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R Schmid

Publications and source records attributed to R Schmid.

At least 91 records · Page 5Linked to original sources
PubMed1998

cDNA sequence and heterologous expression of monomeric spinach pullulanase: multiple isomeric forms arise from the same polypeptide.

The spinach pullulanase gene was cloned and sequenced using peptide sequences of the purified enzyme as a starting point and employing PCR techniques and cDNA library screening. Its open reading frame codes for a protein of 964 amino acids which represents a precursor of the pullulanase. The N-terminal transit peptide consists of 65 amino acids, and the mature protein, comprising 899 amino acids, has a calculated molecular mass of 99kDa. Pullulanase is a member of the alpha-amylase family. In addition to a characteristic catalytic (beta/alpha)8-barrel domain, it contains a domain, F, that is specific for branching and debranching enzymes. Pullulanase cDNA was expressed in Escherichia coli, and the purified protein was compared with the enzyme from spinach leaves. Identity of the two proteins was confirmed in terms of catalytic properties, N-terminal amino acid sequences and molecular masses. The pullulanase produced by E. coli showed the same microheterogeneity as the spinach leaf enzyme: it could be resolved into two substrate-induced forms by electrophoresis in amylopectin-containing polyacrylamide gels, and, in the absence of substrate, into several free forms (charge isomers) by isoelectric focusing or chromatofocusing. Rechromatofocusing of single free forms resulted in the originally observed pattern of molecular forms. However, heterogeneity of the protein disappeared on isoelectric focusing under completely denaturing conditions when only one protein band was observed. Post-translational modifications such as glycosylation and phosphorylation could be excluded as potential explanations for the protein heterogeneity. Therefore the microheterogeneity of spinach leaf pullulanase results from neither genetic variation nor post-translational modifications, but is a property of the single unmodified gene product. The different interconvertible forms of the pullulanase represent protein populations of different tertiary structure of the same polypeptide.

Amino Acid Sequence↗
PubMed1998

Mapping and identification of Corynebacterium glutamicum proteins by two-dimensional gel electrophoresis and microsequencing.

As a prerequisite for proteome analyses of Corynebacterium glutamicum separation of the cytoplasm and the membrane fraction was optimized and two-dimensional (2-D) gel electrophoresis was established. The resulting 2-D protein maps revealed over 1000 silver-stained protein spots separated by isoelectric point and molecular mass for cytoplasmic proteins and approximately 700 silver-stained spots for proteins of the membrane fraction. Proposing a mean size of 1 kbp per gene the complete C. glutamicum genome of 3 Mbp encodes 3000 different proteins; more than half of these can be located using the maps which are presently available. In this study 10 proteins were identified by N-terminal microsequencing, namely the 35 kDa antigen, antigen 84, ATP synthase subunits alpha, gamma and delta, cysteine synthase, elongation factor G and Ts, enolase, and rotamase. For seven sequences, corresponding proteins could not be identified. Additionally, two proteins were specifically detected by immunoblotting, a corynebacterial porin and the cytoplasmic protein threonine dehydratase. The methods and 2-D maps established in this study will be the basis for comparative studies of protein expression and a detailed proteome analysis of C. glutamicum.

Amino Acid Sequence↗
PubMed1998

Changes in protein synthesis as a consequence of heme depletion in Escherichia coli.

Two-dimensional gel electrophoresis and N-terminal amino acid sequence determination were used to compare the protein synthesis of exponentially growing Escherichia coli with heme-deficient cells. Mutation of the E. coli hemA gene encoding glutamyl-tRNA reductase resulted in the absence of detectable amounts of heme. As a consequence of heme deficiency, the induction of tryptophanase (trpA), citrate synthase (gltA), and aldehyde dehydrogenase (aldA) and the repression of enolase (eno) and phosphoglycerate kinase (pgk) were observed. All induced genes are under the control of the catabolite repressor protein Crp. The observed changes in gene expression as a consequence of heme depletion are discussed.

