PUB - Publikationen an der Universität Bielefeld

The molecular cloning of the first subunit C of the plant vacuolar NC-ATPase is reported, Tonoplast vesicles were purified from barley leaves by sucrose gradient centrifugation, and the tonoplast polypeptides were separated by two-dimensional (2-D) gel electrophoresis, Using an anti-ATPase holoenzyme antibody, a polypeptide was recognized in the molecular mass range of 40 kDa with an isoelectric point of about 6.0, and tentatively identified as subunit C, The polypeptide spot was excised from about 50 2-D gels and subjected to endo Lys C proteolysis, Two proteolytic peptides were sequenced and the amino acid sequences were used to design degenerated oligonucleotides, followed by PCR amplification with cDNA template and screening elf a cDNA library synthesized from Hordeum vulgare poly A mRNA of epidermis strips. The full length clone of 1.5 kbp contains an open reading frame of 1062 bp encoding a polypeptide of 354 amino acids with a molecular mass of 39 982 Da and an isoelectric point of 6.04, Amino acid identity with sequences of SUC from animals and fungi is in the range of 36.7 to 38.5%, Expression of the cloned gene was demonstrated by Northern blotting and RT-PCR, (C) 1999 Federation of European Biochemical Societies.