Influence of readily metabolizable carbon on pentachlorophenol metabolism by a pentachlorophenol-degrading Flavobacterium sp.

The influence of high concentrations of pentachlorophenol (PCP) and readily metabolizable carbon on the activity and viability of a PCP-degrading Flavobacterium sp. was examined in a mineral salts medium. Lags preceding PCP removal by glutamate-grown Flavobacterium cells were greatly attenuated by the addition of glutamate, aspartate, succinate, acetate, glucose, or cellobiose. The effect of these supplementary carbon sources on the apparent lag was not mediated entirely through the stimulation of growth since PCP metabolism accompanied the onset of growth. The specific activity of PCP-degrading cells in the absence of supplementary carbon was 1.51 x 10(-13) +/- 0.08 x 10(-13) g of PCP per cell per h and in the presence of supplementary carbon was 0.92 x 10(-13) +/- 0.09 x 10(-13) g of PCP per cell per h. Glutamate in combination with glucose or cellobiose partially repressed PCP metabolism. PCP removal by PCP-induced, glutamate-grown cells suspended in the presence of 4 g of sodium glutamate per liter was sensitive to shock loads of PCP, with a Ki of about 86.8 micrograms/ml. Subsequent removal rates, however, were more resistant to PCP. Optimal stimulation of PCP removal by sodium glutamate required 3.0 g/liter, about the same concentration as that which saturated growth in the absence of PCP. PCP removal rates decayed within minutes following the transfer of PCP-induced, glutamate-grown cells to media containing PCP without supplementary carbon, and increasing PCP concentrations accelerated the decay.(ABSTRACT TRUNCATED AT 250 WORDS)     

Hydrolysis of peptides by carboxypeptidase A: equilibrium trapping of the ES2 intermediate

The cobalt absorption and electron paramagnetic resonance (EPR) spectra of cobalt carboxypeptidase undergo unique variations on formation of catalytic peptide and ester intermediates as previously recorded in cryoenzymologic experiments employing rapid-scanning spectroscopy and cryotrapping [Geoghegan, K. F., Galdes, A., Martinelli, R. A., Holmquist, B., Auld, D.S., & Vallee, B. L. (1983) Biochemistry 22, 2255-2262]. We here describe a means of stabilizing these intermediates, which we have termed "equilibrium trapping". It allows peptide intermediates to be observed for longer periods (much greater than 1 min) at ambient as well as subzero temperatures. The reaction intermediate with the rapidly turned over peptide substrate Dns-Ala-Ala-Phe is trapped when the cobalt enzyme (greater than 10 microM) has catalyzed the attainment of chemical equilibrium between high concentrations of the hydrolysis products Dns-Ala-Ala, 10 mM, and L-phenylalanine, 50 mM, and the product of their coupling Dns-Ala-Ala-Phe. Under these conditions, Dns-Ala-Ala-Phe is present in the equilibrated substrate-product reaction mixture at a level that exceeds the one predicted on the basis of K'eq for hydrolysis of this substrate and is close to the enzyme concentration. Other pairs of peptide hydrolysis products yield similar results. Visible absorption and EPR spectra of the cobalt enzyme show that the synthesized peptide binds to the active site in the mode previously recognized as the ES2 catalytic intermediate in peptide hydrolysis. Equilibrium trapping of the ES2 intermediate allows analysis of its physicochemical properties by methods that could not be employed readily under cryoenzymological conditions, e.g., circular dichroic and magnetic circular dichroic spectra.(ABSTRACT TRUNCATED AT 250 WORDS)     

Controls on calcium ion fluxes in injured or shocked corn root cells: Importance of proton pumping and cell membrane potential

Passive influx of ⁴⁵Ca²⁺ into non-growing corn root tissue ( Zea mays L.) was increased as a result of actions (cutting, rubbing, chilling, heating, acidifying) or agents (cyanide, uncouplers) known to depolarize the cell membrane, and was decreased by actions (washing) or agents (fusicoccin) known to hyperpolarize it. These responses indicate the presence of Ca²⁺ channels which are voltage controlled. If the injuries were extensive, however, voltage control was lost and hyperpolarization with fusicoccin was expressed by increased ⁴⁵Ca²⁺ influx. Control could be regained by tissue washing, and millimolar levels of external Ca²⁺ would protect against loss of control. Influx of Ca²⁺ was strongly inhibited by La³⁺, but only weakly by verapamil. Intact roots showed greater cold shock sensitivity in maturing cells than in growing cells. We conclude that corn roots normally restrict Ca²⁺ influx by a mechanism linked to hyper-polarization of the plasmalemma.
Calcium ions which enter cold-shocked tissue are partially extruded during the early phase of recovery by a process stimulated by fusicoccin and subject to uncoupling.     

Immunoglobulin G antibody bound to the C1q subcomponent of human complement exhibits segmental flexibility

The rotational dynamics of rabbit immunoglobulin G (IgG) anti-dansyl antibodies bound to the C1q subcomponent of human complement were studied by nanosecond fluorescence spectroscopy. Deconvoluted anisotropy decays of IgG-C1q mixtures were fitted to a two-exponential expression and were corrected for the effects of unbound IgG, which was determined with an analytical ultracentrifuge. Compared with the anisotropy parameters for free IgG, the pre-exponential weighting factors and the short correlation time of the C1q-bound antibody were nearly unchanged, and the long correlation time increased by only about 45 nanoseconds. These results, together with rotational diffusion calculations, indicate that the Fab arms of the C1q-bound antibody exhibited considerable flexibility. This finding may have biological relevance because it suggests that C1q can bind to the Fc segments of IgG molecules anchored in an immune complex, even though the angles between the two Fab arms of the different antibodies may vary. The results of this study also support our earlier interpretation that both the short and long correlation times of IgG principally represent flexible motions of the Fab segments.     

Construction of broad-host-range cosmid cloning vectors: identification of genes necessary for growth of Methylobacterium organophilum on methanol.

Four new cloning vectors have been constructed from the broad-host-range cloning vector pRK290. These vectors, pLA2901, pLA2905, pLA2910, and pLA2917, confer resistance to kanamycin and tetracycline. The latter two are cosmid derivatives of pLA2901. The new vectors can be mobilized into, and are stably maintained in, a variety of gram-negative bacteria. A Sau3A genomic bank of Methylobacterium organophilum strain xx DNA has been constructed in pLA2917, and complementation analysis, with a variety of mutants unable to grow on methanol, revealed at least five separate regions necessary for growth on methanol. Complementation analysis and Tn5 mutagenesis data suggest that at least three genes are responsible for expression of active methanol dehydrogenase.