Aldehyde Oxidoreductases↗
PubMed1998

Comparison of three different experimental methods for the assessment of peripheral compartment pharmacokinetics in humans.

In many cases the concentration reached in a peripheral effect compartment rather than in plasma determines the clinical outcome of therapy. Therefore, several experimental approaches have been developed for direct assessment of drug kinetics in peripheral compartments. Particularly saliva sampling, skin blister fluid sampling, and in vivo microdialysis are frequently employed for measuring peripheral drug concentrations. However, data derived from these techniques have never been directly compared. In the present study, the tissue kinetics of theophylline were measured following single dose administration simultaneously in cantharides induced skin blisters, saliva and microdialysates of subcutaneous- and skeletal muscle- tissue and compared to plasma concentrations. Theophylline was administered to 9 healthy volunteers as an i.v. infusion of 240 mg. Mean ratio (AUCsaliva/AUCplasma) was 0.63 +/- 0.05, mean ratio (AUCblister/AUCplasma) was 0.69 +/- 0.12, mean ratio (AUCmuscle/AUCplasma) was 0.41 +/- 0.10, mean ratio (AUCsubcutaneous/AUCplasma) was 0.34 +/- 0.07. The time course of the concentration(peripheral)/concentration(plasma)-ratios showed that tissue concentrations obtained by microdialysis were closely correlated to free plasma levels, whereas saliva- and cantharides blister data overestimated the corresponding free plasma concentrations. It is concluded that microdialysis represents a reliable technique for the measurement of unbound peripheral compartment concentrations and is superior to saliva- and skin blister concentration measurements.

Adult↗
PubMed1998

Characterization of high affinity progesterone-binding membrane proteins by anti-peptide antiserum.

A chemically synthesized 15-mer oligopeptide derived from the N terminus of high affinity progesterone-binding membrane site(s) from porcine liver was used to generate site-specific antibodies. Western blotting experiments confirmed the specificity of the anti-peptide serum obtained. In further investigations this antiserum was used for the identification of the native progesterone-binding membrane protein complex that represents an oligomer with an apparent molecular mass of about 200 kDa. In temperature-induced Triton X-114 phase separation experiments combined with Western-blotting, the progesterone-binding site was identified as an hydrophobic (integral) membrane protein. In addition, in Western blotting analyses the antiserum reacted with the progesterone-binding or related proteins in membrane fractions from a wide array of different tissues in various species.

Amino Acid Sequence↗
PubMed1998

Soft tissue texture analysis by B-mode-ultrasound in the evaluation of impairment in chronic low back pain.

OBJECTIVE: Patients complaints in chronic low back pain do not always correspond to the results of X-ray and MRI investigations. The paraspinal muscle is very sensitive to all structural disorders of the spine. We used B-mode-ultrasound to investigate the change of the ultrasonic texture of the paraspinal lumbar muscle in people suffering from low back pain. METHODS: The quantitative texture analysis was defined by the ratio of the mean echo level of a unilateral cross-section (L2) of the paraspinal muscle and the level of a central area (L1) of the same cross-section (square 1 x 1 cm, defined by the ultrasonic device). In the lumbar spine there were five cross sections on either side starting at L1 through L5. In the first study healthy males were investigated (n=30). In the second study, 20 males with MRI controlled lumbar disc disorders were investigated. Finally screening was performed with a group of 40 male patients. The last investigation was a pathohistologic cross-check from the ultrasonic findings in five individual shortly after exitus. RESULTS: The ratio showed results >0.6 in the healthy group. The group with lumbar disc disorders showed at the affected levels ratios <0.5. All patients (with one exception) with a lumbar spinal history were detected. Pathohistology confirmed the ultrasonic findings. CONCLUSIONS: The ultrasonic texture analysis of the paraspinal lumbar muscle allows a rapid and easy to perform investigation for relating discal (actually all structural) disorders of the lumbar spine to the reported pain or disability.

