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Biomedical subjects

D A Gage

Publications and source records attributed to D A Gage.

At least 55 records · Page 3Linked to original sources
PubMed1996

Mapping cross-linking sites in modified proteins with mass spectrometry: an application to cross-linked hemoglobins.

The combined use of trypsin digestion and peptide mass mapping by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) is reported here as an effective and rapid means for identifying the cross-linking sites in human oxy hemoglobin A (HbA) cross-linked with either bis(3,5-dibromosalicyl)-succinate or -glutarate. MALDI-MS analysis of a nondigested sample of oxy HbA modified with bis(3,5-dibromosalicyl)-glutarate showed that cross-linking only occurred between the beta 1- and beta 2-protomers and not between alpha 1- and alpha 2- or alpha- and beta-protomers, along with a modification reaction on an un-cross-linked beta-chain. Results of the MALDI tryptic peptide mass maps of cross-linked hemoglobins showed several cross-linked peptides having masses consistent with: beta Val67-Lys95-XL-beta Val67-Lys95, beta Val67-Lys95-XL-beta Val67-Arg104, beta Val67-Arg104-XL-beta Val67-Arg104, where XL represents the succinyl or glutaryl bridging span moiety. Each of these peptides contains Lys82, the targeted residue for these reagents, substantiating the cross-linking sites at beta 1Lys82-beta 2Lys82. This approach in general will enable rapid identification of the cross-linking sites in engineered proteins or intracellularly recombinant cross-linked proteins when the mass of the cross-linker and the protein primary structure are known.

Amino Acid Sequence↗
PubMed1996

Fetal globin expression in New World monkeys.

Reverse phase chromatography of the globin chains of adult, newborn, and fetal erythrocytes from three species of New World monkeys (Cebus apella, Aotus azarae, and Callithrix jacchus) representing three of the seven platyrrhine clades showed that gamma-globin expression was fetal in these animals. The globins were identified by a combination of chemical sequencing and mass spectrometric analysis. Since gamma-globin expression is fetal in the other major simian branch, the catarrhines, but embryonic in prosimian primates and nonprimate placental mammals, the evolution of fetal recruitment can now be assigned to the period between the simian-prosimian divergence (55 million years ago) and the platyrrhine-catarrhine divergence (35 million years ago). The gamma-globin gene underwent tandem duplication during the same evolutionary epoch, in accord with a model that suggests that the downstream duplicated gamma-gene (gamma2) was free to acquire the mutations necessary for fetal recruitment. Mass spectrometric analysis of tryptic digests of the gamma-globins verified the amino acid sequences deduced from genomic sequencing. Detailed analysis of high performance liquid chromatography and matrix-assisted laser desorption/ionization mass spectrometry data showed that gamma2-globin in Cebus was expressed to a far greater extent than gamma1-globin, supporting inferences drawn from a study of the promoter sequences. A "pre-gamma"-globin was observed in C. apella and shown to be primarily the glutathionyl adduct. The other species, A. azarae and C. jacchus, also express only one gamma-globin polypeptide. This work provides biochemical evidence of an evolutionary trend in the platyrrhines to alter the duplicated gamma-globin gene locus so that only one gamma-globin polypeptide is expressed.

Abortion, Veterinary↗
PubMed1996

The inhibitor protein of phosphorylated nitrate reductase from spinach (Spinacia oleracea) leaves is a 14-3-3 protein.

The inhibitor protein (IP) that inactivates spinach leaf NADH:nitrate reductase (NR) has been identified for the first time as a member of the eukaryotic 14-3-3 protein family based on three lines of evidence. First, the sequence of an eight amino acid tryptic peptide, obtained from immunopurified IP, matched that of a highly conserved region of the 14-3-3 proteins. Second, an authentic member of the 14-3-3 family, recombinant Arabidopsis GF14omega, caused inactivation of phospho-NR in a magnesium-dependent manner identical to IP. Third, an anti-GF14 monoclonal antibody cross-reacted with IP and anti-IP monoclonal antibodies cross-reacted with GF14omega.

