Events Surrounding the Early Development of Euglena Chloroplasts: VI. Action Spectra for the Formation of Chlorophyll, Lag Elimination in Chlorophyll Synthesis, and Appearance of TPN-dependent Triose Phosphate Dehydrogenase and Alkaline DNase Activities

Action spectra derived from dose-response curves measured for various processes associated with chloroplast development in Euglena gracilis var. bacillaris are presented. The action spectrum for chlorophyll synthesis during the first 36 hours of continuous illumination of dark-grown resting cells resembles the absorption spectrum of protochlorophyll(ide). The action spectrum for the preillumination phase of potentiation, during which preillumination followed by a dark period brings about lag elimination in chlorophyll synthesis when the cells are subsequently exposed to postilluminating light, shows a high peak in the blue region (at about 433 nm) with a small peak in the yellow-orange region (at about 597 nm); the postillumination phase yields an action spectrum very similar to that obtained for chlorophyll synthesis in continuous light in normal, unpotentiated cells, with peaks at 433 and 631 nm. Alkaline DNase and TPN-linked triose phosphate dehydrogenase, two plastid enzymes which are synthesized outside the chloroplast, yield action spectra which are consistent with protochlorophyll(ide) being the major light receptor. The action spectra which implicate pigments resembling protochlorophyll(ide) holochrome have blue to red peak ratios in the vicinity of 5:1 as does the absorption spectrum of the protochlorophyllide holochrome from beans; the action spectrum is not identical with the holochrome spectrum indicating that the Euglena holochrome may differ from the bean pigment in details of its absorption spectrum. The action spectrum for preillumination, shows a ratio of the blue peak to the red effectiveness of about 24:1. This suggests that preillumination is controlled by a photoreceptor different from the protochlorophyll(ide) holochrome.     

The chemical synthesis of polysaccharides : Part III. Synthesis of 6-O-α-D-mannopyranosyl-D-mannose and 4-O-α-D-mannopyranosyl-D-mannose. Isolation of A (1→6)-linked D-mannan

When 1,2,3,4-tetra-O-acetyl-α-D-mannopyranose was fused with a catalytic amount of toluene-p-sulphonic acid, 6-O-α-D-mannopyranosyl-D-mannose and 4-O-α-D-mannopyranosyl-D-mannose were isolated after deacetylation of the reaction mixture. No β-D-linked disaccharide was detected in the reaction mixture. When the corresponding β-D-tetra-acetate was fused with zinc chloride as catalyst, higher oligomers were formed, and a D-mannan was isolated and shown to be mainly an α-(1→6)-linked polymer having d.p. of 10. With 5% of zinc chloride, the α-D-tetra-acetate showed oligosaccharide formation, and yielded a smaller proportion of a (1→6)-linked D-mannan.     

Prophage Induction of Noninducible Coliphage 186

Coliphage 186 has been regarded as a member of the noninducible group of coliphages. Evidence that prophage 186 is induced by ultraviolet irradiation or by treatment with nalidixic acid or mitomycin C is now presented. The phage yields were similar to those from lysogens of the inducible phage lambda, and the induction required a recA(+) host. A noninducible mutant of 186 was isolated from its heat-inducible derivative, 186cIts, that was no longer inducible by ultraviolet irradiation but remained heat inducible. That zygotic induction of 186 after transfer from a lysogenic male to a non-lysogenic recipient did not occur is indicated by the following findings: (i) there was only a slight increase in phage titer; (ii) similar levels of recombinants were obtained for markers adjacent or distal to the phage integration site, whether the recipient was lysogenic or not, and there was no effect on the gradient of marker transfer; (iii) lysogenic recombinants were readily found and the co-transfer of 186 with adjacent markers was the same to lysogenic or non-lysogenic recipients. Thus, 186 formed an inducible prophage that did not display zygotic induction. Nevertheless, it shared many properties with the noninducible phage P2 as outlined in the discussion.     

Anthranilate synthase/anthranilate 5-phosphoribosyl 1-pyrophosphate phosphoribosyltransferase from Aerobacter aerogenes

1. Anthranilate synthase and phosphoribosyltransferase from Aerobacter aerogenes purify simultaneously and sediment together on sucrose gradients, showing that they occur as an enzyme aggregate. Both activities of the intact aggregate are subject to inhibition by tryptophan. 2. By using appropriate auxotrophic mutants it was shown that an intact active enzyme aggregate is formed when the components come from separate mutant strains. An intact active aggregate can also be formed when one component is from Escherichia coli and the other from A. aerogenes. 3. Phosphoribosyltransferase of A. aerogenes is active when not in an aggregate with anthranilate synthase, but is not subject to tryptophan inhibition, indicating that the inhibitor site is on the anthranilate synthase component. 4. Anthranilate synthase can be active and sensitive to tryptophan inhibition when complexed with an inactive phosphoribosyltransferase. 5. Kinetic studies on the anthranilate synthase activity show that tryptophan is a competitive inhibitor with respect to chorismate and a non-competitive inhibitor with respect to either glutamine or NH(4) (+) ions. This is consistent with a sequential mechanism of the ordered type in which chorismate is the first reactant.     

