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J P Rosenbusch

Publications and source records attributed to J P Rosenbusch.

16 recordsLinked to original sources
PubMed1978

Paracrystalline arrays of protein-synthesis elongation factor Tu. Comparison with polymerized actin.

Homogeneous protein synthesis elongation factor Tu from Escherichia coli forms aggregates at high concentrations of ammonium sulfate which have a filamentous appearance in the light microscope. Electron microscopy of negatively stained preparations shows that these aggregates are paracrystalline, including three different forms. On the basis of analyses by optical diffraction, this polymorphism can be explained in terms of three different tubular foldings of the same basic two-dimensional surface lattice. This can be compared with that underlying the structure of actin filaments, thus providing a crucial test of the putative relationship between the elongation factor and actin [Rosenbusch, J. P. et al. (1976) J. Supramol. Struct. 5, 391-396]. The differences between the surface lattices, in conjunction with the negative results of sensitive immunochemical tests for possible cross-reactivities between the two proteins, suggest that any such relationship is very remote.

Actins↗
PubMed1978

Matrix protein from Escherichia coli outer membranes forms voltage-controlled channels in lipid bilayers.

Matrix protein from Escherichia coli was integrated into planar lipid bilayers. The incorporated protein generates aqueous channels across these membranes. Channels are induced irreversibly by voltage, and their number is proportional to the protein content of the membrane and stays constant over hours. They are uniform in size, with a diameter of about 1 nm and a single-channel conductance of 0.14 nS in 0.1 M NaCl. In addition to ionic conductance, the channels allow free diffusion of small, uncharged molecules. Channels assume either an open or a closed state. Membrane potentials shift this two-state equilibrium distribution in favor of closed channels, an observation that explains both negative resistance and inactivation at high potentials. Channels are not randomly distributed in the membrane but interact cooperatively within aggregates. The smallest entity inducible consists of three channels.

Cell Membrane↗
PubMed1977

Asymmetry of binding and physical assignments of CTP and ATP sites in aspartate transcarbamoylase.

The allosteric effectors of aspartate transcarbamoylase from Escherichia coli, CTP and ATP, associate with both the regulatory and the catalytic moieties of the enzyme. Studies with isolated, active subunits yield one binding site per regulatory dimer and one per catalytic trimer. Investigations of effector association with hybrid enzymes, containing either the three regulatory dimers or the two catalytic trimers in inactivated forms, indicate that the data obtained with isolated subunits can be used to analyze the binding patterns of these ligands to the native hexamer. Thus, the nonlinear Scatchard plots, characteristic of the binding of CTP and ATP to the native enzyme, can be interpreted in terms of three effector molecules associating with the regulatory subunits, and two binding to the catalytic moiety of the enzyme. Results with native protein in the presence of saturating concentrations of active site ligands support these assignments. The differences between the binding isotherms of CTP and ATP to the enzyme are due to their different affinities to the two types of subunits. The apparent half-of-the-site saturation of the regulatory moiety of aspartate transcarbamoylase supports the concept that this protein has a tendency to exist in an asymmetric state.

Adenosine Triphosphate↗
PubMed1977

Limited proteolysis of elongation factor Tu from Escherichia coli, Multiple intermediates.

Limited proteolysis of native elongation factor Tu (Mr 44 000) by trypsin occurs in at least three distinct steps. The first intermediate arises through cleavage at a site about 65 residues from the amino-terminal end of the protein. It is functionally active [Jacobson, G. R. & Rosenbusch, J. P. (1976) Biochemistry, 15, 5105-5110] and is partially protected from further degradation by the antibiotic kirromycin. The second step converts this intermediate to one of similar size (Mr 37 000) which now is partially inactivated. It is likely to be identical with the intermediate described by Arai et al. [(1976) J. Biochem. Tokyo, 79, 69-83]. In the third step, the partially inactive intermediate is cleaved without any apparent change in the functional properties tested. The resulting two trypsin-resistant fragments have molecular weights of 24 000 and 14 000, and remain associated under nondenaturing conditions. When either of these polypeptides, after isolation in 8 M urea, is allowed to renature, no significant reactivation of GDP binding is observed unless the isolated fragments are mixed before renaturation. These results show that the two fragments are structurally and functionally interdependent.

