PubMed Health⌕ Search

Biomedical subjects

G D Prestwich

Publications and source records attributed to G D Prestwich.

At least 37 records · Page 2Linked to original sources
PubMed2000

Fluorescently labeled neomycin as a probe of phosphatidylinositol-4, 5-bisphosphate in membranes.

Phosphatidylinositol-4,5-bisphosphate (PI(4,5)P(2)), a minor component of the plasma membrane, is important in signal transduction, exocytosis, and ion channel activation. Thus fluorescent probes suitable for monitoring the PI(4,5)P(2) distribution in living cells are valuable tools for cell biologists. We report here three experiments that show neomycin labeled with either fluorescein or coumarin can be used to detect PI(4,5)P(2) in model phospholipid membranes. First, addition of physiological concentrations of PI(4,5)P(2) (2%) to lipid vesicles formed from mixtures of phosphatidylcholine (PC) and phosphatidylserine (PS) enhances the binding of labeled neomycin significantly (40-fold for 5:1 PC/PS vesicles). Second, physiological concentrations of inositol-1,4,5-trisphosphate (10 microM I(1,4,5)P(3)) cause little translocation of neomycin from PC/PS/PI(4,5)P(2) membranes to the aqueous phase, whereas the same concentrations of I(1,4,5)P(3) cause significant translocation of the green fluorescent protein/phospholipase C-delta pleckstrin homology (GFP-PH) constructs from membranes (Hirose et al., Science, 284 (1999) 1527). Third, fluorescence microscopy observations confirm that one can distinguish between PC/PS vesicles containing either 0 or 2% PI(4, 5)P(2) by exposing a mixture of the vesicles to labeled neomycin. Thus fluorescently labeled neomycin could complement GFP-PH constructs to investigate the location of PI(4,5)P(2) in cell membranes.

Acetates↗
PubMed2000

Attachment of hyaluronic acid to polypropylene, polystyrene, and polytetrafluoroethylene.

Surfaces of polypropylene (PP), polystyrene (PS) and polytetrafluoroethylene (PTFE) were activated with radio frequency plasmas Ar and NH3 to aminate the polymer surface and were subsequently reacted with hyaluronic acid (HA) in one of the three different attachment schemes. Results show that ammonia plasma treated polymers were more reactive toward HA attachment. The three chemistry schemes consisted of two distinct approaches: (1) direct attachment of the HA to the aminated surface, and (2) extending the reactive group away from the surface with succinic anhydride and then reacting the newly formed carboxylic acid group with an adipic dihydrazide modified HA (HA-ADH). The latter scheme proved to be more effective, suggesting that steric effects were involved with the reactivity of the HA with surface groups. These HA-coated polymers are a candidate for cell attachment and growth.

Ammonia↗
PubMed2000

Using photolabile ligands in drug discovery and development.

Photoactivatable ligands are important tools used in drug discovery and drug development. These ligands enable researchers to identify the targets of drugs, to determine the affinity and selectivity of the drug-target interaction, and to identify the binding site on the target. Examples are presented from three fundamentally different approaches: (1) photoaffinity labeling of target macromolecules; (2) photoactivation and release of 'caged ligands'; and (3) photoimmobilization of ligands onto surfaces.

Drug Design↗
PubMed2000

Site-directed mutagenesis of squalene-hopene cyclase: altered substrate specificity and product distribution.

