MgATP-dependent activation by phosphoenolpyruvate of the E187A mutant of Escherichia coli phosphofructokinase

Using enzymatic assays and steady-state fluorescence emission, we performed a linkage analysis of the three-ligand interaction of fructose 6-phosphate (Fru-6-P), phosphoenolpyruvate (PEP), and MgATP on E187A mutant Escherichia coli phosphofructokinase (PFK). PEP allosterically inhibits Fru-6-P binding to E. coli PFK. The magnitude of antagonism is 90-fold in the absence and 60-fold in the presence of a saturating concentration of MgATP [Johnson, J. J., and Reinhart, G. D. (1997) Biochemistry 36, 12814-12822]. Substituting an alanine for the glutamate at position 187, located in the allosteric site (i.e., mutant E187A), activates Fru-6-P binding and inhibits the maximal rate of enzyme turnover [Lau, F. T.-K., and Fersht, A. R. (1987) Nature 326, 811-812]. The allosteric action of PEP appears to depend on the presence of the cosubstrate MgATP. In the presence of a saturating concentration of MgATP, PEP enhances the binding of Fru-6-P to the enzyme by a modest 2-fold. Decreasing the concentration of MgATP mitigates the extent of activation. At MgATP concentrations approaching 25 microM, PEP becomes insensitive to the binding of Fru-6-P. At MgATP concentrations < 25 microM, PEP "crosses over" and becomes antagonistic toward substrate binding. The present study examines the role of Glu 187 at the allosteric site in the binding of Fru-6-P and offers a more complex explanation of the mechanism than that described by traditional allosteric mechanistic models.     

The unipolar Shigella surface protein IcsA is targeted directly to the bacterial old pole: IcsP cleavage of IcsA occurs over the entire bacterial surface

Shigella flexneri is an intracellular pathogen that is able to move within the cytoplasm of infected cells by the continual assembly of actin onto one pole of the bacterium. IcsA, an outer membrane protein, is localized to the old pole of the bacterium and is both necessary and sufficient for actin assembly. IcsA is slowly cleaved from the bacterial surface by the protease IcsP (SopA). Absence of IcsP leads to an alteration in the distribution of surface IcsA, such that the polar cap is maintained and some IcsA is distributed along the lateral walls of the bacillus. The mechanism of unipolar localization of IcsA and the role of IcsP in its unipolar localization are incompletely understood. Here, we demonstrate that cleavage of IcsA occurs exclusively in the outer membrane and that IcsP is localized to the outer membrane. In addition, we show that IcsA at the old pole is susceptible to cleavage by IcsP and that native IcsP is active at the pole. Taken together, these data indicate that IcsP cleaves IcsA over the entire bacterial surface. Finally, we show that, immediately after induction from a tightly regulated promoter, IcsA is expressed exclusively at the old pole in both the icsP- icsA- and the icsA- background. These data demonstrate that unipolar localization of IcsA results from its direct targeting to the pole, followed by its diffusion laterally in the outer membrane.     

Precultivation technique for studies of microorganisms exhibiting overflow metabolism

A precultivation technique for microorganisms exhibiting overflow metabolism is presented. It is based on a low initial medium volume in the fermenter. The medium feed contains all the nutrients, but is diluted with respect to sugar, the concentration of which determines the biomass concentration at the end of the preculture. By this method, effects of varying activity of the inocula from shake flask cultures are minimised and the metabolic state, i.e. oxido-reductive, oxidative growth on sugar plus overflow metabolite or oxidative growth on sugar alone, can be controlled at the start of the main fermentation.     

An in vivo study of antifreeze protein adjuvant cryosurgery

Cryosurgery employs freezing to destroy undesirable tissue. However, under certain thermal conditions, frozen tissues survive. The survival of frozen undesirable tissue may lead to complications, such as recurrence of cancer. In a study of nude mice with subcutaneous metastatic prostate tumors, we showed that the preoperative injection of a phosphate-buffered saline solution with 10 mg/ml antifreeze protein of type I into the tumor prior to freezing enhances destruction under thermal conditions which normally yield cell survival. This suggests that the adjunctive use of antifreeze proteins in cryosurgery may reduce the complications from undesirable tissues that survive freezing.     