Escherichia coli↗
PubMed1977

Ultrastructure of a periodic protein layer in the outer membrane of Escherichia coli.

Matrix protein (36,500 daltons), one of the major polypeptides of the Escherichia coli cell envelope, is arranged in a periodic monolayer which covers the outer surface of the peptidoglycan. Although its association with the peptidoglycan layer is probably tight, the periodic structure of the peptidoglycan. Although its association with the peptidoglycan later is probably tight, the periodic structure is maintained in the absence of peptidoglycan, and is therefore based on strong protein-protein interactions. A detailed analysis of the ultrastructure of the matrix protein array by electron microscopy and image processing of specimens prepared by negative staining or by freeze-drying and shadowing shows that the molecules are arranged according to three fold symmetry on a hexagonal lattice whose repeat is 7.7 nm. The most pronounced feature of the unit cell, which probably contains three molecules of matrix protein, is a triplet of indentations, each approx. 2 nm in diameter, with a center-to-center spacing of 3nm. They are readily penetrated by stain and may represent channels which span the protein monolayer.

Bacterial Proteins↗
PubMed1976

Heterogeneity of sites in isolated catalytic subunits of aspartate transcarbamoylase.

Carbamoyl phosphate, a substrate of aspartate transcarbamoylase from Escherichia coli, binds with different modes of association in 3 sites in the unmodified catalytic subunits. Over a narrow pH range (6.6--8.0), positive, negative or no interactions are observed. Several substrate analogues also bind to 3 sites in the catalytic trimer. The association of pyridoxal phosphate, CTP and ATP, all competitive inhibitors of carbamoyl phosphate, exhibit negative interactions. Binding of succinate, an analogue of the second substrate, aspartate, is also characterized by heterogeneity. Dissociation constants to high and low-affinity sites differ by factors of 10-100. These observations clearly indicate that, although not observed kinetically, the active sites in the catalytic subunits of aspartate transcarbamoylase are heterogenous.

Adenosine Triphosphate↗
PubMed1976

Determination of ligand binding: partial and full saturation of aspartate transcarbamylase. Applicability of a filter assay to weakly binding ligands.

Carbamyl phosphate and succinate each bind to six sites in the hexameric aspartate transcarbamylase from Escherichia coli when both ligands are present in saturating concentrations. Their respective dissociation constants are 2.4 and 1400 muM. Positive homotropic interaction, shown earlier for the association of succinate with the enzyme in the presence of carbamyl phosphate (Changeux, J.-P., Gerhart, J.C., and Schachamn, H.K. (1968) Biochemistry 7, 513-538), is also found for carbamyl phosphate binding in the presence of succinate. Apparent half-of-the-sites saturation, previously described for carbamyl phosphate binding in the absence of succinate (Rosenbusch, J.P., and Griffin, J.H. (1973) J. Biol, Chem. 248, 5063-5066), also occurs when succinate binds to the enzyme in the absence of carbamyl phosphate. A second class of three low affinity sites for carbamyl phosphate could be detected in the enzyme when succinate was absent. These results indicate that aspartate transcarbamylase exists in an asymmetric state under several defined conditions. The results reported were obtained with a highly sensitive filter bonding assay, modified to allow the study of the protein-ligand interactions with dissociation constants in the millimolar range. The assay is described in detail. Its validity is demonstrated by the good correlation of the results obtained with those observed with independent methods.

Binding Sites↗
PubMed1976

Properties of a major protein released from Escherichia coli by osmotic shock.

A large fraction of a constitutively synthesized polypeptide, comprising 5% of the total Escherichia coli protein, is released when plasmolysed cells are subjected to osmotic shock into ice-cold water. Since the protein is not liberated by the conversion of cells to spheroplasts, it is not a typical periplasmic protein. A complex pattern of association with the cell envelope indicates that it is bound to this structure in vivo. Its susceptibility to trypsin and its interaction with specific antibodies vary with the type of preparations used. Based on these observations, we postulate a peripheral location at the inner surface of the plasma membrane. The protein has been purified to homogeneity from osmotic shock fluid. It has a mass of 44 000 daltons. Some of its physical and chemical properties have been investigated. Most remarkable are its strongly aggregating and adhesive characteristics and its precipitation by vinblastine and calcium ions. These unusual properties, its presumed location, and the observation that it is present in large amounts (approximately 70 000 molecules per cell) suggest a structural role for this protein.

