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Biomedical subjects

H Kleinkauf

Publications and source records attributed to H Kleinkauf.

At least 73 records · Page 4Linked to original sources
PubMed1983

Gramicidin S synthetase. Stability of reactive thioester intermediates and formation of 3-amino-2-piperidone.

The reactive thioester complexes of gramicidin S synthetase with substrate amino acids and intermediate peptides are slowly hydrolyzed in neutral buffer solutions under mild conditions. Fully active enzyme is recovered. These processes are strongly accelerated by certain thiol protective agents. In the presence of 1 mM dithioerythritol the half-life times of these hydrolysis reactions are in the range of 1-90 h at 3 degrees C. The thioester complex of gramicidin S synthetase 2 (GS2, the heavy enzyme) with the tripeptide DPhe-Pro-Val is distinguished by the highest stability of all these intermediates. A different decomposition pattern is observed for the thioester complex of GS2 with LOrn. Here 3-amino-2-piperidone (cyclo-LOrn) is formed in a rapid cyclization reaction. This product specifically blocks the activation center of GS2 for LOrn at the thioester binding site. All other activation reactions of gramicidin S synthetase are unaffected. A procedure for a specific labelling of the reaction centers of the multienzyme is outlined.

Amino Acid Isomerases↗
PubMed1983

Mechanism of depsipeptide formation catalyzed by enniatin synthetase.

Covalently bound intermediates of enniatin B synthesis could be isolated from enniatin synthetase by treatment with performic acid. By comparison with products of mild alkaline cleavage of authentic enniatin B they could be identified as the dipeptide D-2-hydroxyisovaleryl-N-methylvaline and the corresponding tetrapeptide. Synthesis of enniatins apparently proceeds via condensation of dipeptides. This was confirmed by the use of the substrate analogue isovaleric acid, which has shown to be a strong inhibitor for enniatin synthesis by formation of N-isovaleryl-N-methyl valine.

Anti-Bacterial Agents↗
PubMed1983

Studies of protoplast fusion in Streptomyces chrysomallus.

Conditions for the regeneration of cells from protoplasts of Streptomyces chrysomallus, a producer of the peptide antibiotic actinomycin, are described. Regeneration of fusion products was most efficient at 27-30 degrees C on regeneration R2 medium (Okanishi et al., 1974) containing 0.25 M-sucrose. The addition of phosphate (150-300 mg 1(-1) to the medium and incubation at 23 degrees C proved to be optimal for the regeneration of individual strains. Highest recombination frequencies after protoplast fusion were obtained by fusing protoplasts in the presence of 45% (w/v) polyethylene glycol 6000. With strains that produce no, or little antibiotic, protoplasts must be present in excess in fusion mixtures in order to overcome inhibition of regeneration by the antibiotic-producing partner.

Dactinomycin↗
PubMed1983

Cell-free synthesis of the depsipeptide beauvericin.

The enzymatic formation of the cyclodepsipeptide beauvericin was demonstrated in cell-free extracts from Beauveria bassiana. In analogy to the enniatin synthetase system formation of beauvericin is strictly dependent on the presence of the constituent amino and hydroxy acid, S-adenosylmethionine, and ATP/Mg2+. Synthesizing activity could be enriched about 12-fold by fractional ammonium sulfate precipitation. Besides the enniatin synthetase system this represents another example of the cell-free synthesis of a depsipeptide from eucaryotic origin.

Anti-Bacterial Agents↗
PubMed1983

A new polyene with antimycotic activity produced by a streptomycete from the rhizosphere of an indoor plant.

From the rhizosphere of a potted Sanseviera trifasciata, a Streptomyces griseus strain with a broad antifungal activity was isolated; in the soil of this plant, a sole hyphomycete, Aspergillus niger, was found in pure culture at 37 degrees C. This streptomycete produced a polyene, belonging to the pentaene group. It was found to inhibit, in vitro, the growth of all the 123 strains tested; the strains belonging to the genera Aspergillus, Cryptococcus, Candida, Trichophyton, Epidermophyton, Microsporum, Penicillium and Mucor. An antimycotic activity of this substance was also found to be present after i.v. administration to white mice infected with Cr. neoformans. Toxicological tests will have to show if this polyene could be considered as a candidate antimycotic drug. The soil of potted indoor plants is discussed as a possible habitat for streptomycetes with defined antifungal activities.

Animals↗
PubMed1982

Biosynthesis of carnosine and related peptides by glial cells in primary culture.

