Search PubMed⌕ Search

Biomedical subjects

D A Gage

Publications and source records attributed to D A Gage.

At least 37 records · Page 2Linked to original sources
PubMed1998

Hetero-oligomerization-dependent binding of pig oocyte zona pellucida glycoproteins ZPB and ZPC to boar sperm membrane vesicles.

The zona pellucida surrounding the pig oocyte contains two Mr 55,000 glycoproteins, pZPB and pZPC, which are orthologues of mouse zona proteins ZP1 and ZP3, respectively. We previously reported that isolated boar sperm membrane vesicles possess high affinity binding sites for partially purified pZPB, but not pZPC. Interestingly, co-incubation experiments also implicated pZPB-pZPC complexes as potential ligands. We now report that when depleted of a minor pZPC contaminant by size exclusion chromatography, pZPB lacks independent binding activity. In solid phase binding assays employing immobilized boar sperm membranes, pZPB failed to compete with biotin-(pZPB+pZPC) probe, and biotin-labeled pZPB yielded negligible binding. However, when co-incubated with pZPC prior to the binding assays, pZPB acted as a potent competitor, and biotin-labeled pZPB exhibited high affinity, saturable binding. Binding activity was attributed to pZPB-pZPC heterocomplexes, which were detected in co-incubation mixtures by size exclusion chromatography and Western blot analysis. In the pig, therefore, sperm membranes possess a zona-binding protein with high affinity sites for pZPB-pZPC heterocomplexes, but not free glycoprotein subunits. Consequently, associative interactions between zona molecules can contribute toward both the assembly of the zona matrix and generation of ligands important for sperm-zona interactions.

Animals↗
PubMed1998

Synthesis and confirmation of structure for a new gibberellin, 2 beta-hydroxy-GA12 (GA110), from spinach and oil palm.

The identity of a new gibberellin (GA) in spinach and oil palm sap has been confirmed as 2 beta-hydroxy-GA12 (GA110) by comparisons of GC-mass spectral data obtained for the trimethylsilyl ether methyl ester derivatives with those of a synthetic sample prepared by means of a 24 step sequence from gibberellic acid; 2 beta-hydroxy-GA24 was also prepared. Experimental details for the latter part of the syntheses are described.

Gas Chromatography-Mass Spectrometry↗
PubMed1998

The endogenous choline supply limits glycine betaine synthesis in transgenic tobacco expressing choline monooxygenase.

Certain plants produce glycine betaine (GlyBet) in the chloroplast by a two-step oxidation of choline. Introducing GlyBet accumulation into plants that lack it is a well-established target for metabolic engineering because GlyBet can lessen damage from osmotic stress. The first step in GlyBet synthesis is catalyzed by choline mono-oxygenase (CMO), a stromal enzyme with a Rieske-type [2Fe-2S] center. The absence of CMO is the primary constraint on GlyBet production in GlyBet-deficient plants such as tobacco, but the endogenous choline supply is also potentially problematic. To investigate this, we constructed transgenic tobacco plants that constitutively express a spinach CMO cDNA. The CMO protein was correctly compartmented in chloroplasts and was enzymatically active, showing that its [2Fe-2S] cluster had been inserted. Salinization increased CMO protein levels, apparently via a post-transcriptional mechanism, to as high as 10% of that in salinized spinach. However, the GlyBet contents of CMO+ plants were very low (0.02-0.05 mumol g-1 fresh weight) in both unstressed and salinized conditions. Experiments with [14C]GlyBet demonstrated that this was not due to GlyBet catabolism. When CMO+ plants were supplied in culture with 5 mM choline or phosphocholine, their choline and GlyBet levels increased by at least 30-fold. The choline precursors mono- and dimethylethanolamine also enhanced choline and GlyBet levels but ethanolamine did not, pointing to a major constraint on flux to choline at the first methylation step in its synthesis. The extractable activity of the enzyme mediating this step in tobacco was only 3% that of spinach. We conclude that in GlyBet-deficient plants engineered with choline-oxidizing genes, the size of the free choline pool and the metabolic flux to choline need to be increased to attain GlyBet levels as high as those in natural accumulators.

