Acetohydroxyacid synthase: a proposed structure for regulatory subunits supported by evidence from mutagenesis.

Valine inhibition of acetohydroxyacid synthase (AHAS) plays an important role in regulation of biosynthesis of branched-chain amino acids in bacteria. Bacterial AHASs are composed of separate catalytic and regulatory subunits; while the catalytic subunits appear to be homologous with several other thiamin diphosphate-dependent enzymes, there has been no model for the structure of the small, regulatory subunits (SSUs). AHAS III is one of three isozymes in Escherichia coli. Its large subunit (encoded by ilvI) by itself has 3-5 % activity of the holoenzyme and is not sensitive to inhibition by valine. The SSU (encoded by ilvH) associates with the large subunit and is required for full catalytic activity and valine sensitivity. The isolated SSU binds valine. The properties of several mutant SSUs shed light on the relation between their structure and regulatory function. Three mutant SSUs were obtained from spontaneous Val(R) bacterial mutants and three more were designed on the basis of an alignment of SSU sequences from valine-sensitive and resistant isozymes, or consideration of the molecular model developed here. Mutant SSUs N11A, G14D, N29H and A36V, when reconstituted with wild-type large subunit, lead to a holoenzyme with drastically reduced valine sensitivity, but with a specific activity similar to that of the wild-type. The isolated G14D and N29H subunits do not bind valine. Mutant Q59L leads to a valine-sensitive holoenzyme and isolated Q59L binds valine. T34I has an intermediate valine sensitivity. The effects of mutations on the affinity of the large subunits for SSUs also vary. D. Fischer's hybrid fold prediction method suggested a fold similarity between the N terminus of the ilvH product and the C-terminal regulatory domain of 3-phosphoglycerate dehydrogenase. On the basis of this prediction, together with the properties of the mutants, a model for the structure of the AHAS SSUs and the location of the valine-binding sites can be proposed.

Micrometrically controlled surface modification of Teflon by redox catalysis: electrochemical coupling between Teflon and a gold band ultramicroelectrode

Carbon-fluorine bonds of Teflon (polytetrafluoroethylene, PTFE) can be reduced electrochemically with the purpose of modifying its adhesive and wetting surface properties by micrometrically controlled surface carbonization of the material. This can be performed adequately by redox catalysis provided that the redox mediator couple has a sufficiently negative reduction potential. The process is investigated kinetically with benzonitrile as the mediator and a gold-band ultramicroelectrode mounted adjacent to a PTFE block, though separated from it by an insulating micrometric mylar gap. For moderate fluxes of reduced mediator, the whole device behaves as a generator-collector double-band assembly with a constant current amplification factor. This is maintained over long periods of time, during which the carbonized PTFE zones extends over distances that are much wider than the slowly expanding cylindrical diffusion layer generated at the gold-microband electrode. This establishes that the overall redox catalysis proceeds through electronic conduction in the n-doped carbonized material. Thus, carbonization progresses at the external edge of the freshly carbonized surface in a diffusion-like fashion (dependence on the square root of time), while the redox-mediator oxidized form is regenerated at the carbonized PTFE edge facing to the gold ultramicroelectrode, so that the overall rate of carbonization is controlled by solution diffusion only. For larger fluxes of mediator, the heterogeneous rate of reduction and doping of PTFE becomes limiting, and the situation is more complex. A conceptually simple model is developed which predicts and explains all the main dynamic features of the system under these circumstances and allows the determination of the heterogeneous rate constant of carbon-fluorine bonds at the interface between the carbonized zone and the fresh PTFE. This model can be further refined to account for the effect of ohmic drop inside the carbonized zone on the heterogeneous reduction rate constants and henceforth gives an extremely satisfactory quantitative agreement with the experimental data.

Optical Properties of Ag-TiO(2) Nanocermet Films Prepared by Cosputtering and Multilayer Deposition Techniques.

