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C Webster

Publications and source records attributed to C Webster.

12 recordsLinked to original sources
PubMed1992

Identification, purification and characterization of a novel phosphatidylinositol-specific phospholipase C, a third member of the beta subfamily.

The partial sequence of a novel PtdIns-specific phospholipase C of the beta subfamily (PtdIns-PLC beta 3) is described. Based upon the predicted protein sequence, monospecific antibodies have been raised and used to identify a suitable source for purification of the protein. Fractionation of HeLa S3 cells revealed that immunoreactive PtdIns-PLC beta 3 is membrane associated; purification (approximately 1000-fold) from this fraction yielded a single immunoreactive protein of 158 kDa, with a specific activity of 136 mumol.min-1.mg-1, with PtdIns 4,5-bisphosphate as substrate. Substrate specificity and Ca2+ dependence of this purified PtdIns-PLC are characteristic of the PtdIns-PLC beta subfamily.

Amino Acid Sequence↗
PubMed1992

Studies on the binding of the Escherichia coli MelR transcription activator protein to operator sequences at the MelAB promoter.

Escherichia coli MelR protein binds to two sites located upstream of the melAB transcription start site. Although both sites are required for optimal melibiose-dependent expression from the melAB promoter, some MelR-dependent expression is found if the upstream site is deleted or if the spacing between the two sites is altered. Gel retardation assays have been exploited to study MelR binding to a DNA fragment carrying just the upstream site. Methylation interference analysis was used to identify one guanine (at -104) which is important for MelR binding. Mutational analysis confirmed the importance of this base and revealed a second position (at -110) where mutations interfere with melAB promoter activity. Experiments using potassium permanganate as a probe suggested that the DNA sequence around -110 adopts a distorted conformation. We propose that the mutation at -104 alters MelR binding by interfering with a direct contact, whereas the mutation at -110 primarily affects DNA conformation. The binding of purified MelR protein to a melAB promoter fragment carrying both binding sites has also been studied: binding results in four retarded bands in gel assays. Methylation interference experiments have been exploited to identify the binding sites occupied in each complex. Although both binding sites share a common 18 bp sequence, MelR binding to the more upstream site is stronger. We could find no evidence for co-operative interactions between MelR and RNA polymerase and no major effects of melibiose. Some evidence for melibiose-dependent distortion in complexes between MelR and the melAB promoter is discussed.

Autoradiography↗
PubMed1992

Identification of an isozyme form of protein synthesis initiation factor 4F in plants.

We showed previously that wheat germ extracts contain two forms of protein synthesis initiation factor 4F that have very similar functional properties (Browning, K. S., Lax, S. R., and Ravel, J. M. (1987) J. Biol. Chem. 262, 11228-11232). One form, designated eIF-4F, is a complex containing two subunits, p220 and p26. The other form, designated eIF-(iso)4F, is a complex containing two subunits, p82 and p28, which are antigenically distinct from the subunits of eIF-4F. Both the p26 subunit of eIF-4F and the p28 subunit of eIF-(iso)4F are m7G cap-binding proteins. In this investigation, affinity-purified antibodies to the p220 and p26 subunits of wheat germ eIF-4F and to the p82 and p28 subunits of wheat germ eIF-(iso)4F were used to determine if isozyme forms of eIF-4F are present in maize and cauliflower. Extracts from wheat germ, maize root tips, and cauliflower inflorescences were partially purified by adsorption on m7GTP-Sepharose and elution with m7GTP (MGS eluate). Analysis by sodium dodecyl sulfate gel electrophoresis and immunoblotting with antibodies to the subunits of the wheat germ factors showed that the MGS eluate from maize contains polypeptides that react with antibodies to the p82 and p28 subunits of wheat eIF-(iso)4F, as well as polypeptides that react with antibodies to the p220 and p26 subunits of wheat eIF-4F. The MGS eluate from cauliflower also contains polypeptides that reacted with antibodies to the subunits of wheat eIF-(iso)4F. These results indicate that both maize and cauliflower contain the isozyme form of eIF-4F. In addition, it was found that the factors in the MGS eluate from maize support polypeptide synthesis in a system from wheat deficient in eIF-4F and eIF-(iso)4F, whereas the factors in the MGS eluate from cauliflower support polypeptide synthesis only to a small extent.

