of the latter increases on aging, and on exposing the solution to ultrasonic vibration, in a manner similar to that known to occur at nearly isoelectric pH values. 2. 2. The extrapolated sedimentation coefficients of F 1 - and F 2 -actin are 40 S and 98 S, respectively. The value for F 1 -actin is independent of centrifugal field. 3. 3. Assuming F 1 -actin to consist of elongated linear molecules, their diameter is calculated as 62 ± 5 A , for an assumed axial ratio of between 50 and 200. It is shown to be likely that F 2 -actin is a side-to-side dimer of F 1 -actin. 4. 4. Gel-actin undergoes reversible depolymerisation, and combines with myosin to give an ATP-sensitive complex, similar to the “gel-component” of actomyosin systems. 5. 5. A small amount of non-polymerised material was always present in polymerised solutions. Its sedimentation-concentration dependence was anomalous, the sedimentation coefficient increasing with concentration.

Further characterization of the subunit of Tamm-Horsfall mucoprotein

Abstract The subunit of Tamm-Horsfall mucoprotein has a molecular weight of approx. 100 000. The amino acid analyses are in fair agreement with previously reported results, with the exceptions of those for alanine, half-cystine, and tryptophan residues. Endgroup analysis reveals tyrosine to be the only detectable amino-terminal amino acid. The existence of heterogeneous fragments resulting from performic acid oxidation suggests that the subunit is in turn composed of 3 or 4 peptide chains of similar size, connected by interchains disulfide bonds.

Bromoacetyl-L-carnitine: biochemical and antitrypanosomal actions against Trypanosoma brucei brucei.

One of the causative agents of the African Trypanosamiases, Trypanosoma brucei brucei is able to use high intracellular carnitine concentrations and a high carnitine acetyl transferase (CAT) activity to stimulate ATP production. This paper reports that a carnitine analogue, bromoacetyl-L-carnitine, is an irreversible inhibitor of CAT from T.brucei, non-competitively inhibits carnitine uptake by T.brucei and has a potent in vitro effect against T.brucei motility and infectivity. An in vivo action in T.brucei infected mice is also reported. These results represent a new area of investigation in the important search for new antitrypanosomal agents.

A comparative study of the glutamate dehydrogenases isolated from bovine and chicken livers

Abstract A comparative study of the glutamate dehydrogenases isolated from bovine and chicken liver has been made by sedimentation in the ultracentrifuge and by optical rotatory dispersion measurements. It became apparent that the very different tendencies of the enzymes to associate molecularly in solution was not reflected in the secondary structures as indicated by optical rotatory dispersion. In the latter respect the two enzymes show similar but by no means identical behaviour. The molecular weight of the active subunit of the chicken liver enzyme was found to be very similar to that of the bovine liver and dogfish liver enzymes (about 326 000). It was therefore concluded that the different tendencies to associate must be due to differences of fine structure beyond the reach of the physical methods used.