BACKGROUND: Two regions of squalene-hopene cyclase (SHC) were examined to define roles for motifs posited to be responsible for initiation and termination of the enzyme-catalyzed polyolefinic cyclizations. Specifically, we first examined the triple mutant of the DDTAVV motif, a region deeply buried in the catalytic cavity and thought to be responsible for the initiation of squalene cyclization. Next, four mutants were prepared for Glu45, a residue close to the substrate entrance channel proposed to be involved in the termination of the cyclization of squalene. RESULTS: The DDTAVV motif in SHC was changed to DCTAEA, the corresponding conserved region of eukaryotic oxidosqualene cyclase (OSC), by the triple mutation of D377C/V380E/V381A; selected single mutants were also examined. The triple mutant showed no detectable cyclization of squalene, but effectively cyclized 2,3-oxidosqualene to give mono- and pentacyclic triterpene products. Of the Glu45 mutants, E45A and E45D showed reduced activity, E45Q showed slightly increased activity, and E45K was inactive. A normal yield of pentacyclic products was produced, but the ratio of hopene 2 to hopanol 3 was significantly changed in the less active mutants. CONCLUSIONS: Initiation and substrate selectivity may be determined by the interaction of the DDTAVV motif with the isopropylidene of squalene (for SHC) and of the DCTAEA motif with the epoxide of oxidosqualene (for OSC). This is the first report of a substrate switch determined by a central catalytic motif in a triterpenoid cyclase. At the termination of cyclization, the product ratio may be largely controlled by Glu45 at the entrance channel to the active site.

Amino Acid Motifs↗
PubMed2000

A hyaluronic acid-taxol antitumor bioconjugate targeted to cancer cells.

A cell-targeted polymeric prodrug prepared from Taxol and chemically modified hyaluronic acid (HA) was evaluated in vitro. Herein we report four results in support of the selective uptake and targeted toxicity of the HA-Taxol prodrug. First, a fluorescently labeled HA-Taxol (FITC-HA-Taxol) was synthesized and used to demonstrate cell-specific binding and uptake using flow cytometry and confocal microscopy. Second, the selective cytotoxicity of FITC-HA-Taxol allowed direct correlation of uptake with selective cytotoxicity. Third, the rapid uptake and selective cytotoxicity of HA-Taxol bioconjugates could be blocked by either excess HA or by an anti-CD44 antibody, but not by chondroitin sulfate (CS). Finally, the release of free Taxol from HA-Taxol in human plasma or in cell culture media revealed that the free drug was hydrolytically released from the bioconjugate by cleavage of the labile 2' ester linkage. Taken together, these data support the notion that the targeted cytotoxicity of HA-Taxol bioconjugates requires receptor-mediated cellular uptake of the bioconjugate followed by hydrolytic release of free Taxol.

3T3 Cells↗
PubMed1999

Expression cloning of protein targets for 3-phosphorylated phosphoinositides.

The phosphatidylinositol 3-kinase (PI 3'-K) family of lipid kinases play a critical role in cell proliferation, survival, vesicle trafficking, motility, cytoskeletal rearrangements, and oncogenesis. To identify downstream effectors of PI 3'-K, we developed a novel screen to isolate proteins that bind to the major products of PI 3'-K: phosphatidylinositol-3,4-bisphosphate (PtdIns-3,4-P(2)) and PtdIns-3,4,5-trisphosphate (PtdIns-3,4,5-P(3)). This screen uses synthetic biotinylated analogs of these lipids in conjunction with libraries of radiolabeled proteins that are produced by coupled in vitro transcription/translation reactions. The feasibility of the screen was initially demonstrated using avidin-coated beads prebound to biotinylated PtdIns-3,4-P(2) and PtdIns-3,4,5-P(3) to specifically isolate the pleckstrin homology domain of the serine/threonine kinase Akt. We then demonstrated the utility of this technique in isolating novel 3'-phosphorylated phosphatidylinositol (3'-PPI)-binding proteins through the preliminary screening of in vitro transcribed/translated cDNAs from a small pool expression library derived from mouse spleen. Three proteins were isolated that bound specifically to 3'PPIs. Two of these proteins have been previously characterized as PIP3BP/p42(IP4) and the PtdIns-3,4,5-P(3)-dependent serine/threonine kinase phosphoinositide-dependent kinase 1. The third protein is a novel protein that contains only a Src homology 2 domain and a pleckstrin homology domain; this protein has a higher specificity for both PtdIns-3,4,5-P(3) and PtdIns-3,4-P(2) than for PtdIns-4, 5-bisphosphate. Transcripts of this novel gene are present in every tissue analyzed but are most prominently expressed in spleen. We have renamed this new protein PHISH for 3'-phosphoinositide-interacting Src homology-containing protein. This report demonstrates the utility of this technique for isolating and characterizing 3'-PPI-binding proteins and has broad applicability for the isolation of binding domains for other lipid products.

