SummaryProtoplasts of corn coleoptiles and Arabidopsis hypocotyls respond to the plant hormone auxin with a rapid change in volume. We checked the effect of antibodies directed against epitopes of auxin-binding protein 1 from Arabidopsis thaliana (AtERabp1) and Zea mays (ZmERabp1), respectively. Antibodies raised against the C-terminus of AtERabp1 inhibited the response to auxin, while antibodies raised against a part of box a, the putative auxin-binding domain, induced a swelling response similar to that caused by auxin treatment. Synthetic C-terminal oligopeptides of ZmERabp1 also caused a swelling response. These effects occurred regardless of whether the experiments were carried out with homologous (anti-AtERabp1 antibodies on Arabidopsis protoplasts or anti-ZmERabp1 antibodies in maize protoplasts) or heterologous immunological tools. The results indicate that the auxin signal for protoplast swelling is perceived by extracellular ABP1.

Numerous protocols for isolation of mitochondria are available. Here, three methods for the isolation of intact mitochondria from mouse liver tissues are compared with regard to yield, purity and activity. Mitochondria were isolated by sucrose density gradient ultracentrifugation, free-flow electrophoresis or a commercially available kit-based method. Our analyses show that the sophisticated (and most expensive) free-flow electrophoresis method enables isolation of intact mitochondria with an enrichment of approximately 70%. Using the classical density centrifugation method is very laborious and time-consuming, but delivers about 57% intact mitochondria. Using standard laboratory equipment in a quick and simple procedure, the kit provides approximately 50% intact mitochondria, suitable for most standard investigations.

The major auxin-binding protein (ZmERabp1) from maize (Zea mays L.) has been structurally characterized. We determined the position of a disulfide bridge in ZmERabp1 by mass-spectrometric analysis. We show that Cys2 and Cys61 are covalently linked and that residue Cys155 bears the free sulfhydryl group. By making use of electrospray mass spectrometry, the molecular mass of ZmERabp1 was determined to be 20 243 Da comprising a sugar moiety of 1865 Da, corresponding to a high mannose-type glycan structure. Due to the high homology among all characterized ABPs, the information on the disulfide bonds will be important for functional analysis of recombinantly expressed ABP1. ß 2001 Published by Elsevier Science B.V. on behalf of the Federation of European Biochemical Societies.