Betaine↗
PubMed1998

Salinity promotes accumulation of 3-dimethylsulfoniopropionate and its precursor S-methylmethionine in chloroplasts.

Wollastonia biflora (L.) DC. plants accumulate the osmoprotectant 3-dimethylsulfoniopropionate (DMSP), particularly when salinized. DMSP is known to be synthesized in the chloroplast from S-methylmethionine (SMM) imported from the cytosol, but the sizes of the chloroplastic and extrachloroplastic pools of these compounds are unknown. We therefore determined DMSP and SMM in mesophyll protoplasts and chloroplasts. Salinization with 30% (v/v) artificial seawater increased protoplast DMSP levels from 4.6 to 6.0 mumol mg-1 chlorophyll (Chl), and chloroplast levels from 0.9 to 1.9 mumol mg-1 Chl. The latter are minimum values because intact chloroplasts leaked DMSP during isolation. Correcting for this leakage, it was estimated that in vivo about one-half of the DMSP is chloroplastic and that stromal DMSP concentrations in control and salinized plants are about 60 and 130 mM, respectively. Such concentrations would contribute significantly to chloroplast osmoregulation and could protect photosynthetic processes from stress injury. SMM levels were measured using a novel mass-spectrometric method. About 40% of the SMM was located in the chloroplast in unsalinized W. biflora plants, as was about 80% in salinized plants; the chloroplastic pool in both cases was approximately 0.1 mumol mg-1 Chl. In contrast, > or = 85% of the SMM was extrachloroplastic in pea (Pisum sativum L.) and spinach (Spinacia oleracea L.), which lack DMSP. DMSP synthesis may be associated with enhanced accumulation of SMM in the chloroplasm.

Amino Acids↗
PubMed1998

Dimethylsulfoniopropionate biosynthesis in Spartina alterniflora1. Evidence that S-methylmethionine and dimethylsulfoniopropylamine are intermediates.

The osmoprotectant 3-dimethylsulfoniopropionate (DMSP) occurs in Gramineae and Compositae, but its synthesis has been studied only in the latter. The DMSP synthesis pathway was therefore investigated in the salt marsh grass Spartina alterniflora Loisel. Leaf tissue metabolized supplied [35S]methionine (Met) to S-methyl-l-Met (SMM), 3-dimethylsulfoniopropylamine (DMSP-amine), and DMSP. The 35S-labeling kinetics of SMM and DMSP-amine indicated that they were intermediates and, consistent with this, the dimethylsulfonium moiety of SMM was shown by stable isotope labeling to be incorporated as a unit into DMSP. The identity of DMSP-amine, a novel natural product, was confirmed by both chemical and mass-spectral methods. S. alterniflora readily converted supplied [35S]SMM to DMSP-amine and DMSP, and also readily converted supplied [35S]DMSP-amine to DMSP; grasses that lack DMSP did neither. A small amount of label was detected in 3-dimethylsulfoniopropionaldehyde (DMSP-ald) when [35S]SMM or [35S]DMSP-amine was given. These results are consistent with the operation of the pathway Met --> SMM --> DMSP-amine --> DMSP-ald --> DMSP, which differs from that found in Compositae by the presence of a free DMSP-amine intermediate. This dissimilarity suggests that DMSP synthesis evolved independently in Gramineae and Compositae.

Computer Simulation↗
PubMed1998

Phosphoglycerylethanolamine posttranslational modification of plant eukaryotic elongation factor 1alpha.

