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W T Moore

Publications and source records attributed to W T Moore.

At least 37 records · Page 2Linked to original sources
PubMed1991

Recent advances in liquid chromatography-mass spectrometry and capillary zone electrophoresis-mass spectrometry for protein analysis.

The utility of the combination of separations techniques, such as liquid chromatography and capillary zone electrophoresis, with mass spectrometry in applications involving protein analysis is discussed. The use of continuous-flow fast atom bombardment and electrospray ionization mass spectrometry is compared for the analysis of tryptic digests. For liquid chromatography, both microbore and slurry-packed capillary bore columns were used to separate peptides from proteolytic digests.

Chromatography, Liquid↗
PubMed1991

Monoclonal antibody-defined epitope map of expressed rubella virus protein domains.

An expanded library of murine monoclonal antibodies (MAbs) was generated by infecting BALB/C mice with the Therien strain of rubella virus (RV) and selecting secreting hybrids by enzyme-linked immunosorbent assay (ELISA) using purified virion targets. A panel of plasmids containing specified RV cDNA fragments was also constructed by using a variety of strategies with pGE374- and pGE374-derived expression vectors. Hybrid RecA-RV-beta-galactosidase (LacZ)- or RecA-RV-truncated LacZ-containing proteins collectively representing the entire open reading frame of the structural proteins of RV were overexpressed in Escherichia coli. Bacterial lysates were then probed by ELISA with selected MAbs and by immunoblot following separation by electrophoresis under denaturing conditions. With this approach, MAbs that appeared to react with linear determinants defined epitopes localized within the following domains: MAbs C-1, C-2, and C-8 bind epitopes within the predicted amino-terminal 21 amino acids of the capsid region C9 to C29; MAb C-9 binds to a domain bounded by C64 and C97; MAbs E2-1 through E2-6 bind to the E2 glycoprotein backbone region from E2(1) to E2(115); MAbs E1-18 and E1-20 bind to the E1 glycoprotein region from E1(202) to E1(283). MAb E1-18 neutralizes RV infectivity; MAb E1-20 neutralizes infectivity and modestly inhibits hemagglutination. Analyses with selected synthetic peptides have confirmed several of the molecular domains deduced with the expressed proteins. These plasmid constructions and peptides have proven useful in beginning to unravel the molecular organization of several antigenic sites of this human pathogen.

Animals↗
PubMed1989

Coupling capillary zone electrophoresis and continuous-flow fast atom bombardment mass spectrometry for the analysis of peptide mixtures.

Combined capillary zone electrophoresis (CZE)-continuous-flow fast atom bombardment (CF-FAB) mass spectrometry is described for the analysis of mixtures of peptides. A 90 cm x 50 microns I.D. fused-silica capillary column was used for electrophoretic separations and was connected to the CF-FAB probe via an interface which allows a total flow into the mass spectrometer of about 5 microliters/min. Solutions of peptides were pneumatically loaded onto the CZE capillary, providing sample amounts of 0.1-20 pmol. The magnetic mass spectrometer was scanned over the desired mass range, usually between m/z 500 and 2500. Results are shown for separation and analysis of mixtures of synthetic peptides and also for protease digests of recombinant human growth hormone and horse heart cytochrome c.

Electrophoresis↗
PubMed1989

Synthesis of phosphopeptides containing O-phosphoserine or O-phosphothreonine.

Peptides containing phosphoserine or phosphothreonine were synthesized by solid phase methods. Phosphoserine and phosphothreonine were incorporated into peptides using Boc-diphenylphosphono esters of serine and threonine and standard DCC/HOBt coupling. The phenylphosphoesters were not removed when the peptides were cleaved from the resin by HF or by trifluoromethane sulfonic acid, but were subsequently removed by catalytic hydrogenation. Phosphopeptides were purified by HPLC and by Fe+3-Chelex chromatography and their identity verified by mass spectrometry. Two peptides, Leu-Arg-Arg-Ala-Ser(P)-Leu-Gly and Leu-Arg-Arg-Ala-Thr(P)-Leu-Gly, were prepared by both enzymatic and chemical methods and had identical properties.

