Uptake of CO(2) and bicarbonate by intact cells and chloroplasts of Tetraedron minimum and Chlamydomonas noctigama.

Using a mass-spectrometric disequilibrium technique, net uptake of HCO(3)(-) and CO(2) during steady-state photosynthesis was studied in whole cells and chloroplasts from the green algae Tetraedron minimum and Chlamydomonas noctigama, grown in air enriched with 5% (v/v) CO(2) (high-CO(2) cells) or in air [0.035% (v/v) CO(2); low-CO(2) cells]. High- and low-CO(2) cells of both species were able to take up CO(2) and HCO(3)(-), with maximum rates being largely unaffected by the growth conditions. High- and low-CO(2) cells of T. minimum showed a pronounced preference for HCO(3)(-) while the rates of net HCO(3)(-) and CO(2) uptake were similar in C. noctigama. The most significant differences between high- and low-CO(2) cells of the two species were the 5- to 6-fold increase in the apparent affinities of net HCO(3)(-) uptake and CO(2) uptake after acclimation to air. The high-affinity uptake systems for inorganic carbon were almost completely induced within 4 h in both algae. Photosynthetically active chloroplasts isolated from both species were also able to take up CO(2) and HCO(3)(-). As in whole cells, HCO(3)(-) was the dominant carbon species taken up by chloroplasts from T. minimum while CO(2) and HCO(3)(-) were taken up at similar rates in plastids from C. noctigama. In addition, high-affinity uptake systems for CO(2) and HCO(3)(-) were detected in chloroplasts preparations after acclimation of the parent cells to air. Isolation of ribulose-1,5-bisphosphate carboxylase/oxygenase revealed K(m) values of 13 and 42 micro M CO(2) for the enzymes from T. minimum and C. noctigama, respectively. These results are consistent with the presence of inducible and energy-dependent high-affinity HCO(3)(-) and CO(2) uptake systems associated with chloroplasts, indicating that these organelles play an important role in the CO(2)-concentrating mechanism.

Identification and localization of a thylakoid-bound carbonic anhydrase from the green algae Tetraedron minimum (Chlorophyta) and Chlamydomonas noctigama (Chlorophyta).

In order to broaden our understanding of the eukaryotic CO2-concentrating mechanism the occurrence and localization of a thylakoid-associated carbonic anhydrase (EC 4.2.1.1) were studied in the green algae Tetraedron minimum and Chlamydomonas noctigama. Both algae induce a CO2-concentrating mechanism when grown under limiting CO2 conditions. Using mass-spectrometric measurements of 18O exchange from doubly labelled CO2, the presence of a thylakoid-associated carbonic anhydrase was confirmed for both species. From purified thylakoid membranes, photosystem I (PSI), photosystem II (PSII) and the light-harvesting complex of the photosynthetic apparatus were isolated by mild detergent gel. The protein fractions were identified by 77 K fluorescence spectroscopy and immunological studies. A polypeptide was found to immunoreact with an antibody raised against thylakoid carbonic anhydrase (CAH3) from Chlamydomonas reinhardtii. It was found that this polypeptide was mainly associated with PSII, although a certain proportion was also connected to light harvesting complex II. This was confirmed by activity measurements of carbonic anhydrase in isolated bands extracted from the mild detergent gel. The thylakoid carbonic anhydrase isolated from T. minimum had an isoelectric point between 5.4 and 4.8. Together the results are consistent with the hypothesis that thylakoid carbonic anhydrase resides within the lumen where it is associated with the PSII complex.

Purification of the Fusicoccin-Binding Protein from Oat Root Plasma Membrane by Affinity Chromatography with Biotinylated Fusicoccin.

Fusicoccin (FC), a fungal phytotoxin, evokes a number of physiological responses after binding to the FC-binding protein (FCBP). For characterization of this plasma membrane protein and elucidation of the signal transduction pathway, we purified active FCBP from oat (Avena sativa L. cv Valiant) root plasma membranes using avidin-biotin affinity chromatography. For the binding of FCBP to immobilized avidin, a bifunctional FC derivative (FC-biotin, FCBio) was synthesized. FCBio retained high binding affinity for the FCBP (KD = 70 nM), it elicited a biological response comparable to FC, and it was bound by avidin. The purification of the FCBP involved three important steps. First, FCBio was bound to the FCBP in purified plasma membrane vesicles. Next, plasma membrane proteins were solubilized in detergent, and part of the solubilized proteins was precipitated by decreasing the detergent concentration below the critical micelle concentration. The FCBP remained in the soluble fraction, and this fraction was loaded on a "low-affinity" avidin column. Proteins, bound through a biotin moiety to the column, were specifically eluted with excess biotin. This resulted in fractions active in [3H]FC binding and two bands on sodium dodecyl sulfate-polyacrylamide gel electrophoresis of 31 and 30 kD. The nonhydrophobic behavior of the FCBP was confirmed by means of phase separation with Triton X-114, wherein the FCBP migrated to the hydrophilic phase. Purification of the FCBP in active form using this novel affinity technique opens the possibility to study other features of the FCBP necessary for inducing physiological responses in plant cells.

Affinity purification of GTPase proteins from oat root plasma membranes using biotinylated GTP.

Biotinylated GTP was synthesized and it was demonstrated that this ligand was bi-functional: it competed with [3H]Gpp(NH)p for binding to membrane proteins and it bound to immobilized avidin. Peripheral plasma membrane proteins were solubilized in a low-salt wash, incubated with GTP-biotin and biotinylated proteins were coupled to an avidin column. Elution with excess biotin yielded 10 polypeptides as seen with a silver stained SDS-PAGE gel. Antisera raised against Ras, a small GTPase, strongly interacted with three proteins with MW of 38, 27 and 25 kDa and also with 6 other proteins. G alpha-common antibodies interacted with proteins of MW = 66 and 38 kDa. This method enables the rapid purification of GTP-binding proteins and opens the possibility to assign a role to specific GTPases in signal transduction pathways.