Eukaryotic elongation factor 1alpha (eEF-1A) is a multifunctional protein. There are three known posttranslational modifications of eEF-1A that could potentially affect its function. Except for phosphorylation, the other posttranslational modifications have not been demonstrated in plants. Using matrix-assisted laser desorption/ionization-mass spectrometry and peptide mass mapping, we show that carrot (Daucus carota L.) eEF-1A contains a phosphoglycerylethanolamine (PGE) posttranslational modification. eEF-1A was the only protein labeled with [14C]ethanolamine in carrot cells and was the predominant ethanolamine-labeled protein in Arabidopsis seedlings and tobacco (Nicotiana tabacum L.) cell cultures. In vivo-labeling studies using [3H]glycerol, [32P]Pi, [14C]myristic acid, and [14C]linoleic acid indicated that the entire phospholipid phosphatidylethanolamine is covalently attached to the protein. The PGE lipid modification did not affect the partitioning of eEF-1A in Triton X-114 or its actin-binding activity in in vitro assays. Our in vitro data indicate that this newly characterized posttranslational modification alone does not affect the function of eEF-1A. Therefore, the PGE lipid modification may work in combination with other posttranslational modifications to affect the distribution and the function of eEF-1A within the cell.

Amino Acid Sequence↗
PubMed1997

Structural characterization of an abnormally cross-linked muropeptide dimer that is accumulated in the peptidoglycan of methicillin- and cefotaxime-resistant mutants of Staphylococcus aureus.

Laboratory mutants of Staphylococcus aureus strain ATCC 8325 (27S) selected for increased minimal inhibitory concentration (MIC) values to methicillin and cefotaxime showed increased rates of cell wall turnover and detergent-induced autolysis in virtual parallel with the increasing MIC for the antibiotic. Also in parallel with the increasing MICs for the particular antibiotic used in the selection was the gradual accumulation of an unusual muropeptide in the peptidoglycan of the mutants, muropeptide 12, which is a minor component of the cell wall of the parental strain. Analysis of muropeptide 12, its peptide derivative, and its lysostaphin degradation products by high pressure liquid chromatography, Edman degradation, and mass spectrometry suggests that muropeptide 12 is a dimer in which the two monomeric components are interlinked by two pentaglycyl cross-bridges, thus generating a 14-member macrocyclic ring structure. This unusual cross-linked structure may be the product of the abnormal activity of penicillin-binding protein 2 which has grossly reduced antibiotic binding capacity in the mutant staphylococci.

Amino Acid Sequence↗
PubMed1997

A new route for synthesis of dimethylsulphoniopropionate in marine algae.

The 3-dimethylsulphoniopropionate (DMSP) produced by marine algae is the main biogenic precursor of atmospheric dimethylsulphide (DMS). This biogenic DMS, formed by bacterial and algal degradation of DMSP, contributes about 1.5 x 10(13) g of sulphur to the atmosphere annually, and plays a major part in the global sulphur cycle, in cloud formation and potentially in climate regulation. Although DMSP biosynthesis has been partially elucidated in a higher plant, nothing is known about how algae make DMSP except that the whole molecule is derived from methionine. Here we use in vivo isotope labelling to demonstrate that DMSP synthesis in the green macroalga Enteromorpha intestinalis proceeds by a route entirely distinct from that in higher plants. From methionine, the steps are transamination, reduction and S-methylation to give the novel sulphonium compound 4-dimethylsulphonio-2-hydroxybutyrate (DMSHB), which is oxidatively decarboxylated to DMSP. The key intermediate DMSHB was also identified in three diverse phytoplankton species, indicating that the same pathway operates in other algal classes that are important sources of DMS. The fact that a transamination initiates this pathway could help explain how algal DMSP (and thereby DMS) production is enhanced by nitrogen deficiency.

Chlorophyta↗
PubMed1997

Specific oxidative cleavage of carotenoids by VP14 of maize.

The plant growth regulator abscisic acid (ABA) is formed by the oxidative cleavage of an epoxy-carotenoid. The synthesis of other apocarotenoids, such as vitamin A in animals, may occur by a similar mechanism. In ABA biosynthesis, oxidative cleavage is the first committed reaction and is believed to be the key regulatory step. A new ABA-deficient mutant of maize has been identified and the corresponding gene, Vp14, has been cloned. The recombinant VP14 protein catalyzes the cleavage of 9-cis-epoxy-carotenoids to form C25 apo-aldehydes and xanthoxin, a precursor of ABA in higher plants.