Amino Acids↗
PubMed1988

Microbore high-performance liquid chromatography-mass spectrometry for the analysis of proteolytic digests by continuous-flow fast-atom bombardment mass spectrometry.

Microbore high-performance liquid chromatographic (HPLC) techniques have been combined with fast atom bombardment mass spectrometry (FAB-MS) for the mass-specific detection of mixtures of peptides produced by proteolytic hydrolysis of proteins. The continuous-flow FAB interface has been utilized for direct coupling of the microbore HPLC system and the mass spectrometer. Conditions are reported for the effective separation of 100 pmol of peptides at flow-rates of 5 microliters/min with acetonitrile gradients and 1-mm I.D. C8 columns. A comparison is also made between columns of 5 and 25 cm lengths for the separation of peptide mixtures. Data are presented for the separation of peptides on a slurry-packed C18 fused-silica capillary column with the continuous-flow HPLC-FAB-MS interface at flow-rates of 3 microliters/min.

Chromatography, High Pressure Liquid↗
PubMed1987

Microbore HPLC/mass spectrometry for the analysis of peptide mixtures using a continuous flow interface.

Microbore HPLC techniques have been combined with fast atom bombardment mass spectrometry to provide HPLC/MS capabilities for the analysis of mixtures of peptides and small proteins. The interface between the liquid chromatograph and mass spectrometer is a continuous flow direct insertion probe which contains a fused silica capillary that delivers the eluting solvent to the FAB source of the mass spectrometer at a rate of 5-10 microL/min. Data are presented for the analysis of several mixtures of peptides ranging in molecular weights from about 900 to 6000 daltons. In addition, the analysis of 100 pmol of a tryptic digest of whale myoglobin is shown where 16 of the possible 19 peptides were identified in the mass range m/z 2200-250. The advantages of this approach to HPLC/MS are a relatively high sensitivity because of the low flow rates and low background, and the ability to detect high molecular weight compounds.

Animals↗
PubMed1987

Structural studies on equine glycoprotein hormones. Amino acid sequence of equine chorionic gonadotropin beta-subunit.

The complete amino acid sequence of the beta-subunit of equine chorionic gonadotropin (eCG beta) has been established by both automated Edman and manual 5-dimethylaminonaphthalene-1-sulfonyl-Edman degradations. Specific fragments were produced by cleavage with Staphylococcus aureus V8 protease, trypsin, or dilute HCl. For the sequence analyses of the heavily glycosylated COOH-terminal portion, a chemical deglycosylation procedure with trifluoromethanesulfonic acid was employed. The peptide chain of eCG beta consists of 149 amino acid residues. Five or more oligosaccharide chains are attached to the protein, 1 unit linked by an N-glycosidic bond to asparagine at residue 13 and four or more units linked by O-glycosidic bonds to serine or threonine at residues in the COOH-terminal portion. The carbohydrate-bearing hydroxy amino acids have not yet been rigorously established. As compared to the beta-subunits of the pituitary gonadotropin hormones, lutropin, follitropin, and thyrotropin, eCG beta possesses a glycosylated COOH-terminal extension of about 30 amino acid residues, as does the human chorionic gonadotropin beta-subunit (hCG beta). When the comparison is restricted inside the disulfide bond-containing core (residues 1-110), the beta-subunit of eCG is highly homologous to hCG beta (66%). On the other hand, although the overall structural features closely resemble each other, much less homology exists in the COOH-terminal extensions of eCG beta and hCG beta.

Amino Acid Sequence↗
PubMed1986

Cyclic AMP potentiates the retinoic acid-induced expression of tissue transglutaminase in peritoneal macrophages.

