S. Brian Wilson

Identification of plant mitochondrial proteins: A procedure linking two-dimensional gel electrophoresis to protein sequencing from PVDF membranes using a FastBlot cycle

Identification of the 329 spots visible in 2D gels of plant mitochondrial proteins is a challenge. This paper describes a 2D mini-gel protocol involving free-radical scavengers and purified reagents to make it compatible with protein sequencing, and evaluates its performance. The paper also describes a “FastBlot” sequencing cycle with the cycle time for protein sequencing from PVDF membranes reduced to less than 29 min with femtomole sensitivity. Other benefits of the cycle include reduced lag, reduced background, reduced loss of labile residues, and increased initial and repetitive yields. The procedure gave excellent results with maize mitochondrial proteins: of six protein spots that we tried to sequence, only one was blocked. The other spots yielded considerable sequence information. One spot was identified from the sequence as superoxide dismutase, while another spot corresponded to an unidentified cDNA from rice. The results of these experiments show that modifications of our previous procedures can provide good N-terminal protein sequencing from individual spots on 2D gels. The technique makes it possible to obtain sequence data, prepare gene probes, and identify many of the polypeptides in the 2D-gel map for plant mitochondria.

The F1-ATPase from turnip (Brassica napus L.) mitochondria : purification, subunit composition and properties

This paper describes the purification of solvent released turnip (Brassica napus L.) F1-ATPase. The specific activity of the purified ATPase was 135–194 μmol/min per mg when assayed in the presence of the activating anion, sulphite, or 40–55 μmol/min per mg in the absence of sulphite, values similar to those of conventionally isolated mammalian F1-ATPase. The isolated turnip F1-ATPase is cold-labile but can be stored in 30% v/v glycerol+4 mM ATP. Turnip ATPase (Mr 420 000) has six subunits with Mr 55 000, 52 000, 30 500 24 500 19 900 and 9100, respectively. Turnip ATPase has optimal activity at pH 8.0 and is more active with Ca2+ than Mg2+ as dependent cation. The calcium-dependent activity increases relative to the magnesium-ATPase activity when the ATPase is released from the membranes. Excess free Ca2+ stimulates whereas free Mg2+ inhibits turnip FI activity. The activity of turnip F1 is stimulated by chloride and markedly stimulated by oxy-anions, particularly sulphite. The nucleotide specificity in the presence of magnesium and sulphite is GTP>ATP>ITP>CTP>UTP; without sulphite the relative activities towards ATP and ITP are reversed. Turnip ATPase requires higher concentrations of classical ATPase inhibitors for inhibition than the bovine heart-muscle ATPase and is particularly susceptible to inhibition by the calmodulin antagonist Calmidazolium (R24571) in the presence of calcium.

A Plant Mitochondrial ATPase/Synthase

The membrane-bound proton transporting ATPase/ ATP-synthase of bacteria, mitochondria and chloroplasts catalyses the reversible synthesis/hydrolysis of ATP from ADP and Pi. The mitochondrial enzyme is the major source of cellular ATP in the absence of photosynthesis. It is believed that the electro-chemical gradient produced during electron transport is utilised by the ATPase during ATP synthesis in a process involving proton translocation. The enzyme has a requirement for a divalent cation for activity. Normally this requirement is fulfilled by magnesium ions although the chloroplast (Vambutas and Racker 1965) and pea mitochondrial enzymes are also activated by calcium ions (Grubmeyer and Spencer 1979).