Amino Acids↗
PubMed1976

Does a bacterial elongation factor share a common evolutionary ancestor with actin?

Protein synthesis elongation factor Tu from E. coli shares several physical, chemical, and functional properties with actin-like proteins. Limited tryptic degradation indicates that the two polypeptides have a similar molecular architecture. These observations suggest that they could have evolved from a common ancestor, although more information will be necessary to prove or disprove this hypothesis. A partial sequence, comprising 22 aminoacid residues from the aminoterminal end of the large tryptic fragment of elongation factor Tu is presented.

Actins↗
PubMed1976

ATP binding to a protease-resistant core of actin.

Actin can be cleaved by trypsin or chymotrypsin into a large, autonomous fragment with approximately 80% of the mass of the undegraded polypeptide. The protease-resistant cores obtained with either enzyme are very similar. Although the fragment does not bind calcium ions and fails to polymerize to the filamentous form of actin or to stimulate myosin adenosine triphosphatase (ATP phosphohydrolase, EC 3.6.1.3) activity, it retains the full capacity to bind ATP. This observation suggests that it represents an independent functional unit. Cleavage of globular actin with either trypsin or chymotrypsin occurs with half-times of 3 min, while that of filamentous actin proceeds with reaction half-times of 20 min for trypsin and nearly 2 hr for chymotrypsin. Denaturation and renaturation of the trypsin-resistant core shows that approximately 20% of the molecules refold to functional forms which indicates that the fragment can be considered as an independent unit of folding as well.

Actins↗
PubMed1975

Modification of Escherichia coli membranes in the prereplicative phase of phage T4 infection. Specificity of association and quantitation of bound phage proteins.

Reinfection of Escherichia coli with the bacterial virus T4 causes modifications of the properties of the host cell envelope during the preeplicative phase of the lytic cycle. These changes include altered densities cell enveloped and their subfractions, morphological modifications of membrane vesicles, and association of newly synthesized proteins with the host cell envelope. Polypeptide analysis by high resolution electrophoresis on polyacrylamide slab gels in dodecyl sulfate revealed that most of some 30 prereplicative phage-coded polypeptides are attached to this structure. Different means of cell disruption and selective extraction procedures, such as variations of ionic strength, removal of divalent cations, and the addition of chaotropic agents or detergents were used to study the characteristics of these attachments. Many proteins appeared to be artifactually absorbed or weakly bound to the envelope, Separation of cell walls from plasma membranes showed that all of the tightly bound proteins were associated withthe cell membrane fraction. The partitioning of phage proteins between the different fractions was monitored using 12 polypeptides which were identified as products of distinct phage genes. Of these, 8 were eliminated as potential membrane markers. Four polypeptides, the products of genes rIIA, rIIB, 39, and 52 were operationally defined as membrane proteins. The number of molecules of the 12 identified phage gene products, synthesized during a single lytic cycle, was determined. The results allowed the estimation of the concentration in the membrane of those proteins which were found to be quantitatively associated with that structure. Association of phage proteins with the cell envelope was found to be unaffected by mutations in any of the identified phage polypeptides.

Amino Acids↗
PubMed1975

Topology of binding sites for carbamyl phosphate in aspartate transcarbamylase from Escherichia coli. The use of pyridoxal phosphate as covalent probe.

Pyridoxal phosphate, a competitive inhibitor of aspartate transcarbamylase, binds to six sites in the catalytic and to twelve sites in the regulatory subunits of this hexameric protein. The properties of its association to the active sites of the enzyme are very similar to those observed with one of its substrates, carbamyl phosphate. It tightly binds to one half of the sites in the absence of succinate, an analogue of the second substrate. Since pyridoxal phosphate can be linked covalently to the protein by reduction, the distribution of the high affinity binding sites on catalytic trimers was studied after dissociation of modified holoenzyme. Electrophoresis of isolated subunits under non-denaturing conditions revealed four distinct bands, corresponding to trimers containing 0 to 3 pyridoxal phosphate derivatives. The distribution among the four species as a function of ligand concentration in the absence of succinate indicates that in the native oligomer, pyridoxal phosphate (and by extrapolation, carbamyl phosphate) binds to both catalytic trimers, rather than to three sites on a single subunit.

Aspartate Carbamoyltransferase↗