Adult↗
PubMed1998

Direct assessment of peripheral pharmacokinetics in humans: comparison between cantharides blister fluid sampling, in vivo microdialysis and saliva sampling.

AIMS: Skin blister fluid sampling, in vivo microdialysis and saliva sampling are commonly employed as surrogates for the measurement of drug concentrations in peripheral compartments. Although expected to exhibit comparable results, data derived from these techniques have never been directly compared. Thus, the aim of the present study was to evaluate the comparability of these techniques. METHODS: Paracetamol, a model drug with low protein binding, was administered to seven healthy volunteers at an oral dose of 2000 mg. Subsequently, tissue kinetics were measured simultaneously in cantharides induced skin blisters, microdialysates of subcutaneous- and skeletal muscle-tissue and saliva and compared to serum concentrations. RESULTS: Mean ratio (AUCblister/AUCserum) was 0.88 (95% CI, 0.50-1.26), mean ratio (AUCmuscle/AUCserum) was 1.08 (0.67-1.49), mean ratio (AUCsubcutaneous/AUCserum) was 0.96 (0.41-1.51) and mean ratio (AUCsaliva/AUCserum) was 1.83 (1.39-2.27). In this study the concentration profiles after single oral administration differed among the three methods. The time course of the concentration (peripheral compartment)/concentration (serum)-ratios showed that cantharides blister and microdialysate concentrations closely paralleled serum levels. An equilibration period of less than 2 h had to be taken into account for blister measurements. In contrast, saliva concentrations were significantly higher than corresponding serum concentrations. CONCLUSIONS: Skin blister sampling and microdialysis closely mirrored corresponding serum concentrations and, thus, proved to be suitable techniques for the assessment of peripheral compartment pharmacokinetics. In contrast, saliva data overestimated the corresponding serum concentrations.

Acetaminophen↗
PubMed1998

Moderate activation of the apoptosis inhibitor bcl-xL worsens the prognosis in pancreatic cancer.

OBJECTIVE: To analyze the expression of the antiapoptotic gene bcl-xL in human pancreatic cancer and to correlate the results with clinical patient parameters. SUMMARY BACKGROUND DATA: Bcl-xL belongs to the bcl-2-related gene family and acts as a broad antiapoptotic factor to extend both normal and tumor cell survival. Recent findings indicate that tumor cell death induced by chemotherapy and radiotherapy is mediated by the activation of apoptosis. The fact that pancreatic cancer has an extremely malignant potential and that it is resistant to most anticancer treatment modalities suggests that mechanisms are activated that increase the viability of pancreatic cancer cells. METHODS: Seventy-four pancreatic cancer tissue samples were obtained from 32 female and 42 male patients undergoing surgery for exocrine pancreatic cancer. Normal human pancreatic tissue samples were available from 11 organ donors and 4 patients without pancreatic disease. The levels of bcl-xL mRNA expression were analyzed by Northern blot analysis. The exact site of bcl-xL mRNA transcription was determined by nonradioactive in situ hybridization. In addition, immunohistochemistry using specific polyclonal antibodies was used to localize the protein. RESULTS: Northern blot analysis indicated that, in comparison with the normal pancreas, bcl-xL mRNA was markedly overexpressed in 54% of the pancreatic cancer samples. Densitometric analysis revealed that pancreatic adenocarcinomas exhibited a mean 3.4-fold increase (p < 0.01) in bcl-xL mRNA levels in comparison with normal controls. With in situ hybridization, bcl-xL mRNA was found to be highly expressed in the cancer cells of tumor samples that exhibited increased mRNA expression by Northern blot analysis. Immunohistochemical analysis revealed bcl-x immunostaining in 88% of the cancer samples. Correlation of the molecular data with clinical patient parameters revealed that patients whose tumors exhibited no, faint, or weak bcl-xL expression lived significantly longer after tumor resection (median 12 months) than patients whose tumors exhibited moderate bcl-xL mRNA expression (median 5 months) (p < 0.05). However, 5 patients whose tumors exhibited intense bcl-xL mRNA expression tended to live longer (median 14 months). CONCLUSION: Enhanced expression of the antiapoptotic gene bcl-xL in pancreatic cancer and its association with shorter patient survival suggests that this factor may enhance the viability of pancreatic cancer cells in vivo. Inhibition of apoptotic pathways might be one of the reasons why pancreatic cancer shows only limited sensitivity to anticancer treatment.

