The nature of the stable noncovalent dimers of band 3 protein from erythrocyte membranes in solutions of Triton X-100.

Stable noncovalent dimers of band 3 protein from human erythrocyte membranes, in which state the protein is thought to exist after solubilization by the nonionic detergent Triton X-100, do not occur when purified batches of the detergent are used. Instead, the protein is in a monomer/dimer/tetramer association equilibrium. The stable dimers do appear, however, when the detergent has been 'aged'. They thus seem to be artifacts.

Analysis of chemically reacting systems by sedimentation-diffusion equilibrium.

A novel procedure to evaluate equilibrium constants from sedimentation-diffusion equilibrium data of analytical ultracentrifuge runs is proposed. It is shown that, by comparison of a reacting mixture at chemical equilibrium with a non-reacting but equally composed one, the sum of the mean concentrations of the reaction products can immediately be taken from optical absorption or from interferometric measurements. In most but not in all cases the use of stacked double-sector centerpieces is required.

Taxonomy of alkaliphilic Bacillus strains.

The DNA base compositions of 78 alkaliphilic Bacillus strains were determined. These strains were grouped as follows: DNA group A, guanine-plus-cytosine (G+C) content of 34.0 to 37.5 mol% (17 strains); DNA group B, G+C content of 38.2 to 40.8 mol% (33 strains); and DNA group C, G+C content of 42.1 to 43.9 mol% (28 strains). DNA group A includes the type strain of Bacillus alcalophilus Vedder 1934. DNA-DNA hybridization studies with DNA group A strains revealed that only one strain, strain DSM 2526, exhibited a high level of DNA homology with B. alcalophilus DSM 485T (T = type strain). Neither strain DSM 485T nor any other DNA group A strain is homologous to any of the Bacillus type strains with comparable base compositions. Six strains formed a distinct group containing three highly homologous strains and three strains exhibiting greater than 50% DNA homology.

Isolation and binding properties of leucyl-tRNA synthetase from Escherichia coli MRE 600.

A procedure for the large-scale isolation of leucyl-tRNA synthetase from E. cole MRE 600 is described: The enzyme was purified about 320-fold to homogeneity by precipitation with cetyl-trimethyl-ammonium bromide, two consecutive chromatographies on DEAE-cellulose and three on hydroxyapatite with an over-all yield of 4%. The molecular weight of leucyl-tRNA synthetase from E. coli MRE 600 was found to be 99 000 daltons. Bindings studies by ultracentrifugation and equilibrium partition showed that the enzyme binds leucine, leucyl-adenylate and tRNA Leu, each in a 1 : 1 stoichiometry. For ATP only a very weak binding to the enzyme could be observed, which did not allow the evaluation of the complex stoichiometry. The presence of ATP was not required for the binding of leucine or tRNA to leucyl-tRNA synthetase from E. coli MRE 600.

Affinity partitioning: Development of mathematical model describing behavior of biomolecules in aqueous two-phase systems.

A mathematical model describing the affinity partitioning of macromolecules in aqueous two-phase systems has been derived. The model was used to calculate binding parameters that were compared against values deter mined by means of ultracentrifugation and fluorescence titration. The mathematical model and its modifications were found to describe satisfactorily the partition behavior of macromolecules with differing numbers of binding sites. It could be shown that in solutions containing PEG the binding behavior of FDH is changed fundamentally. The dissociation constants of FDH with PEG-blue in the presence and absence of PEG are different.

Purification and properties of formaldehyde dehydrogenase and formate dehydrogenase from Candida boidinii.

Formaldehyde hydrogenase and formate dehydrogenase were purified 130-fold and 19-fold respectively from Candida boidinii grown on methanol. The final enzyme preparations were homogenous as judged by acrylamide gel electrophoresis and by sedimentation in an ultracentrifuge. The molecular weights of the enzymes were determined by sedimentation equilibrium studies and calculated as 80000 and 74000 respectively. Dissociation into subunits was observed by treatment with sodium dodecylsulfate. The molecular weights of the polypeptide chains were estimated to be 40000 and 36000 respectively. The NAD-linked formaldehyde dehydrogenase specifically requires reduced glutathione for activity. Besides formaldehyde only methylglyoxal served as a substrate but no other aldehyde tested. The Km values were found to be 0.25 mM for formaldehyde, 1.2 mM for methylglyoxal, 0.09 mM for NAD and 0.13 mM for glutathione. Evidence is presented which demonstrates that the reaction product of the formaldehyde-dehydrogenase-catalyzed oxidation of formaldehyde is S-formylglutathione rather than formate. The NAD-linked formate dehydrogenase catalyzes specifically the oxidation of formate to carbon dioxide. The Km values were found to be 13 mM for formate and 0.09 mM for NAD.

