Differential effects of suramin on the coupling of receptors to individual species of pertussis-toxin-sensitive guanine-nucleotide-binding proteins.

The anti-helminthic drug suramin inhibited the basal high-affinity GTPase activity of both C6 BU1 glioma and NG 108-15 neuroblastoma x glioma hybrid-cell membranes with an IC50 (concentration causing half-maximal inhibition) value close to 30 micrograms/ml. This effect was shown to occur via a non-competitive mechanism in which the binding affinity of the G-proteins for GTP was not altered, but the maximal velocity of the subsequent hydrolysis was reduced. In NG 108-15 membranes, both opioid peptides and foetal-calf serum stimulated high-affinity GTPase activity in a pertussis-toxin-sensitive manner. These effects have previously been shown to be mediated by different G-proteins [McKenzie, Kelly, Unson, Spiegel & Milligan (1988) Biochem. J. 249, 653-659]. Suramin completely prevented the opioid-peptide-stimulated increase in GTP hydrolysis, but did not prevent the opioid peptide from binding to its receptor. Suramin, however, did not block the foetal-calf-serum-stimulated GTPase response. This selective action of suramin provides further evidence for distinct roles for two separate pertussis-toxin-sensitive G-proteins in signal transduction in NG 108-15 membranes and provides the first evidence for a selective effect of a drug on the functions of different G-proteins.     

Modes of integration of heterologous plasmid DNA into the chromosome of Streptococcus pneumoniae.

We compared the efficiencies of two different processes that can direct integration of plasmids into chromosomes of recipient cells during transformation. A donor-recipient system was constructed to allow a single donor plasmid to use either flanking homology, involving an apparent double crossover, or the insertion duplication process that has been described as due to a "Campbell-like" single crossover between the chromosome and a circular duplex. The latter process gave 600-fold fewer insertions that did the former, confirming expectations from prior work showing that insertion of heterologous DNA by use of flanking homology is highly efficient. It has some advantages for cloning and mapping purposes and can be exploited once it is recognized.     

Isolation and characterization of three new classes of transformation-deficient mutants of Streptococcus pneumoniae that are defective in DNA transport and genetic recombination

A bifunctional enzyme, L-(+)-tartrate dehydrogenase-D-(+)-malate dehydrogenase (decarboxylating) (EC 1.1.1.93 and EC 1.1.1. . . , respectively), was discovered in cells of Rhodopseudomonas sphaeroides Y, which accounts for the ability of this organism to grow on L-(+)-malate. The enzyme was purified 110-fold to homogeneity with a yield of 51%. During the course of purification, including ion-exchange chromatography and preparative gel electrophoresis, both enzyme activities appeared to be in association. The ratio of their activities remained almost constant [1:10, L-(+)-tartrate dehydrogenase/D-(+)-malate dehydrogenase (decarboxylating)] throughout all steps of purification. Analysis by polyacrylamide gel electrophoresis revealed the presence of a single protein band, the position of which was coincident with both L-(+)-tartrate dehydrogenase and D-(+)-malate dehydrogenase (decarboxylating) activities. The apparent molecular weight of the enzyme was determined to be 158,000 by gel filtration and 162,000 by ultracentrifugation. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis yielded a single polypeptide chain with an estimated molecular weight of 38,500, indicating that the enzyme consisted of four subunits of identical size. The isoelectric point of the enzyme was between pH 5.0 and 5.2. The enzyme catalyzed the NAD-linked oxidation of L-(+)-tartrate as well as the oxidative decarboxylation of D-(+)-malate. For both reactions, the optimal pH was in a range from 8.4 to 9.0. The activation energy of the reaction (delta Ho) was 71.8 kJ/mol for L-(+)-tartrate and 54.6 kJ/mol for D-(+)-malate. NAD was required as a cosubstrate, and optimal activity depended on the presence of both Mn2+ and NH4+ ions. The reactions followed Michaelis-Menten kinetics, and the apparent Km values of the individual reactants were determined to be: L-(+)-tartrate, 2.3 X 10(-3) M; NAD, 2.8 X 10(-4) M; and Mn2+, 1.6 X 10(-5) M with respect to L-(+)-tartrate; and D-(+)-malate, 1.7 X 10(-4) M; NAD, 1.3 X 10(-4); and Mn2+, 1.6 X 10(-5) M with respect to D-(+)-malate. Of a variety of compounds tested, only meso-tartrate, oxaloacetate, and dihydroxyfumarate were effective inhibitors. meso-Tartrate and oxaloacetate caused competitive inhibition, whereas dihydroxyfumarate caused noncompetitive inhibition. The Ki values determined for the inhibitors were, in the above sequence, 1.0, 0.014, and 0.06 mM with respect to L-(+)-tartrate and 0.28, 0.012, and 0.027 mM with respect to D-(+)-malate.     