Brassica↗
PubMed1992

Effect of phorbol esters on protein kinase C-zeta.

Protein kinase C-zeta (PKC-zeta) is a member of the protein kinase C gene family which using in vitro preparations has been described as being resistant to activation by phorbol esters. PKC-zeta was found to be expressed in several cell types as an 80-kDa protein. In vitro translation of a full-length PKC-zeta construct also yielded as a primary translation product an 80-kDa protein. In the U937 cell, PKC-zeta was slightly more abundant in the cytosol than in the particulate fraction. Acute exposure of U937 cells to tetradecanoyl-phorbol-13-acetate (TPA), phorbol dibutyrate, mezerin, or diacylglycerol derivatives did not induce translocation of this isoform to the particulate fraction. Chronic exposure to 1 microM TPA failed to translocate or down-regulate PKC-zeta in U937, HL-60, COS, or HeLa-fibroblast fusion cells. To examine whether PKC-zeta was activated by TPA, PKC activity was evaluated in COS cells transiently over-expressing this isoform. In non-transfected cells, two peaks of phospholipid- and TPA-dependent kinase activity were observed. Eluting at a lower salt concentration was a peak of activity associated with PKC-alpha. PKC-zeta eluted with the second peak of activity and at a higher salt concentration. In transfected cells which expressed PKC-zeta at 4-10-fold over endogenous levels, there was only a slight increase in activity associated with the second peak. The activity and quantity of PKC-zeta did not strictly correlate. Treatment with TPA under conditions that did not alter PKC-zeta content abolished detection of the second peak of PKC activity eluting from the Mono Q column. Thus, PKC-zeta does not translocate or down-regulate in response to phorbol esters or diacylglycerol derivatives. However, for reasons discussed these studies do not resolve the issue of whether this isoform is activated by TPA.

Amino Acid Sequence↗
PubMed1992

A maize ribosome-inactivating protein is controlled by the transcriptional activator Opaque-2.

Although synthesis of the cytosolic maize albumin b-32 had been shown to be controlled by the Opaque-2 regulatory locus, its function was unknown. We show here that b-32 is a member of the large and widely distributed class of toxic plant proteins with ribosome-inactivating activity. These ribosome-inactivating proteins (RIPs) are RNA N-glycosidases that remove a single base from a conserved 28S rRNA loop required for elongation factor 1 alpha binding. Cell-free in vitro translation extracts were used to show that both maize and wheat ribosomes were resistant to molar excesses of b-32 but not to the dicotyledonous RIP gelonin. We extracted RIP activity from kernels during seed maturation and germination. The amount of RIP activity increased during germination, although the amount of b-32 protein remained fairly constant. Expression of a maize RIP gene under the control of an endosperm-specific transcriptional regulatory may be an important clue prompting investigation of the biological basis for RIP expression in seeds of other plants.

Amino Acid Sequence↗
PubMed1992

Efficient repression by a heterodimeric repressor in Escherichia coli.

Previous studies with purified variants of the 434 repressor having different operator-binding specificities have demonstrated the interactions of a heterodimeric repressor with a hybrid operator site. The present study investigates the interactions between the 434 repressor and its operator site. The optimum 434 operator half-site is used with a P22 operator half-site to create a hybrid 434/P22 operator. We show that this hybrid operator can be efficiently bound by a heterodimeric repressor, consisting of one wild-type 434 repressor monomer and one 434 repressor monomer with the binding specificity of the P22 repressor, to bring about repression in Escherichia coli.

