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M Hecker

Publications and source records attributed to M Hecker.

At least 217 records · Page 12Linked to original sources
PubMed1987

Products, kinetics, and substrate specificity of homogeneous thromboxane synthase from human platelets: development of a novel enzyme assay.

Homogeneous thromboxane synthase from human platelets converted prostaglandin H2 (PGH2) to thromboxane A2 (measured as thromboxane B2, TxB2), 12(L)-hydroxy-5,8,10-heptadecatrienoic acid (HHT), and malondialdehyde (MDA) in equimolar amounts under a variety of experimental conditions. PGG2 was transformed to MDA and corresponding 15- and 12-hydroperoxy products. PGH1 was enzymatically transformed into 12(L)-hydroxy-8,10-heptadecadienoic acid (HHD) and PGH3 into TxB3 and 12(L)-hydroxy-5,8,10,14-heptadecatetraenoic acid (delta 14-HHT) as earlier reported for solubilized and partially purified thromboxane synthase preparations. The ratio of thromboxane to C17 hydroxy fatty acid formation was 1:1 with PGG2, PGH2, and PGH3 as substrates. These results confirm and extend earlier observations with partially purified enzyme that the three products are formed in a common enzymatic pathway (Diczfalusy, U., Falardeau, P., and Hammarström, S. (1977) FEBS Lett. 84, 271-274). A convenient spectrophotometric assay for thromboxane synthase activity measuring the ultraviolet light absorption of the C17 hydroxy acid formed (e.g., HHT) was developed. The validity of the assay was determined employing specific inhibitors for thromboxane synthase. The substrate specificity of thromboxane synthase was determined using this assay. PGG2 and PGH3 showed Vmax and KM values similar to those of PGH2. The KM value of PGH1 was also identical to that of PGH2 but the Vmax value PGH1 was more than twice as high as that of PGH2.

Blood Platelets↗
PubMed1986

Spectral studies on structure-activity relationships of thromboxane synthase inhibitors.

Thromboxane A2 synthase is a cytochrome P450-type enzyme and its interaction with imidazole or pyridine-based inhibitors could be studied by absolute and difference spectroscopy with the solubilized as well as the purified enzyme. Nitrogenous bases shift the 418-nm Soret absorption by 4-6 nm to the red and among them the best inhibitors of enzyme activity showed a stoichiometric binding to the enzyme. The structural and energetic prerequisites for such high binding affinities were primarily the liganding of the basic nitrogen to the hemin but also the attachment of a hydrophobic carboxylic side chain to the active site at an about 1 nm distance from the nitrogen. In addition, the side chain seemed to be oriented almost parallel to the plane of the heme. If this geometry was changed, a decrease in affinity was observed and if the ligand binding was sterically hindered, a spectral shift to a five-coordinated complex absorbing at 390 nm occurred. This is best explained by the displacement of an endogenous oxygen ligand, presumably water, from the sixth coordination position of the heme. From these results it can be concluded that the inhibitors mimic the binding of prostaglandin H2 (PGH2) with its carboxylic group at the carboxyl side chain and the endoperoxide oxygen atom at C9 as previously reported. The methyl side chain of PGH2 does not seem to play a role in the formation of the enzyme-substrate complex.

Blood Platelets↗
PubMed1986

Synthesis of poly(A)+-containing RNA during growth in Escherichia coli relA+ and relA- strains.

