The Path of Carbon Flow during NO(3)-Induced Photosynthetic Suppression in N-Limited Selenastrum minutum

Nitrate addition to nitrate-limited cultures of Selenastrum minutum Naeg. Collins (Chlorophyta) resulted in a 70% suppression of photosynthetic carbon fixation. In ¹⁴CO₂ pulse/chase experiments nitrate resupply increased radiolabel incorporation into amino and organic acids and decreased radiolabel incorporation into insoluble material. Nitrate resupply increased the concentration of phosphoenolpyruvate and increased the radiolabeling of phosphoenolpyruvate, pyruvate and tricarboxylic acid cycle intermediates, notably citrate, fumarate, and malate. Furthermore, nitrate also increased the pool sizes and radiolabeling of most amino acids, with alanine, aspartate, glutamate, and glutamine showing the largest changes. Nitrate resupply increased the proportion of radiolabel in the C-4 position of malate and increased the ratios of radiolabel in aspartate to phosphoenolpyruvate and in pyruvate to phosphoenolpyruvate, indicative of increased phosphoenolpyruvate carboxylase and pyruvate kinase activities. Analysis of these data showed that the rate of carbon flow through glutamate (10.6 μmoles glutamate per milligram chlorophyll per hour) and the rate of net glutamate production (7.9 μmoles glutamate per milligram chlorophyll per hour) were both greater than the maximum rate of carbon export from the Calvin cycle which could be maintained during steady state photosynthesis. These results are consistent with the hypothesis that nitrogen resupply to nitrogen-limited microalgae results in a transient suppression of photosynthetic carbon fixation due, in part, to the severity of competition for carbon skeletons between the Calvin cycle and nitrogen assimilation (IR Elrifi, DH Turpin 1986 Plant Physiol 81: 273-279).     

Chlorophyll a Fluorescence Predicts Total Photosynthetic Electron Flow to CO(2) or NO(3)/NO(2) under Transient Conditions

A model which predicts total photosynthetic electron flow from a linear regression of the relationship between corrected steady-state quantum yield and nonphotochemical quenching (E Weis, JA Berry [1987] Biochem Biophys Acta 894: 198-208) was formulated for N-limited cells of the green alga Selenastrum minutum. Unlike other models based on net CO₂ fixation, our model is based on total photosynthetic electron flow measured as gross O₂ evolution. This allowed for the prediction of total photosynthetic electron flow from water to both CO₂ fixation and NO₃⁻/NO₂⁻ reduction. The linear regression equation predicting electron flow is of the form: J = I · Q_q[0.4777-0.3282 Q_NP] (where J = gross photosynthetic electron flow, I = incident PAR, Q_q = photochemical quenching, Q_NP = nonphotochemical quenching). During steady-state photosynthesis, over a range of irradiance, the model predicted a photosynthetic light saturation curve which was well correlated with that observed. Although developed under steady-state conditions, the model was tested during nonsteady-state photosynthesis induced by transient nitrogen assimilation. The model predicted transient rates of gross O₂ evolution which were in excellent agreement with the rates observed under a variety of conditions regardless of whether CO₂ or NO₃⁻/NO₂⁻ served as the physiological electron acceptor. The fluorescence transients resulting from ammonium and nitrate assimilation are discussed with respect to metabolic demands for reductant and ATP.     

Demonstration of Both a Photosynthetic and a Nonphotosynthetic CO(2) Requirement for NH(4) Assimilation in the Green Alga Selenastrum minutum

Nitrogen-limited and nitrogen-sufficient cell cultures of Selenastrum minutum (Naeg.) Collins (Chlorophyta) were used to investigate the dependence of NH₄⁺ assimilation on exogenous CO₂. N-sufficient cells were only able to assimilate NH₄⁺ maximally in the presence of CO₂ and light. Inhibition of photosynthesis with 3-(3,4-dichlorophenyl)-1,1-dimethylurea, diuron also inhibited NH₄⁺ assimilation. These results indicate that NH₄⁺ assimilation by N-sufficient cells exhibited a strict requirement for photosynthetic CO₂ fixation. N-limited cells assimilated NH₄⁺ both in the dark and in the light in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea, diuron, indicating that photosynthetic CO₂ fixation was not required for NH₄⁺ assimilation. Using CO₂ removal techniques reported previously in the literature, we were unable to demonstrate CO₂-dependent NH₄⁺ assimilation in N-limited cells. However, employing more stringent CO₂ removal techniques we were able to show a CO₂ dependence of NH₄⁺ assimilation in both the light and dark, which was independent of photosynthesis. The results indicate two independent CO₂ requirements for NH₄⁺ assimilation. The first is as a substrate for photosynthetic CO₂ fixation, whereas the second is a nonphoto-synthetic requirement, presumably as a substrate for the anaplerotic reaction catalyzed by phosphoenolpyruvate carboxylase.     