Amino Acid Sequence↗
PubMed1991

Hypoxia enhances phosphorylation of eukaryotic initiation factor 4A in maize root tips.

We have identified two isoforms of initiation factor 4A (eIF-4A) in maize root tips, with distinct isoelectric points and similar molecular mass (approximately 50 kDa). Both isoforms of maize eIF-4A cross-react with antibodies raised against wheat germ eIF-4A, and one of the maize proteins (higher pI isoform) comigrates with purified wheat germ eIF-4A on two-dimensional gels. The two maize eIF-4As were indistinguishable by comparative peptide fingerprint analysis, which also showed a very strong similarity between eIF-4A in maize roots and wheat germ. Maize eIF-4As copurify with eIF-4F and eIF-(iso)4F on a 7-methyl-GTP-Sepharose affinity column, indicating that they are part of the 5'-cap-binding complex. Two-dimensional gel electrophoresis and immunoblotting of proteins from 32P-labeled maize root tips revealed that the lower pI isoform of eIF-4A is phosphorylated. Two-dimensional phosphopeptide maps of trypsin-digested eIF-4A contained one principal phosphorylated fragment; phosphoamino acid analysis indicated phosphorylation of threonine. In oxygenated maize root tips, the ratio of phosphorylated to nonphosphorylated eIF-4A is approximately 0.2. This ratio increases to approximately 1 within 20 min following the onset of hypoxia, due to interconversion between the two maize eIF-4A isoforms. The hypoxia-induced phosphorylation of eIF-4A is discussed with respect to metabolic responses, and the translational control of gene expression, in hypoxic plant tissues.

Electrophoresis, Gel, Two-Dimensional↗
PubMed1990

Effector-dependent conformational changes in protein kinase C gamma through epitope mapping with inhibitory monoclonal antibodies.

Three monoclonal antibodies (mAb) directed against the regulatory domain of the protein kinase C gamma (PKC gamma); 15G4, 5A2 and 36G9, were shown to display distinct properties with respect to PKC gamma kinase activity [Cazaubon, S., Marais, R., Parker, P. & Strosberg, A.D. (1989) Eur. J. Biochem. 182, 401-406]. The mAb 5A2 and 36G9, which act as potent inhibitors of the cofactor-dependent kinase activity, can no longer bind PKC gamma in the presence of phosphatidylserine and phosphatidylserine/phorbol ester, respectively; 15G4 binding is not influenced by effectors. Due to this functional relationship between the inhibitory mAb- and cofactor-binding sites, we sought to localize the mAb epitopes with respect to the functional sites of PKC gamma. For this purpose, several deletions were introduced at the 5' end of the PKC gamma cDNA and the mutant proteins were expressed in Escherichia coli. The determination of the immunoreactivity of the deleted PKC gamma proteins shows that the amino acid residues essential to the binding of 5A2 and 36G9 are directly adjacent to the second cysteine-rich motif: these are contained in the sequences at positions 151-163 and 164-197, respectively. In addition, various deletions around the C1 region of the regulatory domain allowed the identification of the second cysteine-rich motif as a functional binding site for phorbol dibutyrate. These deletion studies thus demonstrate that the epitopes recognized by the inhibitory mAbs 5A2 and 36G9 are distinct from the cofactor-binding sites. This suggests that the binding of phosphatidylserine and phorbol ester induce conformational changes in the regulatory domain of PKC, which are thus responsible for the loss of the 5A2 and 36G9 immunoreactivity of the native protein. In this conformational state, PKC gamma conserves its ability to interact with the non-inhibitory mAb 15G4. By using synthetic peptides, the 15G4 epitope was localized to the sequence 297-310 in the V3 variable region. This indicates that the flexibility of the V3 region, which delimits the C-terminus of the regulatory domain, may not be necessary for the allosteric activation of PKC. In view of these results, we propose that PKC activation by its cofactors results in intramolecular changes which allow the enzyme to bind exogenous substrates.

Amino Acid Sequence↗