Deaggregation and proteolytic modification of a galactosyltransferase of Poterioochromonas malhamensis

We isolated hybridomas that produced monoclonal antibodies specific for the UDP-galactose: sn -glycerol-3-phosphate α-D-galactosyltransferase (IFP synthase, EC 2.4.1.96), an enzyme involved in the volume regulation of Poterioochromonas malhamensis Peterfi. Western blotting of native gradient gels with the most reactive antibody S 162 revealed several immunoreactive proteins in crude homogenates suggesting the occurrence of multiple molecular mass species of the galactosyltransferase. The amount of the presumed enzyme monomer (64 kDa under native conditions) was strongly increased by a pH shift of crude homogenates from pH 8 to 6. During activation of the galactosyltransferase in the cell homogenate and also by shrinking the cells, the presumed enzyme monomer appeared to be proteolytically degraded generating stepwise products of 52 and 40 kDa. We assume that the proteolytically processed enzyme becomes highly active, but is very susceptible to further proteolytic degradation.     

Photosynthesis and apparent affinity for dissolved inorganic carbon by cells and chloroplasts of Chlamydomonas reinhardtii grown at high and low CO2 concentrations

Chloroplasts with high rates of photosynthetic O₂ evolution (up to 120 mol O₂· (mg Chl)^-1·h^-1 compared with 130 mol O₂· (mg Chl)^-1·h^-1 of whole cells) were isolated from Chlamydomonas reinhardtii cells grown in high and low CO₂ concentrations using autolysine-digitonin treatment. At 25° C and pH=7.8, no O₂ uptake could be observed in the dark by high- and low-CO₂ adapted chloroplasts. Light saturation of photosynthetic net oxygen evolution was reached at 800 mol photons·m^-2·s^-1 for high- and low-CO₂ adapted chloroplasts, a value which was almost identical to that observed for whole cells. Dissolved inorganic carbon (DIC) saturation of photosynthesis was reached between 200–300 M for low-CO₂ adapted chloroplasts, whereas high-CO₂ adapted chloroplasts were not saturated even at 700 M DIC. The concentrations of DIC required to reach half-saturated rates of net O₂ evolution (K_m(DIC)) was 31.1 and 156 M DIC for low- and high-CO₂ adapted chloroplasts, respectively. These results demonstrate that the CO₂ concentration provided during growth influenced the photosynthetic characteristics at the whole cell as well as at the chloroplast level.Abbreviations Chl chlorophyll - DIC dissolved inorganic carbon - K_m(DIC) coneentration of dissolved inorganic carbon required for the rate of half maximal net O₂ evolution - PFR photon fluence rate - SPGM silicasol-PVP-gradient medium     

Biochemical Plant Responses to Ozone : II. Induction of Stilbene Biosynthesis in Scots Pine (Pinus sylvestris L.) Seedlings

Formation of the stilbenes pinosylvin and pinosylvin 3-methyl ether, as well as the activity of the biosynthetic enzyme stilbene synthase (pinosylvin-forming), were induced several hundred- to thousandfold in primary needles of 6-week-old pine (Pinus sylvestris L.) seedlings upon exposure to a single pulse of ozone of at least 0.15 microliters per liter. The seedlings required 4 hours of exposure as a minimum for the induction of stilbene biosynthesis when exposed to 0.2 microliters per liter ozone. Both stilbene synthase activity and stilbene accumulation increased with the duration of ozone treatment. The activity of phenylalanine ammonia-lyase and the activity of chalcone synthase, a key enzyme of the flavonoid pathway that uses the same substrates as stilbene synthase, were also stimulated about twofold by ozone. Stilbene biosynthesis appears to represent the first example of a dose-dependent biochemical response to ozone in a conifer species and may serve as a useful biomarker to study stress impacts on pine trees.     

A simple model relating photoinhibitory fluorescence quenching in chloroplasts to a population of altered Photosystem II reaction centers

A model is presented describing the relationship between chlorophyll fluorescence quenching and photoinhibition of Photosystem (PS) II-dependent electron transport in chloroplasts. The model is based on the hypothesis that excess light creates a population of inhibited PS II units in the thylakoids. Those units are supposed to posses photochemically inactive reaction centers which convert excitation energy to heat and thereby quench variable fluorescence. If predominant photoinhibition of PS II and cooperativity in energy transfer between inhibited and active units are presumed, a quasi-linear correlation between PS II activity and the ratio of variable to maximum fluorescence, F_VF_M, is obtained. However, the simulation does not result in an inherent linearity of the relationship between quantum yield of PS II and F_VF_M ratio. The model is used to fit experimental data on photoinhibited isolated chloroplasts. Results are discussed in view of current hypotheses of photoinhibition.Abbreviations F_M maximum total fluorescence - F₀ initial fluorescence - F_V maximum variable fluorescence - PS Photosystem - Q_A, Q_B primary and secondary electron acceptors of Photosystem II     

Crustacean cardioactive peptide-immunoreactive neurons in the ventral nerve cord and the brain of the meal beetle Tenebrio molitor during postembryonic development

Summary By use of an antiserum against the crustacean cardioactive peptide (CCAP) several types of bilaterally symmetrical neurons have been mapped quantitatively in the ventral nerve cord and in the brain of the meal beetle, Tenebrio molitor. The general architecture of these neurons was reconstructed from peroxidase-antiperoxidase-labelled whole-mount preparations. From the subesophageal to the seventh abdominal ganglia two types of neurons show a repetitive organization of contralateral projection patterns in each neuromere. The first type has few branches in the central neuropil and a distinct peripheral projection. The second type is characterized by an elaborate central branching pattern, which includes ascending and descending processes. Some of its peripheral branches were found to supply peripheral neurohemal areas. In the protocerebrum, 10 CCAP-immunoreactive neurons occur with projections into the superior median protocerebrum and the tritocerebrum. Immunopositive neurons were mapped in larval and various pupal stages, as well as in the adult. All types of identified neurons were found to persist throughout metamorphosis maintaining their essential structural and topological characteristics. The CCAP-immunoreactive neurons of T. molitor are compared with those described for the locust. Putative structural homologies of subsets of neurons in both species are discussed.     