Synthesis of carnosine (beta-alanylhistidine) and related peptides by glial cells in primary culture could be demonstrated. After incubation with [3H]beta-alanine, the radiolabeled dipeptides could be isolated from the cell extract and the culture medium. With gamma-amino[3H]butyric acid, however, rapid degradation of the tracer without significant synthesis of homocarnosine (gamma-aminobutyrylhistidine) was observed. Aminooxyacetic acid, a known inhibitor of gamma-aminobutyryl:alpha-ketoglutarate aminotransferase, inhibits the degradation of gamma-amino[3H]butyric acid very strongly and of [3H]beta-alanine partially. After preincubation of the cells with this inhibitor, incorporation of gamma-amino[3H]butyric acid into homocarnosine and related peptides could be demonstrated.

Alanine↗
PubMed1982

Enniatin synthetase, a novel type of multifunctional enzyme catalyzing depsipeptide synthesis in Fusarium oxysporum.

Enniatin synthetase, a multifunctional enzyme catalyzing depsipeptide formation in Fusarium oxysporum was purified to 98% homogeneity as judged by analytical disc gel electrophoresis. The enzyme consists of a single polypeptide chain of a molecular weight of about 250 000. Similar to a number of peptide synthetases and to fatty acid synthetase the enzyme contains 4'-phosphopantetheine as a prosthetic group. Studies on substrate specificity revealed that the enzyme is capable of synthesizing enniatins A--C and also mixed-type enniatins containing more than one species of amino acid. A linear dependence of rate of enniatin synthesis on enzyme concentration was observed, indicating that depsipeptide formation is an intramolecular process. Omission of the methyl donor S-adenosyl-L-methionine resulted in the formation of unmethylated enniatins with a reaction rate of about 10% of that observed in the case of enniatins. Sulfhydryl-directed reagents generally had an inhibitory influence on enniatin synthesis. However, inhibition studies with iodoacetamide revealed that it behaves differently toward the hydroxy acid site(s) and amino acid site(s). This indicates the presence of chemically distinct thiol groups in the active sites for the two substrates.

Amino Acids↗
PubMed1982

Antibacterial action of gramicidin S and tyrocidines in relation to active transport, in vitro transcription, and spore outgrowth.

The cyclopeptide antibiotic gramicidin S or tyrocidine in concentrations of 2 to 4 mumol/mg of membrane protein inhibited the active transport of [3H]alanine and [3H]uridine in membrane vesicles isolated from Bacillus brevis and Bacillus subtilis. We used one analog of gramicidin S and two of tyrocidine A to study the relationship between peptide structure and antibacterial action as seen in inhibiting active transport and in vitro transcription and in delaying spore outgrowth. The data showed that [Ser2,2']-gramicidin S, in which the two ornithine residues were replaced by two serines, was at least 50 times less active antibacterially and gave a low response in transport inhibition and delay of spore outgrowth compared with the natural peptide. The antibacterial activity of [Val6]-tyrocidine A was twice lower than that of tyrocidine A, and it also showed a considerable reduction in transport and transcription inhibition. [Orn7]-tyrocidine A containing two ornithine residues in positions corresponding to those in gramicidin S was almost inactive in all functions tested. The correlation between peptide structure and activity is discussed.

Alanine↗
PubMed1982

Characterization of an intracellular serine protease from sporulating cells of Bacillus brevis.

Sporulating cells of Bacillus brevis ATCC 9999 produced a high level of an intracellular serine protease when grown in nutrient medium. The protease activity in the crude extracts of this strain appeared at hour 5 (t5) after the end of exponential growth and increased gradually during sporulation, reaching a maximum at t12 to t13. The enzyme isolated in a partially purified state showed a pH optimum between 7.3 and 9.0 and had an apparent molecular weight of about 60,000. The activity was completely inhibited by phenylmethylsulfonyl fluoride, diisopropyl fluorophosphate, EDTA, and ethylene glycol-bis(beta-aminoethyl ether)-N,N-tetraacetic acid. The protease possessed a high activity for azocoll and low activities for azocasein and 14C-labeled hemoglobin. It cleaved the cyclic decapeptide gramicidin S specifically at the peptide linkage between valine and ornithine and hydrolyzed the oxidized insulin B-chain mainly at peptide bonds 4-5 (Glu-His), 6-7 (Leu-CysSO3H), and 15-16 (Leu-Tyr). No catalysis of bond cleavage by the enzyme on a variety of small peptides or esters was detected. Unlike other Bacillus species, B. brevis ATCC 9999 grown in nutrient medium excreted no extracellular proteases.

Bacillus↗
PubMed1981

Specificity of a serum peptidase hydrolyzing thyroliberin at pyroglutamyl-histidine bone.

