The lutein-epoxide cycle (Lx cycle) is an auxiliary xanthophyll cycle known to operate only in some higher-plant species. It occurs in parallel with the common violaxanthin cycle (V cycle) and involves the same epoxidation and de-epoxidation reactions as in the V cycle. In this study, the occurrence of the Lx cycle was investigated in the two major families of mistletoe, the Loranthaceae and the Viscaceae. In an attempt to find the limiting factor(s) for the occurrence of the Lx cycle, pigment profiles of mistletoes with and without the Lx cycle were compared. The availability of lutein as a substrate for the zeaxanthin epoxidase appeared not to be critical. This was supported by the absence of the Lx cycle in the transgenic Arabidopsis plant lutOE, in which synthesis of lutein was increased at the expense of V by overexpression of epsilon-cyclase, a key enzyme for lutein synthesis. Furthermore, analysis of pigment distribution within the mistletoe thylakoids excluded the possibility of different localizations for the Lx- and V-cycle pigments. From these findings, together with previous reports on the substrate specificity of the two enzymes in the V cycle, we propose that mutation to zeaxanthin epoxidase could have resulted in altered regulation and/or substrate specificity of the enzyme that gave rise to the parallel operation of two xanthophyll cycles in some plants. The distribution pattern of Lx in the mistletoe phylogeny inferred from 18S rRNA gene sequences also suggested that the occurrence of the Lx cycle is determined genetically. Possible molecular evolutionary processes that may have led to the operation of the Lx cycle in some mistletoes are discussed.

We have measured Ca2+ uptake and Ca2+ release in isolated permeabilized pancreatic acinar cells and in isolated membrane vesicles of endoplasmic reticulum prepared from these cells. Ca2+ uptake into cells was monitored with a Ca2+ electrode, whereas Ca2+ uptake into membrane vesicles was measured with 45Ca2+. Using inhibitors of known action, such as the H+ ATPase inhibitors NBD-Cl and NEM, the Ca2+ ATPase inhibitor vanadate as well as the second messenger inositol 1,4,5-trisphosphate (IP3) and its analog inositol 1,4,5-trisphosphorothioate (IPS3), we could functionally differentiate two nonmitochondrial Ca2+ pools. Ca2+ uptake into the IP3-sensitive Ca2+ pool (IsCaP) occurs by a MgATP-dependent Ca2+ uptake mechanism that exchanges Ca2+ for H+ ions. In the absence of ATP Ca2+ uptake can occur to some extent at the expense of an H+ gradient that is established by a vacuolar-type MgATP-dependent H+ pump present in the same organelle. The other Ca2+ pool takes up Ca2+ by a vanadate-sensitive Ca2+ ATPase and is insensitive to IP3 (IisCaP). The IsCaP is filled at "higher" Ca2+ concentrations (approximately 10(-6) mol/liter) which may occur during stimulation. The low steady-state [Ca2+] of approximately 10(-7) mol/liter is adjusted by the IisCaP. It is speculated that both Ca2+ pools can communicate with each other, the possible mechanism of which, however, is at present unknown.

Isolated acinar cells from the rat exocrine pancreas were loaded with 6-carboxyfluorescein diacetate (CFDA), and the intracellular pH (pHi) was estimated from the pH-dependent fluorescence intensity of trapped 6-carboxyfluorescein liberated from CFDA by intracellular esterases. The intracellular fluorescence intensity was calibrated by equilibrating the internal and external pH with nigericin in K+ buffers. In the absence of Na+ (130 mmol/l K+) a pHi of 6.86 +/- 0.04 was found; in its presence (130 mmol/l Na+) a pHi of 7.17. Acute addition of Na+ increased intracellular pH with increasing Na+ concentrations, reaching a maximum at 150 mmol/l with an apparent Km of approximately 40 mmol/l. Of the different cations tested on pHi, such as Li+, K+, Rb+, and Cs+, only Li+ showed an effect on pHi similar to that of Na+. Amiloride dose dependently inhibited both Na+- and Li+-induced alkalinization (apparent Km approximately 10(-5) mol/l). In the presence of ouabain pHi was decreased by 0.2 pH units. Intracellular acidification induced by permeable buffers such as acetic acid-acetate or CO2-HCO3- was dissipated more rapidly in the presence of Na+ compared with K+ or with Na+ and amiloride in the medium. In Li+-preincubated cells intracellular acidification was higher in the absence of Li+ in the extracellular medium than in its presence. This Li+ gradient-induced acidification was dependent on the extracellular pH, was highest at an extracellular pH of 7.05, and decreased with increasing pH to 7.5. The results allow the conclusion that a coupled Na+-H+ exchange is present in pancreatic acinar cells and that the intracellular pH rather than the extracellular Na+ concentration regulates this transport mechanism.

In microsomal vesicles, as isolated from exocrine pancreas cells, MgATP-driven H+ transport was evaluated by measuring H+-dependent accumulation of acridine orange (AO). Active H+ uptake showed an absolute requirement for ATP with simple Michaelis-Menten kinetics (Km for ATP 0.43 mmol/liter) with a Hill coefficient of 0.99. H+ transport was maximal at an external pH of 6.7, generating an intravesicular pH of 4.8. MgATP-dependent H+ accumulation was abolished by protonophores, such as nigericin (10(-6) mol/liter) or CCCP (10(-5) mol/liter), and by inhibitors of nonmitochondrial H+ ATPases, such as NEM or NBD-Cl, at a concentration of 10(-5) mol/liter. Inhibitors of both mitochondrial and nonmitochondrial H+ pumps, such as DCCD (10(-5) mol/liter) or Dio 9 (0.25 mg/ml), reduced microsomal H+ transport by about 90%. Vanadate (2 x 10(-3) mol/liter), a blocker of those ATPases, which form a phosphorylated intermediate, did not inhibit H+ transport. The stilbene derivative DIDS (10(-4) mol/liter), which inhibits anion transport systems, abolished H+ transport completely. MgATP-dependent H+ transport was found to be anion dependent in the sequence Cl- greater than Br- greater than gluconate-; in the presence of SO2-4, CH3COO- or No-3, no H+ transport was observed. MgATP-dependent H+ accumulation was also cation dependent in the sequence K+ greater than Li+ greater than Na+ = choline+. As shown by dissipation experiments in the presence of different ion gradients and ionophores, both a Cl- and a K+ conductance, as well as a small H+ conductance, were found in the microsomal membranes. When membranes containing the H+ pump were further purified by Percoll gradient centrifugation (ninefold enrichment compared to homogenate), no correlation with markers for endoplasmic reticulum, mitochondria, plasma membranes, zymogen granules or Golgi membranes was found. The present data indicate that the H+ pump located in microsomes from rat exocrine pancreas is a vacuolar- or "V" -type H+ ATPase and has most similarities to that described in endoplasmic reticulum, Golgi apparatus or endosomes.

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