Adaptor Proteins, Signal Transducing↗
PubMed1999

Distinct specificities of inwardly rectifying K(+) channels for phosphoinositides.

Activation of several inwardly rectifying K(+) channels (Kir) requires the presence of phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P(2)). The constitutively active Kir2.1 (IRK1) channels interact with PtdIns(4,5)P(2) strongly, whereas the G-protein activated Kir3.1/3.4 channels (GIRK1/GIRK4), show only weak interactions with PtdIns(4,5)P(2). We investigated whether these inwardly rectifying K(+) channels displayed distinct specificities for different phosphoinositides. IRK1, but not GIRK1/GIRK4 channels, showed a marked specificity toward phosphates in the 4,5 head group positions. GIRK1/GIRK4 channels were activated with a similar efficacy by PtdIns(3,4)P(2), PtdIns(3,5)P(2), PtdIns(4,5)P(2), and PtdIns(3,4,5)P(3). In contrast, IRK1 channels were not activated by PtdIns(3,4)P(2) and only marginally by high concentrations of PtdIns(3,5)P(2). Similarly, high concentrations of PtdIns(3,4,5)P(3) were required to activate IRK1 channels. For either channel, PtdIns(4)P was much less effective than PtdIns(4,5)P(2), whereas PtdIns was inactive. In contrast to the dependence on the position of phosphates of the phospholipid head group, GIRK1/GIRK4, but not IRK1 channel activation, showed a remarkable dependence on the phospholipid acyl chains. GIRK1/GIRK4 channels were activated most effectively by the natural arachidonyl stearyl PtdIns(4,5)P(2) and much less by the synthetic dipalmitoyl analog, whereas IRK1 channels were activated equally by dipalmitoyl and arachidonyl stearyl PtdIns(4,5)P(2). Incorporation of PtdInsP(2) into the membrane is necessary for activation, as the short chain water soluble diC(4) PtdIns(4,5)P(2) did not activate either channel, whereas activation by diC(8) PtdIns(4, 5)P(2) required high concentrations.

Animals↗
PubMed1999

Identification of a phosphoinositide binding motif that mediates activation of mammalian and yeast phospholipase D isoenzymes.

Phosphoinositides are both substrates for second messenger-generating enzymes and spatially localized membrane signals that mediate vital steps in signal transduction, cytoskeletal regulation and membrane trafficking. Phosphatidylcholine-specific phospholipase D (PLD) activity is stimulated by phosphoinositides, but the mechanism and physiological requirement for such stimulation to promote PLD-dependent cellular processes is not known. To address these issues, we have identified a site at which phosphoinositides interact with PLD and have assessed the role of this region in PLD function. This interacting motif contains critical basic amino acid residues that are required for stimulation of PLD activity by phosphoinositides. Although PLD alleles mutated at this site fail to bind to phosphoinositides in vitro, they are membrane-associated and properly localized within the cell but are inactive against cellular lipid substrates. Analogous mutations of this site in yeast PLD, Spo14p, result in enzymes that localize normally, but with catalytic activity that has dramatically reduced responsiveness to phosphoinositides. The level of responsiveness to phosphoinositides in vitro correlated with the ability of PLD to function in vivo. Taken together, these results provide the first evidence that phosphoinositide regulation of PLD activity observed in vitro is physiologically important in cellular processes in vivo including membrane trafficking and secretion.