There are very different results on the synthesis of poly(A)+-containing RNA in bacteria. We, therefore, studied the influence of growth and amino acid starvation on the synthesis of poly(A)+-RNA in a relA+ and relA- strains of E. coli. Only the relA+ strains is able to respond to an amino acid limitation by production of ppGpp which causes a strong reduction of stable RNA transcription. During growth we observed significant alterations of the percentage of [3H]-uridine labelled total RNA which bound to poly(U)-sepharose (% poly(A)-RNA). It was mainly influenced by drastic changes of the synthesis of non-polyadenylated stable RNA (rRNA, tRNA) during growth and, therefore, it did not reflect the actual synthesis of polyadenylated mRNA. An amino acid starvation induced in the relA+ strain a stronger and more rapid reduction of the transcription of non-polyadenylated RNA as well as of poly(A)+-RNA than in the relA- strain which did not produce ppGpp under these conditions. Therefore, we conclude that ppGpp inhibited not only the synthesis of the non-polyadenylated stable RNA but also that of poly(A)+-containing mRNA, although the latter was apparently less affected.

Amino Acids↗
PubMed1986

Physiological studies on pBR 322 DNA amplification in an Escherichia coli relA strain.

Amino acid limitation leads in E. coli relA cells which cannot synthesize guanosine tetraphosphate (ppGpp) under these conditions to an amplification of pBR 322 DNA. We previously proposed that ppGpp produced in E. coli relA+ cells subjected to amino acid limitation inhibits pBR 322 DNA replication (Hecker et al. 1983). In further experiments it was established that an E. coli relA strain shows plasmid amplification during amino acid limitation (arginine, threonine, leucine or histidine) only in the presence of sufficient concentrations of phosphate, ammonia and glucose. Plasmid amplification does not occur if ammonia or phosphate is depleted. We suggest that glucose, ammonia and phosphate are needed for nucleotide and deoxynucleotide synthesis as the essential prerequisite for plasmid amplification. The activity of beta-lactamase was determined as an indicator for the expression of plasmid-encoded genes. The enzyme activity remains on a low level during plasmid amplification because of arginine exhaustion. A remarkable increase in the activity of beta-lactamase was observed after resumption of growth of relA cells containing amplified plasmid DNA. The plasmid content decreased as the cells continued to grow. We found that plasmid amplification and expression of plasmid-localized genes are opposite reactions which do not occur at the same time.

Amino Acids↗
PubMed1986

Role of relA mutation in the survival of amino acid-starved Escherichia coli.

Amino acid-starved cells of Escherichia coli relA+, which contain a large number of glycogen particles, are able to survive in phosphate buffer for a longer time period than their relaxed counterparts. With regard to NH4+ starvation differences in the survival of both strains were not found. NH4+ starved cells of E. coli relA are able to synthesize glycogen but amino acid-starved cells of the relA strain are not. We suggest that the synthesis of glycogen triggered by guanosine tetraphosphate during amino acid starvation is responsible for the prolonged viability of the E. coli relA+ strain.

Amino Acids↗
PubMed1985

[Synthesis of alkaline phosphatase in a stringent and a relaxed strain of Escherichia coli under amino acid and phosphate limitation].

Synthesis of alkaline phosphatase in Escherichia coli is derepressed under phosphate starvation in the stringent strain CP78 as well as in its relaxed counterpart CP79. During limitation of phosphate as well as of amino acids a decrease of enzyme activity is observed, especially in the relaxed strain, which can not produce ppGpp under this conditions. After phosphate limitation synthesis of ppGpp is not stimulated and the kinetics of RNA synthesis is similar in both strains. We suggest that ppGpp is not directly involved in the regulation of gene expression during phosphate starvation.

Alkaline Phosphatase↗
PubMed1985

Transformation of Candida maltosa and Pichia guilliermondii by a plasmid containing Saccharomyces cerevisiae ARG4 DNA.

Saccharomyces cerevisiae, Candida maltosa and Pichia guilliermondii have been transformed by the plasmid pYe(ARG4)411, which contains the S. cerevisiae ARG4 gene inserted into pBR322. In all transformants argininosuccinate lyase as well as beta-lactamase were detected. The ARG+ phenotype of transformants is mitotically unstable. Closed circular pYe(ARG4)411 DNA was detected in transformant DNA preparations by hybridization to pBR322 DNA and by transformation of E. coli to ampicillin resistance.

