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G Bauw

Publications and source records attributed to G Bauw.

46 records · Page 3Linked to original sources
PubMed1989

Purification and determination of the NH2-terminal amino acid sequence of uracil-DNA glycosylase from human placenta.

Uracil-DNA glycosylase has been purified approximately 130,000-fold from extracts of human placenta. Although all of the uracil-DNA glycosylase activity coeluted through six chromatographic steps, at least four distinct peaks of activity were resolved in the final purification on a Mono S column. Each of the peaks containing uracil-DNA glycosylase activity contained two peptides of Mr = 29,000 and Mr = 26,500, respectively, as analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Experimental evidence indicated that the Mr = 29,000 peptide was the uracil-DNA glycosylase enzyme. The amino-terminal sequence of each peptide was determined after blotting of the peptides from the gel onto Polybrene GF/C paper. The sequences were not related to each other, and neither was any significant homology to other proteins found. Uracil-DNA glycosylase had a molecular turnover number of approximately 600/min and apparent Km value of 2 microM. The enzyme is a basic protein and was stimulated about 10-fold by 60-70 mM NaCl whereas higher concentrations were inhibitory.

Amino Acid Sequence↗
PubMed1989

Molecular cloning and structural analysis of a gene from Zea mays (L.) coding for a putative receptor for the plant hormone auxin.

The major auxin-binding protein from maize coleoptiles was purified to homogeneity. The protein has an apparent mol. wt of 22 kd and binds 1-naphthylacetic acid with a KD of 2.40 x 10(-7) M. Additional antigenically related proteins, present in very low amounts, could be demonstrated in maize coleoptiles using immunodetection. Extensive protein sequence analysis of the major auxin-binding protein allowed the construction of several synthetic oligonucleotide probes which were used to isolate a cDNA coding for this protein. The cDNA corresponds to a mRNA with a 3'-poly(A)+ sequence and a single, long open reading frame of 603 bases. The open reading frame, starting 34 residues from the 5' end of the cDNA, predicts a 21,990 Dalton protein of 201 amino acids. Comparison of this deduced amino acid sequence with the partial amino acid sequences of purified auxin-binding protein, revealed a perfect match, involving a total of 53 amino acid residues. The primary amino acid sequence includes a 38-amino-acid-long N-terminal hydrophobic leader sequence which could represent a signal for translocation of this protein to the endoplasmic reticulum. An additional signal is located at the C-terminal end, consisting of the amino acids KDEL known to be responsible for preventing secretion of proteins from the lumen of the endoplasmic reticulum in eucaryotic cells. The primary sequence contains a N-glycosylation site (-asp133-thr-thr-). This site was found to be glycosylated by a high-mannose-type oligosaccharide.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1989

Protein-electroblotting and -microsequencing strategies in generating protein data bases from two-dimensional gels.

Coomassie blue-stained, heat-dried, and computer-imaged two-dimensional gels used to develop comprehensive human protein data bases served as the protein source to generate partial amino acid sequences. The protein spots were collected from multiple gels, rehydrated, concentrated by stacking into a new gel, electroblotted onto inert membranes, and in situ-digested with trypsin. Peptides eluting from the membranes were separated by HPLC and sequenced. Using this procedure, it was possible to generate partial sequences from 13 human proteins recorded in the amnion cell protein data base. Eight of these sequences matched those of proteins stored in data bases, demonstrating that a systematic analysis of proteins by computerized two-dimensional gel electrophoresis can be directly linked to protein microsequencing methods. The latter technique offers a unique opportunity to link information contained in protein data bases derived from the analysis of two-dimensional gels with forthcoming DNA sequence data on the human genome.

Amino Acid Sequence↗
PubMed1989

Characterization of vacuolar and extracellular beta(1,3)-glucanases of tobacco: Evidence for a strictly compartmentalized plant defense system.

beta(1,3)-Glucanases are a class of hormonally and developmentally regulated plant hydrolytic enzymes, which are also induced upon pathogen infection, suggesting a role of these hydrolases in the defense response to pathogens. We have purified several beta(1,3)-glucanases present in tobacco leaves from control plants and from plants treated with salicylic acid or infected with Pseudomonas syringae and studied in detail the subcellular localization of the different isoforms. Partial protein sequence analysis demonstrated that each of the different isoforms had a unique amino acid sequence and was therefore encoded by a different gene. We have also demonstrated that two of these isoforms, similar to the cytokinin/auxin-regulated isoforms previously isolated from tobacco cell suspensions, are located in the central vacuole. Upon salicylic acid treatment or P. syringae infection, three secreted isoforms are induced, belonging to the so-called pathogenesis-related proteins. These pathogenesis-related beta(1,3)-glucanases are all distinct from each other and also different from the vacuolar isoforms. We demonstrate that the vacuolar isoforms are not secreted to the extracellular spaces of the plant following pathogen infection, suggesting that any function they play in the plant defense response is restricted to an intracellularly coordinated defense process.

Journal Article↗
PubMed1989

A plant manganese superoxide dismutase is efficiently imported and correctly processed by yeast mitochondria.

In the plant Nicotiana plumbaginifolia, manganese superoxide dismutase (MnSOD) is synthesized in the cytoplasm as a preprotein and is subsequently translocated to the mitochondrial matrix with corresponding cleavage of an NH2-terminal leader sequence. To determine whether the plant enzyme could replace the endogenous SOD activities of Escherichia coli and yeast, constructions have been made in appropriate vectors for expression of the preprotein and the mature MnSOD. These were introduced into SOD-deficient strains for complementation studies. In E. coli, both forms of the protein were shown to be active and able to complement SOD deficiency to different degrees. Expression of the preprotein in a yeast strain lacking a mitochondrial MnSOD resulted in a restoration of wild-type growth, only possible if the plant protein was being targeted to the mitochondria. Subsequent studies revealed that the protein was processed and that the leader sequence was cleaved at the identical position as recognized by the mitochondrial peptidase of plants. The components mediating mitochondrial import thus appear to be highly conserved between plants and yeast.