Fresh serum and retinoids induce the expression of tissue transglutaminase in cultured mouse resident peritoneal macrophages. Analogues of cyclic AMP, such as dibutyryl cyclic AMP, and agents that increase intracellular cyclic AMP levels enhance the induction. Dibutyryl cyclic AMP alone has little effect on transglutaminase expression, but it increases the sensitivity of macrophages to low concentrations of either serum or retinoic acid. Dibutyryl cyclic AMP potentiates the transglutaminase-inducing activity of both free retinoic acid and retinoic acid bound to the serum retinol-binding protein. Pretreating macrophages with dibutyryl cyclic AMP or retinoic acid does not prime the cells to respond to the other agent; instead, both agents must be present simultaneously to obtain the synergistic induction of transglutaminase. Our studies suggest that the modulation of intracellular cyclic AMP levels may have pronounced effects on retinoic acid-induced gene expression in myeloid cells.

Animals↗
PubMed1985

Retinoic acid-induced expression of tissue transglutaminase in human promyelocytic leukemia (HL-60) cells.

Addition of retinoic acid to human promyelocytic leukemia cells results in a dramatic increase in cellular transglutaminase activity. This increase is due to the induction of a specific intracellular transglutaminase, tissue transglutaminase. Retinoic acid-induced expression of tissue transglutaminase is potentiated by analogues of cyclic AMP. The induction of the enzyme can be detected within 6 h of the addition of the retinoid to the cell and results in increases of the enzyme of at least 50-fold. The induction of HL-60 transglutaminase is a specific response of the cells to retinoic acid and is not seen with other agents that induce HL-60 differentiation. We believe that the induction of tissue transglutaminase is a useful index of the early events in retinoid-regulated gene expression in both normal and transformed cells.

Acyltransferases↗
PubMed1985

Priming procedure and hormone preparations influence rat granulosa cell response.

The FSH activity in equine (e) FSH, eLH, eCG, and ovine LH were examined and compared to that in the standard reference preparation NIH FSH-S13 by three types of assay: the FSH radioreceptor assay with rat testicular homogenate and the stimulation of plasminogen activator production and steroidogenic activity in granulosa cells from diethylstilbestrol (DES)- or eCG-primed donor rats. The difference in the two types of granulosa cells was that the eCG-primed cells have already acquired significant aromatase activity. With the exception of oLH, which showed very little FSH activity (approximately 0.03-0.08 X NIH FSH-S13) throughout the three assays, the equine gonadotropins exhibited great variations in activity with respect to each assay. eFSH, the most active molecule in these assays, had an activity of 44 X NIH FSH-S13 in the receptor binding assay, 8.75 X NIH FSH-S13 in plasminogen activator production, and 4-5 X NIH FSH-S13 in steroid production when assayed in the DES-primed granulosa cells. In the eCG-primed cells, eFSH showed an activity of 4.2 X NIH FSH-S13 in plasminogen activator production and 8.2 X NIH FSH-S13 in progesterone production. eLH had an activity of 10 X NIH FSH-S13 in FSH radioreceptor assay, but showed very little activity and behaved like oLH in stimulation of the cellular responses of DES-primed granulosa cells. However, when eLH was assayed in the eCG-primed cells, it did show stimulating activity with respect to the production of plasminogen activator and progesterone; however, the dose-response curves were not parallel to those of eFSH and eCG. eCG had much less FSH receptor-binding activity (0.29 X NIH FSH-S13) than eLH. It behaved like a LH molecule in DES-primed granulosa cells, but did show activity (approximately 1 X NIH FSH-S13) in stimulating the production of plasminogen activator and progesterone in eCG-primed granulosa cells. From these results, we conclude that under our culture conditions, neither eLH nor eCG was active in the DES-primed granulosa cells, but both were active in the eCG-primed cells, and that the choice of assay conditions and reference standards is very important. Different types of assay may give rise to completely different comparisons for the same molecules. The equine gonadotropins provide a particularly dramatic example of such differences.

