Ammonia was excreted at high rates in the presence of L-methionine sulfoximine (L-MSO) from Chlorella cells which have been grown and analyzed at normal CO 2 partial pressure (330 ppm ). If these cells are analyzed at high CO 2 -concentration (3% CO 2 in air) only little ammonia is excreted in the presence of L-MSO. In the absence of L-MSO no ammonia is excreted under either condition. In agreem ent with this observation Chlorella cells grown under high CO 2 partial pressure (3% CO 2 in air) but tested under normal CO 2 partial pressure excreted only very little ammonia. Under these conditions neither “High CO 2 -cells” nor “Low CO 2 -cells” exhibited any glycolate excretion. However, glycolate excretion was observed in the presence of a-HPMS (a-hydroxy-2-pyridyl methanesulfonate) an inhibitor of glycolate dehydrogenase or INH (isonicotinyl hydrazide) an inhibitor of the glycine-serine am inotransferase, irrespective of the presence or absence of L-M SO. INH inhibited ammonia excretion. The above described high ammonia excretion in “Low CO 2 -cells” in the presence of L-MSO was suppressed or substantially reduced by 0.1 mм ethoxyzolamide an inhibitor of carbonic anhydrase which, however, at the same time caused a substantial excretion of glycolate into the medium. The same qualitative effect of ethoxyzolam ide was observed in “High CO 2 -cells” (tested under normal CO 2 partial pressure) although the amount of glycolate excreted in this type of culture was very small. It was generally noted that glycolate excretion caused by ethoxyzolamide was stoichiometrically always more important than the rate of ammonia excretion which was inhibited. This shows that excretion and therefore most probably also the formation of glycolate are enhanced by ethoxyzolamide. The experiments seem to show that due to the inhibition of carbonic anhydrase the affinity of the ribulose-1,5-bisphosphate carboxylase/oxygenase system is increased towards oxygen, which leads to a stimulation of the photorespiratory carbon cycle.
Abstract. Strains of the coccolithophore Emiliania huxleyi (Haptophyta) collected from the subarctic North Pacific and Arctic Oceans during the R/V MIRAI cruise in 2010 (MR10-05) were established as clone cultures and have been maintained in the laboratory at 15 °C and 32 ‰ salinity. To study the physiological responses of coccolith formation to changes in temperature and salinity, growth experiments and morphometric investigations were performed on two strains of MR57N isolated from the northern Bering Sea (56°58' N, 167°11' W) and MR70N at the Chukchi Sea (69°99' N, 168° W). This is the first report of a detailed morphometric and morphological investigation of Arctic Ocean coccolithophore strains. The specific growth rates at the logarithmic growth phases in both strains markedly increased as temperature was elevated from 5 to 20 °C, although coccolith productivity (the percentage of calcified cells) was similar at 10–20 % at all temperatures. On the other hand, the specific growth rate of strain MR70N was affected less by changes in salinity in the range 26–35 ‰, but the proportion of calcified cells decreased at high and low salinities. According to scanning electron microscopy (SEM) observations, coccolith morphotypes can be categorized into Type B/C on the basis of their biometrical parameters, such as length of the distal shield (LDS), length of the inner central area (LICA), and the thickness of distal shield elements. The central area elements of coccoliths varied from grilled type to closed type when temperature was increased or salinity was decreased, and coccolith size decreased simultaneously. Coccolithophore cell size also decreased with increasing temperature, although the variation in cell size was slightly greater at the lower salinity level. This indicates that subarctic and arctic coccolithophore strains can survive in a wide range of seawater temperatures and at lower salinities due to their marked morphometric adaptation ability. Because all coccolith biometric parameters followed the scaling law, the decrease in coccolith size was caused simply by the reduced calcification. Taken together, our results suggest that calcification productivity may be used to predict future oceanic environmental conditions in the Polar Regions.
Previous studies suggested that certain protein(s) other than carbonic anhydrase might play an important role in the facilitated transport of dissolved inorganic carbon (DIC) from the medium to the site of CO2 fixation by ribulose-1,5-bisphosphate carboxylase/oxygenase in the unicellular green alga Chlorella regularis adapted to low-CO2 (ordinary air) conditions [Shiraiwa et al. (1991) Jpn. J. Phycol. 39: 355; Satoh and Shiraiwa (1992) Research in Photosynthesis, Vol. III, p. 779]. The proteins that might be involved in this facilitated transport of DIC were investigated by pulse-labeling of induced proteins with 35S-sulfate during adaptation of cells grown under high-CO2 conditions to low CO2. Analysis by SDS-PAGE revealed that synthesis of two polypeptides, with molecular masses of 98 and 24 kDa, respectively, was induced under low-CO2 conditions. The 24-kDa polypeptide was induced at pH 5.5 but not at pH 8.0, whereas the 98-kDa polypeptide was induced at both pH 5.5 and pH 8.0. The possible role of these polypeptides in the facilitated transport of DIC in Chlorella regularis is discussed.
Mannitol-1-phosphate (M1P) dehydrogenase (M1PDH; EC 1.1.1.17), an enzyme catalyzing the reduction of Fru-6-phosphate (F6P) to M1P in algal mannitol biosynthesis, was purified to homogeneity from a cell homogenate of the eulittoral red alga Caloglossa continua (Okamura) King et Puttock. The enzyme was a monomer with an apparent molecular mass of 53 kD, as determined by gel filtration and SDS-PAGE, and exhibited an pI of approximately 5.5. The substrate specificity was very high toward F6P and M1P for respective reductive and oxidative reactions. The enzyme was found to be a sulfhydryl-type, because its activity was inhibited by N-ethylmaleimide and p-hydroxymercuribenzoate, and the inhibition by p-hydroxymercuribenzoate was rescued by 2-mercaptoethanol. Some unknown factors in the extract may also have inhibited the activity, because the total activity was greatly increased through the purification procedure. The optimum pH for F6P reduction was changed from 6.0 or lower to 7.2 by the addition of 200 mm NaCl. The reduction of F6P showed strong substrate inhibition above 0.5 mm. However, Km(F6P) of M1PDH was increased eight times by the addition of 200 mm NaCl, whereas Vmax was in a similar range with the avoidance of substrate inhibition by F6P. These results indicate that the enzyme was finely and directly regulated by the salt concentration without the requirement for gene expression. M1PDH can therefore be a key enzyme for regulating mannitol biosynthesis when the alga is stressed by a salinity change.