Adult↗
PubMed1998

Identification of OmpT as the protease that hydrolyzes the antimicrobial peptide protamine before it enters growing cells of Escherichia coli.

The influence of extracytoplasmic proteases on the resistance of Escherichia coli to the antimicrobial peptide protamine was investigated by testing strains with deletions in the protease genes degP, ptr, and ompT. Only DeltaompT strains were hypersusceptible to protamine. This effect was abolished by plasmids carrying ompT. Both at low and at high Mg2+ concentrations, ompT+ strains cleared protamine from the medium within a few minutes. By contrast, at high Mg2+ concentrations, protamine remained present for at least 1 h in the medium of an ompT strain. These data indicate that OmpT is the protease that degrades protamine and that it exerts this function at the external face of the outer membrane.

Escherichia coli↗
PubMed1998

Electroencephalogram and clinical focalities in juvenile myoclonic epilepsy.

Partial seizures and asymmetric abnormalities seen on electroencephalogram (EEG) are infrequent in juvenile myoclonic epilepsy, but when present, can lead to a misdiagnosis of partial seizures. We report four patients with juvenile myoclonic epilepsy who had generalized spike or polyspike and wave discharges on EEG in addition to clinical and EEG evidence of focality. The clinical course and response to therapy was similar to that in typical juvenile myoclonic epilepsy.

Adolescent↗
PubMed1997

The NAD synthetase NadE (OutB) of Bacillus subtilis is a sigma B-dependent general stress protein.

The identification of sigma B-dependent general stress proteins is a useful strategy to understand the physiological role of the unspecific stress response in Bacillus subtilis. By N-terminal sequencing of B. subtilis stress proteins Gsp38 was identified as the NAD-synthetase (NadE). NadE was previously characterized as spore outgrowth factor B (OutB) conferring a temperature-sensitive spore outgrowth defective phenotype. Transcriptional studies showed that nadE is strongly induced in response to heat, ethanol and salt stress or after starvation for glucose in a sigma B-dependent manner. Two promoters are involved in transcriptional initiation, the sigma A-dependent upstream promoter contributes to the basal level during growth, whereas the sigma B-dependent downstream promoter is induced after different stress conditions.

Amino Acid Sequence↗
PubMed1997

A new glycosylated lipopeptide incorporated into the cell wall of a smooth variant of Gordona hydrophobica.

A cell wall component of a smooth variant of Gordona hydrophobica 1775/15 was isolated and purified, and its structure was determined by various chemical methods, including chemical synthesis of part structures, Edman degradation, gas chromatography/mass spectrometry analysis, matrix-assisted laser desorption ionization-post-source decay (MALDI-PSD) tandem mass spectrometry, and 1H and 13C NMR using one- and two-dimensional, homo- and heteronuclear correlated spectroscopy. The cell wall component was found to be a (mono-) glycosylated peptidolipid (GPL) consisting of a tridecapeptide interlinked by a beta-hydroxylated fatty acid (3-hydroxyeicosanoic acid, 20:0 (3-OH)) to form a cyclic lactone ring structure. The main fraction of GPL, for which we propose the name gordonin, was identified as 3-hydroxyeicosanoyl-L-seryl-L-phenylalanyl-L-seryl- L-seryl-D-alanyl-L -(O-beta-D-glucopyranosyl)-threonyl-glycyl-D-leucyl-L-valyl-L-seryl-L -phenylalanyl-glycyl-L-valyl lactone. The other GPLs constitute structural variations within the nature of the beta-hydroxylated fatty acid (20:0(3-OH) versus 22:1(3-OH)) in a ratio of about 1:0.9 as well as within one amino acid (D-Leu versus L-Phe) in about 30%. Sequence information was obtained in part by Edman degradation as well as gas chromatography/mass spectrometry analysis of di- and tripeptide fragments. However, the complete amino acid sequence could only be established by MALDI-PSD from the linear molecule, i.e. after ring opening of the lactone. In contrast, rough variants of G. hydrophobica 1775/15 lack these peptidolipids or synthesize them to a much lesser extent indicating that gordonin contributes significantly to the physicochemical character of the cell surface.

