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The H$ sp+$-translocating inorganic pyrophosphatase from vacuolar membranes of red beet storage roots (Beta vulgaris L.) was purified after solubilization in Triton X-100 through a combination of anion-exchange and size exclusion chromatographies. SDS-PAGE showed strong correlation between a 67 kDa polypeptide and pyrophosphatase activity. Radiation-inactivation studies of the H$ sp+$-PPase indicate a functional size of 91 kDa for hydrolysis and 320 kDa for H$ sp+$ translocation, suggesting an oligomeric structure for the holoenzyme. Affinity purified antibodies were used to screen cDNA libraries of Arabidopsis thaliana yielding clones which contained sequences matching amino acid sequences obtained from tryptic fragments of the 67 kDa hydrolytic subunit. The predicted protein is highly hydrophobic with a molecular size of 81 kDa. Southern analyses show a single copy for the H$ sp+$-PPase in Arabidopsis. The lack of sequence identities between the H$ sp+$-PPase and other known proteins implies a novel class of ion translocases.

Thesis (MSc)--University of Stellenbosch, 2005. / Bibliography. Also available via the Internet.

Thesis (MSc (Plant Biotechnology))--University of Stellenbosch, 2005. / Vacuolar Pyrophosphatase (V-PPase) has never been studied in sugarcane before and to date nothing is known about V-PPase in sugarcane, except for the sequences of a few expressed sequence tags (ESTs). The aim of this project was to characterize V-PPase expression in several hybrid sugarcane varieties that differ significantly in sucrose content, with the main objective of the study to assess whether V-PPase is correlated in any way to the sucrose storage phenotype. Therefore, the goals of this project were to (i) develop molecular tools for the detection and quantification of V-PPase on a DNA, RNA, protein and enzyme level and (ii) to use these tools to characterize the expression of V-PPase within the culm of the three hybrid varieties. The cDNA sequence of the catalytic subunit of the sugarcane V-PPase gene was cloned, expressed in a bacterial system and the V-PPase peptide was purified. This peptide was used for the immunization of mice and the production of polyclonal anti-VPPase antiserum. Anti-VPPase antiserum reacted specifically with a single polypeptide among vacuolar membrane proteins. Moreover, anti-VPPase antiserum recognized V-PPase from various monocotyledons and dicotyledons. The anti-VPPase antiserum was used for the establishment of an ELISA system to determine V-PPase protein content in vacuolar membrane preparations. This system proved to have several advantages over the protein blotting technique and shared a strong linear relation with V-PPase specific activity, showing that these two tests are compatible and reliable. The optimisation of sugarcane V-PPase zero-order kinetics was fundamental in order to measure V-PPase specific activity accurately. It had a relative broad pH optimum, retaining more than 90% of its maximum activity between pH 6.50 and 7.25. V-PPase required both Mg2+ and K+, in addition to PPi, for maximum activity in vitro. The reported kinetic variables are within range of previous data determined for other species, including mung bean, red beet and sugar beet. V-PPase protein level and specific activity within the sugarcane culm followed a similar trend , withoiofofoenaobserved for sucrose accumulation rates observed in sugarcane. Moreover, V-PPase protein contents and specific activity share the same general trend as total sucrose content in a specific tissue compared among the three varieties. No significant differences were observed in V-ATPase activity among the three varieties. Our findings suggest that V-PPase may play a role in sucrose accumulation in sugarcane.