Candida↗
PubMed1984

[Protein biosynthesis following heat shock in Bacillus subtilis].

Heat-activated spores of Bacillus subtilis synthesize during the early outgrowth special proteins which are absent from vegetative cells (Hecker 1983). Some of these outgrowth-specific proteins may be synthesized in response to heat activation of spores. In vegetative cells of B. subtilis synthesis of several proteins is markedly induced when temperature is shifted up from 30 to 44 degrees C. None of these putative heat shock proteins is synthesized during early outgrowth of heat-activated spores at 30 degrees C.

Bacillus subtilis↗
PubMed1984

[3H-thymidine incorporation following isoleucine limitation in stringent and relaxed controlled strains of Escherichia coli].

Stringent and relaxed strains of E. coli subjected to isoleucine starvation were examined by follow-wing the incorporation of 3H-thymidine into chromosomal DNA. After valine treatment to trigger an isoleucine deprivation (p)ppGpp is synthesized in the stringent strain only. Remarkable differences in the morphology of the amino acid starved cells of the stringent and relaxed strains can be observed. Upon isoleucine limitation 3H-thymidine incorporation into DNA is reduced in both strains, but this inhibition is remarkably delayed in the relaxed strain. Our result show that the reduction of chromosomal DNA synthesis during amino acid limitation occurs also without ppGpp, but in the presence of ppGpp this process is accelerated.

Chloramphenicol↗
PubMed1984

[Formation of plasmid pBR322 oligomers as depending on a tetracycline concentration].

Escherichia coli K12 strains containing the plasmid pBR322 often show varying contents of plasmid oligomers, in which the monomer units are arranged in tandem. When the concentration of the plasmid-selective antibiotic tetracycline in the medium becomes increased selection of cells containing largely higher oligomers occurs. The number of monomer units organized in the oligomers increases with tetracycline concentration. recA- mutants are unable to generate oligomers under the same conditions and show lower tetracycline resistance. This observations suggest a selective advantage of oligomer containing cells in the presence of tetracycline as a result of higher gene dosage. But E. coli cells transformed with monomers, dimers, trimers, as well as tetramers of pBR322 are characterized by roughly the same plasmid DNA content as well as plasmid coded beta-lactamase and resistance to tetracycline.

Culture Media↗
PubMed1983

[Replication and expression of plasmid pBR 322 during discontinuous culture of a stringent and relaxed Escherichia coli strain].

The E. coli strains CP78 and CP79 carrying the plasmid pBR 322 display similar growth kinetics in discontinuous culture. During limitation of amino acids the stringent strain CP78 is able to synthesize guanosine-5'diphosphate-3'-diphosphate (ppGpp) and guanosine-5'-triphosphate-3' diphosphate, but the relaxed strain can not produce highly phosphorylated guanosine nucleotides. During the logarithmic phase of growth both strains contain similar amounts of plasmid DNA. During amino acid starvation plasmid DNA is amplified in the relaxed strain only, whereas in the stringent strain the plasmid content per cell remains constant. In stationary phase cells of CP78 a higher activity of plasmid-coded beta-lactamase than in CP79 cells was detected. Furthermore, remarkable differences between both strains were observed in the composition of proteins derived from the periplasmic fraction and separated by polyacrylamide gel electrophoresis. Our results might indicate a negative control of pBR 322 DNA replication by ppGpp during amino acid starvation.

Amino Acids↗
PubMed1983

[Molecular biology of the germination of Bacillus spores].

The review deals with recent results and problems of gene expression during germination of Bacillus spores. Three problems were selected: 1. The activation of metabolism as a prerequisite for the synthesis of nucleic acids and proteins. 2. The activation of nucleic acid and protein synthesis during germination. 3. The gene expression programme of germinating spores. Using the highly sensitive two-dimensional polyacrylamide gel analysis three major classes of proteins were distinguished, depending on the time of onset and duration of their syntheses: a) proteins made throughout germination (main class), b) proteins whose synthesis started only after a lag phase and then continued throughout germination, and c) proteins which are synthesized only during the early phases of germination. The programme of protein synthesis is an indicator for the control of gene expression during germination. The regulation of expression of these major gene groups during spore outgrowth is discussed.