Involvement of various organs in the initial plasma clearance of differently glycosylated rat liver secretory proteins

The initial plasma clearance and organ distribution of alpha 1-acid glycoprotein and alpha 2-macroglobulin carrying different types of oligosaccharide, side chains was studied in rats. The differently glycosylated proteins were synthesized by rat hepatocytes in culture in the presence of tunicamycin (unglycosylated form), swainsonine (hybrid type), or 1-deoxymannojirimycin (high-mannose type). Deglycosylated glycoproteins (Asn-GlcNAc) were obtained by endoglucosaminidase H treatment of high-mannose-type glycoproteins. Ten minutes after intravenous injection 3% of complex type, 26% of hybrid type, 84% of high-mannose type. 64% of unglycosylated and 80% of deglycosylated alpha 1-acid glycoprotein disappeared from the plasma. The respective values for alpha 2-macroglobulin were 26%, 42%, 59% and 67%. When the clearance of total hepatic secretory proteins was examined, major differences between glycosylated and unglycosylated (glyco)proteins were found, particularly in the case of low-molecular-mass polypeptides. Whereas complex-type alpha 1-acid glycoprotein and alpha 2-macroglobulin showed no accumulation in various organs, hybrid-type alpha 1-acid glycoprotein and alpha 2-macroglobulin were present in spleen and liver. High-mannose-type alpha 1-acid glycoprotein and alpha 2-macroglobulin also accumulated mainly in spleen and liver. Spleen had the highest specific activity; liver, due to its larger organ mass, represented the major organ for the uptake of high-mannose-type glycoproteins. Competition experiments with mannan and GlcNAc-bovine-serum-albumin showed a mannose/GlcNAc receptor-mediated removal. Whereas unglycosylated alpha 1-acid glycoprotein was taken up by the kidney, unglycosylated alpha 2-macroglobulin was found in the spleen. Deglycosylated glycoproteins (Asn-GlcNAc) were removed from the plasma via two different mechanisms: firstly, clearance by the kidney similar to the unglycosylated glycoproteins; secondly, clearance by a mannose/GlcNAc receptor-mediated uptake mainly into the spleen. We conclude that N-linked oligosaccharide side chains are important for the plasma survival of hepatic secretory glycoproteins and that unphysiologically glycosylated forms are cleared by different mechanisms.     

Biosynthesis and secretion of alpha 1 acute-phase globulin in primary cultures of rat hepatocytes

Experimental inflammation in rats led to a sevenfold increase in serum levels of alpha 1 acute-phase globulin. This increase is correlated with elevated levels of translatable mRNA for alpha 1 acute-phase globulin in the liver. Biosynthesis and secretion of alpha 1 acute-phase globulin were studied in rat hepatocyte primary cultures. An intracellular form of alpha 1 acute-phase globulin with an apparent relative molecular mass of 63 500 and a secreted form of 68 000 were found. The intracellular form of alpha 1 acute-phase globulin could be deglycosylated by endoglucosaminidase H treatment indicating that its oligosaccharide chains were of the high-mannose type. The secreted form of alpha 1 acute-phase globulin was not sensitive to endoglucosaminidase H, but was susceptible to the action of sialidase reflecting carbohydrate side-chains of the complex type. Pulse-chase experiments revealed a precursor-product relationship for the high-mannose and the complex type alpha 1 acute-phase globulin. In the hepatocyte medium newly synthesized alpha 1 acute-phase globulin was detected 30 min after the pulse. Unglycosylated alpha 1 acute-phase globulin was found in the cells as well as in the medium when the transfer of oligosaccharide chains onto the polypeptide chains was blocked by tunicamycin. Tunicamycin led to a marked delay in alpha 1 acute-phase globulin secretion.     

Effect of light intensity on ammonia assimilation in maize leaves

The effect of light on the metabolism of ammonia was studied by subjecting detached maize leaves to 150 or 1350 mol m^–2 s^–1 PAR during incubation with the leaf base in 2 mM ¹⁵NH₄Cl. After up to 60 min, leaves were extracted. Ammonia, glutamine, glycine, serine, alanine, and aspartate were separated by isothermal distillation and ion exchange chromatography. ¹⁵N enrichments were analyzed by emission spectroscopy. The uptake of ammonium chloride did not influence CO₂ assimilation (8.3 and 17.4 mol m^–1 s^–1 at 150 and 1350 mol m^–2 s^–1 PAR, respectively). Leaves kept at high light intensity contained more serine and less alanine than leaves from low light treatments. Within 1 h of incubation the enrichment of ammonia extracted from leaves rose to approximately 20% ¹⁵N. In the high light regime the amino acids contained up to 15% ¹⁵N, whereas in low light ¹⁵N enrichments were small (up to 6%). The kinetics of ¹⁵N incorporation indicated that NH₃ was firstly assimilated into glutamine and then into glutamate. After 15 min ¹⁵N was also found in glycine, serine and alanine. At high light intensity nearly half of the ¹⁵N was incorporated in glycine. On the other hand, at low light intensity alanine was the predominant ¹⁵N sink. It is concluded that light influences ammonia assimilation at the glutamine synthetase reaction.