PubMed Health⌕ Search

Biomedical subjects

J Markwell

Publications and source records attributed to J Markwell.

15 recordsLinked to original sources
PubMed2001

Identification of IgE-binding proteins in soy lecithin.

BACKGROUND: Soy lecithin is widely used as an emulsifier in processed foods, pharmaceuticals and cosmetics. Soy lecithin is composed principally of phospholipids; however, it has also been shown to contain IgE-binding proteins, albeit at a low level. A few clinical cases involving allergic reactions to soy lecithin have been reported. The purpose of this investigation is to better characterize the IgE-binding proteins typically found in lecithin. METHODS: Soy lecithin proteins were isolated following solvent extraction of lipid components and then separated on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The separated lecithin proteins were immunoblotted with sera from soy-sensitive individuals to determine the pattern of IgE-binding proteins. The identity of IgE-reactive bands was determined from their N-terminal sequence. RESULTS: The level of protein in six lecithin samples obtained from commercial suppliers ranged from 100 to 1,400 ppm. Lecithin samples showed similar protein patterns when examined by SDS-PAGE. Immunoblotting with sera from soy-sensitive individuals showed IgE binding to bands corresponding to 7, 12, 20, 39 and 57 kD. N-terminal analysis of these IgE-binding bands resulted in sequences for 3 components. The 12-kD band was identified as a methionine-rich protein (MRP) and a member of the 2S albumin class of soy proteins. The 20-kD band was found to be soybean Kunitz trypsin inhibitor. The 39-kD band was matched to a soy protein with unknown function. CONCLUSIONS: Soy lecithin contains a number of IgE-binding proteins; thus, it might represent a source of hidden allergens. These allergens are a more significant concern for soy-allergic individuals consuming lecithin products as a health supplement. In addition, the MRP and the 39-kD protein identified in this study represent newly identified IgE-binding proteins.

Adolescent↗
PubMed2001

Oxidative responses of resistant and susceptible cereal leaves to symptomatic and nonsymptomatic cereal aphid (Hemiptera: Aphididae) feeding.

The impact of the leaf-chlorosis-eliciting Russian wheat aphid, Diuraphis noxia (Mordvilko), and the nonchlorosis-eliciting bird cherry-oat aphid, Rhopalosiphum padi (L.), feeding on D. noxia-susceptible and -resistant cereals was examined during the period (i.e., 3, 6, and 9 d after aphid infestation) that leaf chlorosis developed. After aphid number, leaf rolling and chlorosis ratings, and fresh leaf weight were recorded on each sampling date, total protein content, peroxidase, catalase, and polyphenol oxidase activities of each plant sample were determined spectrophotometrically. Although R. padi and D. noxia feeding caused significant increase of total protein content in comparison with the control cereal leaves, the difference in total protein content between R. padi and D. noxia-infested leaves was not significant. Although R. padi-feeding did not elicit any changes of peroxidase specific activity in any of the four cereals in comparison with the control leaves, D. noxia feeding elicited greater increases of peroxidase specific activity only on resistant 'Halt' wheat (Triticum aestivum L.) and susceptible 'Morex' barley (Hordeum vulgare L.), but not on susceptible 'Arapahoe' and resistant 'Border' oat (Avena sativa L.). D. noxia-feeding elicited a ninefold increase in peroxidase specific activity on Morex barley and a threefold on Halt wheat 9 d after the initial infestation in comparison with control leaves. Furthermore, D. noxia feeding did not elicit any differential changes of catalase and polyphenol oxidase activities in comparison with either R. padi feeding or control leaves. The findings suggest that D. noxia feeding probably results in oxidative stress in plants. Moderate increase of peroxidase activity (approximately threefold) in resistant Halt compared with susceptible Arapahoe wheat might have contributed to its resistance to D. noxia, whereas the ninefold peroxidase activity increase may have possibly contributed to barley's susceptibility. Different enzymatic responses in wheat, barley, and oat to D. noxia and R. padi feeding indicate the cereals have different mechanisms of aphid resistance.

