Utilization of Diammonium Citrate by Adult Rats

In the 1st experiment, the utilization of diammonium citrate (DAC) as a non-essential nitrogen source was studied in comparison with glutamic acid. Adult rats fed the amino acid diet containing DAC in place of glutamic acid as a nonessential amino acid maintained their body weights and had nitrogen balances almost equal to those of rats on the glutamic acid containing diets.

In the 2nd experiment, DAC-¹⁵N was orally administered after the rats were fed the DAC diet for a month, and the distribution of ¹⁵N in the rats’ bodies and excreta was examined at the 6th, 12th, 24th and 48th hr after administration.

At the 6th hr, 85% of ¹⁵N intake was retained and 60% of ¹⁵N intake was found as protein-¹⁵N. At the 48th hr, retained ¹⁵N was 83% and protein-¹⁵N increased 5% above that at the 6th hr. The ¹⁵N concentration of non-protein fractions was higher and changed more rapidly than that of protein fractions, especially in the blood, liver and small intestine.

These results seem to indicate that DAC was utilized by rats fed a diet, in which non- essential amino acids were completely replaced by DAC. Gastrointestinal microbes might hardly play any role in the utilization of dietary non-protein nitrogen under these experimental conditions.     

Heat shock regulation in the ftsH null mutant of Escherichia coli: dissection of stability and activity control mechanisms of σ32in vivo

The heat shock response of Escherichia coli is regulated by the cellular level and the activity of σ³², an alternative sigma factor for heat shock promoters. FtsH, a membrane-bound AAA-type metalloprotease, degrades σ³² and has a central role in the control of the σ³² level. The ftsH null mutant was isolated, and establishment of the Δ ftsH mutant allowed us to investigate control mechanisms of the stability and the activity of σ³² separately in vivo . Loss of the FtsH function caused marked stabilization and consequent accumulation of σ³² (≈20-fold of the wild type), leading to the impaired downregulation of the level of σ³². Surprisingly, however, Δ ftsH cells express heat shock proteins only two- to threefold higher than wild-type cells, and they also show almost normal heat shock response upon temperature upshift. These results indicate the presence of a control mechanism that downregulates the activity of σ³² when it is accumulated. Overproduction of DnaK/J reduces the activity of σ³² in Δ ftsH cells without any detectable changes in the level of σ³², indicating that the DnaK chaperone system is responsible for the activity control of σ³² in vivo . In addition, CbpA, an analogue of DnaJ, was demonstrated to have overlapping functions with DnaJ in both the activity and the stability control of σ³².     

ATP and other nucleotide interaction with model compounds of amino acids.

With the use of a new liquid scintillation technique, the partitioning and interfacial interaction of ³H-ATP and other adenine nucleotides to various amphiphiles has been studied. The amphiphiles consisted of lipophilic groups attached to amino acid residues or certain functional groups and were as follows: trimethylindole, octadecylamine, octadecyl alcohol, dodecylguanidine, stearylamide, acetododecyl amide, p-nonyl phenol, stearyl mercaputan, stearic acid, and imidazole. A toluene solution containing the lipophilic substance and fluor molecules was layered onto aqueous solutions containing a ³H-nucleotide, and the interaction followed by the liquid scintillation technique. A strong interaction with the nucleotides occurred with dodecylguanidine, octadecylamine, and trimethylindole, while the interaction with the other amphiphiles was slight or absent. The time course of the interaction followed first order kinetics irrespective of the number of reactive species; the rate of diffusion of the nucleotide being the rate-determining step. The technique permitted a simple analysis of formation constants; which for ATP were 1–2 orders of magnitude greater than for ADP and AMP. In the presence of Ca²⁺, but not Mg²⁺, an interaction occurred between ATP and either stearic acid, stearyl mercaputan, and p-nonyl phenol. On the other hand, in the presence of Mg²⁺, but not Ca²⁺, an interaction occurred between stearic acid and polyadenylic acid. The results are discussed in relation to ATP interactions with proteins and lipids.     

Structural and functional studies on an FtsH inhibitor from Bacillus subtilis

The small 3 kDa SpoVM protein is essential for development of the spore in Bacillus subtilis. Genetic and biochemical experiments have shown that the function of SpoVM is to inhibit the proteolytic activity of FtsH during sporulation. We have used a combination of genetic and biophysical techniques to characterise the role of this small polypeptide. SpoVM was found to be widespread in Bacillus as well as in two Clostridia species, suggesting that SpoVM provides a common mechanism for inactivating the FtsH protease during spore differentiation. Using site-specific mutagenesis, we have identified C-terminal residues of SpoVM essential for biological activity. Analysis of SpoVM’s structure showed that it is able to assume an α-helical conformation in the presence of a lipid interface which may be important in interacting with FtsH.