Search PubMed⌕ Search

Biomedical subjects

J Reichert

Publications and source records attributed to J Reichert.

At least 37 records · Page 2Linked to original sources
PubMed1992

Characterization of EntF as a serine-activating enzyme.

EntF is the enzyme responsible for serine activation during the biosynthesis of enterobactin (a cyclic trimer of N-dihydroxybenzoyl serine) in Escherichia coli. EntF has been overexpressed and purified to > 90% homogeneity. The enzyme has been shown to complement the entF- MK1 strain in the synthesis of 2,3-dihydroxybenzoyl serine derivatives and exhibits L-serine-dependent ATP[32P] pyrophosphate exchange activity with a Km for serine of 260 mM and a turnover number of 760 min-1. Release of PPi during incubation of EntF with serine and ATP was observed, but with a low turnover number of 1.0 min-1. These results suggested the presence of an enzyme-bound intermediate, which has been shown by gel filtration analysis to be (L-serine)adenylate.

Adenosine Triphosphate↗
PubMed1992

Purification and characterization of a tryptic peptide of Borrelia burgdorferi flagellin, which reduces cross-reactivity in immunoblots and ELISA.

In man the early immune response in Lyme disease is primarily directed against the endoflagellin antigen. Isolated flagellar protein of Borrelia burgdorferi suggests itself as a suitable test antigen. However, cross-reactivity between flagellins of B. burgdorferi, Escherichia coli, Bacillus subtilis, Proteus mirabilis and Salmonella typhimurium was demonstrated by immunoblotting and ELISA with polyclonal rabbit-hyperimmune-sera. Tryptic cleavage of recombinant B. burgdorferi 41 kDa flagellin, expressed in E. coli, produced a peptide fragment which was recognized exclusively by antisera to Borrelia species. This peptide was designated as the 14 kDa fragment due to its migratory behaviour in SDS-PAGE. The fragment is part of the variable region of the flagellin, as proven by amino acid sequencing. The flagellin peptide was employed as an antigen in ELISA and immunoblot assays, testing the polyclonal sera mentioned above. The specificity was superior to that obtained with the intact recombinant flagellin.

Amino Acid Sequence↗
PubMed1991

The LIM region of a presumptive Caenorhabditis elegans transcription factor is an iron-sulfur- and zinc-containing metallodomain.

The cysteine-rich LIM motif is highly conserved between invertebrates and mammals. This motif shows similarity both to proteins that bind zinc and to ferredoxins, which contain iron-sulfur clusters. Two tandem copies of the LIM motif are found in a number of presumptive transcription factors, including the protein product of the Caenorhabditis elegans cell-lineage gene lin-11. To investigate the possible metal-binding properties of the LIM region of the lin-11 protein, we expressed and purified a 151-amino acid peptide containing the tandem LIM motifs. The purified peptide binds both zinc (two atoms per protein molecule) and iron (as a redox-active iron-sulfur cluster, with four atoms of iron and four atoms of inorganic sulfide per protein molecule). These observations suggest that the LIM motif is a metallodomain that might function in a redox-sensitive regulation of transcription.

Amino Acid Sequence↗
PubMed1991

Biosynthesis of the Escherichia coli siderophore enterobactin: sequence of the entF gene, expression and purification of EntF, and analysis of covalent phosphopantetheine.

The sequence of the entF gene which codes for the serine activating enzyme in enterobactin biosynthesis is reported. The gene encodes a protein with a calculated molecular weight of 142,006 and shares homologies with the small subunits of gramicidin S synthetase and tyrocidine synthetase. We have subcloned and overexpressed entF in a multicopy plasmid and attempted to demonstrate L-serine-dependent ATP-[32P]PPi exchange activity and its participation in enterobactin biosynthesis, but the overexpressed enzyme appears to be essentially inactive in crude extract. A partial purification of active EntF from wild-type Escherichia coli, however, has confirmed the expected activities of EntF. In a search for possible causes for the low level of activity of the overexpressed enzyme, we have discovered that EntF contains a covalently bound phosphopantetheine cofactor.

Amino Acid Isomerases↗
PubMed1987

Proton concentration jumps and generation of transmembrane pH-gradients by photolysis of 4-formyl-6-methoxy-3-nitrophenoxyacetic acid.

Proton concentration gradients across membranes are important for many biological energy transducing processes. The kinetics of proton dependent processes can be studied by pH-jump methods in which protons are photochemically released. In the following we describe the synthesis and the properties of photolabile 4-formyl-6-methoxy-3-nitrophenoxyacetic acid, a 'caged proton'. The synthesis is based on vanillin, which is alkylated with chloroacetic acid to give a carboxylic acid (pK = 2.72). In a second step a nitro group ortho to the formyl group is introduced. Photochemical proton release occurs by a reaction mechanism analogous to the well known photochemical formation of 2-nitrosobenzoic acid from 2-nitrobenzaldehyde. The pK values of the photoproduct are 0.75 and 2.76, respectively, thus allowing the use of the compound in a wide pH-range. The quantum yield is 0.18, lower than in the case of the 2-nitrobenzaldehyde/2-nitrosobenzoic acid system (phi = 0.5). The release of the proton in a flash photolysis experiment occurs within less than 1 microseconds. The spectrum of photolabile compound has absorption maxima at 263 nm and 345 nm, respectively. Its permeability across a lipid bilayer membrane is very low (permeability coefficient Pd approximately equal to 10(-9) cm.s-1 at pH 8) so that transmembrane proton concentration gradients can be generated.

Glycolates↗
PubMed1985

Light-induced activation of the rod phosphodiesterase leads to a rapid transient increase of near-infrared light scattering.