Animals↗
PubMed2000

Formate dehydrogenase in Arabidopsis thaliana: characterization and possible targeting to the chloroplast.

Formate dehydrogenase (E.C. 1.2.1.2) is a mitochondrial-localized NAD-requiring enzyme in green plants. The enzyme activity and corresponding mRNA in leaves of Arabidopsis thaliana are induced by treatment with one-carbon metabolites. The cDNA for the Arabidopsis formate dehydrogenase is similar to that of other plants except for the N-terminal region, which is predicted to target chloroplasts as well as mitochondria. The specific of activity of the enzyme in isolated chloroplasts suggests it is targeted to both mitochondria and chloroplasts in Arabidopsis. Formate dehydrogenase from Arabidopsis was partially purified and K(m) values for formate and NAD(+) were determined to be 10 mM and 65 µM, respectively; the K(i) for NADH was 17 µM. We conclude that formate dehydrogenase is normally present in Arabidopsis chloroplasts and that sensitivity to inhibition by NADH may play a role in whether cellular formate is assimilated or dissimilated.

Journal Article↗
PubMed1997

Purification of a protein phosphatase from chloroplast stroma capable of dephosphorylating the light-harvesting complex-II.

A protein phosphatase was purified from the stroma of Pea (Pisum sativum L.) chloroplasts that is capable of dephosphorylating synthetic phosphopeptides. Following chromatographic purification of greater than 400-fold, two-dimensional electrophoresis indicated that the stromal protein phosphatase is a 29-kD protein. A similar molecular size was determined for the protein-phosphatase activity using gel-permeation chromatography, indicating that the stromal protein phosphatase is probably a monomer. The purified enzyme was able to dephosphorylate synthetic phosphopeptides, which mimic the thylakoid light-harvesting complex II (LHC-II) N terminus, as well as LHC-II in thylakoid membranes, but did not dephosphorylate the major 64-kD phosphoprotein in the stroma. The stromal protein phosphatase did not discriminate between dephosphorylation of phosphothreonine and phosphoserine residues in synthetic peptide substrates, providing further evidence that this enzyme is distinct from the protein phosphatase localized in thylakoid membranes. The exact physiological role of the stromal protein phosphatase has yet to be determined, but it may function in the dephosphorylation of LHC-II.

Amino Acid Sequence↗
PubMed1993

Phosphopeptides as substrates for thylakoid protein phosphatase activity.

Lack of a suitable substrate has been a major obstacle in studying the chloroplastic thylakoid membrane protein phosphatase activity. In this study, the suitability of synthetic phosphopeptides for this purpose was investigated. Phosphothreonine-containing phosphopeptides mimicking the N-terminal phosphorylation site of the major thylakoid phosphoprotein, the light-harvesting chlorophyll a/b-binding protein (LHCP-II), were dephosphorylated by isolated peak thylakoid membranes. Phosphopeptides representing unrelated sequences or in which the target phosphothreonine had been changed to a phosphoserine were not dephosphorylated. The dephosphorylation of phosphopeptides by thylakoid membranes was similar to the dephosphorylation of endogenous LHCP-II in its pH-dependence profile, sensitivity to inhibitors, and bivalent cation requirement. The same phosphopeptide analogs of the LHCP-II phosphorylation site inhibited endogenous LHCP-II dephosphorylation in isolated thylakoids, whereas the dephospho-analogs and nonsubstrate phosphopeptides had no effect. Collectively, these results suggest that phosphopeptides mimicking a thylakoid phosphoprotein dephosphorylation site can be exploited for further study of the thylakoid protein phosphatase activity.

Amino Acid Sequence↗
PubMed1993

Characterization of mutation-induced changes in the maize (Zea mays L.) ADH1-1S1108 alcohol dehydrogenase.