Ag-TiO(2) nanocermet thin films, deposited for optical filtering applications by two sputtering techniques, codeposition and multilayer deposition, exhibit surface plasmon absorption in the spectral range 450-500 nm. The cosputtering technique induces a columnar growth, whereas multilayer deposition produces a more-random distribution of silver inclusions. Both films have large, flat silver grains at the air-cermet interface. An optical double-heterogeneous layer model based on the experimental morphological parameters of the films accounts well for their experimental transmittance, notably for extra absorption near 700 nm, which is attributed to a surface plasmon in the flat silver grains of the surface.

Optical properties of Ag -TiO 2 nanocermet films prepared by cosputtering and multilayer deposition techniques.

Ag -TiO2 nanocermet thin films, deposited for optical filtering applications by two sputtering techniques, codeposition and multilayer deposition, exhibit surface plasmon absorption in the spectral range 450 -500 nm. The cosputtering technique induces a columnar growth, whereas multilayer deposition produces a more-random distribution of silver inclusions. Both films have large, flat silver grains at the air -cermet interface. An optical double-heterogeneous layer model based on the experimental morphological parameters of the films accounts well for their experimental transmittance, notably for extra absorption near 700 nm, which is attributed to a surface plasmon in the flat silver grains of the surface.

Isolation and characterization of subunits of acetohydroxy acid synthase isozyme III and reconstitution of the holoenzyme.

The separately cloned large and small subunits of AHAS isozyme III from Escherichia coli have been isolated and purified. The essentially pure small subunit (17 kDa ilvH product) was obtained by a procedure exploiting its low solubility. The large, catalytic subunit (62 kDa ilvI product) was isolated by standard techniques, to > or = 95% purity. The large subunit has low catalytic activity relative to holoenzyme (approximately 5%) but shows similar substrate specificity and qualitatively similar cofactor dependence and inhibition by a sulfonylurea herbicide. Its activity is insensitive to valine, and the protein does not bind valine. The small subunit binds valine with Kd = 0.2 mM. Reconstitution of the holoenzyme from its subunits leads to a complex with the properties of the native protein, including valine inhibition of activity with Ki = 12 microM. Reconstitution titrations confirm the 1:1 stoichiometry of subunit assembly and a tendency to dissociation (about 50% dissociation near 0.1 microM subunit). Size exclusion HPLC indicates that either subunit alone is largely monomeric, and that assembly of the holoenzyme (two large + two small subunits, 150-160 kDa) requires FAD. On the basis of its homology with pyruvate oxidase and pyruvate decarboxylase, we suggest that the active sites of AHAS III are located at the interface of a dimer of catalytic subunits. Our experiments suggest that such a dimer is not stable except in the presence of the small subunits. The association of valine with sites on the regulatory subunits presumably influences the active sites by an allosteric conformational effect.

Subunit association in acetohydroxy acid synthase isozyme III.

Acetohydroxy acid synthase isozyme III (AHAS III) from Escherichia coli is composed of large and small subunits (encoded by the genes ilvI and ilvH) in an alpha 2 beta 2 structure. The large (61-kDa) subunit apparently contains the catalytic machinery of the enzyme, while the small (17-kDa) subunit is required for specific stabilization of the active conformation of the large subunit as well as for valine sensitivity. The interaction between subunits has been studied by using purified enzyme and extracts containing subcloned subunits. The association between large and small subunits is reversible, with a dissociation constant sufficiently high to have important experimental consequences: the activity of the enzyme shows a concentration dependence curve which is concave upward, and this dependence becomes linear upon the addition of excess large or small subunits. We estimate that at a concentration of 10(-7) M for each subunit (7 micrograms of enzyme ml-1), the large subunits are only half associated as the I2H2 active holoenzyme. This dissociation constant is high enough to cause underestimation of the activity of AHAS III in bacterial extracts. The true activity of this isozyme in extracts is observed in the presence of excess small subunits, which maintain the enzyme in its associated form. Reexamination of an E. coli K-12 ilvBN+ ilvIH+ strain grown in glucose indicates that AHAS III is the major isozyme expressed. As an excess of small subunits does not influence the apparent Ki for valine inhibition of the purified enzyme, it is likely that valine binds to and inhibits I2H2 rather than inducing dissociation. AHAS I and II seem to show a much lower tendency to dissociate than does AHAS III.