14-3-3 Proteins↗
PubMed1996

A strategy to locate cysteine residues in proteins by specific chemical cleavage followed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

A simple methodology has been developed to characterize the number and location of free cysteine and cystine groups in peptides and proteins, using chemical modification and matrix-assisted laser desorption/ ionization time-of flight mass spectrometry (MALDITOF MS). This new approach employs a specific reaction between free sulfhydryls and 2-nitro-5-thiocyanobenzoic acid (NTCB) to selectively cyanylate cysteine thiols. The N-terminal peptide bond of the modified cysteinyl residue can then be cleaved under alkaline conditions to form an amino-terminal peptide and a series of 2-iminothiazolidine-4-carboxylyl peptides which can be mapped to the sequence by MALDI-MS. The number and location of cysteines can be deduced from mass analysis of the peptide mixture resulting from NTCB chemical processing of the original protein of known sequence under nonreducing conditions. Similar experiments are then performed following disulfide bond reduction to further characterize both cysteine and cystine groups. Experimental conditions are described for protein disulfide bond reduction, sulfhydryl cyanylation, and cleavage reactions performed both in solution and on Zetabind membranes.

Cysteine↗
PubMed1996

Identification of three C20-gibberellins: GA97 (2 beta-hydroxy-GA53), GA98 (2 beta-hydroxy-GA44) and GA99 (2 beta-hydroxy-GA19).

Three new C20-gibberellins, GA97 (2 beta-hydroxy-GA53), GA98 (2 beta-hydroxy-GA44) and GA99 (2 beta-hydroxy-GA19), have all been isolated from spinach, GA97 also from tomato root cultures and pea pods, and GA98 from maize pollen. The structures of these compounds were established by GC-mass spectrometric comparisons of the trimethylsilylated methyl esters with authentic samples prepared from gibberellic acid (GA3).

Gibberellins↗
PubMed1996

Molecular cloning and photoperiod-regulated expression of gibberellin 20-oxidase from the long-day plant spinach.

Spinach (Spinacia oleracea L.) is a long-day (LD) rosette plant in which stem growth under LD conditions is mediated by gibberellins (GAs). Major control points in spinach are the later steps of sequential oxidation and elimination of C-20 of C20-GAs. Degenerate oligonucleotide primers were used to obtain a polymerase chain reaction product from spinach genomic DNA that has a high homology with GA 20-oxidase cDNAs from Cucurbita maxima L. and Arabidopsis thaliana Heynh. This polymerase chain reaction product was used as a probe to isolate a full-length cDNA clone with an open reading frame encoding a putative 43-kD protein of 374 amino acid residues. When this cDNA clone was expressed in Escherichia coli, the fusion protein catalyzed the biosynthetic sequence GA53-->GA44-->GA19-->GA20 and GA19-->GA17. This establishes that in spinach a single protein catalyzes the oxidation and elimination of C-20. Transfer of spinach plants from short day (SD) to LD conditions caused an increase in the level of all GAs of the early-13-hydroxylation pathway, except GA53, with GA20, GA1, and GA8 showing the largest increases. Northern blot analysis indicated that the level of GA 20-oxidase mRNA was higher in plants in LD than in SD conditions, with highest level of expression in the shoot tips and elongating stems. This expression pattern of GA 20-oxidase is consistent with the different levels of GA20, GA1, and GA8 found in spinach plants grown in SD and LD conditions.

Amino Acid Sequence↗
PubMed1996

Phosphorylation of serine-15 of maize leaf sucrose synthase. Occurrence in vivo and possible regulatory significance.

Experiments were conducted to determine whether sucrose synthase (SuSy) was phosphorylated in the elongation zone of maize (Zea mays L.) leaves. The approximately 90-kD subunit of SuSy was 32P-labeled on seryl residue(s) when excised shoots were fed [32P]orthophosphate. Both isoforms of SuSy (the SS1 and SS2 proteins) were phosphorylated in vivo, and tryptic peptide-mapping analysis suggested a single, similar phosphorylation site in both proteins. A combination of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and automated Edman sequencing analysis unequivocally identified the phosphorylation site in the maize SS2 protein as serine-15. This site was phosphorylated in vitro by endogenous protein kinase(s) in a strictly Ca(2+)-dependent manner. A synthetic peptide, based on the phosphorylation site sequence, was used to identify and partially purify an endogenous Ca(2+)-dependent protein kinase(s) from the maize leaf elongation zone and expanding spinach leaves. Phosphorylation of SuSy in vitro selectively activates the cleavage reaction by increasing the apparent affinity of the enzyme for sucrose and UDP, suggesting that phosphorylation may be of regulatory significance. Conservation of the phosphorylation site, and the sequences surrounding it, among plant species suggests that phosphorylation of SuSy may be widespread, if not universal, in plants.

