Molecular dissection of the epsilon subunit of the chloroplast ATP synthase of spinach.

The gene encoding the epsilon subunit (atpE) of the chloroplast ATP synthase of Spinacia oleracea has been overexpressed in Escherichia coli. The recombinant protein can be solubilized in 8 M urea and directly diluted into buffer containing ethanol and glycerol to obtain epsilon that is as biologically active as epsilon purified from chloroplast-coupling factor 1 (CF1). Recombinant epsilon folded in this manner inhibits the ATPase activity of soluble and membrane-bound CF1 deficient in epsilon and restores proton impermeability to thylakoid membranes reconstituted with CF1 deficient in epsilon. Site-directed mutagenesis was used to generate truncations and single amino acid substitutions in the primary structure of epsilon. In the five mutants tested, alterations that weaken ATPase inhibition by recombinant epsilon affect its ability to restore proton impermeability to a similar extent, with one exception. Substitution of histidine-37 with arginine appears to uncouple ATPase inhibition and the restoration of proton impermeability. As in the case of E. coli, it appears that N-terminal truncations of the epsilon subunit have more profound effects than C-terminal deletions on the function of epsilon. Recombinant epsilon with six amino acids deleted from the C terminus, which is the only region of significant mismatch between the epsilon of spinach and the epsilon of Pisum sativum, inhibits ATPase activity with a reduced potency similar to that of purified pea epsilon. Four of the six amino acids are serine or threonine. These hydroxylated amino acids may be important in epsilon-CF1 interactions.

Characterization of the Activation of Membrane-Bound and Soluble CF(1) by Thioredoxin.

The activation of spinach (Spinacia oleracea) chloroplast coupling factor 1 (CF(1)) by thioredoxin (ThR) was characterized using membrane-bound and soluble CF(1). Light generates an electrochemical proton gradient across the thylakoid membrane, which increases the accessibility of the disulfide bond on the gamma-subunit of CF(1) to reduced ThR. The proton gradient substantially accelerates the activation of CF(1) compared with thylakoids incubated in the dark with similar concentrations of dithiothreitol and ThR. The interaction of soluble CF(1) with ThR was studied using fluorescent probes. CF(1) in solution, with and without its associated epsilon-subunit, was labeled at Cys-322 of the gamma-subunit with fluoresceinyl maleimide. ThR from Escherichia coli was labeled with eosin isothiocyanate. Labeled ThR and CF(1) showed normal activities. Fluorescence energy transfer between donor fluoresceinyl maleimide and acceptor eosin isothiocyanate, manifested by a quenching of the donor fluorescence, was detected, suggesting that ThR and CF(1) form an intermolecular complex. When the epsilon-subunit was absent, quenching of donor fluorescence was approximately doubled, indicating that labeled ThR could approach more closely to the gamma-subunit of CF(1). The distance between the fluorescent probes on CF(1) and ThR was calculated to be approximately 65 A when epsilon-subunit was present and 52 A when epsilon was absent. These values are consistent with other distance measurements and energy transfer values reported previously for fluorescent probes on CF(1). Whereas the extent of quenching increased by removal of the epsilon-subunit, the apparent dissociation constant was unchanged. The quenching effect was reversed when the epsilon-subunit was added back to the titration mixture. Similarly, the addition of unlabeled ThR decreased donor quenching.

Decline of activity and quantity of ribulose bisphosphate carboxylase/oxygenase and net photosynthesis in ozone-treated potato foliage.

The effect of ozone (O(3)) on ribulose bisphosphate carboxylase/oxygenase (Rubisco) activity and quantity and net photosynthesis in greenhouse-grown Solanum tuberosum L. cv ;Norland' foliage was studied in relation to oxidant-induced premature senescence. Plants, 26 days old, were exposed to 0.06 to 0.08 microliters per liter O(3) from 1000 to 1600 hours for 4 days in a controlled environment chamber. On day 5, plants were exposed to a 6-hour simulated inversion in which O(3) peaked at 0.12 microliters per liter. Net photosynthesis declined in response to O(3) but recovered to near control levels 3 days after the exposure ended. Rubisco activity and quantity in control potato foliage increased and then decreased during the 12-day interval of the study. In some experiments foliage studied was physiologically mature and Rubisco activity had peaked when O(3) exposure commenced. In those cases, O(3) accelerated the decline in Rubisco activity. When less mature foliage was treated with O(3), the leaves never achieved the maximal level of Rubisco activity observed in control foliage and also exhibited more rapid decline in initial and total activity. Percent activation of Rubisco (initial/total activity) was not affected significantly by treatment. Quantity of Rubisco decreased in concert with activity. The decrease in activities is most likely due to a decrease in available protein rather than a decrease in the percentage of Rubisco activated in vivo. The reduction in the quantity of Rubisco, an important foliar storage protein, could contribute to premature senescence associated with toxicity of this air pollutant.

University of Pennsylvania Smell Identification Test: a rapid quantitative olfactory function test for the clinic.

Despite the fact that clinical otolaryngologists are often presented with complaints of olfactory dysfunction, they have limited means to diagnose these problems. A major reason has been the lack of a clinically-useful and reliable quantitative test of olfactory function. Recent work at our Clinical Research Center has resulted in the development of such a test. This test--the University of Pennsylvania Smell Identification Test (UPSIT)--can be self-administered and uses microencapsulated odorants which are released by scratching standardized odor-impregnated test booklets. As indicated in this paper, studies have demonstrated that the UPSIT can identify most malingerers and is sensitive to age, gender, smoking habits, and a wide variety of olfactory disorders. A description of this new test, along with some of its applications, is presented.