Adenosine 3'',5''-cyclic monophosphate-dependent release of prolactin from GH3 pituitary tumour cells. A quantitative analysis.

The involvement of cyclic AMP in mediating regulatory peptide-controlled prolactin release from GH3 pituitary tumour cells was investigated. Cholera toxin and forskolin elicited concentration-dependent increases in both GH3 cell cyclic AMP content and prolactin release. The maximum rise in prolactin release with these agents was 2-fold over basal. 8-Bromo-cyclic AMP produced a similar stimulation of prolactin release. The phosphodiesterase inhibitor isobutylmethylxanthine also produced an increase in prolactin release and GH3 cell cyclic AMP content. However, the magnitude of the stimulated prolactin release exceeded that obtained with any other agent. Thyrotropin-releasing hormone (thyroliberin) and vasoactive intestinal polypeptide produced a concentration-dependent rise in both cell cyclic AMP content and prolactin release. However, only vasoactive intestinal polypeptide elicited an increase in cell cyclic AMP content at concentrations relevant to the stimulation of prolactin release. Vasoactive intestinal polypeptide and thyrotropin-releasing hormone, when used in combination, were additive with respect to prolactin release. Vasoactive intestinal polypeptide and forskolin, at concentrations that were maximal upon prolactin release, were, when used in combination, synergistic upon GH3 cell cyclic AMP content but were not additive upon prolactin release. In conclusion the evidence supports a role for cyclic AMP in the mediation of vasoactive intestinal polypeptide- but not thyrotropin-releasing hormone-stimulated prolactin release from GH3 cells. A quantitative analysis indicates that a 50-100% rise in cyclic AMP suffices to stimulate cyclic AMP-dependent prolactin release fully.     

Number of Transformable Units Per Cell in Diplococcus pneumoniae

Analysis of frequencies of single and random multiple transformations in Diplococcus pneumoniae showed that there are at least two transformable units per cell of the total population in highly competent cultures. If 100% of the cells are competent in these cases, the units may be interpreted as the strands of one duplex deoxyribonucleic acid recipient chromosome. The theory is developed to allow for extension to more complex situations.     

Pathway of plasmid transformation in Pneumococcus: open circular and linear molecules are active.

We have extended the analysis of plasmid transformation in Streptococcus pneumoniae by finding that monomeric and dimeric open circular and linear forms of pMV158 were active in transformation. Their efficiencies were at least 35-fold lower than those of the corresponding closed circular forms. The evidence came largely from analysis of S1 nuclease-digested plasmid deoxyribonucleic acid by combinations of dye-buoyancy, gel electrophoresis, and sedimentation velocity methods. As with closed circular forms, monomer open circular forms gave second-order kinetics and dimer forms gave first-order kinetics. Unique linear products of digestion by either of two restriction enzymes were inactive, but a mixture of the two digests was active, as was the mixture of linear monomer deoxyribonucleic acids produced by S1 nuclease. Absolute efficiencies of transformation were low even for closed circular donors. All of the results, including the low efficiencies, were consistent with the interpretation that plasmid replicons were assembled in the recipient cell by pairing of fragments of single strands that had entered the cell separately from duplex donors that had been cut on the cell surface.     

Monomer plasmid DNA transforms Streptococcus pneumoniae

Summary The covalently closed (CC) monomer form of plasmid pMV158 was found to transform pneumococcus (Streptococcus pneumoniae) and to do so with two-hit kinetics. The evidence came from analysis of the behavior of the transforming activity in fractions from preparative gel electrophoresis. Activity in the first major peak to elute (i) co-eluted with monomer CC as detected on analytical gels, (ii) banded as CC in dye-buoyancy gradients, (iii) sedimented with the velocity expected for monomer CC, and (iv) gave two-hit kinetics as functions of both concentration and time of exposure of the cells to DNA. A second major peak of activity behaved physicall as though mostly due to dimer CC forms and gave single-hit response curves. Because almost no dimer was detectable optically on analytical gels of starting preparations, its specific activity was high relative to that of the monomers.