Studies on Chloroplast Development and Replication in Euglena: III. A Study of the Site of Synthesis of Alkaline Deoxyribonuclease Induced during Chloroplast Development in Euglena gracilis

During chloroplast development in Euglena, the activity of a specific DNase, Euglena alkaline DNase, increases in a manner similar to that of chlorophyll synthesis, but without the lag customarily associated with the early hours of chlorophyll synthesis. The increase in Euglena alkaline DNase activity is not inhibited by chloramphenicol or by streptomycin, but is inhibited by cycloheximide. Euglena alkaline DNase activity is present in a group of aplastidic substrains which contain carotenoids. These results are interpreted to mean that this chloroplast-related DNase is synthesized in the cytoplasm, and that the genetic information for this enzyme is probably nuclear.     

New n.m.r.-spectroscopic approaches for structural studies of polysaccharides: application to the Haemophilus influenzae type a capsular polysaccharide

The extension of several modern nuclear magnetic resonance (n.m.r.) spectroscopic techniques to polysaccharides is discussed and illustrated, using the native Haemophilus influenzae type a capsular polysaccharide. These techniques provide for the unambiguous assignment of all n.m.r. resonances (1H, 13C, and 31P) via high-sensitivity homonuclear and 1H-detected heteronuclear correlations, and they are capable of locating the intersaccharide linkages (both O-linked and phosphoric diester-linked) and appended groups (e.g. O-acetyl groups). To illustrate the power and sensitivity of these methods, a 10-mg sample of the H. Influenzae type a polysaccharide (repeat unit mol. wt. = 376) was studied. The combined acquisition time for the two-dimensional 1H-13C correlation data (one-bond and multiple-bond), the 1H-31P correlation data, and the 1H-1H (homonuclear Hartmann-Hahn) data was approximately 18 h.     

Differential effects of prostaglandin synthetase stimulators on inhibition of cyclooxygenase.

The different effects of prostaglandin synthetase stimulators on inhibition of the cyclooxygenase by structurally distinct classes of nonsteroidal antiinflammatory agents suggest that the enzyme is altered by interaction with these stimulators. Reversible stimulation of prostaglandin synthetase activity by phenols and some other compounds and the relative influence of these stimulators on inhibitors of the cyclooxygenase were determined quantitatively. Two distinct classes of inhibitors were established. The fenamates were relatively weak inhibitors alone but were much more potent in the presence of phenolic compounds. In contrast, ibuprofen, indomethacin, and flurbiprofen were more potent than the fenamates and were reduced in effectiveness by the stimulators, as expected on the basis of two opposing actions. The relative potency of the cyclooxygenase stimulators (phenol greater than norepinephrine greater than tryptophan greater than benzoquinone greater than anisole) paralleled their synergistic action on the fenamates and their antagonist action on the nonfenamates. This correlation suggests that an enzyme alteration which leads to cyclooxygenase stimulation may also result in increased sensitivity to fenamates and decreased sensitivity to the other inhibitors, possibly by altering their capacity to bind.     

Outer-membrane damage in sublethally heated Escherichia coli K-12.

Exponentially grown cells of Escherichia coli K-12 heated at 48 degrees C in potassium phosphate buffer at pH 7.0 were structrually injured before death. During heating for 60 min about 20% of the cellular lipopolysaccharide (LPS) was released from the outer membrane into the heating medium. Removal of 30% of the cellular LPS, by washing the cells in buffer containing ethylenediaminetetraacetic acid (EDTA), caused no significant increase in the rate of death and structural injury produced by heating. The addition of EDTA to the heating medium produced only a slight increase in the rate of thermal death but a large increase in the rate of structural injury. By a combination of heating at 48 degrees C and washing with EDTA, a maximum of 50% of the LPS was released from cells. These results taken together suggest that structural injury and loss of LPS are not the direct causes of death. The addition of 5 m M Mg2+ to the heating medium protected the cells from death and structural injury caused by heating at 48 degrees C.     

Energetics of Microbacterium thermosphactum in glucose-limited continuous culture.

Microbacterium thermosphactum was grown at 25 degrees C in glucose-limited continuous culture under aerobic (greater than 120 microM oxygen) and anaerobic (less than 0.2 microM oxygen) conditions. The end products of the anaerobic metabolism of glucose were identified as L-lactate and ethanol. Together these compounds accounted for between 85 and 90% of the glucose utilized over the full range of growth rates studied. In addition, 4% of the glucose utilized was incorporated into cellular material. Under anaerobic conditions the molar growth yield was 40 g (dry weight) of cells per mol of glucose utilized, and the maintenance energy coefficient was 0.4 mmol of glucose utilized per g (dry weight) of cells per h. For cells grown under aerobic conditions in the corresponding values were 73 g/mol and 0.2 mmol/g per h, respectively. The molar growth yield with respect to adenosine 5'-triphosphate varied with the growth rate of the culture, and the true molar growth yield with respect to adenosine 5'-triphosphate was found to be 20 g/mol of adenosine 5'-triphosphate.