Abscisic Acid↗
PubMed1997

Structural characterization of peptidoglycan muropeptides by matrix-assisted laser desorption ionization mass spectrometry and postsource decay analysis.

In this study we report the development of matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS)-based methods for the structural characterization of muropeptides derived from peptidoglycan. Prior to analysis, peptidoglycan samples were subjected to enzymatic digestion with muramidase and the resulting muropeptides were purified by HPLC. A new matrix, 5-chloro-2-mercaptobenzothiazole, was employed for the MALDI-MS analysis. The results have demonstrated that sub-picomole to femtomole detection can be achieved in both positive mode and negative mode, allowing unambiguous determination of the molecular masses of monomeric and oligomeric muropeptides. Structural information from monomeric muropeptides was obtained by further postsource decay (PSD) analysis. Fragmentation patterns in positive mode and negative mode PSD were complementary for the elucidation of the peptide chain sequence. Lysostaphin digestion was also incorporated with MALDI mass mapping analysis for determination of peptide chain cross-linking patterns of muropeptide oligomers from Staphylococcus aureus strains.

Amino Acid Sequence↗
PubMed1997

Identification of in vivo phosphorylation sites of CD45 protein-tyrosine phosphatase in 70Z/3.12 cells.

Phosphorylation of CD45, a transmembrane protein-tyrosine phosphatase (PTPase), has been proposed to mediate docking of signaling proteins and to modulate PTPase activity. To study the role of phosphorylation in CD45, in vivo phosphorylation sites of CD45 from 70Z/3.12 cells were identified using 32P labeling, trypsin digestion, two-dimensional peptide mapping, high performance liquid chromatography, phosphoamino acid analysis, matrix-assisted laser desorption/ionization mass spectrometry, and specific enzymatic degradation. Eight phosphopeptides, a through h, were isolated and four phosphorylation sites were identified. All four phosphorylation sites were in the membrane-distal PTPase domain (D2) and the C-terminal tail and none were in the membrane-proximal PTPase domain (D1). One site, Ser(P)939 peptide h, was in the D2 domain and, by comparison to the three-dimensional structure of PTP1B, is predicted to lie at the apex of the substrate binding loop. Ser939 was the only in vitro phosphorylation site for protein kinase C among the phosphorylation sites identified. Four of the C-terminal peptides identified (d, e, f, and g) spanned the same sequence and were derived from the same phosphorylation site in the C-terminal tail, Ser1204. Peptide a was derived from the intact C terminus and comprised a mixture of monophosphorylated peptides containing either Ser(P)1248 or Thr(P)1246. Knowledge of the precise phosphorylation sites of CD45 will lead to the design of experiments to define the role of phosphorylation in PTPase activity and in signaling.

Amino Acid Sequence↗
PubMed1997

Accumulation of sulfoquinovosyl-1-O-dihydroxyacetone in a sulfolipid-deficient mutant of Rhodobacter sphaeroides inactivated in sqdC.

The biosynthesis of the sulfolipid sulfoquinovosyl diacylglycerol in the purple bacterium Rhodobacter sphaeroides requires at least four genes:sqdA, sqdB, sqdC, and sqdD. As part of our strategy aimed at the elucidation of the function of the different sqd gene products, we insertionally inactivated sqdC of R. sphaeroides. The resulting sqdC null mutant showed only a 90% reduction in sulfolipid content. Apparently, the sqdC gene product is required for optimal sulfolipid biosynthesis, but either catalyzes no essential reaction in the pathway or can be functionally replaced to a certain extent by a different protein. The mutant accumulated a 35S-labeled compound that was purified to homogeneity from cell extracts. Matrix-assisted laser desorption mass spectrometry and nuclear magnetic resonance spectroscopy provided conclusive structural evidence to identify the compound as alpha-D-sulfoquinovosyl-1-O-dihydroxyacetone that exists in two interconvertible, keto and hemiacetal forms. Incubation of wild-type protein extracts with the labeled compound did not result in the incorporation into sulfolipid as would be expected for an intermediate of the pathway. Based on our results we propose that the sqdC gene product mediates the substrate specificity of the UDP-sulfoquinovose:diacylglycerol sulfoquinovosyltransferase that is encoded by sqdD and that catalyzes the final reaction of sulfolipid biosynthesis.