Chromophoric cinnamic acid substrates as resonance Raman probes of the active site environment in native and unfolded alpha-chymotrypsin

Chromophoric [4-(dimethylamino)cinnamoyl]imidazole reacts with the serine protease alpha-chymotrypsin to form an acyl enzyme. At pHs below 4.0, the acyl enzyme turns over very slowly to yield the free acid. During this slow deacylation it is possible to obtain a very good resonance Raman spectrum of the acyl intermediate by using the 350.7-nm line of the krypton laser. The resonance Raman carbonyl frequency of the covalently bonded substrate and its wavelength at maximum intensity in the absorption spectrum of the acyl enzyme have been taken and used to monitor the active site environment. A comparison has been made of the absorption and Raman spectra of the acyl enzyme and those of the corresponding chromophoric methyl ester, aldehyde, and imidazole model compounds. A linear correlation is found between the wavelength of maximum absorption and the Raman frequency of the carbonyl group over a wide range of solvent conditions for each of the model compounds. By combining the Raman carbonyl frequency with the absorption maximum, we can determine that the bond order changes in the carbonyl bond of the bound substrate are not due to changes in the solvent, since the carbonyl frequency and the absorption maximum of the acyl enzyme do not fall on any of the linear correlations for the model compounds. The unusual spectroscopic properties of the bound substrate appear to be due to some specific enzyme-induced change in the substrate when it is bound at the active site. Thermal unfolding of the acyl enzymes changes both the carbonyl frequency of the acyl enzyme and its absorption maximum to completely different values.(ABSTRACT TRUNCATED AT 250 WORDS)     

A study of the thermal stability of the Z form of (m5dC-dG)3 by resonance Raman spectroscopy.

The thermal stability of the hexanucleoside pentaphosphate m5dCpdGpm5dCpdGpm5 dCpdG has been studied by resonance Raman spectroscopy with 257 nm excitation wavelength. At low temperature and in 3M NaClO4, the Raman spectrum resembles that of poly(dG-dC).poly(dG-dC) in the Z conformation. As the temperature is increased, the position and the intensity of several bands (1312 cm-1, 1482 cm-1, 1584 cm-1 and 1632 cm-1) are modified. The variation of intensity versus temperature is biphasic. Analysis of the results suggests that the increase of temperature induces first a transition from the Z form to an intermediate stable form which then melts. These results and those previously obtained by circular dichroism and 31P nuclear magnetic resonance suggest that the intermediate form belongs to the left family but with changes in the stacking of the bases and the geometry of the phosphate groups as compared to the canonical Z form.     

Immunological and chemical characterization of hamster brain polyribosomes-cytomatrix complexes

We have studied the cytoskeletal nature of a brain subcellular fraction previously shown to contain polyribosomes. We have identified the major proteins of this fraction by electrophoretic comparison to a standard cytoskeletal fraction and by immunodetection. These methods have shown the presence of actin, glial fibrillary acidic protein, and neurofilament triplet proteins. We have also studied the effect of various ions and nonionic detergents on the stability of this structure. It was stable in presence of Triton X-100 up to 2% but disrupted by 200 mM K⁺ acetate.Abbreviations CMT cytomatrix - CSK cytoskeleton - DOC sodium deoxycholate - DTT dithiothreitol - EGTA ethylenglycolbis (-Ether)-N,N-N-N-Tetraacetic Acid - GFAP glial fibrillary acidic protein - PR polyribosome - PRCMC polyribosomes-cytomatrix complex     

Fructose 1,6-Bisphosphatase in the Green Alga Selenastrum minutum: I. Evidence for the Presence of Isoenzymes

Two isoforms of fructose 1,6-bisphosphatase are present in the green alga Selenastrum minutum. The isoenzymes can be separated with ionexchange chromatography or acid precipitation. The stability of the two isoenzymes differ largely. The acid insoluble enzyme exhibits properties similar to that of the enzyme from the chloroplasts of higher plants, i.e. an alkaline pH optima in the absence of reductant, a lower affinity for substrate, strong inhibition by phosphate, and a low sensitivity to fructose-2,6-bisphosphate and AMP. The more abundant form of the enzyme exhibits several properties indicative of heterotrophic fructose 1,6 bisphosphatases, i.e. a high affinity for substrate and sensitivity toward fructose-2,6-bisphosphate and AMP. but is absolutely dependent on a reductant for stability and activity. Evidence is provided indicating that previously reported purification protocols cause inactivation of one of the isoenzymes which could lead to the erroneous conclusion that algae have a single fructose 1,6-bisphosphatase isoenzyme.