Amino Acid Sequence↗
PubMed1995

Mechanism-based inactivation of cytochrome P450 2B1 by 9-ethynylphenanthrene.

The 7-ethoxycoumarin O-deethylase activity of rat cytochrome P450 (P450) 2B1 was inactivated by 9-ethynylphenanthrene (9EPh) in a time- and NADPH-dependent manner, and the loss of activity followed pseudo-first-order kinetics. At 20 degrees C, the extrapolated maximal rate constant of inactivation (kinactivation) was 0.45 min-1 and the inactivator concentration required for half-maximal inactivation (KI) was 138 nM. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and HPLC analysis demonstrated that [2'-3H]-9EPh was irreversibly bound to the protein moiety of P450 2B1 and the stoichiometry of binding was determined to be 0.82 mol of inactivator bound per mole of P450 2B1. A radiolabeled peptide of approximately 3.0 kDa was identified by autoradiography after Tricine SDS-PAGE analysis of the peptides generated from a cyanogen bromide cleavage of [2'-3H]9EPh-inactivated P450 2B1. After HPLC separation of these peptides, the fraction containing the most radioactivity was analyzed by matrix-assisted laser desorption ionization-mass spectrometry (MALDI-MS) and peaks at m/z 2720.9 and 2939.9 were detected. The lower mass peak represents the molecular ion (MH+) for the peptide Ile290 to Met314 (theoretical 2722.2), while the higher mass peak corresponds to the MH+ of the modified peptide (theoretical 2940.5). The difference in mass (approximately 219) would correspond to the addition of a phenanthrylacetyl group to the peptide. When the fraction containing the modified and unmodified peptides was further digested with pepsin and reanalyzed by MALDI-MS, the site of attachment could be assigned to one of the amino acids contained in the peptide Phe297 to Leu307.

7-Alkoxycoumarin O-Dealkylase↗
PubMed1995

Structural analysis of cyclamen seed xyloglucan oligosaccharides using cellulase digestion and spectroscopic methods.

Xyloglucan polymers have been isolated from cyclamen seeds and characterized by both liquid and CP-MAS 13C NMR spectroscopy. Treatment of the polysaccharides with cellulase from Trichoderma viride afforded XG oligomers which have been studied with both mass spectrometry and NMR spectroscopy. The repeating unit in the intact polymers and the most abundant hydrolysis product correspond to the Gal1 Xyl3 Glc4 (XXLG) fragment. However, detection of notable amounts of Xyl3 Glc4 (XXXG) and Gal2 Xyl3 Glc4 (XLLG) indicates that the galactose distribution in xyloglucan from cyclamen is irregular. FAB-MS analysis of a new derivative, prepared by forming the glycosamine of m-tetrafluoroethoxy aniline, has led to unambiguous sequence information for the XXLG oligomer.

Carbohydrate Conformation↗
PubMed1995

Oxygenation mechanism of ribulose-bisphosphate carboxylase/oxygenase. Structure and origin of 2-carboxytetritol 1,4-bisphosphate, a novel O2-dependent side product generated by a site-directed mutant.

