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The effect of temperature on Hill activity has been compared in chilling-sensitive and chilling-resistant plants. The Arrhenius activation energy (Ea) for the photorednction of 2,6-dichlorophenolindophenol by chloroplasts isolated from two chilling-sensitive plants, mung bean (Vigna radiata L. var. Mungo) and maize (Zea mays L. cv. PX 616), increased at low temperatures, below 17 C for mung bean and below 11 C for maize. However, the Ea for this reaction in pea ( (6, throughout 13). It has been reported that a fundamental difference in the response to these low temperatures exists between mitochondrial and other cellular membranes of chilling-sensitive plants and membranes of plants which are resistant to chilling temperatures (7,13,14). The Ea' of membrane-bound respiratory enzymes in mitochondria of chilling-sensitive plants increased below a critical chilling temperature, typically between 10 C to 15 C (7), and this change was correlated with a temperature-induced change in the molecular ordering of the mitochondrial lipids as detected with the aid of spin-labeled compounds (16). The membrane lipids of chloroplasts from chilling-sensitive plants also exhibited similar changes below the chilling temperature (14,15) and there was an increase in Ea for photoreduction of NADP+ although not of other Hill oxidants (18). Neither mitochondria nor chloroplasts isolated from chilling-resistant plants showed any of the temperature-dependent changes in membrane properties at temperatures close to or within the chilling-temperature range (7,13).Recently, we have investigated the effect of temperature on the Hill activity of chloroplasts isolated from barley (12). Although barley is a chilling-resistant plant, the Ea for photoreduc-I Abbreviations: DCIP: 2,6-dichlorophenolindophenol; Ea: Arrhenius activation energy. tion of either DCIP or ferricyanide increased at temperatures below 9 C. The change was reversible and additional changes in Ea took place at 20 and 29 C. Thus, there were at least four different values of Ea as the temperature was varied within the physiological temperature range for growth of barley. The alterations in Ea of Hill activity by the temperature became apparent only when a rate-limiting reaction in coupled barley chloroplasts was circumvented by the addition of uncouplers or by other means.Since the studies with barley indicated that changes in membrane properties and function induced by low temperatures were, at least for chloroplasts, not confined to chilling-sensitive plants, the effect of temperature on Hill activity was compared in two chilling-sensitive plants, mung bean and maize, and another chilling-resistant plant, pea. Chloroplasts isolated from all of these plants showed a distinct increase in the Ea for Hill activity at low temperatures, as well as another change in Ea at temperatures above 25 C. These results, together with those previously obtained with barley (12), showed that the Ea for Hill activity was not constant as the temperature was varied throughout the physiologi...

Bacillus stearothermophilus contains two carbamoyl-phosphate synthetases (CPS), one specific for pyrimidine biosynthesis and the other for arginine biosynthesis. The pyrimidine-specific CPS is repressed by exogenous pyrimidines, and its activity is inhibited by UMP and activated by 5-phospho-a-~-ribosyI diphosphate. The arginine-specific CPS is similarly repressed by exogenous arginine but its activity is not sensitive to these or other potential effectors. Each of the two enzymes consist of two unequal subunits, as is the case for other microbial CPS ; however, the large subunit for the arginine-specific CPS is smaller than that for the pyrimidine-specific enzyme. Comparison of the derived amino acid sequence for the cloned large subunit of the arginine-specific CPS with those for subunits from pyrimidine-sensitive CPS showed significant similarity throughout the polypeptides except at the carboxy terminus, which was identified by other laboratories to contain the binding site for the pyrimidine effector. Unlike the results previously reported for CPS from an enteric mesophile, the kinetic properties of the arginine-specific CPS were not affected by growth of B. stearothermophilus at temperatures near the minimal growth temperature. Furthermore, calorimetric studies showed that the thermal stability of cloned CPS was identical regardless of the growth temperature of B. stearothermophilus between 42°C and 63°C. The thermal stability of cloned CPS was not affected by expression at 37OC in Bacillus subtilis or Escherichia coli. In contrast, the thermal stabilities for CPS and other proteins were higher in extracts of cells grown at higher temperatures. These results indicate that cellular factors, probably chaperonins, are necessary for thermal stability of proteins at and below the optimal temperature for this thermophile.Keywords: thermal stability ; growth temperature; carbamoylphosphate synthetase; kinetics.Carbamoyl-phosphate synthetase (CPS) catalyzes the synthesis of carbamoyl phosphate from ATP, bicarbonate and either ammonia or glutamine. Bacterial CPS consist of two unequal subunits ; the large subunit alone catalyzes the synthesis of carbamoyl phosphate from MgATP, bicarbonate and ammonia; the small subunit functions as a glutamine amidotransferase (Glansdorff, 1996). In gram-negative bacteria, a single CPS catalyzes the synthesis of carbamoyl phosphate as a precursor for arginine and pyrimidines (Glansdorff, 1996). In contrast, studies with Bacillus subtilis showed that this gram-positive bacterium possesses two CPS, each specific for arginine or pyrimidine biosynthesis and each under separate control (Paulus and Switzer, 1979). The nucleotide sequences for the two CPS from B. subtilis have been determined (Quinn et al., 1991;O'Reilly and Devine, 1994) and limited studies with partially purified enzymes have been reported (Paulus and Switzer, 1979 Enzymes. Carbamoyl-phosphate synthetase (glutaniine-hydrolyzing) (EC 6.3.5.5); glyceraldehyde-3-phosphate dehydrogenase (phosphorylating) (EC 1.2.1.12).Note....