Interacting binding sites of isoleucyl-tRNA synthetase from Escherichia coli studied by equilibrium partition.

The binding of tRNAIIe to isoleucyl-tRNA synthetase in the presence of isoleucine or ATP was investigated using the equilibrium partition method. Isoleucine decreased the affinity of tRNAIIe for the enzyme by a factor of about 5. For the free standard energy of interaction a value of about 1 kcal/mol (4.2 kJ/mol) was calculated. ATP exhibits qualitatively the same effect as isoleucine. A binding of two molecules isoleucine per molecule of enzyme could not be demonstrated even in the presence of ATP and pyrophosphatase.

On the binding of aminoalkyl adenylates to isoleucyl-tRNA synthetase from Escherichia coli MRE 600.

The binding of nine aminoalkyl adenylates to isoleucyl-tRNA synthetase from Escherichia coli MRE 600 was measured and compared with the binding of the cognate amino acids. It was found that they bind rather tightly to the enzyme, the Kd's ranging from 3.1.10(-4) M with glycinol-AMP ester to 3.7.10(-9) M with L-isoleucinol-AMP ester. The binding is not affected by magnesium. It is shown that the free energies of binding of the esters can be calculated adding a constant contribution of the AMP-moiety of about - 4.1 (- 17) kcal/mole (kJ/mole) to the free energies of binding of the cognate amino acids, which we have reported earlier (19, 25, 26).

Influences of amino acid, ATP, pyrophosphate and tRNA on binding of aminoalkyl adenylates to isoleucyl-tRNA synthetase from Escherichia coli MRE 600.

Aminoalcohol-AMP esters, structurally related to the assumed intermediates of the amino acid activation reaction, behave as competitive inhibitors both with respect to the amino acid and ATP, when tested in the ATP-(32P) PPi-exchange or the tRNA-charging reaction. However, closer investigation of the binding of norvalinyl adenylate to isoleucyl-tRNA synthetase from Escherichia coli MRE 600 by an equilibrium method shows that only the amino acid is a true competitor, while ATP cannot displace the ester from binding. Pyrophosphate enhances the stability of the ester-enzyme complex whereas tRNA is without detectable influence.

ATP-analogues as substrates for the leucyl-tRNA synthetase from Escherichia coli MRE 600.

No analogous nucleoside triphosphate was found which acts as well as ATP in binding to and supporting catalysis of leucyl-tRNA synthetase from Escherichia coli MRE 600. However, there are numerous nucleotides which are able to replace ATP, but with lower efficiency. The 6-amino group of the adenine ring and the 2'-hydroxyl group of the ribose ring are essential for binding and catalytic activity. Alterations in the triphosphate moiety of the molecule can cause drastic changes in Km and/or Vmax, whereas alterations of the imidazole ring and substitutions at the 8-position of the adenine ring cause only minor losses of catalytic activity.

Influence of side-chain structure of aliphatic amino acids on binding to isoleucyl-tRNA synthetase from Escherichia coli MRE 600.

The binding of 10 isomeric alpha-amino-heptanoic acids, of two isomeric alpha-amino-octanoic acids, of isoleucinol and valinol, and of various alpha-hydroxy acids to isoleucyl-tRNA synthetase from Escherichia coli MRE 600 has been investigated by an ultracentrifuge method. It was found that the enzyme requires a primary amino group together with a not-too-small side chain as prerequisites for ligand recognition. Though the enzyme is absolutely specific for the L isomers, it is fairly tolerant against replacement of the carboxyl group of the natural substrate by more or less hydrophobic substituents. These findings can be explained in terms of Ogston's three-point-attachment model, if it is additionally assumed that there is no further space available in the binding region normally occupied by the alpha-hydrogen atom to accept other substituents which are as bulky as the carboxyl moiety. Similarly, the architecture of the binding region of the aliphatic side chain is discussed. Our measurements show that the free energy of binding strongly depends on the size and the structure of the remainder of the molecule. None of the isoleucine analogues employed is bound as tightly as the natural substrate itself, but there is a clear preference for side chains branched at the beta-carbon atom. The functioning of the side-chain recognition site is best understood by imaging a two-finger glove, of which one finger is tailored to a methyl and the other to an ethyl group. Both these fingers, together with the binding region for the glycine moiety and a steric barrier against a fourth substituent bulkier than hydrogen, are responsible for a high steric specificity towards the one side chain over its Cbeta epimer.