Cloning, Molecular↗
PubMed1988

The homology between the serum proteins PO2 in pig, Xk in horse and alpha 1B-glycoprotein in human.

1. Pig serum Po2 protein and horse Xk protein were purified by FPLC, non-denaturing 2D agarose-PAGE and 2D IPG-PAGE. 2. The separated fractions were electroblotted to poly(4-vinyl-N-methylpyridinium iodide) coated GF/C glass fiber sheets. 3. The partial amino acid sequences and amino acid compositions of different genetic variants of the proteins were determined. 4. The results proved that previously reported polymorphic serum post-albumins in each of these species were homologous to human plasma alpha 1B-glycoprotein.

Amino Acid Sequence↗
PubMed1987

Alterations in the phenotype of plant cells studied by NH(2)-terminal amino acid-sequence analysis of proteins electroblotted from two-dimensional gel-separated total extracts.

Phenotypic alterations induced by the cytokinin 6-benzylaminopurine in cell suspensions of Nicotiana plumbaginifolia were studied at the level of the NH(2)-terminal sequence of the constituent proteins. Total protein extracts were separated by classical two-dimensional PAGE, and the proteins were recovered by electroblotting onto support materials allowing direct gas-phase sequence analysis of the immobilized proteins. The systems used consist of an efficient electrotransfer buffer (50 mM Tris borate, pH 8.3) in combination with either glass-fiber sheets to which poly(4-vinyl-N-methylpyridinium iodide) is adsorbed or with membranes of polyvinylidene difluoride. The former is an improved version of our previously reported Polybrene-coated glass-fiber sheets and was found to be at least twice as efficient as the polyvinylidene difluoride blots. Thirteen proteins were selected for analysis. They were either induced, repressed, or independent of cytokinin. Ten proteins yielded a sequence, ranging from 10 to 38 residues. Three of the studied Nicotiana proteins show a degree of homology higher than 85% with the amino acid sequences of other eukaryotic proteins-triose-phosphate isomerase, Mn superoxide dismutase, and (1,3)-beta-glucanase. The latter enzyme was repressed by the plant hormone. This study demonstrates that proteins associated with phenotypic variations in cells can now be sequenced by a straightforward procedure involving two-dimensional gel separation of total cellular proteins, recovery by electroblotting, and gas-phase sequence analysis of the immobilized proteins.

Journal Article↗
PubMed1985

Protein-blotting on Polybrene-coated glass-fiber sheets. A basis for acid hydrolysis and gas-phase sequencing of picomole quantities of protein previously separated on sodium dodecyl sulfate/polyacrylamide gel.

A procedure has been developed which allows the immobilization on glass-fiber sheets coated with the polyquaternary amine, Polybrene, of proteins and protein fragments previously separated on sodium-dodecylsulfate-containing polyacrylamide gels. The transfer is carried out essentially as has been used for protein blotting on nitrocellulose membranes [Towbin, H., Staehelin, T. and Gordon, J. (1979) Proc. Natl Acad. Sci. USA 76, 4350-4354], but is now used to determine the amino acid composition and partial sequence of the immobilized proteins. Protein transfer could be carried out after staining the proteins in the gels with Coomassie blue, by which immobilized proteins are visible as blue spots, or without previous staining, after which transferred proteins are detected as fluorescent spots following reaction with fluorescamine. The latter procedure was found to be more efficient and yielded binding capacities of +/- 20 micrograms/cm2. Fluorescamine detection was of equal or higher sensitivity than the classical Coomassie staining of proteins in the gel. Immobilized proteins could be hydrolyzed when still present on the glass fiber and reliable amino acid compositions were obtained for various reference proteins immobilized in less than 100 pmol quantities. In addition, and more importantly, glass-fiber-bound proteins could be subjected to the Edman degradation procedure by simply cutting out the area of the sheet carrying the immobilized protein and mounting the disc in the reaction chamber of the gas-phase sequenator. Results of this immobilization-sequencing technique are shown for immobilized myoglobin (1 nmol) and two proteolytic fragments of actin (+/- 80 pmol each) previously separated on a sodium-dodecylsulfate-containing gel.

Amino Acid Sequence↗
PubMedDate not supplied

Separation and characterization of needle and xylem maritime pine proteins.

Two-dimensional gel electrophoresis (2-DE) and image analysis are currently used for proteome analysis in maritime pine (Pinus pinaster Ait.). This study presents a database of expressed proteins extracted from needles and xylem, two important tissues for growth and wood formation. Electrophoresis was carried out by isoelectric focusing (IEF) in the first dimension and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in the second. Silver staining made it possible to detect an average of 900 and 600 spots on 2-DE gels from needles and xylem, respectively. A total of 28 xylem and 35 needle proteins were characterized by internal peptide microsequencing. Out of these 63 proteins, 57 (90%) could be identified based on amino acid similarity with known proteins, of which 24 (42%) have already been described in conifers. Overall comparison of both tissues indicated that 29% and 36% of the spots were specific to xylem and needles, respectively, while the other spots were of identical molecular weight and isoelectric point. The homology of spot location in 2-DE patterns was further validated by sequence analysis of proteins present in both tissues. A proteomic database of maritime pine is accessible on the internet (http://www.pierroton.inra.fr/genetics/2D/).

Acrylic Resins↗