Biomedical subjects

E P Beers

Publications and source records attributed to E P Beers.

9 recordsLinked to original sources

PubMed2001

Regulation and execution of programmed cell death in response to pathogens, stress and developmental cues.

Recent studies have expanded our view of the interactions between small molecule signals that regulate the hypersensitive response and other forms of cell suicide in plants. The mitochondrion has received increasing support as a mediator of at least some forms of programmed cell death in plants. In addition, new information provides a glimpse of how plant hormone signaling may be integrated with extensive autolysis, sensitivity to reactive oxygen intermediates and cell death.

Adaptation, Physiological↗

PubMed2000

Plant proteolytic enzymes: possible roles during programmed cell death.

Proteolytic enzymes are known to be associated with developmentally programmed cell death during organ senescence and tracheary element differentiation. Recent evidence also links proteinases with some types of pathogen- and stress-induced cell suicide. The precise roles of proteinases in these and other plant programmed cell death processes are not understood, however. To provide a framework for consideration of the importance of proteinases during plant cell suicide, characteristics of the best-known proteinases from plants including subtilisin-type and papain-type enzymes, phytepsins, metalloproteinases and the 26S proteasome are summarized. Examples of serine, cysteine, aspartic, metallo- and threonine proteinases linked to animal programmed cell death are cited and the potential for plant proteinases to act as mediators of signal transduction and as effectors of programmed cell death is discussed.

Apoptosis↗

PubMed2000

Exploiting secondary growth in Arabidopsis. Construction of xylem and bark cDNA libraries and cloning of three xylem endopeptidases.

The root-hypocotyl of Arabidopsis produces a relatively large amount of secondary vascular tissue when senescence is delayed by the removal of inflorescences, and plants are grown at low population density. Peptidase zymograms prepared from isolated xylem and phloem revealed the existence of distinct proteolytic enzyme profiles within these tissues. cDNA libraries were constructed from isolated xylem and bark of the root-hypocotyl and screened for cDNAs coding for cysteine, serine, and aspartic peptidases. Three cDNAs, two putative papain-type cysteine peptidases (XCP1 and XCP2) and one putative subtilisin-type serine peptidase (XSP1), were identified from the xylem library for further analysis. Using RNA gel blots it was determined that these peptidases were expressed in the xylem and not in the bark. Quantitative reverse transcriptase-polymerase chain reaction confirmed the RNA gel-blot results and revealed high levels of XCP1 and XCP2 mRNA in stems and flowers of the infloresence. A poly-histidine-tagged version of XCP1 was purified from Escherichia coli by denaturing metal-chelate chromatography. Following renaturation, the 40-kD recombinant XCP1 was not proteolytically active. Activation was achieved by incubation of recombinant XCP1 at pH 5.5 and was dependent on proteolytic processing of the 40-kD inactive polypeptide to a 26-kD active peptidase.

Amino Acid Sequence↗

PubMed1997

Programmed cell death during plant growth and development.

This review describes programmed cell death as it signifies the terminal differentiation of cells in anthers, xylem, the suspensor and senescing leaves and petals. Also described are cell suicide programs initiated by stress (e.g., hypoxia-induced aerenchyma formation) and those that depend on communication between neighboring cells, as observed for incompatible pollen tubes, the suspensor and synergids in some species. Although certain elements of apoptosis are detectable during some plant programmed cell death processes, the participation of autolytic and perhaps autophagic mechanisms of cell killing during aerenchyma formation, tracheary element differentiation, suspensor degeneration and senescence support the conclusion that nonapoptotic programmed cell death pathways are essential to normal plant growth and development. Heterophagic elimination of dead cells, a prominent feature of animal apoptosis, is not evident in plants. Rather autolysis and autophagy appear to govern the elimination of cells during plant cell suicide.

Journal Article↗

PubMed1993

Utility of polyhistidine-tagged ubiquitin in the purification of ubiquitin-protein conjugates and as an affinity ligand for the purification of ubiquitin-specific hydrolases.

The purification and biochemical characterization of protein substrates of the ubiquitin-dependent pathway of proteolysis is made difficult in part by the low steady state levels of ubiquitin-protein conjugates. We report here on the use of a polyhistidine-tagged ubiquitin molecule (HisUb) for the purification of ubiquitin-protein conjugates by metal chelate chromatography. When Escherichia coli extracts containing expressed HisUb were passed through a nitrilotriacetic acid-agarose column containing immobilized Ni2+ ions (Ni-NTA column), HisUb was retained. After washing to remove unbound and nonspecifically bound proteins, a pH 4.5 wash was used to elute highly purified HisUb. Purified HisUb and wild-type ubiquitin were tested for their ability to form Ni(2+)-binding ubiquitin-protein conjugates in a wheat germ in vitro conjugation reaction. In some experiments, wheat germ extracts were preincubated with iodoacetamide to inhibit ubiquitin activating and conjugating enzymes. Only those conjugation assays containing HisUb and an ATP-regenerating system not pretreated with iodoacetamide produced significant levels of multiple Ni(2+)-binding ubiquitin-protein conjugates. We also examined the potential of HisUb as an affinity ligand for the purification of higher plant ubiquitin-specific hydrolases. As a test, a crude lysate of E. coli expressing a yeast ubiquitin-specific hydrolase (Yuh1) was passed through a Ni-NTA column containing bound HisUb. Yuh1 was retained on the column and was specifically eluted when the column was equilibrated with buffer containing wild-type ubiquitin.