Solution structure of the cytoplasmic domain of the human immunodeficiency virus type 1 encoded virus protein U (Vpu).

The HIV-1-specific Vpu protein is an 81 amino acid class I integral membrane phosphoprotein that induces degradation of the virus receptor CD4 in the endoplasmic reticulum and enhances the release of virus particles from infected cells. Vpu is of amphipathic nature and consists of a hydrophobic N-terminal membrane anchor proximal to a polar C-terminal cytoplasmic domain. In our recent work, focussed on the structural analysis of the cytoplasmic tail, we established an alpha-helix-flexible-alpha-helix-turn model. Now we present the experimental solution structure of the Vpu cytoplasmic domain which has been elucidated in aqueous 50% trifluoroethanol solution by 2D 1H NMR spectroscopy, and restrained molecular dynamics and energy minimization calculations. Under these conditions the peptide, Vpu32-81, is predominantly monomeric and adopts a well defined helix-interconnection-helix-turn conformation, in which the four regions are bounded by residues 37-51, 52-56, 57-72 and 73-78. The presence of the cis isomer of Pro-75 manifests itself as a doubling of cross peaks of neighbouring residues in the 2D spectra. A related variant peptide, Vpum32-81, in which the Vpu-phosphoacceptor sites Ser52 and Ser56 were exchanged for Asn, adopts a very similar structure and, taken together, provides evidence that the second helix and the turn form a comparatively rigid region. Both helices are amphipathic in character, but show different charge distributions. In general the cytoplasmic region is N-terminally positively charged, passes through a region of alternating charges in helix 1 and then becomes negatively charged. The flexibility of the interconnection permits orientational freedom of the two helices. The motif found here is the first experimentally refined solution structure of the cytoplasmic domain of Vpu, and it is conceivable that these alpha-helices are important for a previously defined physical interaction with an alpha-helical Vpu-responsive element located within the cytoplasmic tail of CD4.

Thermodynamic studies on the specificity of L-isoleucine-tRNA ligase of Escherichia coli MRE 600. Calorimetric investigations on binding of amino acids and isoleucinol to the enzyme.

The association enthalpies, delta Ha, involved in the reactions between L-isoleucine:tRNA ligase (AMP-forming) from Escherichia coli MRE 600 (EC 6.1.1.5) and various amino acids have been determined calorimetrically in 50 mM potassium phosphate buffer, at pH 7.5, in the presence of 1 mM dithioerythritol. The delta Ha values for binding of L-isoleucine, L-leucine, L-valine, L-norvaline and L-2-amino-3S, 4-dimethyl pentanoic acid agree within the limits of experimental error in magnitude (3.7 +/- 0.9 kcal mol-1 or 15.5 +/- 3.8 kJ mol-1 at 25 degrees C) and variation with temperature (delta cp = -430 +/- 20 cal mol-1 K-1 or 1799 +/- 84 J mol-1 K-1). In view of the large differences in the equilibrium constants for the corresponding binding equilibria, the identical association enthalpies suggest that the enthalpic contribution to the Gibbs free energy of binding, delta Ga, cannot be responsible for the specificity of the interaction of the enzyme with the amino acids. It has rather to be inferred that the entropic term, delta Sa, is decisive in discriminating the correct amino acid. Analogous calorimetric binding studies on the reaction between L-isoleucinol and the enzyme suggest that the absence of the carboxyl group renders the association enthalpy more positive (by 4-5 kcal mol-1 or 16.7-20.9 kJ mol-1) with respect to that of the amino acids. The variation with temperature of the delta Ha values, however, practically parallels that of the amino acids.