Bacterial Proteins↗
PubMed1997

Choline monooxygenase, an unusual iron-sulfur enzyme catalyzing the first step of glycine betaine synthesis in plants: prosthetic group characterization and cDNA cloning.

Plants synthesize the osmoprotectant glycine betaine via the route choline --> betaine aldehyde --> glycine betaine. In spinach, the first step is catalyzed by choline monooxygenase (CMO), a ferredoxin-dependent stromal enzyme that has been hypothesized to be an oligomer of identical subunits and to be an Fe-S protein. Analysis by HPLC and matrix-assisted laser desorption ionization MS confirmed that native CMO contains only one type of subunit (Mr 42,864). Determination of acid-labile sulfur and nonheme iron demonstrated that there is one [2Fe-2S] cluster per subunit, and EPR spectral data indicated that this cluster is of the Rieske type--i.e., coordinated by two Cys and two His ligands. A full-length CMO cDNA (1,622 bp) was cloned from spinach using a probe generated by PCR amplification for which the primers were based on internal peptide sequences. The ORF encoded a 440-amino acid polypeptide that included a 60-residue transit peptide. The deduced amino acid sequence included two Cys-His pairs spaced 16 residues apart, a motif characteristic of Rieske-type Fe-S proteins. Larger regions that included this motif also showed some sequence similarity (approximately 40%) to Rieske-type proteins, particularly bacterial oxygenases. Otherwise there was very little similarity between CMO and proteins from plants or other organisms. RNA and immunoblot analyses showed that the expression of CMO in leaves increased several-fold during salinization. We conclude that CMO is a stress-inducible representative of a new class of plant oxygenases.

Amino Acid Sequence↗
PubMed1997

A picomole-scale method for charge derivatization of peptides for sequence analysis by mass spectrometry.

A highly activated ester containing a fixed positive charge, S-pentafluorophenyl [tris(2,4,6-trimethoxyphenyl)phosphonium]acetate bromide (TMPP-AcSC6F5 bromide), has been synthesized as a reagent for N-terminal modification of peptides. Stable in aqueous acetonitrile solution during extended storage, TMPP-AcSC6F5 bromide reacts with unprotected peptides through p-(dimethylamino)pyridine (DMAP)-promoted amidation in aqueous acetonitrile (15 min, ambient temperature) to form N-TMPP-Ac derivatives of peptides. These peptide derivatives are readily amenable to analysis by fast atom bombardment (FAB) and matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. Greater than 90% conversion has been observed in transforming low-nanomole quantities of analyte using molar ratios of 1:5:10 (peptide/reagent/ DMAP). For reactions at the picomole level a slightly modified stoichiometry, with molar ratios of 1:10:500, is employed. Owing to the high reaction efficiency and the tolerance to moderate excess reagent and base during analysis by FAB- and MALDI-MS, the reaction mixture containing the modified peptides can be analyzed directly in most cases, without sample cleanup. Examples of the preparation and analysis of a variety of N-TMPP-acetyl-peptides (TMPP-Ac-peptides) ranging from hexamers to 15-mers are given. Collisionally activated dissociation tandem mass spectrometry of TMPP-Ac-derivatives showed dominant a-type ions, accompanied by d- and c-type ions in some cases, allowing sequence determination to be made in a straightforward manner.

Amino Acid Sequence↗
PubMed1997

Mechanism-based inactivation of cytochrome P450 2B1 by N-benzyl-1-aminobenzotriazole.