Site-directed mutagenesis has implicated active-site Lys329 of Rhodospirillum rubrum ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) in promoting the reaction of CO2 with the 2,3-enediol of ribulose bisphosphate and in stabilizing carboxylation intermediates [Hartman, F. C., & Lee, E. H. (1989) J. Biol. Chem. 264, 11784-11789; Lorimer, G. H., Chen, Y.-R., & Hartman, F. C. (1993) Biochemistry 32, 9018-9024]. Although the K329A mutant is greatly impaired in carboxylation, it catalyzes formation of the enediol, which is misprocessed to an O2-dependent side product [Harpel, M. R., & Hartman, F. C. (1994) Biochemistry 33, 5553-5561]. We now identify this novel side product as 2-carboxytetritol 1,4-bisphosphate (CTBP) by mass spectrometry, 1H-, 13C-, and 31P-NMR spectroscopy, and periodate oxidation. H2O2 accumulates during formation of CTBP, which we show to be derived from a transient precursor, the dicarbonyl D-glycero-2,3-pentodiulose 1,5-bisphosphate. The isolated dicarbonyl bisphosphate is processed by K329A to CTBP. These results, combined with isotope-labeling studies, suggest that CTBP arises by H2O2 elimination from an improperly stabilized peroxy adduct of the enediol intermediate, followed by rearrangement of the resulting dicarbonyl. Therefore, normal oxygenation, as catalyzed by wild-type Rubisco, is not a spontaneous reaction but must involve stabilization of the peroxy intermediate to mitigate formation of the dicarbonyl bisphosphate and subsequently CTBP. CTBP formation verifies the identity of Rubisco's previously invoked oxygenase intermediate, provides additional mechanistic insight into the oxygenation reaction, and shows that Lys329 promotes oxygenation as well as carboxylation. These results may be relevant to other oxygenases, which also exploit substrate carbanions rather than organic cofactors or transition metals for biological oxygen utilization.

Hydrogen Peroxide↗
PubMed1995

Fatty acid substrate specificities of human prostaglandin-endoperoxide H synthase-1 and -2. Formation of 12-hydroxy-(9Z, 13E/Z, 15Z)- octadecatrienoic acids from alpha-linolenic acid.

Human prostaglandin-endoperoxide H synthase-1 and -2 (hPGHS-1 and hPGHS-2) were expressed by transient transfection of COS-1 cells. Microsomes prepared from the transfected cells were used to measure the rates of oxygenation of several 18- and 20-carbon polyunsaturated fatty acid substrates including eicosapentaenoic, arachidonic, dihomo-gamma-linolenic > alpha-linolenic (delta 9, 12, 15), gamma-linolenic, and linoleic acids. Comparisons of kcat/Km values indicate that the order of efficiency of oxygenation is arachidonate > dihomo-gamma-linolenate > linoleate > alpha-linolenate for both isozymes; while the order of efficiency was the same for hPGHS-1 and hPGHS-2, alpha-linolenate was a particularly poor substrate for hPGHS-1. Gamma-Linolenate and eicosapentaenoate were poor substrates for both isozymes, but in each case, these two fatty acids were better substrates for hPGHS-2 than hPGHS-1. These studies of substrate specificities are consistent with previous studies of the interactions of PGHS isozymes with nonsteroidal anti-inflammatory drugs that have indicated that the cyclooxygenase active site of PGHS-2 is somewhat larger and more accommodating than that of PGHS-1. The major products formed from linoleate and alpha-linolenate were characterized. 13-Hydroxy-(9Z,11E)-octadecadienoic acid was found to be the main product formed from alpha-linoleate by both isozymes. The major products of oxygenation of alpha-linolenate were determined by mass spectrometry to be 12-hydroxy-(9Z,13E/Z,15Z)-octadecatrienoic acids. This result suggests that alpha-linolenate is positioned in the cyclooxygenase active site with a kink in the carbon chain such that hydrogen abstraction occurs from the omega 5-position in contrast to abstraction of the omega 8-hydrogen from other substrates.

Animals↗
PubMed1995

The GA5 locus of Arabidopsis thaliana encodes a multifunctional gibberellin 20-oxidase: molecular cloning and functional expression.