Properties of subcloned subunits of bacterial acetohydroxy acid synthases.

The acetohydroxy acid synthase (AHAS) isozymes from enterobacteria are each composed of a large and small subunit in an alpha 2 beta 2 structure. It has been generally accepted that the large (ca. 60-kDa) subunits are catalytic, while the small ones are regulatory. In order to further characterize the roles of the subunits as well as the nature and the specificities of their interactions, we have constructed plasmids encoding the large or small subunits of isozymes AHAS I and AHAS III, each with limited remnants of the other peptide. The catalytic properties of the large subunits have been characterized and compared with those of extracts containing the intact enzyme or of purified enzymes. Antisera to the isolated subunits have been used in Western blot (immunoblot) analyses for qualitative and semiquantitative determinations of the presence of the polypeptides in extracts. The large subunits of AHAS isozymes I and III have lower activities than the intact enzymes: Vmax/Km is 20 to 50 times lower in both cases. However, for AHAS I, most of this difference is due to the raised Km of the large subunit alone, while for AHAS III, it is due to a lowered Vmax. The substrate specificities, R, of large subunits are close to those of the intact enzymes. The catalytic activity of the large subunits of AHAS I is dependent on flavin adenine dinucleotide (FAD), as is that of the intact enzyme, although the apparent affinities of the large subunits alone for FAD are 10-fold lower. Isolated subunits are insensitive to valine inhibition. Nearly all of the properties of the intact AHAS isozyme I or III can be reconstituted by mixing extracts containing the respective large and small subunits. The mixing of subunits from different enzymes does not lead to activation of the large subunits. It is concluded that the catalytic machinery of these AHAS isozymes is entirely contained within the large subunits. The small subunits are required, however, for specific stabilization of an active conformation of the large subunits as well as for value sensitivity.

Absolute reflectivity measurements at 44.79 A of sputter deposited multilayer x-ray mirrors.

Multilayer x-ray mirrors have been deposited using a dc triode sputtering system, which incorporates an accurate method of thickness monitoring based on the dependence of the deposition rate on the target current. Thickness can be controlled with an accuracy of better than 0.1 A. High efficiency W-C and Ni-C multilayer mirrors have been synthesized and tested at 1.54-A (CuKoalpha) and 44.79-A (CKalpha). Absolute reflectivity measurements at lambda = 44.79-A (CKalpha) have been carried out. In this case the incident beam is previously polarized by a premonochromator equipped with a pair of parallel-plane multilayer mirrors fixed at an angle close to the Brewster (theta ? 45 degrees ). Thus the measured reflectivities are not affected by a progressive variation of the P-component.

Corrosion protection of metal implants by hard biocompatible ceramic coatings deposited by radio-frequency sputtering.

Most metals used for orthopaedic and stomatology implants and prostheses belong to the families of titanium or nickel-based and cobalt-based superalloys designed for advanced technology industries (e.g. space, aeronautic and nuclear industries). Ideal materials should be as insoluble and biologically compatible as possible. In the present paper the corrosion behaviour of Ni-Cr and Co-Cr alloys in biological media is evaluated through potentiodynamic polarization tests. It is shown that these metals exhibit some minor release of the component elements and degradation products, which may induce cytotoxic and allergic effects. The corrosion resistance of these alloys can be strongly enhanced by hard ceramic coatings deposited by radio-frequency sputtering. The biocompatibility of coated and uncoated metals is compared from differentiated human cell cultures.