The kinetics of inactivation of cytochrome P450 2B1, the major phenobarbital inducible rat hepatic P450, by N-benzyl-1-aminobenzotriazole (BBT) were characterized. Purified, reconstituted P450 2B1 7-ethoxy-4-(trifluoromethyl)coumarin (7-EFC) O-deethylase activity was inhibited by BBT in a mechanism-based manner. The loss of O-deethylase activity followed pseudo-first-order kinetics and was NADPH and BBT dependent. After a 5 min incubation, greater than 90% of the 2B1 activity was lost, whereas more than 70% of the ability of the reduced enzyme to bind CO was maintained. Inclusion of 10 mM glutathione in the inactivation reaction lowered the rate of inactivation (k(inactivation)) and increased the partition ratio without significantly affecting the inactivator concentration required for half-maximal inactivation (K(I)). The maximal rate constant for inactivation at 23 degrees C was 0.24 min(-1) without and 0.15 min(-1) with glutathione. The apparent K(I) was 2 microM in both cases. The extrapolated partition ratios were 4 and 9 without and with 10 mM glutathione, respectively. Consistent with mechanism-based inactivation, the loss of 7-EFC O-deethylase activity was irreversible, was not due to product inhibition, was saturable, and could be slowed by including increasing concentrations of competing substrate. However, the inactivated P450 2B1 was still able to metabolize substrate if iodosobenzene was used as an alternate oxidant. Inactivation of 2B1 with either N-[14C]-7-benzyl-1-aminobenzotriazole (BBT) or N-benzyl-1-amino-[14C]-2,3-benzotriazole resulted in the incorporation of covalent radiolabel into the apoprotein. The stoichiometry of labeled metabolite adduct to protein was approximately 0.4:1 in both cases. Identification of metabolites revealed the formation of 1-aminobenzotriazole, benzotriazole, benzaldehyde, and a new metabolite (27) during catalysis of BBT by P450 2B1. Together, these data suggest that P450 2B1 could be inactivated and labeled by more than one metabolite.

Animals↗
PubMed1997

Gibberellins and stem growth in Arabidopsis thaliana. Effects of photoperiod on expression of the GA4 and GA5 loci.

Arabidopsis thaliana (L.) Heynh. is a quantitative long-day (LD) rosette plant in which stem growth is mediated by gibberellins (CAs). Application of GAs to plants in short-day (SD) conditions resulted in rapid stem elongation and flower formation, with GA4 and GA9 being equally effective, and GA1 showing lower activity. The effects of photoperiod on the levels of endogenous GAs were measured by combined gas chromatography-mass spectrometry with selected ion monitoring. When plants were transferred from SD to LD conditions there was a slight decrease in the level of GA53 and an increase in the levels of C19-GAs, GA9, GA20, GA1, and GA8, indicating that GA 20-oxidase activity is stimulated in LD conditions. Expression of GA5, which encodes GA 20-oxidase, was highest in elongating stems and was correlated with the rate of stem elongation. By contrast, GA4, which encodes 3 beta-hydroxylase, showed low expression in stems and its expression was not correlated with the rate of stem elongation. We conclude that stem elongation in LD conditions is at least in part due to increased expression of GA5, whereas expression of GA4 is not under photoperiodic control.

Arabidopsis↗
PubMed1997

Identification of pre-gamma-globin.

A pre-gamma-globin species was identified by high performance liquid chromatography in platyrrhine primates. Although pre-gamma-globin has not been observed in human hemoglobin, its identification in platyrrhine hemoglobin was facilitated by the functional inactivation of one of the duplicated gamma-globin genes in platyrrhines, which simplified the high performance liquid chromatography elution pattern. Part, but not all, of the pre-gamma was glutathionyl gamma 2-globin, and matrix-assisted laser desorption/ionization mass spectrometry was used to demonstrate that the glutathionyl residue was located on cysteine 93. As this residue is invariant in primates, it is predicted that the formation of glutathionyl gamma-globin will be seen in all primate hemoglobins under appropriate conditions.

Amino Acid Sequence↗