The biosynthesis of gibberellins (GAs) after GA12-aldehyde involves a series of oxidative steps that lead to the formation of bioactive GAs. Previously, a cDNA clone encoding a GA 20-oxidase [gibberellin, 2-oxoglutarate:oxygen oxidoreductase (20-hydroxylating, oxidizing), EC 1.14.11.-] was isolated by immunoscreening a cDNA library from liquid endosperm of pumpkin (Cucurbita maxima L.) with antibodies against partially purified GA 20-oxidase. Here, we report isolation of a genomic clone for GA 20-oxidase from a genomic library of the long-day species Arabidopsis thaliana Heynh., strain Columbia, by using the pumpkin cDNA clone as a heterologous probe. This genomic clone contains a GA 20-oxidase gene that consists of three exons and two introns. The three exons are 1131-bp long and encode 377 amino acid residues. A cDNA clone corresponding to the putative GA 20-oxidase genomic sequence was constructed with the reverse transcription-PCR method, and the identity of the cDNA clone was confirmed by analyzing the capability of the fusion protein expressed in Escherichia coli to convert GA53 to GA44 and GA19 to GA20. The Arabidopsis GA 20-oxidase shares 55% identity and > 80% similarity with the pumpkin GA 20-oxidase at the derived amino acid level. Both GA 20-oxidases share high homology with other 2-oxoglutarate-dependent dioxygenases (2-ODDs), but the highest homology was found between the two GA 20-oxidases. Mapping results indicated tight linkage between the cloned GA 20-oxidase and the GA5 locus of Arabidopsis. The ga5 semidwarf mutant contains a G-->A point mutation that inserts a translational stop codon in the protein-coding sequence, thus confirming that the GA5 locus encodes GA 20-oxidase. Expression of the GA5 gene in Ara-bidopsis leaves was enhanced after plants were transferred from short to long days; it was reduced by GA4 treatment, suggesting end-product repression in the GA biosynthetic pathway.

Amino Acid Sequence↗
PubMed1995

Accumulation of a novel glycolipid and a betaine lipid in cells of Rhodobacter sphaeroides grown under phosphate limitation.

Cells of the photosynthetic bacterium Rhodobacter sphaeroides grown under phosphate-limiting conditions accumulated nonphosphorous glycolipids and lipids carrying head groups derived from amino acids. Concomitantly, the relative amount of phosphoglycerolipids decreased from 90 to 22 mol% of total polar lipids in the membranes. Two lipids, not detectable in cells grown under standard conditions, were synthesized during phosphate-limited growth. Fast atom bombardment mass spectroscopy, exact mass measurements, 1H NMR spectroscopy, sugar composition analysis, and methylation analysis of the predominant glycolipid led to the identification of the novel compound 1,2-di-O-acyl-3-O-[alpha-D-glucopyranosyl-(1-->4)-O-beta-D-galactopyr anosyl]glycerol. The second lipid was identified as the betaine lipid 1,2-di-O-acyl-[4'-(N,N,N-trimethyl)-homoserine]glycerol by cochromatography employing an authentic standard from Chlamydomonas reinhardtii, fast atom bombardment mass spectroscopy, exact mass measurements, and 1H NMR spectroscopy. Prior to this observation, the occurrence of this lipid was thought to be restricted to lower plants and algae. Apparently, these newly synthesized nonphosphorous lipids, in addition to the sulfo- and the ornithine lipid also found in R. sphaeroides grown under optimal conditions, take over the role of phosphoglycerolipids in phosphate-deprived cells.

Betaine↗
PubMed1995

Localization of epitopes for monoclonal antibodies at the N-terminus of the porcine zona pellucida glycoprotein pZPC.

Zona pellucida glycoproteins play an important role in fertilization. In this study, attempts have been made to identify and define epitopes of monoclonal antibodies (mAbs) possessing contraceptive efficacy in vitro. The porcine zona glycoprotein pZPC, a homologue of mouse/human ZP3, was reduced and alkylated and subsequently digested with trypsin. Reverse-phase HPLC of the tryptic digest yielded twenty two peaks (T1-T22). When tested against mAbs reactive against sequential determinants on pZPC, T11 was immunoreactive with two mAbs, mAb-455 and mAb-467, as shown by antigen inhibition ELISA. IC50 values of 3.1 nM and 8.6 nM were recorded versus mAb-455 and mAb-467 respectively, and approximated the IC50 values obtained with intact pZPC. Amino acid analysis, Edman degradation, and FAB-MS identified T11 as the N-blocked decapeptide pyro-Gln-Pro-Val-Trp-Gln-Asp-Glu-Gly-Gln-Arg derived from the N-terminus of pZPC. Synthesis of overlapping octapeptides further identified VWQDE and WQDE as the minimum motifs with antigenic activity for mAb-455 and mAb-467, respectively. Glycine replacement peptides confirmed residues W,Q,E as critical for binding mAb-455 and W,Q,D,E as critical for binding mAb-467. Both mAbs inhibited binding of boar sperm to zona-encased porcine oocytes. These results, the first to define peptide epitopes of porcine zona glycoprotein, will assist in the design of an immunocontraceptive vaccine based on synthetic peptides corresponding to pZPC or its homologues in other species.