Subunit interactions provide a significant contribution to the stability of the dimeric four-alpha-helical-bundle protein ROP.

Detailed thermodynamic and spectroscopic studies were carried out on the ColE1-ROP protein in order to establish a quantitative basis for the contribution of noncovalent interactions to the stability of four-helix-bundle proteins. The energetics of both heat- and GdnHCl-induced denaturation were measured by differential scanning microcalorimetry (DSC) and/or by following the change in circular dichroism in the far-UV range. Sedimentation equilibrium analyses were performed to characterize the state of aggregation of the protein. No intermediate species could be detected during thermal unfolding of the dimer in the absence of GdnHCl. Under these conditions ROP unfolding exhibits a strict two-state behavior. The thermodynamic parameters for the reaction N2<->2D are delta HD = 580 +/- 20 kJ.(mol of dimer)-1, delta Cp = 10.3 +/- 1.3 kJ.(mol of dimer)-1.K-1, and Tm = 71.0 +/- 0.5 degrees C. The corresponding Gibbs energy change of unfolding is delta GD degree = 71.7 kJ.(mol of dimer)-1 at 25 degrees C and pH 6. In the presence of 2.5 M GdnHCl, however, ROP dissociates into monomers at elevated temperatures, as the loss of the concentration dependence of Tm and the decreased molecular weight demonstrate. The corresponding transition parameters are delta HD (2.5 M GdnHCl) = 130 +/- 10 kJ.(mol of monomer)-1 and Tm = 51.6 +/- 0.3 degrees C. Isothermal unfolding studies at 19 degrees C using GdnHCl as denaturant yielded a Gibbs energy change of unfolding of 22.4 kJ.(mol of monomer)-1. This extrapolated value is 38% lower than the corresponding delta GD degree value of 35.85 kJ.(mol of monomer)-1 calculated from thermal unfolding for the monomer in the absence of GdnHCl, where the protein is known to be a dimer. These results suggest that subunit interactions are an important source of stabilization of the native four-helix-bundle structure of ROP.

Mycoplasma phocarhinis sp. nov. and Mycoplasma phocacerebrale sp. nov., two new species from harbor seals (Phoca vitulina L.).

A total of 120 mycoplasma strains were recovered from 97 of 265 diseased seals investigated during the seal epidemic in the North Sea and in the Baltic Sea in 1988. Mycoplasmas were isolated from the respiratory tracts (including lungs), hearts, brains, and eyes of the seals. Thirty strains were filter cloned and investigated for their morphological, biochemical, and serological characteristics compared with the characteristics of previously described species. The results of an indirect immunofluorescence test, a growth inhibition test, and an immunobinding assay showed that these strains belong to two new species, for which the names Mycoplasma phocarhinis and Mycoplasma phocacerebrale are proposed. M. phocarhinis (17 strains) did not ferment glucose or hydrolyze arginine but did reduce tetrazolium chloride and potassium tellurite and produced films and spots. M. phocacerebrale (13 strains) metabolized arginine but not glucose and produced phosphatase but did not reduce tetrazolium chloride and potassium tellurite. Both species lysed sheep erythrocytes but did not absorb sheep or guinea pig erythrocytes. The type strain of M. phocarhinis is strain 852 (= ATCC 49639), and the type strain of M. phocacerebrale is strain 1049 (= ATCC 49640).

Mycoplasma crocodyli sp. nov., a new species from crocodiles.

Organisms with the typical characteristics of mycoplasmas were isolated from joints and lungs of crocodiles. The results of growth inhibition tests and immunobinding assays showed that the 24 mycoplasma strains isolated were identical and distinct from previously described Mycoplasma, Entomoplasma, Mesoplasma, and Acholeplasma species. These organisms represent a new species, for which the name Mycoplasma crocodyli is proposed. M. crocodyli ferments glucose and maltose, does not produce films and spots, does not hydrolyze arginine, esculin, and urea, reduces tetrazolium chloride, and possesses phosphatase activity. It lyses and adsorbs bovine, ovine, and rabbit erythrocytes. Cholesterol or serum is required for growth. The optimum growth temperature is 37 degrees C. The G + C content of the DNA is 27.6 mol%. This organism causes exudative polyarthritis in crocodiles. The type strain of M. crocodyli is strain MP145 (= ATCC 51981).