Iron was resupplied to tobacco that was severely iron-deficient and the chloroplasts investigated for chlorophyll, iron content, photochemical, and development aberrations and photosystem components. Ferredoxin was purified from normal and iron deficient tobacco leaves. Lower ferredoxin and chlorophyll content in iron-deficient leaves were found compared to normal leaves. The ferredoxin from normal and iron-deficient leaves had the same molecular weight as spinach ferredoxin (Sigma Chemical Co.) and shared a similar antigenic property.Photosystem I (PSI) acti vity increased by about 100% during the first 10 days of regreening on a leaf surface area comparison. Only small increases in activity were found with PSII. Purified PSI particles were spectrophotometrically examined for any changes in P700, chlorophyll, cytochrome and protein components on a leaf area basis. With greening the individual components increased but the ratios remained constant. There were 6 polypeptide bands in the PSI particles when examined by electrophoresis. All bands stained with equal intensity with coomassie blue except for the ISkD band at day 0 of greening. Data suggest that iron stress is involved in the regulation of PSI development, possibly by the direct regulation of a low-molecular weight protein required for system assembly. Discussion is presented suggesting this protein is ferredoxin and that limitation of this component would directly affect chlorophyll biosynthesis and chloroplast structure.
The enzyme pyruvate kinase which catalyzes the reversible transfer of phosphate from phosphoenolpvruvate (PEPA) to adenosine diphosphate (ADP) yielding pyruvate and adenosine triphosphate (ATP) is widely distributed in living organisms (2, 3, 4, 8, 9, 11, 15). Lohmann and AMeverhof (11) in 1934 demonstrated that the activity of pyrtuvate kinase from rat muscle was dependent on the presence of Mg++ in the assay medium. Utter and Werkman (22) reported that extracts from Escherichia coli contained pyruvate kinase and that it was activated by Mg++ or \In++. In their experiments Ni++, Na+ and K+ were either without effect or were inhibitory. The K+ activation of pyruvate kinase from rat muscle was shown initially by Boyer, Lardy and Phillips (3, 4) and late; by Lardy and Ziegler (9). The complete dependence of the activity of the enzyme on univalent cations was demonstrated first bv the work of Kachmar and Boyer (8). This requirement was in addition to that for divalent cations. K+, NH4' or Rb+ satisfied the univalent cation requirement but Na+, Li' or Ca++ counteracted the activation. In the absence of K+, Na+ additions resulted in a small btit significant stimulation. Boyer (2) reported that extracts from eleven different vertebrate and invertebrate organisms contained pyruvate kinase and that the enzyme from all sources was strongly activated by K+. The optimum concentrations of univalent cations in molarity were 0.15, 0.01 and 0.05 for activity of the enzyme from muscles of rabbit, Anodonta and Limulus, respectively. Introductory studies (14) on the fermentation of phosphoglyceric acid by dialyzed yeast extracts showed no univalent cation activation. Later, Seitz (18) reported that yeast extracts that had been thoroughly dialyzed failed to ferment phosphoglyceric acid unless K+ or NH4+ was supplied. In the absence of univalent cations the phosphoglyceric acid fermentation stopped at the phosphoenolv-pyruvate stage. With this system NH4+ was more effective than K+ as an activator which is in contrast to observations with the enzyme from other sources. Stumpf (20) and Tewfik and Stumpf (21) have provided indirect evidence for the presence of pyruvate kinase in higher plants. Extracts of an acetone powder of pea seed or leaves contained enzymes that catalyzed the conversion of phosphoglyceric acid to p)yruvate. When dialyzed extracts were used, the ad-1 Received February 5, 1957. 2 Contribution from the Faculty of Botany, North Carolina Agricultural Experiment Station and published with the approval of the Director as paper No. 786. This research was supported in part by a grant from the Chilean Nitrate Educational Bureau.
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