Microbial production of L-[15N]glutamic acid and its gas chromatography-mass spectrometry analysis

L-[15N]Glutamic acid was prepared in high yields via a fermentative process. Brevibacterium lactofermentum, growing on a medium containing 97% enriched 15NH4Cl as a sole isotopic precursor, excreted mostly L-[15N]glutamic acid. The L-[15N]glutamic acid was purified and identified. Gas chromatography-mass spectrometry analysis was performed to demonstrate its usefulness in clinical studies.     

Degradation of ornithine decarboxylase in reticulocyte lysate is ATP-dependent but ubiquitin-independent

Reticulocyte lysate contains all the components of the ubiquitin-dependent proteolytic system. Several proteins are degraded in reticulocyte lysate in a ubiquitin-dependent manner. However, none of the proteins studied has a short intracellular half-life. We have investigated the degradation of ornithine decarboxylase (ODC), one of the most labile proteins in mammalian cells. ODC is efficiently degraded in reticulocyte lysate depleted of the ubiquitin activating enzyme, E1, in fraction II of reticulocyte lysate completely lacking ubiquitin, and in fraction II depleted of the entire complex of enzymes responsible for the ligation of ubiquitin to target proteins. The degradation of ODC is ATP dependent. Therefore, our results demonstrate that in addition to the ubiquitin-dependent proteolytic pathway, reticulocyte lysate contains at least one additional ATP-dependent proteolytic pathway. In vitro synthesized ODC served as a substrate in the present degradation study. Its successful utilization establishes a general strategy for investigating the degradation of short-lived proteins (for which a corresponding cDNA is available), that constitute a very small fraction of cellular proteins and for which purification is difficult or impossible. In contrast to ODC synthesized in vitro, that isolated from cells was not degraded by the reticulocyte lysate degradation system, suggesting that post-translational modifications may be involved in regulating ODC degradation.     

Characterization of sequences involved in mediating degradation of ornithine decarboxylase in cells and in reticulocyte lysate

Mouse ornithine decarboxylase is a 461-amino-acid protein that is extremely labile. A set of contiguous in-frame deletions were introduced into its C-terminal hydrophilic region. The resulting mutant proteins were expressed in cos monkey cells using an expression vector based on simian virus 40 (SV40) or by in vitro translation in reticulocyte lysate. The degradation of wild-type and mutant proteins was determined in transfected cos cells and in a degradation system based on reticulocyte lysate. Deletion mutants lacking segments of the C-terminus (amino acids 423-461, 423-435, 436-449 and 449-461) were converted into stable proteins in both experimental systems. The mutant lacking amino acids 295-309 was significantly stabilized in transfected cos cells, but was rapidly degraded in reticulocyte-lysate-based degradation mix. Our results suggest that the carboxyl-terminal region encompassing amino acids 423-461 and perhaps also amino acids 295-309 may constitute a signal recognized by the proteolytic machinery that degrades ornithine decarboxylase.     

A simple spectroscopic method for studying erythrocyte ghost resealing.

A novel spectroscopic method is described for following the kinetics of resealing of hemolysed erythrocyte ghosts. The procedure is based on the broadening of the EPR spectrum of nitroxyl radicals by paramagnetic ions. The method is used to study the effect of Ca2+, Mg2+ and dimethonium ion on the kinetics of resealing.     

The interaction between Sendai virus and cell membranes: A quantitative analysis of 125I-Sendai virus particles' association with human red blood cells

Intact Sendai virus particles were radiolabeled by the use of chloramine-T and Na ¹²⁵I. The method described is reproducible, efficient and appropriate for the preparation of large quantities of biologically active virus with relatively high specific activity. Gel electrophoresis analysis of the radiolabeled virus revealed that approx. 50% of the total ¹²⁵I incorporated in the virus are associated with the two viral envelope glycoproteins, while the remaining 50% are evenly distributed throughout the other viral polypeptides. The ¹²⁵I-virus particles were used to study some of the kinetic parameters of the interaction between Sendai virus particles and human erythrocytes. Binding of virus particles at 4 °C is irreversible, non-cooperative and exhibits a characteristic saturation curve. A maximum of 1–2 × 10³ virus particles bound per cell was derived from the saturation curve. Non-radioactive native virus particles as well as isolated glycophorin molecules competitively inhibit binding of the ¹²⁵I-virus particles to human erythrocytes. Incubation at 37 °C of the virus-erythrocyte complex resulted in the release of about 33% of the bound virus to the surrounding medium.     

The natively helical chain segment 169-188 of Escherichia coli adenylate kinase is formed in the latest phase of the refolding transition

The refolding transition of Escherichia coli adenylate kinase (AK) was investigated by monitoring the refolding kinetics of a selected 20 residue helical segment in the CORE domain of the protein. Residues 169 and 188 were labeled by 1-acetamido-methyl-pyrene, and by bimane, respectively. The experiment combines double-jump stopped-flow fast mixing initiation of refolding and time-resolved F?rster energy transfer spectroscopy for monitoring the conformational transitions (double-kinetics experiment). Two kinetic phases were found in the denaturant-induced unfolding of AK. In the first phase, the fluorescence quantum yields of both probes decreased. The distribution of the distances between them transformed from the native state's narrow distribution with the mean distance corresponding to the distance in the crystal structure, to a distribution compatible with an unordered structure. In the second, slow step of denaturation, neither the fluorescence parameters of the probes nor the distance distribution between them changed. This step appeared to be a transformation of the fast-folding species formed in the first phase, to the slow-folding species. Refolding of the fast-folding species of the denatured state of AK was also a two-phase process. During the first fast phase, within less than 5ms, the fluorescence emission of both probes increased, but the distance distribution between the labeled sites was unchanged. Only during the second slow refolding step did the intramolecular distance distribution change from the characteristic of the denatured state to the narrow distribution of the native state. This experiment shows that for the case of the CORE domain of AK, the large helical segment of residues 169-188 was not formed in the first compaction step of refolding. The helical conformation of this segment is established only in the second, much slower, refolding phase, simultaneously with the completion of the native structure.