The homodimeric alcohol dehydrogenase gene produce of maize (Zea mays L.) Adh1-1S1108 mutation was purified and compared with the parental Adh1-1S enzyme. The mutant alcohol dehydrogenase activity had pH optima and substrate specificity similar to those of the parental enzyme, but exhibited somewhat increased and decreased Km values for acetaldehyde and NADH, respectively. The mutant enzyme was also markedly less stable than the enzyme from parental tissues to temperatures as low as 50 degrees C. Sequence analysis of a polymerase chain reaction (PCR)-generated cDNA clone revealed a G-to-C mutation at position 406 and a C-to-T mutation at position 974. These would result in residue 103 of each protein subunit being changed from an alanine to a proline and residue 292 being changed from an alanine to a valine. Whether one or both of these changes in primary sequence is responsible for the altered substrate affinities and stability is not yet understood.

Alcohol Dehydrogenase↗
PubMed1992

Isolation of a carotenoid-containing sub-membrane particle from the chloroplastic envelope outer membrane of pea (Pisum sativum).

Chloroplastic envelope membranes isolated from pea (Pisum sativum) leaves are rich in carotenoids, containing approximately 2 micrograms of carotenoid mg-1 protein. We report here that envelopes can be surfactant-solubilized while maintaining association of carotenoids with protein components of the membrane. Treatment of isolated chloroplastic envelope membranes with 0.5% Deriphat 160 (N-lauryl-beta-imminodipropionate) causes general solubilization but preserves an envelope sub-membrane fragment which is fractionated by centrifugation in a sucrose gradient and by chromatography on a column of DEAE-Sephacel. The isolated submembrane complex contained five major proteins with M(r) values equivalent to 75,000, 36,000, 34,000 17,500, and 14,500. Spectroscopic and chromatographic analyses revealed that the complex contains violaxanthin and at least one other carotenoid. Carotenoid content of the fractionated complex was estimated as 4.8 micrograms mg-1 protein. Immunoblot analysis reveals that the constituent proteins of this complex are derived from the chloroplastic outer envelope membrane. These data suggest that at least some of the carotenoids of the chloroplastic envelope may be organized by apoproteins.

Carotenoids↗
PubMed1992

Lack of Types 1 and 2A Protein Serine(P)/Threonine(P) Phosphatase Activities in Chloroplasts.

Protein phosphatase activity in crude leaf extracts and in purified intact chloroplasts of wheat (Triticum aestivum) and pea (Pisum sativum) was analyzed using exogenously supplied phosphoproteins or endogenous thylakoid proteins. Leaf extracts contain readily detectable amounts of protein phosphatase activity measured with either phosphohistone or phosphorylase a, substrates of mammalian protein phosphatases. No significant chloroplast protein phosphatase activity was detected using these exogenous phosphoproteins. The dephosphorylation of endogenous thylakoid light-harvesting chlorophyll a/b binding proteins in situ was inhibited by fluoride, but not by microcystin-LR or okadaic acid, diagnostic inhibitors of mammalian types 1 and 2A protein phosphatases. Additionally, no evidence for a pea chloroplast alkaline phosphatase activity was found using beta-glycerolphosphate or 4-methylum-belliferyl phosphate as substrates. From these results, we conclude that phosphohistone and phosphorylase a are not useful substrates for chloroplast thylakoid protein phosphatase activity and that the chloroplast enzymes may not fit into one of the canonical classifications currently used for protein phosphatases.

Journal Article↗
PubMed1992

Occurrence of Temperature-Sensitive Phenotypic Plasticity in Chlorophyll-Deficient Mutants of Arabidopsis thaliana.

A collection of 75 putative mutants with alterations in leaf pigmentation was visually selected from Arabidopsis thaliana plants (M(2) generation) grown at 26 degrees C from seeds treated with the mutagen ethylmethanesulfonate. Fifty-eight of the plants were found to have chlorophyll contents decreased by at least 10% from the parental Columbia ecotype. These plants were screened for chlorophyll content and the ratio of chlorophyll b/a after growth at 20 or 26 degrees C. Relative to the parental type, a significant number of individuals in which the chlorophyll-deficient phenotype was exacerbated at one of the growth temperatures were identified. We conclude that temperature-sensitive phenotypic plasticity for chlorophyll content is relatively common in mutant populations of higher plants.