Amino Acid Sequence↗
PubMed1995

Matrix-assisted laser desorption ionization mass spectrometry: applications in peptide and protein characterization.

Matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) has quickly gained notoriety as an analytical tool that can accurately determined the molecular weight of proteins up to (and exceeding) 100 kDa from low-picomole amounts of sample. Beyond simple molecular weight determinations, investigators are developing this mass spectrometric technique to obtain more structural information from proteins using a combination of chemical and enzymatic modifications of the analyte prior to analysis by MALDI-MS. Covalent post-translational modifications such as phosphorylation and disulfide bond formation can be identified and localized. MALDI-MS also can be used to rapidly mass-map protein digests for sequence confirmation, often without requiring peptide fractionation. The potential for direct sequencing of peptides and proteins of unknown structure has been successfully demonstrated using several different MALDI-MS approaches. The objective of this report is to introduce MALDI-MS to the non-mass-spectrometrist and to describe applications of this new analytical technique for acquiring information related to peptide and protein structure.

Amino Acid Sequence↗
PubMed1995

Dependence of the murine antibody response to an anti-CDR2 VH peptide on immunogen formulation.

A peptide corresponding to the second complementarity determining region of the heavy chain (CDR2 VH) from a murine anti-CD4 monoclonal antibody, designated L202, was synthesized by solid phase methodology in a number of different antigenic forms, for the purpose of comparing the effectiveness of different adjuvant-carrier systems in the induction of a murine antibody response against the immunizing peptide and parent antibody molecule. Two of the synthetic constructs contained the palmitoyl and N-palmitoyl-cysteinyl-S-(2,3-palmitoyloxy)-propanediol (PAM3Cys) moieties, respectively, attached to the peptide amino terminus with the immunogen comprising liposomal formulations of each. A third immunogen consisted of the CDR2 VH peptide admixed with the PAM3Cys non-covalently and incorporated into liposomes (PAM3Cys + CDR2 VH). A fourth composition comprised the CDR2 VH peptide conjugated to KLH via the sulfhydryl of an added N terminal cysteine (KLH-CDR2 VH) and injected with Complete Freund's adjuvant (CFA). A fifth immunogen consisted of the CDR2 VH peptide synthesized on an octameric, branched polylysine core as a multiple antigenic peptide (MAP-CDR2 VH) injected in the presence of Freund's adjuvant. Groups of five mice were injected intramuscularly with each of these immunogens and bled at two week intervals. The highest anti-peptide gamma-immunoglobulin (IgG) responses (against uncoupled peptide by ELISA) after 56 days were obtained with mice receiving the PAM3Cys-CDR2 VH peptide. However, when screened against the CDR2 V(H) peptide present as the MAP derivative by ELISA, IgG raised against the cognate MAP-CDR2 peptide was much more reactive than IgG raised against the liposomal PAM3Cys-CDR2 VH immunogen. In either case, IgG raised against the KLH-CDR2VH conjugate was poorly reactive. These differences in reactivity to the two forms of the CDR2 VH peptide by ELISA did not correspond to major differences in reactivities to the intact L202 Ab by ELISA. Although the IgG against the MAP immunogen was slightly more reactive than the other antisera against the l202 Ab, all titers were less than 1:100. These data illustrate some limitations of using anti-peptide responses as indicators of potential reactivity against the native protein, but suggest that alternate formulations including lipoidal peptides are more effective than corresponding KLH-peptide conjugates in eliciting Ab responses against poorly immunogenic epitopes.

Amino Acid Sequence↗
PubMed1995

Chemical composition of essential oil from the root bark of Sassafras albidum.

The root bark of Sassafras albidum (Nuttall) Nees (Lauraceae) was extracted at room temperature with hexane and chloroform as solvents. The isolated essential oils were analyzed with GC and GC/MS. Thirty compounds were identified, nine of which have not been previously reported from this species. The major compounds were safrole (85%), camphor (3.25%), and methyleugenol (1.10%). Ten sesquiterpenes were also identified.

Chromatography, Gas↗