The so-called AT-signal described here is a transient light-induced increase of the near-infrared scattering from isolated bovine rod outer segments (ROS). Freshly prepared ROS are permeabilized with 0.01% Triton X-100 immediately before measurement in the presence of 1 mM GTP. The signal amplitude is saturated when approximately 2 rhodopsin molecules out of 30 000 are photo-excited. The signal recovers rapidly (approximately 90 s) and can be repeated in a succession of flashes. The AT-signal can be prevented by pre-activation of the phosphodiesterase (PDE) enzyme cascade at various levels: either at the level of G-protein, using ALF4- in darkness or GTP gamma S plus light; or at the level of the PDE catalytic unit, using protamine as an activator. The light sensitivity and kinetics of the AT-signal are similar to published parameters of PDE activation. These data suggest that light-induced activation of the PDE is the key reaction for the generation of the signal. On the other hand, blocking of the catalytic cGMP binding site by isobutylmethylxanthine only slightly affects the signal. We propose that the AT-signal reflects a structural change linked to the transient removal of the PDE inhibitory subunit from the catalytic unit.

1-Methyl-3-isobutylxanthine↗
PubMed1985

Chemical probing of the light-induced interaction between rhodopsin and G-protein. Near-infrared light-scattering and sulfhydryl modifications.

Near-infrared light scattering and gel electrophoresis were used to monitor the interaction between rhodopsin and G-protein at photoreceptor disc membranes. It is found that substitution of one SH hydrogen at the G alpha-subunit by N-ethylmaleimide or thionitrobenzoate still allows dark binding of the G-unit to the membrane but blocks its light binding to rhodopsin. Repair of the modification restores the interaction of the proteins. Evidence is provided that substitution by the less bulky cyanide is also able to remove the hindrance to light binding. The rhodopsin-G interaction is reduced in proportion to bound N-ethylmaleimide, and the interaction kinetics remain constant over the measurable range. GTP/GDP exchange at the G-protein after interaction with rhodopsin does not reduce the accessibility of the relevant SH group. In contrast to the effect of G-protein modification, even exhaustive modification of rhodopsin has no effect on the dark and light binding of G-protein.

Animals↗
PubMed1984

Sulfhydryl group modification of photoreceptor G-protein prevents its light-induced binding to rhodopsin.

The effect of sulfhydryl modification on the light-induced interaction between rhodopsin and the peripheral GTP-binding protein of the photoreceptor membrane (G-protein) has been investigated by time-resolved near-infrared light-scattering and polyacrylamide gel electrophoresis. It has been found that the modification of rhodopsin with the alkylating agent N-ethylmaleimide (NEM) does not affect its light-induced interaction with the G-protein. Modification of G-protein with NEM or other sulfhydryl agents prevents any light-induced binding to rhodopsin. Dark-association of G to the membrane as well as the light-induced complex with rhodopsin (once formed) is insensitive to NEM.

Alkylation↗
PubMed1981

Kinetics and saturation of light-induced near-infrared scattering changes in isolated bovine rod outer segments.

The axial and radial shrinkage of bovine rod outer segments, monitored by near-infrared scattering changes (P-signal), is investigated in dependence on the intensity of the activating flash. Suspensions of axially oriented and randomly oriented rod outer segments were measured. In the latter case, axial and radial effects are superimposed to another. The following results are obtained: 1. The axial signal (Pa, tau approximately 10 ms) and the radial signal (Pr, tau = 40-100 ms), simultaneously measured on axially oriented rod outer segments, are similarly saturated with a half-saturation at a rhodopsin turnover of 3.5%. 2. For the saturation of the signal amplitude, measured on randomly oriented rod outer segments, a good fit is obtained by: P(rho) approximately 1 - e-beta rho, rho: relative rhodopsin turnover by the flash; beta is found in the range 23 less than or equal to beta less than or equal to 27 in all measurements. 3. The kinetics of the signal, also measured on the isotropic sample, depends on the rhodopsin turnover, the apparent time constant becoming faster with increasing turnover, The distortion of the signal cannot be fitted by a sum of exponentials with a fixed set of time constants. The signals from the isotropic sample are fitted by a phenomenological model. It introduces three first order processes concatenated in series; the first step is assumed as a rhodopsin transition inducing the two further processes. The distortion of the signals with increasing rho is then described assuming a rho-dependent quenching of this induction, according to the measured amplitude saturation. The time constants remain thereby unchanged. The fit yields the values in 2/k = 4, 11, and 45 ms with mean square deviations of 20%.

Animals↗
PubMed1980

History and trends in U.S. bound migration from a Mexican town.

The authors examine "historical trends in U.S. bound migration from a rural Mexican town. The data consist of detailed migration histories collected for all town residents in 1978. From these histories, successive migrant cohorts were constructed for the period 1940-1978." Changes in the age and sex composition of migrants over time are analyzed

Americas↗
PubMed1979

[The significance of x-ray-examination in early diagnosis of colon carcinoma compared with colonoscopy (author's transl)].

The medical literature and our own observations show the importance of x-ray examinations to diagnose colon carcinoma. The conventional barium enema examination without double contrast is not suitable for a general medical check-up. In contrast to the diagnostic value of the conventional procedure, the diagnostic accuracy of double contrast examination is comparable to that of the colonoscopy. In 1108 patients, endoscopically proven polyps smaller than 1 cm were detected in 61%, whereas the percentage for polyps bigger than 1 cm was 87. These results are achieved only in patients with a sufficient preparation. Two of 25 colorectal carcinomas were detected neither by endoscopy nor by the double contrast method.

Barium Sulfate↗