Sugar transport by the bacterial phosphotransferase system. The intrinsic fluorescence of enzyme I

Enzyme I of the bacterial phosphoenolpyruvate: glycose phosphotransferase system has 2 tryptophan residues/monomer, as determined spectrophotometrically. The tryptophan fluorescence has been investigated with the aid of nanosecond time-resolved techniques. The decay of the fluorescence intensity was analyzed in terms of a biexponential function. The contribution of the emission associated with the shorter decay constant increases from 17-19% at 1 degree C to 43-44% at room temperature. Decay-associated spectra obtained with Enzyme I indicate different spectral distributions associated with the two decay constants. The measurement of tumbling of Enzyme I as a function of temperature revealed a transition of rotational rates between 5 and 15.5 degrees C. Global analysis allowed decomposition of the anisotropy decay into a formulation consistent with monomer and dimer rotational contributions.     

The valyl-tRNA synthetase from Bacillus stearothermophilus has considerable sequence homology with the isoleucyl-tRNA synthetase from Escherichia coli

We report the DNA sequence of the valS gene from Bacillus stearothermophilus and the predicted amino acid sequence of the valyl-tRNA synthetase encoded by the gene. The predicted primary structure is for a protein of 880 amino acids with a molecular mass of 102,036. The molecular mass and amino acid composition of the expressed enzyme are in close agreement with those values deduced from the DNA sequence. Comparison of the predicted protein sequence with known protein sequences revealed a considerable homology with the isoleucyl-tRNA synthetase of Escherichia coli. The two enzymes are identical in some 20-25% of their amino acid residues, and the homology is distributed approximately evenly from N-terminus to C-terminus. There are several regions which are highly conservative between the valyl- and isoleucyl-tRNA synthetases. In one of these regions, 15 of 20 amino acids are identical, and in another, 10 of 14 are identical. The valyl-tRNA synthetase also contains a region HLGH (His-Leu-Gly-His) near its N-terminus equivalent to the consensus HIGH (His-Ile-Gly-His) sequence known to participate in the binding of ATP in the tyrosyl-tRNA synthetase. This is the first example of extensive homology found between two different aminoacyl-tRNA synthetases.     

Convenient preparative synthesis of [14C]trehalose from [14C]glucose by intact Escherichia coli cells

At high osmolarity, Escherichia coli synthesizes trehalose intracellularly, irrespective of the nature of the carbon source. Synthesis proceeds via the transfer of UDP-glucose to glucose 6-phosphate, yielding trehalose 6-phosphate, followed by its dephosphorylation to trehalose (H.M. Giaeyer, B.O. Styrvold, I. Kaasen, and A.R. Str?m, J. Bacteriol. 170:2841-2849, 1988). This reaction was exploited to preparatively synthesize [14C]trehalose from exogenous [14C]glucose by using intact bacteria of a mutant (DF214) that could not metabolize glucose. The total yield of radiochemically pure trehalose from glucose was routinely more than 50%.     

Hypothyroidism in rats decreases mitochondrial inner membrane cation permeability

We investigated the cation permeability of liver mitochondria isolated from hypothyroid or euthyroid rats by measuring the rate of swelling of respiring mitochondria in acetate salts as a function of membrane potential. Mitochondria from hypothyroid rats have a decreased permeability of roughly 3-fold in the presence of monovalent cations K and tetramethylammonium at any (measured) membrane potential. Since the monovalent cation leak and the proton leak are known to respond similarly to membrane potential our results support the theory that the difference in non-phosphorylating respiration rate between mitochondria from hypothyroid and euthyroid rats is due to a difference in proton leak.     

A quantitative assessment of the use of 36Cl- distribution to measure plasma membrane potential in isolated hepatocytes

The plasma membrane potential of isolated rat hepatocytes was clamped at different values between 0 and -68 mV by addition of valinomycin in the presence of different extracellular concentrations of K+, and measured by the distribution of 86Rb+ between cells and medium. 36Cl- distribution came to steady state in 10-15 min. This steady-state distribution was compared to the plasma membrane potential over a range of values. 36Cl- distribution provided an accurate measurement of plasma membrane potential between -4 and -40 mV. At higher potentials intracellular chloride concentration is less than 20% of the extracellular concentration and errors due to uncertainties in the measurement of intracellular volume and of the contamination of cell pellets by extracellular medium precluded accurate determination of membrane potential: thus in our experiments 36Cl- underestimated the plasma membrane potential at -68 mV by 8 mV.     

Thyroid-hormone control of state-3 respiration in isolated rat liver mitochondria.

Oxidative phosphorylation can be treated as two groups of reactions; those that generate protonmotive force (dicarboxylate carrier, succinate dehydrogenase and the respiratory chain) and those that consume protonmotive force (adenine nucleotide and phosphate carriers. ATP synthase and proton leak). Mitochondria from hypothyroid rats have lower rates of respiration in the presence of ADP (state 3) than euthyroid controls. We show that the kinetics of the protonmotive-force generators are unchanged in mitochondria from hypothyroid animals, but the kinetics of the protonmotive-force consumers are altered, supporting proposals that the important effects of thyroid hormone on state 3 are on the ATP synthase or the adenine nucleotide translocator.     

Monitoring and control of biotechnological production processes by Bio-FET-FIA-sensors

Summary Single and multisensor field effect transistors (FET) with a pH-sensitive Si/SiO₂/Si₃N₄/Ta₂O₅-gate and reference electrode (for single sensor) were developed and used for manufacturing the following biological (Bio)-FETs: for glucose analysis, glucose oxidase-FET (GOD-FET); for urea analysis, urease-FET; and for cephalosporin C analysis, cephalosporinase-FET. The GOD-FETs were integrated into flow injection analysis (FIA) of the Eppendorf variables analyser (EVA) system and used for monitoring the glucose concentration in microbial cultivation and production processes with recombinant Escherichia coli K12 MF, recombinant E. coli JM103, Saccharomyces cerevisiae H620, and Candida boidinii. Urease-FET-FIA was used to monitor the urea concentration in a simulated cultivation of Cephalosporium acremonium and urease-FET-FIA and GOD-FET-FIA for the monitoring of urea and glucose concentrations in simulated S. cerevisiae cultivations.     

Generating lineage-specific markers to study Drosophila development.

To generate cell- and tissue-specific expression patterns of the reporter gene lacZ in Drosophila, we have generated and characterized 1,426 independent insertion strains using four different P-element constructs. These four transposons carry a lacZ gene driven either by the weak promoter of the P-element transposase gene or by partial promoters from the even-skipped, fushi-tarazu, or engrailed genes. The tissue-specific patterns of beta-galactosidase expression that we are able to generate depend on the promoter utilized. We describe in detail 13 strains that can be used to follow specific cell lineages and demonstrate their utility in analyzing the phenotypes of developmental mutants. Insertion strains generated with P-elements that carry various sequences upstream of the lacZ gene exhibit an increased variety of expression patterns that can be used to study Drosophila development.