The substrate specificity of a serum enzyme which degrades thyroliberin (less than Glu-His-Pro-NH2) into pyroglutamic acid and His-Pro-NH2 has been investigated and compared with that of the pyroglutamyl aminopeptidase from calf liver. The latter enzyme has a broad specificity, causing rapid degradation of thyroliberin, pyroglutamyl beta-naphthylamide and luliberin. In contrast, the serum enzyme causes rapid stereospecific cleavage only of the pyroglutamyl-histidine bond of thyroliberin and closely related peptides. Compounds such as less than Glu-Ala, less than Glu-His and pyroglutamyl beta-naphthylamide, which are known substrates of the pyroglutamyl aminopeptidases (such as the liver enzyme), are not substrates of the serum enzyme, and inhibit it only poorly. Pyroglutamyl-containing peptides such as luliberin and neurotensin and thyroliberin analogues such as LLD-thyroliberin, less than Glu-His-Pro-NHCH3, less than Glu-His-Pro-Gly-NH2 and less than Glu-Phe-Pro-NH2 inhibit effectively the degradation of thyroliberin by the serum enzyme, but are not hydrolyzed by this enzyme. The high specificity of the serum enzyme implies a physiological function.

Aminopeptidases↗
PubMed1981

Characterization of chromosomal and membrane associated plasmid in Bacillus brevis ATCC 9999.

A covalently closed circular (ccc) DNA, with a weight of 44.7 x 10(6) daltons, has been isolated from Bacillus brevis ATCC 999 (a gramicidin S producer) and from the gramicidin S-negative mutant EB16. The ccc DNA in the case of the parent strain, is mainly (99%) attached to the chromosome and membrane fraction. A restriction enzyme map of the plasmid DNA was constructed for the enzymes SalI, SmaI and BamHI, which cleaved the plasmid DNA into two, two and six fragments respectively. Further digestion with the endonucleases EcoRI and HindIII cleaved the plasmid into 17 and 22 fragments.

Anti-Bacterial Agents↗
PubMed1980

Catabolism of thyroliberin by rat adenohypophyseal tissue extract.

Rapid fragmentation of thyroliberin (less than Glu-His-Pro-NH2) by rat adenohypophyseal tissue enzymes could be demonstrated. Based on the identification of the metabolic products and by the demonstration that the individual enzymatic reactions can be preferentially blocked by enzyme inhibitors, specific and sensitive biochemical tests could be developed in order to monitor the enzymatic activities after gel chromatographic fractionation of the tissue extracts. These findings are in agreement with the interpretation that the observed degradation of thyroliberin by hypophyseal tissue extracts may follow the proposed pathways. The primary enzymatic cleavage of thyroliberin is either initiated by the action of a 'thyroliberin-deamidating enzyme' (thyroliberin leads to less than Glu-His-Pro-OH + NH3), or by the action of a pyroglutamate aminopeptidase (thyroliberin leads to less than Glu + His-Pro-NH2). While the pyroglutamate aminopeptidase also catalyzes the subsequent degradation of deamidated thyroliberin (less than Glu-His-Pro-OH leads to less than Glu + His-Pro-OH), the enzymatic deamidation of His-Pro-NH2 is not catalyzed by the 'thyroliberin-deamidating enzyme; but by a post-proline dipeptidyl aminopeptidase. Hydrolysis of the common intermediary metabolite His-Pro-OH to the free amino acids is apparently catalyzed by a proline dipeptidase. In addition to these enzymatic events rapid cyclization of His-Pro-NH2 to histidyl-proline-diketopiperazine His-Pro could be observed. This reaction however is mainly due to the non-enzymatic intramolecular condensation reaction which is characteristic for proline-containing dipeptide derivatives. An enzymatic activity which catalyzes this reaction could not be observed when the enzyme fractions were tested. Enzymatic degradation of His-Pro by hypophyseal tissue extracts could also not be observed.

Aminopeptidases↗
PubMed1979

Biological role of gramicidin S in spore functions. Studies on gramicidin-S-negative mutants of Bacillus brevis ATCC9999.

Gramicidin-S-negative mutants of Bacillus brevis ATCC9999 have been isolated with a remarkly higher yield after ethidium bromide or acridine orange treatment, than after N-methyl-N'-nitro-N-nitrosoguanidine treatment. Four (MIV, Smr170, R5 and EB 16) of 38 isolated mutants were characterized with respect to the lesion in gramicidin-S-synthesizing activity. The mutants sporulate to the same extent as the parental strain except mutant Smr 170 which sporulates less. However, mutant spores were more heat-sensitive and possessed a reduced level of dipicolinic acid content. No significant difference was observed in the germination time of wild-type and mutant spores. All spores germinated after 80--110 min, but the outgrowth time was different: all gramicidin-S-negative mutants grew out immediately after germination whereas wild-type spores required a lag period of 9--10 h. When the mutants were allowed to sporulate in the presence of gramicidin S, the spores were found to be heat-resistant and their outgrowth postponed to the same period as the parent spores. The addition of gramicidin also eliminated the deficiency of dipicolinic acid. A new class of gramicidin-S-negative mutant, R5, which only activates L-valine and L-leucine, is described. A possible biological function of gramicidin S in the heat-resistance and in the timing of spore outgrowth is discussed.

Acridine Orange↗