Actinomycetales↗
PubMed1997

Purification and properties of acetyl-CoA synthetase (ADP-forming), an archaeal enzyme of acetate formation and ATP synthesis, from the hyperthermophile Pyrococcus furiosus.

Acetyl-CoA synthetase (ADP-forming) is an enzyme in Archaea that catalyzes the formation of acetate from acetyl-CoA and couples this reaction with the synthesis of ATP from ADP and Pi (acetyl-CoA + ADP + Pi --> acetate + ATP + CoA) [Schifer, T., Selig, M. & Schonheit, P. (1993) Arch. Microbiol. 159, 72-83]. The enzyme from the anaerobic hyperthermophile Pyrococcus furiosus was purified 96-fold with a yield of 20% to apparent electrophoretic homogeneity. The oxygen-stable enzyme had an apparent molecular mass of 145 kDa and was composed of two subunits with apparent molecular masses of 47 kDa and 25 kDa, indicating an alpha2beta2 structure. The N-terminal amino acid sequences of both subunits were determined; they do not show significant identity to other proteins in databases. The purified enzyme catalyzed the reversible conversion of acetyl-CoA, ADP and Pi to acetate, ATP and CoA. The apparent Vmax value in the direction of acetate formation was 18 U/mg (55 degrees C), the apparent Km values for acetyl-CoA, ADP and Pi were 17 microM, 60 microM and 200 microM, respectively. ADP and Pi could not be replaced by AMP and PPi, defining the enzyme as an ADP-forming rather than an AMP-forming acetyl-CoA synthetase. The apparent Vmax value in the direction of acetyl-CoA formation was about 40 U/mg (55 degrees C), and the apparent Km values for acetate, ATP and CoA were 660 microM, 80 microM and 30 microM, respectively. The purified enzyme was not specific for acetyl-CoA or acetate, in addition to acetyl-CoA (100%), the enzyme accepts propionyl-CoA (110%) and butyryl-CoA (92%), and in addition to acetate (100%), the enzyme accepts propionate (100%), butyrate (92%), isobutyrate (79%), valerate (36%) and isovalerate (34%), indicating that the enzyme functions as an acyl-CoA synthetase (ADP-forming) with a broad substrate spectrum. Succinate, phenylacetate and indoleacetate did not serve as substrates for the enzyme (<3%). In addition to ADP (100%), GDP (220%) and IDP (250%) were used, and in addition to ATP (100%), GTP (210%) and ITP (320%) were used. Pyrimidine nucleotides were not accepted. The enzyme was dependent on Mg2+, which could be partly substituted by Mn2+ and Co2+. The pH optimum was pH 7. The enzyme has a temperature optimum at 90 degrees C, which is in accordance with its physiological function under hyperthermophilic conditions. The enzyme was stabilized against heat inactivation by salts. In the presence of KCI (1 M), which was most effective, the enzyme did not loose activity after 2 h incubation at 100 degrees C.

Acetic Acid↗
PubMed1997

The open conformation of a Pseudomonas lipase.

