Biomedical subjects

M Puype

Publications and source records attributed to M Puype.

23 records · Page 2Linked to original sources

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↗

PubMed1988

Porcine vinculin and metavinculin differ by a 68-residue insert located close to the carboxy-terminal part of the molecule.

Metavinculin is a higher mol. wt variant of vinculin expressed only in muscle tissue. Using amino acid sequencing methods on the intact molecules and their proteolytic subfragments, together with a polyclonal antibody specific only for metavinculin from porcine stomach, we have been able to identify and sequence the difference peptide in the porcine metavinculin molecule. By alignment with the complete sequence of chick fibroblast vinculin (communicated by G.J. Price, P. Jones, M.D. Davison, R. Bendori, S. Griffiths, B. Patel, B. Geiger and D.R. Critchley, prior to publication) the exact location of the insert could be determined. In porcine metavinculin, this insert lies between the 90-kd protease-resistant amino-terminal core and the carboxy terminus of the molecule. It contains 68 amino acids and is flanked by KWSSK sequences, one of which is present in vinculin. The identity of the mapped vinculin and metavinculin sequences outside this difference peptide is consistent with the two proteins arising via alternative splicing at the mRNA level. The lack of reactivity of the porcine metavinculin antibody with metavinculin from chicken as well as the finding of different proteolytic cleavage sites in avian metavinculin indicate a species-specific amino acid sequence in the difference piece of the metavinculin molecule.

Amino Acid Sequence↗

PubMed1986

Protein-chemical analysis of pertussis toxin reveals homology between the subunits S2 and S3, between S1 and the A chains of enterotoxins of Vibrio cholerae and Escherichia coli and identifies S2 as the haptoglobin-binding subunit.

The purified toxin of Bordetella pertussis was dissociated in 5 M urea in the presence of immobilized haptoglobin. The toxin was dissociated in free S1, free S5 and the free complexes S2-S4 and S3-S4, with S2-S4 as the only haptoglobin-binding moiety, identifying S2 as the haptoglobin-binding protein. Partial NH2-terminal amino acid sequences were obtained from the dissimilar toxin subunits, after separation by SDS-polyacrylamide gel electrophoresis followed by electroblotting onto polybrene-coated glass-fiber sheets. The sequences reveal extensive homology of the N-terminal portions of the constitutive subunits S2 and S3 and between S1 and the enterotoxin A chains of Vibrio cholerae and Escherichia coli.

Amino Acid Sequence↗

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↗