-
Biochimica Et Biophysica Acta Oct 1990We compared several features of Na(+)-dependent phosphono[14C]formic acid (PFA) binding and Na(+)-dependent phosphate transport in rat renal brush border membrane...
We compared several features of Na(+)-dependent phosphono[14C]formic acid (PFA) binding and Na(+)-dependent phosphate transport in rat renal brush border membrane vesicles. From kinetic analyses, we estimated an apparent Km for PFA binding of 0.86 mM, an order of magnitude greater than that for phosphate and the high-affinity phosphate transport system. A hyperbolic Na(+)-saturation curve for PFA binding and a sigmoidal Na(+)-saturation curve for phosphate transport were demonstrated; based on these data, we estimated stoichiometries of 1:1 for Na+/PFA and 2:1 for Na+/phosphate. By radiation inactivation analysis, target sizes for brush border membrane protein(s) mediating Na(+)-dependent PFA binding and Na(+)-dependent phosphate transport corresponded to molecular masses of 555 +/- 32 kDa and 205 +/- 36 kDa, respectively. Similar analysis of the phosphate-inhibitable component of Na(+)-dependent PFA binding gave a target size of 130 +/- 28 kDa. We also demonstrated that phosphate deprivation, which elicits a 2.6-fold increase in brush border membrane Na(+)-dependent phosphate transport, had no effect on either Na(+)-dependent PFA binding or on the target size for PFA binding. However, phosphate deprivation appeared to increase the target size for phosphate transport (from 255 +/- 32 to 335 +/- 75 kDa (P less than 0.01]. In summary, we present evidence for several differences between Na(+)-dependent PFA binding and Na(+)-dependent phosphate transport in rat renal brush border membrane vesicles and suggest that PFA may not interact exclusively with the proteins mediating Na(+)-phosphate co-transport.
Topics: Animals; Biological Transport; Foscarnet; Kidney; Male; Microvilli; Phosphates; Phosphonoacetic Acid; Rats; Rats, Inbred Strains; Sodium
PubMed: 2145976
DOI: 10.1016/0005-2736(90)90146-f -
The Journal of Antimicrobial... Feb 1988Potential specific inhibitors of HIV RNA-dependent DNA polymerase (RDDP) were examined in an in-vitro test system containing purified HIV-1 RDDP, or functionally...
Potential specific inhibitors of HIV RNA-dependent DNA polymerase (RDDP) were examined in an in-vitro test system containing purified HIV-1 RDDP, or functionally purified cellular DNA-dependent DNA polymerases alpha, beta and gamma. A wide variety of drugs were tested for their specific inhibitory activity against the viral enzyme to identify substances which, at the same concentration, did not inhibit the cellular DNA polymerases. Phosphonoformic acid and derivatives were found to be the most specific inhibitors, followed by chlortetracycline.
Topics: Antiviral Agents; DNA Polymerase I; DNA Polymerase II; DNA Polymerase III; Foscarnet; HIV; In Vitro Techniques; Microbial Sensitivity Tests; Nucleic Acid Synthesis Inhibitors; Organophosphorus Compounds; Phosphonoacetic Acid; Reverse Transcriptase Inhibitors
PubMed: 2452149
DOI: 10.1093/jac/21.2.151 -
Presse Medicale (Paris, France : 1983) Mar 1992
Topics: Acyclovir; Antiviral Agents; Drug Resistance; Foscarnet; Humans; Immunocompromised Host; Phosphonoacetic Acid; Rimantadine; Virus Diseases; Zidovudine
PubMed: 1533917
DOI: No ID Found -
Biochemical Pharmacology Dec 1989Using the chemical structural analogs of phosphonoacetic acid (PAA) and related phosphonate compounds, we investigated which structural features are required for...
Using the chemical structural analogs of phosphonoacetic acid (PAA) and related phosphonate compounds, we investigated which structural features are required for competitive inhibition of Na+-Pi cotransport in rat renal cortical brush border membrane (BBM) vesicles (BBMV). The effects of compounds on [Nao+ greater than Nai+]-gradient-dependent 32Pi uptake by BBMV were examined using various inhibitor-to-32Pi concentration ratios in the transport assay medium. The replacement of a phosphono-group with an arsono-group in PAA, or the substitution of a carboxylic group in PAA by an amino or hydroxyl group, totally abolished the inhibitory action on Na+-Pi cotransport. Decreased electronegativity of carboxyl in PAA by coupling with hydrazine or hydroxylamine lowered the inhibitory potenty of PAA. Substitution of H at the alpha-carbon of PAA with ethyl or p-Cl-phenyl groups completely abolished the inhibitory activity, whereas alpha-halogenation with Br greatly increased the inhibitory potency of PAA, close to that of phosphonoformic acid (PFA). The inhibition by all the active tested monophosphates was strictly competitive. The tested compounds displaced [14C]PFA pre-bound onto BBMV in the presence of 100 mM NaCl. The ability of monophosphates to inhibit Na+-Pi cotransport across BBM and the binding of [14C]PFA were closely correlated (r = 0.925; P greater than 0.001). These results show that: (a) strong electronegativity at both ends of the PAA molecule is needed for inhibitory action, (b) an alpha-aliphatic or aromatic substituent at the alpha-carbon probably hinders the access of the inhibitor to the Pi-binding site of the Na+-Pi cotransporter in BBM, whereas (c) an alpha-electrophilic substituent--Br--enhances the inhibitory potency of PAA. The tested compounds inhibited Na+-Pi cotransport by binding, in the presence of Na+, on the same site on the luminal surface of BBM as did PFA and, by extension, Pi.
