RESUMEN
The sexuality-inducing glycoprotein of Volvox carteri f. nagariensis was purified from supernatants of disintegrated sperm packets of the male strain IPS-22 and separated by reverse-phase HPLC into several isoforms which differ in the degree of O-glycosylation. Total chemical deglycosylation with trifluoromethanesulphonic acid yields the biologically inactive core protein of 22.5 kDa. This core protein possesses three putative binding sites for N-glycans which are clustered in the middle of the polypeptide chain. The N-glycosidically bound oligosaccharides were obtained by glycopeptidase F digestion and were shown by a combination of exoglycosidase digestion, gaschromatographic sugar analysis and two-dimensional HPLC separation to possess the following definite structures: (A) Man beta 1-4GlcNAc beta 1-4GlcNAc; (B) (Man alpha)3 Man beta 1-4GlcNAc beta 1-4GlcNAc Xyl beta; (C) (Man alpha)2 Man beta 1-4GlcNAc beta 1-4GlcNAc; (D) (Man)2Xyl(GlcNAc)2. Xyl beta Two of the three N-glycosidic binding sites carry one B and one D glycan. The A and C glycans are shared by the third N-glycosylation site. The O-glycosidic sugars, which make up 50% of the total carbohydrate, are short (up to three sugar residues) chains composed of Ara, Gal and Xyl and are exclusively bound to Thr residues.
Asunto(s)
Chlorophyta/análisis , Glicoproteínas/aislamiento & purificación , Oligosacáridos , Feromonas/aislamiento & purificación , Secuencia de Carbohidratos , Cromatografía de Gases , Electroforesis en Gel de Poliacrilamida/métodos , Datos de Secuencia Molecular , Monosacáridos , PéptidosRESUMEN
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)