Animals↗
PubMed1985

Interferon-gamma requires serum retinoids to promote the expression of tissue transglutaminase in cultured human blood monocytes.

The culture of HPBM in serum-containing medium induced a large accumulation of the protein cross-linking enzyme, tissue TGase. Immune IFN enhanced the expression of tissue TGase in cultured monocytes. Enzyme-inducing activity, both in normal and IFN-treated cells, was completely blocked by depleting the serum of the lipid fraction. The readdition of retinol at a physiologic concentration (1 micron) to delipidized serum completely restored the enzyme-inducing activity in cultured monocytes. Thus, serum retinoids seem to play an important regulatory role in the expression of tissue TGase gene in differentiating human monocytes.

Acyltransferases↗
PubMed1984

Retinoic acid-induced expression of tissue transglutaminase in mouse peritoneal macrophages.

The culture of peritoneal macrophages in serum-containing media induces a dramatic increase in the expression of the enzyme tissue transglutaminase. The transglutaminase-inducing activity of serum is abolished by extraction of lipids and fully restored by re-addition of physiological concentrations (1-100 nM) of trans-retinoic acid. Induction of the enzyme is detectable within a 90-min exposure of macrophages to retinoic acid and is completely blocked by actinomycin D, suggesting that the retinoid rapidly increases the rate of transglutaminase gene expression. Delipidized serum is required to elicit the transglutaminase-inducing activity of retinoic acid and this effect is decreased if the serum is depleted of the serum retinol-binding protein. Our studies suggest that retinoic acid and serum retinol-binding protein can directly regulate macrophage gene expression and specifically induce the synthesis of tissue transglutaminase.

Acyltransferases↗
PubMed1980

Pregnant mare serum gonadotropin. Rapid chromatographic procedures for the purification of intact hormone and isolation of subunits.

A method exploiting hydroxylapatite chromatography was developed to purify pregnant mare serum gonadotropin (PMSG or eCG) to high biological activity from partially purified commerical preparations. In addition, an alternative method utilizing chromatography on quaternary aminoethyl (QAE)-Sephadex and Sephadex G-200 is also presented. Both procedures are capable of producing, from commerical material with a potency of approximately 2,500 IU/mg, a product in excess of 12,000 IU/mg. If care is taken in the selection of fractions from the hydroxylapatite chromatography, essentially purified material may be obtained in a single step. The best fraction from the QAE-Sephadex and G-200 chromatography procedure contains a minor impurity. Pregnant mare serum gonadotropin subunits were purified by a single chromatographic step from the foregoing preparations utilizing 6 M guanidine hydrochloride for dissociation, followed by chromatography on Sephadex G-75. Analytical data, including amino acid composition, carbohydrate composition. NH2-terminal amino acid determinations, and electrophoretic behavior of the subunits in sodium dodecyl sulfate polyacrylamide gel electrophoresis are presented.

Amino Acids↗
PubMed1979

Rabbit lutropin: preparation, characterization of the hormone, its subunits and radioimmunoassay.

The purification of rabbit lutropin is described. A product with a potency of 1.53 X NIH-LH-Sl was obtained as assayed by the ovarian ascorbic acid depletion assay. In a homologous radioimmunoassay, which is described, rabbit lutropin has a potency 4.83 X NIH-LH-Sl. In a radioligand assay, utilizing labeled ovine lutropin as the trace, the relative potency was 0.47 X NIH-LH-Sl measured by 50% inhibition comparison since rabbit lutropin response in this system did not parallel ovine lutropin. A counter-current distribution procedure for separation of rabbit lutropin subunits is described. Amino acid composition of the isolated subunits and intact rabbit lutropin was determined. The carbohydrate composition of the latter is presented; only amino sugar determinations are available for the subunits. The NH2-terminal amino acids are phenylalanine (alpha subunit) and alanine (beta subunit). Preliminary data on COOH-terminal amino acids are provided.

Amino Acid Sequence↗