Journal Article↗
PubMed1991

Purification and properties of a 4-nitrophenylphosphatase from Aspergillus niger.

A 4-nitrophenylphosphatase (EC 3.1.3.41) was identified in extracts of Aspergillus niger. The production of this activity was decreased by growth on a phosphate-limiting medium and was greatest in a medium supplemented with corn steep liquor. The phosphatase activity was purified by hydrophobic, ion-exchange, and molecular sieve chromatography. The purified enzyme has a native size of approximately 80,000, polypeptide subunits with sizes of 37,000 upon denaturation, and a pI of 4.6. The activity was optimal at pH 8.0 and was stimulated by Mg2+ and to a lesser extent by Mn2+ but was inhibited by Zn2+ and Ca2+. The enzyme was highly specific for 4-nitrophenyl phosphate as substrate, having a Km of 0.77 mM and a turnover number of 108 s-1. The purified enzyme did not hydrolyze any of 22 sugar phosphates, mononucleotides, or other phosphocompounds tested. A small, but reproducible, amount of activity was measured using 5'-DNA phosphate as a substrate. Although some similarities exist to three previously characterized 4-nitrophenylphosphatases from Saccharomyces cerevisiae, the enzyme from A. niger is distinctly different from at least two of these activities.

4-Nitrophenylphosphatase↗
PubMed1991

How children cope with death.

Zoe and Daniel's parents were killed in a car accident. Jessica Markwell describes their reactions to the trauma and how these two young children came to terms with their sudden and tragic bereavement.

Adaptation, Psychological↗
PubMed1990

Temperature sensitivity as a general phenomenon in a collection of chlorophyll-deficient mutants of sweetclover (Melilotus alba).

A collection of chlorophyll (Chl)-deficient mutants of sweetclover (Melilotus alba) with defects in eight nuclear loci were grown at 17 or 26 degrees C. Plants grown at either temperature were examined for Chl content, Chl a/b ratio, expression of the light-harvesting complex II (LHC-II) apoproteins, and protochlorophyllide (Pchlide) biosynthetic capacity. Except for the ch4 mutant, the parental strain and all mutants accumulate more Chl when grown at 26 degrees C than at 17 degrees C. The ch5 mutants, lacking Chl b under any growth condition, and the ch12 mutant showed little temperature-dependent phenotypic plasticity, whereas this was a marked phenomenon in the other mutants. The ch10 and ch11 mutants demonstrated extreme temperature sensitivity with regard to the production of Chl b and the Chl b-binding LHC-II apoproteins. When excised trifoliolates were supplemented with exogenously supplied delta-aminolevulinic acid, only the ch4 mutant was markedly impaired in the ability to produce Pchlide. These data indicate that temperature-sensitive phenotypic plasticity is a common phenomenon of chlorphyll-deficient mutants and substantiate that only a minority of Chl-deficient mutants is impaired in the biosynthesis of Chl.

Animals↗
PubMed1989

Chloroplast thylakoid protein phosphatase is a membrane surface-associated activity.

Chloroplast thylakoid protein phosphatase activity was measured using (32)P-labeled histone as an exogenous substrate and an assay of the (32)Pi released involving formation of a phosphomolybdate complex and organic extraction. The activity was liberated from wheat (Triticum aestivum) thylakoids by washing the membranes in NaCl-containing solutions followed by centrifugation. The liberated phosphatase activity had a pH optimum of approximately 6.75, was inhibited by addition of 10 millimolar EDTA or EGTA, and was stimulated by addition of millimolar amounts of dithiothreitol, magnesium, manganese, or calcium ions. The rate of thylakoid protein dephosphorylation was decreased following liberation of a portion of the protein phosphatase activity and was increased by addition of salt-liberated phosphatase fraction. These results suggest that at least a portion of wheat thylakoid protein phosphatase is a peripheral, rather than an integral, membrane protein.

Journal Article↗