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UBC Theses and Dissertations

Purification and characterization of chloroplast coupling factor from wheat Moase, Elaine Helen

Abstract

Chloroplast coupling factor (CF₁) from thylakoids of wheat {Triticum aestivum, var Thatcher) was purified by chloroform extraction and subsequent sucrose density gradient centrifugation. The wheat enzyme, in common with most coupling factors, contains five subunits, designated α,β,γ,δ,ε, of molecular weights 57, 55, 39, 25 and 13 kd respectively. Inclusion of proteolysis inhibitors in all steps of the purification prevented the loss of the two smallest subunits during the sucrose gradient step. Although proteolysis inhibitors did not interfere with the extraction of CF₁ by chloroform, they completely prevented CF₁ release from the thylakoid by the hypotonic sucrose method of Strotmann et al. (1973). However, CF₁ release by hypotonic sucrose did not appear to require a proteolytic event in any CF₁ subunit, as no difference in apparent molecular weight of any CF₁ subunit was observed when CF₁ was released by this method. A double band or apparent mw's 37 and 39 kd for the γ subunit in SDS-PAGE was identified as reduced and unreduced forms of the same subunit, suggesting an" internal disulfide bridge in the γ subunit of wheat CF₁. The latent CaATPase of wheat CF₁ is activated by trypsin digestion but not by heat, un1ike spinach CF₁. Wheat CF₁ hydrolyzes GTP at 32% the rate of ATP, but no other nucleotide triphosphate is effective as a substrate. A 1:1 ratio of Ca:ATP gave optimal ATPase activity. K[sub M] and V[sub max] were determined in the presence of a large excess of calcium (10 mM) and with an equimolar amount of calcium and ATP. In the presence of excess calcium, a K[sub M] of 0.125 mM CaATP and a V[sub max] of 18.9 units/mg protein were obtained. Under conditions of equimolar calcium and ATP, the K[sub M] was 0.018 mM CaATP and the V[sub max] was 6.77 units/mg protein. The order of magnitude difference in K[sub M]'s is thought to be due to an alteration of affinity of the enzyme for substrate by the presence or absence of excess calcium. Electron microscopy of negatively-stained wheat CF₁ particles showed a solid hexagonal particle. Escherichia coli F₁ gave a similar structure. Markham rotation supported the conclusion that wheat CF₁ exhibits a six-fold symmetry. Antibodies to wheat CF₁ were generated by injecting the purified enzyme into a rabbit. The resulting antiserum both precipitated wheat CF₁ and inhibited its latent, trypsin-activated CaATPase. The serum cross-reacted with spinach CF₁, showing that there is considerable antigenic similarity between the two enzymes. Crossed immunoelectro-phoresis showed that this antiserum reacts specifically with the α and β subunits of wheat CF₁.

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