BACKGROUND: . The interfacial activation of lipases results primarily from conformational changes in the enzymes which expose the active site and provide a hydrophobic surface for interaction with the lipid substrate. Comparison of the crystallization conditions used and the structures observed for a variety of lipases suggests that the enzyme conformation is dependent on solution conditions. Pseudomonas cepacia lipase (PCL) was crystallized in conditions from which the open, active conformation of the enzyme was expected. Its three-dimensional structure was determined independently in three different laboratories and was compared with the previously reported closed conformations of the closely related lipases from Pseudomonas glumae (PGL) and Chromobacterium viscosum (CVL). These structures provide new insights into the function of this commercially important family of lipases. RESULTS: . The three independent structures of PCL superimpose with only small differences in the mainchain conformations. As expected, the observed conformation reveals a catalytic site exposed to the solvent. Superposition of PCL with the PGL and CVL structures indicates that the rearrangement from the closed to the open conformation involves three loops. The largest movement involves a 40 residue stretch, within which a helical segment moves to afford access to the catalytic site. A hydrophobic cleft that is presumed to be the lipid binding site is formed around the active site. CONCLUSIONS: . The interfacial activation of Pseudomonas lipases involves conformational rearrangements of surface loops and appears to conform to models of activation deduced from the structures of fungal and mammalian lipases. Factors controlling the conformational rearrangement are not understood, but a comparison of crystallization conditions and observed conformation suggests that the conformation of the protein is determined by the solution conditions, perhaps by the dielectric constant.

Amino Acid Sequence↗
PubMed1997

An internal FK506-binding domain is the catalytic core of the prolyl isomerase activity associated with the Bacillus subtilis trigger factor.

Two major families of peptidylprolyl cis-trans-isomerases, the cyclophilins and the structurally unrelated FK506-binding proteins (FKBPs), have been identified as cellular factors involved in protein folding in vitro. Here we report on the biochemical characterization of a second prolyl isomerase of Bacillus subtilis that was purified from a cyclophilin-negative (ppiB null) mutant and was shown to be the trigger factor (TigBS). N-terminal sequencing of 27 amino acid residues of the purified protein revealed 100% identity to the deduced sequence encoded by the tig gene, sequenced as a part of the B. subtilis genome project. The tigBS gene, located at 246 degrees on the genetic map upstream of the clpX and lonA,B genes, encodes an acidic protein (pI 4.3) of 47.5 kDa. Purified and recombinant TigBS-His proteins share the same substrate specificity and catalytic activity (Kcat/K(m) of 1.5 microM-1 s-1); both are inhibited by the macrolide FK506 with IC50 the range of 500 nM. We also demonstrate that the prolyl isomerase activity of TigBS is mediated by an internal domain of about 13 kDa (homologous to FKPB12) that represents the catalytic core of the trigger factor.

Amino Acid Isomerases↗
PubMed1997

First steps from a two-dimensional protein index towards a response-regulation map for Bacillus subtilis.

Data on the identification of proteins of Bacillus subtilis on two-dimensional (2-D) gels as well as their regulation are summarized and the identification of 56 protein spots is included. The pattern of proteins synthesized in Bacillus subtilis during exponential growth, during starvation for glucose or phosphate, or after the imposition of stresses like heat shock, salt- and ethanol stress as well as oxidative stress was analyzed. N-terminal sequencing of protein spots allowed the identification of 93 proteins on 2-D gels, which are required for the synthesis of amino acids and nucleotides, the generation of ATP, for glycolyses, the pentose phosphate cycle, the citric acid cycle as well as for adaptation to a variety of stress conditions. A computer-aided analysis of the 2-D gels was used to monitor the synthesis profile of more than 130 protein spots. Proteins performing housekeeping functions during exponential growth displayed a reduced synthesis rate during stress and starvation, whereas spots induced during stress and starvation were classified as specific stress proteins induced by a single stimulus or a group of related stimuli, or as general stress proteins induced by a variety of entirely different stimuli. The analysis of mutants in global regulators was initiated in order to establish a response regulation map for B. subtilis. These investigations demonstrated that the alternative sigma factor sigma B is involved in the regulation of almost all of the general stress proteins and that the phoPR two-component system is required for the induction of a large part but not all of the proteins induced by phosphate starvation.

Amino Acid Sequence↗