Bacillus↗
PubMed1983

Replication of pBR322 DNA in stringent and relaxed strains of Escherichia coli.

Synthesis of both chromosomal and plasmid (pBR322) DNA was measured in E. coli strains differing in their relA allele (relA+:CP78; relA:CP79). It was found that upon limitation of a required amino acid or after valine treatment to trigger a stringent response synthesis of pBR322 DNA was stimulated only in the relaxed strain and was inhibited in its stringent counterpart. The results suggest that replication of plasmid DNA is negatively controlled by the relA+ allele.

Chloramphenicol↗
PubMed1982

[Activation of protein biosynthesis in outgrowing spores of Bacillus subtilis].

The programme of protein synthesis as an indicator for the control of gene expression was examined during outgrowth of Bacillus subtilis spores. At various stages of outgrowth cells of Bacillus subtilis were labelled with 35S-L-methionine. Extracted proteins were separated on two-dimensional gels according to O'Farrell (1975). Three groups of proteins were synthesized during outgrowth: 1. During all stages of outgrowth a great number of "vegetative genes" is expressed. The programme of protein synthesis of the outgrowing cell is very similar to that of a vegetative cell. 2. Only a few proteins--probably the products of outgrowth-genes--are synthesized especially in outgrowing spores and turned off at different stages of outgrowth. 3. The synthesis of a minor group of vegetative proteins is triggered during different stages of outgrowth. In contrast to earlier assumptions (comp. Torriani and Levinthal 1967, Hansen et al. 1970, Galizzi et al. 1976) we suggest that only a small portion of the genome is activated during outgrowth as a dependent sequence. These results are discussed on the basis of earlier concepts about the regulation of outgrowth as a developmentally regulated gene expression programme.

Bacillus subtilis↗
PubMed1982

[Effect of nalidixic acid on the activation of RNA synthesis in outgrowing spores of Bacillus subtilis].

Outgrowth of B. subtilis spores depends on the action of DNA gyrase (comp. Matsuda and Kameyama 1980). Application of nalidixic acid (100 micrograms/ml) to dormant spores of Bacillus subtilis prevents the outgrowth. Application of nalidixic acid (100 micrograms/ml) during the early outgrowth phase (after a 20 min germination period) does not prevent, but only delay spore outgrowth. Germination of spores is not influenced. Nalidixic acid is an effective inhibitor of RNA synthesis in outgrowing spores, whereas vegetative cells are more resistant. Spores can grow out inspite of a remarkably reduced intensity of RNA synthesis. Nalidixic acid particularly inhibits the synthesis of stable RNA, probably that of ribosomal RNA. We suggest that DNA gyrase-catalyzed alterations in DNA structure are involved in the regulation of the gene expressional program of outgrowing B. subtilis spores.

Bacillus subtilis↗
PubMed1982

[Cell division and macromolecular synthesis in growing spores of a temperature sensitive filamentous mutant of Bacillus subtilis].

A temperature sensitive mutant of Bacillus subtilis SB 19, strain ts 33-6 was characterized. This strain grows at 46 degrees C (restrictive temperature) with reduced intensity without septation processes. Under restrictive conditions DNA- and RNA-synthesis are remarkably reduced. DNA, however, is synthesized continuously under restrictive conditions causing the formation of multinuclear filaments. Septation, induced at permissive temperature, is not prevented under restrictive conditions. That means that under restrictive conditions initiation of septation is blocked whereas formation of septa can be observed. Shift-up experiments have shown that the initiation of septation processes occurs at an early stage of cell cycle.

Bacillus subtilis↗