Amino Acid Sequence↗
PubMed1999

Regulation of AMP deaminase by phosphoinositides.

AMP deaminase (AMPD) converts AMP to IMP and is a diverse and highly regulated enzyme that is a key component of the adenylate catabolic pathway. In this report, we identify the high affinity interaction between AMPD and phosphoinositides as a mechanism for regulation of this enzyme. We demonstrate that endogenous rat brain AMPD and the human AMPD3 recombinant enzymes specifically bind inositide-based affinity probes and to mixed lipid micelles that contain phosphatidylinositol 4,5-bisphosphate. Moreover, we show that phosphoinositides specifically inhibit AMPD catalytic activity. Phosphatidylinositol 4,5-bisphosphate is the most potent inhibitor, effecting pure noncompetitive inhibition of the wild type human AMPD3 recombinant enzyme with a K(i) of 110 nM. AMPD activity can be released from membrane fractions by in vitro treatment with neomycin, a phosphoinositide-binding drug. In addition, in vivo modulation of phosphoinositide levels leads to a change in the soluble and membrane-associated pools of AMPD activity. The predicted human AMPD3 sequence contains pleckstrin homology domains and (R/K)X(n)(R/K)XKK sequences, both of which are characterized phosphoinositide-binding motifs. The interaction between AMPD and phosphoinositides may mediate membrane localization of the enzyme and function to modulate catalytic activity in vivo.

AMP Deaminase↗
PubMed1999

Control of enzymatic degradation of hyaluronan by divalent cations.

Enzymatic degradation of hyaluronan (HA) by testicular hyaluronidase (HAase, hyaluronate 4-glucanohydrolase) requires inclusion of mono- or divalent cations in the reaction mixture. Most divalent cations activated HAase with equal potency; however, Cu2+ suppressed degradation, and Ca2+ showed a concentration-dependent regulation of size of the oligosaccharide products. Careful selection of HAase assay parameters is critical for discovery of novel HAase inhibitors and for preparation of controlled-size oligosaccharide fragments.

Calcium↗
PubMed1999

PDGF induces an early and a late wave of PI 3-kinase activity, and only the late wave is required for progression through G1.

BACKGROUND: Platelet-derived growth factor (PDGF) triggers cytoskeletal rearrangements and chemotaxis within minutes. These events are at least in part due to the activation of phosphoinositide (PI) 3-kinase; there is good temporal correlation between these events and the accumulation of 3-phosphorylated products of the kinase. Prolonged and continuous PDGF exposure results in S-phase entry many hours after the initial burst of activity. Although early signals appear responsible for the early responses, they may not fully account for later responses, such as cell-cycle progression. RESULTS: We assessed when PI 3-kinase products accumulate in PDGF-stimulated cells. In addition to the previously identified early accumulation of products, we detected a second, prolonged wave of accumulation 3-7 hours after stimulation. To determine the relative contribution of each phase to PDGF-dependent DNA synthesis, we first developed an assay in which synthetic 3-phosphorylated lipids were used to rescue DNA synthesis in cells expressing a PDGF-receptor mutant. The lipids rescued DNA synthesis only when added 2-6 hours after PDGF. In addition, PI 3-kinase inhibitors failed to block PDGF-dependent DNA synthesis if added during the first wave of PI 3-kinase activity, but adding them later, in G1 phase, prevented PDGF-dependent cell-cycle progression. CONCLUSIONS: PDGF induces distinct waves of PI 3-kinase activity. The second wave is required for PDGF-dependent DNA synthesis, whereas the initial wave is not. One of the ways in which cells use PI 3-kinase to mediate distinct cellular responses seems to be by regulating when its products accumulate.

Androstadienes↗
PubMed1999

The Ost1p subunit of yeast oligosaccharyl transferase recognizes the peptide glycosylation site sequence, -Asn-X-Ser/Thr-.