Topics: Animals; Binding, Competitive; Biological Transport; Carrier Proteins; Kidney Cortex; Kinetics; Male; Microvilli; Phosphates; Phosphonoacetic Acid; Rats; Rats, Inbred Strains; Sodium-Phosphate Cotransporter Proteins; Structure-Activity Relationship; Symporters
PubMed: 2597189
DOI: 10.1016/0006-2952(89)90514-5 -
The Journal of Biological Chemistry Oct 1994Glu-86, which interacts with the side chain of Arg-54 across the C1-C2 interface of Escherichia coli aspartate transcarbamoylase, tethers the end of the flexible 80's...
Glutamic acid 86 is important for positioning the 80's loop and arginine 54 at the active site of Escherichia coli aspartate transcarbamoylase and for the structural stabilization of the C1-C2 interface.
Glu-86, which interacts with the side chain of Arg-54 across the C1-C2 interface of Escherichia coli aspartate transcarbamoylase, tethers the end of the flexible 80's loop, which moves into the active site during the T to R transition. In order to determine whether this interaction is important for the correct positioning of the 80's loop and Arg-54 at the active site and also for the structural stabilization of the enzyme, a mutant version was created in which Glu-86 was replaced by Gln (Glu-86-->Gln). Although the mutant holoenzyme exhibits almost normal homotropic cooperativity, both the holoenzyme and catalytic subunit exhibit substantial reductions in activity and affinity for aspartate and carbamyl phosphate. Furthermore, the mutant holoenzyme shows a marked decrease in the activation by ATP and by the bisubstrate analog N-(phosphonoacetyl)-L-aspartate, reduced inhibition by CTP, as well as reduced affinities for these ligands. Results from molecular dynamics simulations of the Glu-86-->Gln and Glu-86-->Ala enzymes suggest that the positions of the 80's loop and Arg-54 are significantly perturbed by the introduction of these mutations. Taken together, these results indicate that the interaction between Glu-86 and Arg-54 is important for the formation of the high affinity, high activity form of the enzyme by stabilizing the correct position of the 80's loop and Arg-54 at the active site. Heat inactivation experiments also demonstrated that Glu-86 plays a significant role in the structural stabilization of the C1-C2 interface, since the temperature required for loss of half of the activity of the Glu-86-->Gln catalytic subunit is reduced by 5 degrees C relative to the wild-type catalytic subunit.
Topics: Aspartate Carbamoyltransferase; Aspartic Acid; Binding Sites; Enzyme Stability; Escherichia coli; Glutamic Acid; Kinetics; Mutagenesis, Site-Directed; Phosphonoacetic Acid; Structure-Activity Relationship
PubMed: 7929132
DOI: No ID Found -
Lancet (London, England) Sep 1990
Topics: Antiviral Agents; Capillaries; Crystallization; Foscarnet; Humans; Kidney Glomerulus; Phosphonoacetic Acid
PubMed: 1975929
DOI: 10.1016/0140-6736(90)92253-e -
NatureHuman immunodeficiency virus (HIV) is the causative agent of AIDS (acquired immune deficiency syndrome) a disease which poses a serious challenge to modern medicine. If... (Comparative Study)
Comparative Study
Human immunodeficiency virus (HIV) is the causative agent of AIDS (acquired immune deficiency syndrome) a disease which poses a serious challenge to modern medicine. If we are to conquer this disease we will need a protective vaccine or effective drugs able to block the life cycle of the virus. An early stage in the invasion of the host cell is the conversion of the RNA genome of the virus to a double-stranded DNA intermediate which subsequently becomes integrated into the host cell chromosome. The enzyme reverse transcriptase is crucial in this process and is thus an obvious chemotherapeutic target. In this study we have used site-directed mutagenesis of this enzyme expressed in Escherichia coli to reveal several important functional regions of the protein including putative components of the triphosphate binding site and pyrophosphate exchange sites.
Topics: Amino Acid Sequence; DNA, Viral; Escherichia coli; Foscarnet; HIV; Mutation; Phosphonoacetic Acid; RNA-Directed DNA Polymerase; Reverse Transcriptase Inhibitors; Structure-Activity Relationship; Thymidine; Zidovudine
PubMed: 2439916
DOI: 10.1038/327716a0 -
Genitourinary Medicine Feb 1992
Topics: Acquired Immunodeficiency Syndrome; Adult; Antiviral Agents; Cytomegalovirus Infections; Foscarnet; Ganciclovir; Humans; Phosphonoacetic Acid; Retinitis; Retrospective Studies
PubMed: 1312508
DOI: 10.1136/sti.68.1.60-a -
Lancet (London, England) Jan 1992
Comparative Study
Topics: Acquired Immunodeficiency Syndrome; Antiviral Agents; Cytomegalovirus Infections; Eye Infections, Viral; Foscarnet; Ganciclovir; Humans; Phosphonoacetic Acid; Retinitis; Zidovudine
PubMed: 1346202
DOI: 10.1016/0140-6736(92)90049-9 -
Lancet (London, England) Mar 1990
Topics: Acquired Immunodeficiency Syndrome; Adult; Antiviral Agents; Cytomegalovirus Infections; Foscarnet; Homosexuality; Humans; Male; Penile Diseases; Phosphonoacetic Acid; Skin Ulcer
PubMed: 1968562
DOI: No ID Found