Multistep denaturation of Borrelia burgdorferi OspA, a protein containing a single-layer beta-sheet

Outer surface protein A (OspA) from the Lyme disease spirochete, Borrelia burgdorferi, is a dumbbell-shaped protein in which two globular domains are connected by a three-stranded beta-sheet segment that is solvent-exposed on both faces. Previous studies showed that the whole protein, including the single-layer beta-sheet, is highly rigid. To elucidate the folding mechanism and the role of the central beta-sheet in the formation of the rigid molecule, we investigated the equilibrium thermal denaturation reaction of OspA. We applied differential scanning calorimetry, heteronuclear NMR spectroscopy, and solution small-angle X-ray scattering (SAXS) to characterize the reaction in detail. All three techniques revealed that OspA denatures in two separable cooperative transitions. NMR measurements on OspA specifically 15N-labeled at Lys residues identified the locations of the two folding units and revealed that the C-terminal segment is less stable than the remaining N-terminal segment. The boundary between the two folding units is located within the central beta-sheet. The interconversion among the three folding states (fully folded, C-terminus unfolded, and fully denatured) is slow relative to chemical shift differences (<24 Hz), indicating that there are significant kinetic barriers in the denaturation reactions. SAXS measurements determined the radius of gyration of the native protein to be 25.0 +/- 0.3 A, which increases to 34.4 +/- 1.0 A in the first transition, and then to 56.1 +/- 1.6 A in the second transition. Thus, the intermediate state, in which the C-terminal folding unit is already denatured, is still compact. These results provide a basis for elucidating the folding mechanism of OspA.     

Poly(L-lysine)-mediated immobilisation of oligonucleotides on carboxy-rich polymer surfaces

The immobilisation efficiency of the complexes of oligonucleotide/poly(L-lysine) on two polymeric carboxy-rich surfaces, i.e. poly(styrene/maleic acid) (PSMA) and poly(styrene/maleic anhydride) (PSMAA), has been investigated using X-ray photoelectron spectroscopy, atomic force microscopy (AFM) and fluorescence-based measurements of DNA attachment. A molecularly thin layer of either electrostatically or covalently (via amide bond) bound poly(L-lysine) allows the 'switching' from COOH-based to NH(2)-based surface functionality. The results indicate that approximately 54-57% and 55-62% of the applied oligonucleotides bind to polymeric surfaces via the route of electrostatic adsorption of poly(L-lysine) and covalent bonding of poly(L-lysine), respectively. This system can be applied conveniently for the detection of nucleic acids in both disposable and reusable biosensors.     

Bioconversion of waste office paper to L(+)-lactic acid by the filamentous fungus Rhizopus oryzae

L(+)-lactic acid production was investigated using an enzymatic hydrolysate of waste office automation (OA) paper in a culture of the filamentous fungus Rhizopus oryzae. In 4 d culture, 82.8 g/l glucose, 7 g/l xylose, and 3.4 g/l cellobiose contained in the hydrolysate were consumed to produce 49.1 g/l of lactic acid. The lactic acid yield and production rate were only 0.59 g/g and 16.3 g/l/d, respectively, only 75% and 61% of the results from the glucose medium. The low production rate from waste OA hydrolysate was elucidated by trials using xylose as the sole carbon source; in those trials, the lactic acid production rate was 7.3 g/l/d, only 28% that of glucose or cellobiose. The low lactic acid yield from waste OA hydrolysate was clarified by trials using artificial hydrolysates comprised of 7:2:1 or 7:1:2 ratios of glucose:cellobiose:xylose. For both, the lactic acid production rate of 17.4 g/l/d matched that of waste OA paper, while the lactic acid yield was similar to that of the glucose medium. This indicates that the production rate may be inhibited by xylose derived from hemicellulose, and the yield may be inhibited by unknown compounds derived from paper pulp.     

Extraction of fluorescent cell puncta by adaptive fuzzy segmentation

MOTIVATION: The discrimination and measurement of fluorescent-labeled vesicles using microscopic analysis of fixed cells presents a challenge for biologists interested in quantifying the abundance, size and distribution of such vesicles in normal and abnormal cellular situations. In the specific application reported here, we were interested in quantifying changes to the population of a major organelle, the peroxisome, in cells from normal control patients and from patients with a defect in peroxisome biogenesis. In the latter, peroxisomes are present as larger vesicular structures with a more restricted cytoplasmic distribution. Existing image processing methods for extracting fluorescent cell puncta do not provide useful results and therefore, there is a need to develop some new approaches for dealing with such a task effectively. RESULTS: We present an effective implementation of the fuzzy c-means algorithm for extracting puncta (spots), representing fluorescent-labeled peroxisomes, which are subject to low contrast. We make use of the quadtree partition to enhance the fuzzy c-means based segmentation and to disregard regions which contain no target objects (peroxisomes) in order to minimize considerable time taken by the iterative process of the fuzzy c-means algorithm. We finally isolate touching peroxisomes by an aspect-ratio criterion. The proposed approach has been applied to extract peroxisomes contained in several sets of color images and the results are superior to those obtained from a number of standard techniques for spot extraction. AVAILABILITY: Image data and computer codes written in Matlab are available upon request from the first author.