Other laboratories have established that oligosaccharyl transferase (OST) from Saccharomyces cerevisiae can be purified as a protein complex containing eight different subunits. To identify the OST subunit that recognizes the peptide sites that can be glycosylated, we developed photoaffinity probes containing a photoreactive benzophenone derivative, p-benzoylphenylalanine (Bpa), as part of an 125I-labeled peptide that could be expected to be glycosylated. We found that Asn-Bpa-Thr peptides served as substrates for OST and that photoactivation of these probes in the presence of microsomes abolished the OST activity. Photoactivation of 125I-labeled Asn-Bpa-Thr in the presence of microsomes resulted in specific covalent labeling of a protein doublet of molecular mass 62 and 64 kDa. By carrying out the photoactivation of the probe using microsomes containing epitope-tagged Ost1p, we demonstrated that the 125I-labeled protein was Ost1p. Radiolabeling of this protein was dependent on irradiation at 350 nm. No labeling was detected using a probe containing Ala instead of Thr as the third amino acid residue. We conclude that Ost1p is the subunit of the OST complex that recognizes the peptide sites in the nascent chains that are destined to be glycosylated.

Base Sequence↗
PubMed1999

Photoactivatable peptides based on BMS-197525: a potent antagonist of the human thrombin receptor (PAR-1).

Photoactivatable analogs of the human thrombin receptor (PAR-1) antagonist, N-trans-cinnamoyl-p-fluoroPhe-p-guanidinoPhe-Leu-Arg-NH2 (BMS-197525), were prepared with benzophenone substitutions in the N-terminal, Leu, or Arg position. The analogs retained antagonist activity (with reduced potency); the tritium-labeled isotopomers are potential photoaffinity labels for the receptor. C-Terminal extension of the analogs with ornithine(biotin) did not significantly alter antagonist potency.

Chromatography, High Pressure Liquid↗
PubMed1999

The binding site for an inhibitor of squalene:hopene cyclase determined using photoaffinity labeling and molecular modeling.

BACKGROUND: The squalene:hopene cyclases (SHCs) are bacterial enzymes that convert squalene into hopanoids, a function analogous to the action of oxidosqualene cyclases (OSCs) in eukaryotic steroid and triterpenoid biosynthesis. We have identified the binding site for a selective, potent, photoactivatable inhibitor of an SHC. RESULTS: SHC from Alicyclobacillus acidocaldarius was specifically labeled by [3H]Ro48-8071, a benzophenone-containing hypocholesteremic drug. Edman degradation of a peptide fragment of covalently modified SHC confirmed that Ala44 was specifically modified. Molecular modeling, using X-ray-derived protein coordinates and a single point constraint for the inhibitor, suggested several geometries by which Ro48-8071 could occupy the active site. CONCLUSIONS: A covalent complex of a potent inhibitor with a squalene cyclase has been characterized. The amino acid modification and molecular modeling suggest that Ro48-8071 binds at the junction between the central cavity and substrate entry channel, therefore inhibiting access of the substrate to the active site.

Affinity Labels↗
PubMed1999

Intraspecific nucleotide variation at the pheromone binding protein locus in the turnip moth, Agrotis segetum.

Inter- and intraspecific amino acid variability in the pheromone binding proteins (PBPs) of the Lepidoptera is believed to contribute to a molecular mechanism of pheromone blend discrimination. Messenger RNA coding for PBP sequence in Agrotis segetum (Noctuidae) was cloned, and nucleotide and inferred amino acid variation across a 769-bp region of a PBP locus was studied in two populations. A single gene copy was fully sequenced, revealing an intron/exon structure conserved with distant saturniids. While several nucleotide substitutions are predicted to result in amino acid replacement, tests for the presence of natural selection suggest that the observed variation is neutral. A phylogenetic analysis provides evidence that the two populations are in the process of genetic isolation.

