Ornithine decarboxylase (ornithine carboxy lyase; EC 4.1.1.17) (ODC) from Tetrahymena thermophila was purified 6,300 fold employing fractionated ammonium sulfate precipitation, gel permeation chromatography on Sephadex G-150, ion exchange chromatography on DEAE-Sepharose CL-6B, and preparative isoelectric focussing. The product obtained in 24% yield was a preparation of the specific activity of 10,200 nmol CO2.h-1.mg-1. The purified enzyme was rather stable at 37 degrees C (14% loss of activity within 1 h). The molecular and catalytic properties of this enzyme were investigated. The isoelectric point was 5.7 and the molecular weight (MW) was estimated to be 68,000 under nondenaturing conditions. The pH optimum was between 6.0 and 7.0, the Km for the substrate L-ornithine was 0.11 mM, and the Km for the cofactor pyridoxal 5-phosphate was 0.12 microM; the product of ODC catalysis, putrescine, was a poor inhibitor with an estimated Ki of about 10 mM. The enzyme was inhibited competitively by D-ornithine with a Ki of 1.6 mM and by alpha-difluoromethylornithine with a Ki of 0.15 mM. The latter one, an enzyme activated irreversible inhibitor of mammalian ODC, inactivated the enzyme from T. thermophila at high concentrations with a half life time of 14 min. Other basic amino acids, e.g. L-lysine, L-arginine, and L-histidine, were neither substrates nor inhibitors of the enzyme, as were the diamines 1,3-diaminopropanol and cadaverine, the polyamines spermidine and spermine and the cosubstrate analogues pyridoxal and pyridoxamine-5-phosphate.(ABSTRACT TRUNCATED AT 250 WORDS)

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PubMedDate not supplied

Regulation of ornithine decarboxylase activity in the growth cycle of Tetrahymena thermophila.

Cells of the ciliated protozoan Tetrahymena thermophila, grown in proteose peptone medium up to late logarithmic phase, harvested by centrifugation, and resuspended in fresh medium to almost the same cell density, underwent one more division cycle within 5 h after inoculation, thereafter being definitely in full stationary phase. This growth cycle proved to be a useful tool to investigate the activation and deactivation of ornithine decarboxylase ODC1 in Tetrahymena: In late logarithmic phase the cells contained a very low specific activity of ODC of about 3 nmol CO2.h-1.mg-1 in the soluble protein fraction. After growth stimulation the activity was increased up to 100-fold within 1 h. This high activity was maintained for about 5 h-about as long as division activity-then rapidly declined with a half life time (t1/2) of about 15 min to the original low level. Inhibition assays with cycloheximide and actinomycin D revealed that: i. the rapid increase of ODC activity was biphasic with one component of translation of preexisting mRNA and one component of translation of newly transcribed mRNA; ii. the t1/2 of the mRNA of ODC was estimated to be about 2 h; iii. inhibition of protein biosynthesis before ODC inactivation at 5 h caused a decrease of ODC with a t1/2 of 55 min instead of 15 min. These findings suggest that ODC activity in Tetrahymena is regulated on both levels: transcription and translation and by an inactivating protein factor which is regulated at the level of biosynthesis.

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PubMedDate not supplied

[Microdialysis as a procedure for evaluating intestinal hypoxia--an animal experiment approach].

Usual ICU monitoring of patients with abdominal pathology provides no detailed information about hepatosplanchnic haemodynamics or intestinal metabolism. In our animal experiment, the effects of systemic hypoxia on microdialysis measurements of the peritoneum in comparison with the ischiocrural muscle as reference were investigated in 7 rats. The parameter of interest was the course of glucose metabolism reflecting sympathoadrenergic activity during the experiment. Measurements were obtained at timed intervals at baseline, under hypoxia, and during reoxygenation. After induction of systemic hypoxia, the peritoneal microdialysis showed significantly higher levels of glucose in comparison with the ischiocrural muscle. The results indicate hypermetabolic activity or a hypersympathetic response of the bowel in response to hypoxic stress. In the clinical setting, the bowel has an important role in the development of multiorgan failure. Microdialysis may therefore be an interesting tool for the early detection of hypoxic metabolism during and after abdominal procedures.

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PubMedDate not supplied

[Suspecies criterion in ectoparasites (based on the example of bird lice)].

It has been proved on Mallophaga that the notion "hostal subspecies" can be applied at the level of intraspecies categories to constant parasites possessing, distinct specificity. The notion should be applied in those cases when there are but small differences between groups of parasites from different hosts (e. g. in sizes) and when hosts belong to different species of the same genus.

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