Amino Acid Sequence↗

PubMed1992

Subcellular localization of ubiquitin and ubiquitinated proteins in Arabidopsis thaliana.

Ubiquitin is a highly conserved, 76-amino acid, eukaryotic protein. Its widely accepted role as a proteolytic cofactor depends on its unique ability to covalently ligate to other cellular proteins. While there is good evidence for the existence of such ubiquitinated proteins in the cytosolic and nuclear compartments, relatively little is known about the presence of free ubiquitin and ubiquitinated proteins in other subcellular compartments. This is especially true of higher plants, which have not previously been the subject of extensive biochemical subcellular localizations of ubiquitinated proteins. We extracted cell wall proteins and purified nuclei, vacuoles, chloroplasts, and microsomes from chlorophyllous tissues of Arabidopsis. Immunoblot analyses were used to compare the profiles of ubiquitinated proteins from purified subcellular fractions to those from unfractionated extracts. Purified nuclei contained, in addition to a complex mixture of high molecular mass ubiquitinated proteins, a strongly immunoreactive 28-kDa protein. In the apoplastic extract, we did not detect any ubiquitinated proteins enriched above the background level of those due to cytosolic contamination. Vacuoles appeared to contribute significantly to the ubiquitinated proteins present in the whole protoplast extract. At least three high molecular mass ubiquitinated proteins were unique to the vacuolar extract. Chloroplast stromal proteins did not react specifically with anti-ubiquitin antibodies. When microsomal ubiquitinated proteins were compared to those found in a whole protoplast extract, a distinct pattern was evident. Microsomal ubiquitinated proteins were not visible in the 10,000 x g supernatant used to prepare the 100,000 x g pellet, indicating that they were probably low abundance proteins in the protoplast extract.

Cell Fractionation↗

PubMed1990

Characterization of alpha-Amylase from Shoots and Cotyledons of Pea (Pisum sativum L.) Seedlings.

The most abundant alpha-amylase (EC 3.2.1.1) in shoots and cotyledons from pea (Pisum sativum L.) seedlings was purified 6700-and 850-fold, respectively, utilizing affinity (amylose and cycloheptaamylose) and gel filtration chromatography and ultrafiltration. This alpha-amylase contributed at least 79 and 15% of the total amylolytic activity in seedling cotyledons and shoots, respectively. The enzyme was identified as an alpha-amylase by polarimetry, substrate specificity, and end product analyses. The purified alpha-amylases from shoots and cotyledons appear identical. Both are 43.5 kilodalton monomers with pls of 4.5, broad pH activity optima from 5.5 to 6.5, and nearly identical substrate specificities. They produce identical one-dimensional peptide fingerprints following partial proteolysis in the presence of SDS. Calcium is required for activity and thermal stability of this amylase. The enzyme cannot attack maltodextrins with degrees of polymerization below that of maltotetraose, and hydrolysis of intact starch granules was detected only after prolonged incubation. It best utilizes soluble starch as substrate. Glucose and maltose are the major end products of the enzyme with amylose as substrate. This alpha-amylase appears to be secreted, in that it is at least partially localized in the apoplast of shoots. The native enzyme exhibits a high degree of resistance to degradation by proteinase K, trypsin/chymostrypsin, thermolysin, and Staphylococcus aureus V8 protease. It does not appear to be a high-mannose-type glycoprotein. Common cell wall constituents (e.g. beta-glucan) are not substrates of the enzyme. A very low amount of this alpha-amylase appears to be associated with chloroplasts; however, it is unclear whether this activity is contamination or alpha-amylase which is integrally associated with the chloroplast.

Journal Article↗

PubMed1990

Partial Characterization and Subcellular Localization of Three alpha-Glucosidase Isoforms in Pea (Pisum sativum L.) Seedlings.

Three isoforms of alpha-glucosidase (EC 3.2.1.20) have been extracted from pea (Pisum sativum L.) seedlings and separated by DEAE-cellulose and CM-Sepharose chromatography. Two alpha-glucosidase isoforms (alphaG1 and alphaG2) were most active under acid conditions, and appeared to be apoplastic. A neutral form (alphaG3) was most active near pH 7, and was identified as a chloroplastic enzyme. Together, the activity of alphaG1 and alphaG2 in apoplastic preparations accounted for 21% of the total acid alpha-glucosidase activity recovered from pea stems. The vast majority (86%) of the apoplastic acid alpha-glucosidase activity was due to alphaG1. The apparent K(m) values for maltose of alphaG1 and alphaG2 were 0.3 and 1.3 millimolar, respectively. The apparent K(m) for maltose of alphaG3 was 33 millimolar. The respective native molecular weights of alphaG1, alphaG2, and alphaG3 were 125,000, 150,000, and 110,000.

Journal Article↗

PubMed1988

Localization of alpha-Amylase in the Apoplast of Pea (Pisum sativum L.) Stems.

Most of the activity of an alpha-amylase present in crude pea (Pisum sativum L. cv Laxton's Progress No. 9) leaf preparations cannot be found in isolated pea leaf protoplasts. The same extrachloroplastic alpha-amylase is present in pea stems, representing approximately 6% of total stem amylolytic activity and virtually all of the alpha-amylase activity. By a simple infiltration-extraction procedure, the majority (87%) of this alpha-amylase activity was recovered from the pea stem apoplast without significantly disrupting the symplastic component of the tissue. Only 3% of the beta-amylase activity and less than 2% of other cellular marker enzymes were removed during infiltration-extraction.

Journal Article↗