Amino Acid Sequence↗
PubMed1999

Fluorescent phosphoinositide derivatives reveal specific binding of gelsolin and other actin regulatory proteins to mixed lipid bilayers.

Fluorescent derivatives of phosphatidyl inositol (PtdIns)-(4,5)-P2 were synthesized and used to test the effects of the PtdIns-(4, 5)-P2-regulated proteins gelsolin, tau, cofilin, and profilin on labeled PtdIns-(4,5)-P2 that was either in micellar form or mixed with phosphatidylcholine (PtdCho) in bilayer vesicles. Gelsolin increased the fluorescence of 7-nitrobenz-2-oxa-1,3-diazole (NBD)- or pyrene-labeled PtdIns-(4,5)-P2 and NBD-PtdIns-(3,4,5)-P3. Cofilin and profilin produced no detectable change at equimolar ratios to PtdIns-(4,5)-P2, while tau decreased NBD-PtdIns-(4,5)-P2 fluorescence. Fluorescence enhancement by gelsolin of NBD-PtdIns-(4, 5)-P2 in mixed lipid vesicles depended on the mole fraction of PtdIns-(4,5)-P2 in the bilayer. Specific enhancement of 3% NBD-PtdIns-(4,5)-P2 : 97% PtdCho was much lower than that of 10% PtdIns-(4,5)-P2 : 90% PtdCho, but the enhancement of 3% NBD-PtdIns-(4,5)-P2 could be increased by addition of 7% unlabeled PtdIns-(4,5)-P2. The gelsolin-dependent increase in NBD-PtdIns-(4, 5)-P2 fluorescence was reversed by addition of Ca2+ or G-actin. Significant, but weaker, fluorescence enhancement was observed with the gelsolin N-terminal domain (residues 1-160) and a peptide comprised of gelsolin residues 150-169. Fluorescence energy transfer from gelsolin to pyrene-PtdIns-(4,5)-P2 was much stronger with intact gelsolin than the N-terminal region of gelsolin containing the PtdIns-(4,5)-P2 binding sites, suggesting that PtdIns-(4,5)-P2 may bind near a site formed by the juxtaposition of the N- and C-terminal domains of gelsolin.

4-Chloro-7-nitrobenzofurazan↗
PubMed1999

GCS1, an Arf guanosine triphosphatase-activating protein in Saccharomyces cerevisiae, is required for normal actin cytoskeletal organization in vivo and stimulates actin polymerization in vitro.

Recent cloning of a rat brain phosphatidylinositol 3,4, 5-trisphosphate binding protein, centaurin alpha, identified a novel gene family based on homology to an amino-terminal zinc-binding domain. In Saccharomyces cerevisiae, the protein with the highest homology to centaurin alpha is Gcs1p, the product of the GCS1 gene. GCS1 was originally identified as a gene conditionally required for the reentry of cells into the cell cycle after stationary phase growth. Gcs1p was previously characterized as a guanosine triphosphatase-activating protein for the small guanosine triphosphatase Arf1, and gcs1 mutants displayed vesicle-trafficking defects. Here, we have shown that similar to centaurin alpha, recombinant Gcs1p bound phosphoinositide-based affinity resins with high affinity and specificity. A novel GCS1 disruption strain (gcs1Delta) exhibited morphological defects, as well as mislocalization of cortical actin patches. gcs1Delta was hypersensitive to the actin monomer-sequestering drug, latrunculin-B. Synthetic lethality was observed between null alleles of GCS1 and SLA2, the gene encoding a protein involved in stabilization of the actin cytoskeleton. In addition, synthetic growth defects were observed between null alleles of GCS1 and SAC6, the gene encoding the yeast fimbrin homologue. Recombinant Gcs1p bound to actin filaments, stimulated actin polymerization, and inhibited actin depolymerization in vitro. These data provide in vivo and in vitro evidence that Gcs1p interacts directly with the actin cytoskeleton in S. cerevisiae.

ADP-Ribosylation Factor 1↗