Two approaches currently utilized in studying intracellular regulation of differentiation in D. discoideum have been analyzed. One tends to implicate transcription and translation as the most critical types of control during differentiation. The other approach assumes a priori that no single cellular event or component has inherent causal significance, but rather attempts to assess various factors limiting the rate of differentiation through a kinetic analysis of the relationships between enzymes, metabolites, and in vivo reaction rates. We have attempted to justify our prejudice for the latter approach, and have presented a kinetic analysis of the metabolism involved in the synthesis of carbohydrate end products. In addition, data pertaining to the control of energy metabolism are discussed in considering directions for expansion of the kinetic model.
Abstract The relative activities of lipoteichoic acid (LTA) from four Gram-positive bacteria were compared to different lipopolysaccharide (LPS) preparations for activation of arachidonic acid metabolism in mouse peritoneal macrophages. Total eicosanoid was determined in cultures labeled with [3H]-arachidonic acid. Prostaglandin E2 (PGE2) and leukotriene C4 (LTC4) were determined by EIA analysis. The relative potencies of the different preparations were: smooth LPS from Salmonella abortus ≥ Re-LPS from Salmonella minnesota (R-595) ≥ LTA from Streptococcus pyogenes Streptococcus faecalis Staphylococcus aureus ≥ monophosphoryl lipid A derived from the Re-LPS > > LTA from Bacillus subtilis. Activation of eicosanoid release was inhibited by staurosporin for all of the amphiphiles tested. Treatment of the macrophage cultures with LTA from S. pyogenes, S. faecalis, and S. aureus, either in the presence or absence of indomethacin, desensitized the cells to eicosanoid release on subsequent challenge with LPS. The desensitized cells remained responsive to the phorbol ester phorbol myristate acetate. LPS from Gram-negative bacteria has immunostimulatory and endotoxic activities which result, in part, from the release of eicosanoids and other mediators from activated macrophages. The similarities in the patterns of cell activation by LPS and LTA suggest that lipoteichoic acids might contribute to the pathogenicities of Gram-positive bacteria. J. Leukoc. Biol. 56: 723–728; 1994.
The synthesis of a series of novel analogues of lipid A, the active principle of lipopolysaccharide, is reported. In these compounds, the 1-O-phosphono and (R)-3-hydroxytetradecanoyl moieties of native Salmonella minnesota R595 lipid A have been replaced with hydrogen and the length of the normal fatty acyl residues has been systematically varied. Normal fatty acid chain length in the 3-O-desacyl monophosphoryl lipid A (MLA) series is shown to be a critical determinant of iNOS gene expression in activated mouse macrophages and the induction of proinflammatory cytokines in human peripheral monocytes. Examination of pyrogenicity in rabbits and lethal toxicity in D-galactosamine-treated mice shows that toxic effects in the MLA series can be ameliorated by modifying fatty acid chain length. When used as an adjuvant for tetanus toxoid vaccines, certain MLA derivatives enhance the production of tetanus toxoid-specific antibodies in mice.
Abstract The enzyme UDP-glucose pyrophosphorylase has been purified to electrophoretic homogeneity from both vegetative amoebae and cells at the culmination stage of differentiation in Dictyostelium discoideum. The two purified enzyme protein preparations, having higher specific activities than any preparation previously reported, were clearly resolvable by sodium dodecyl sulfate gel electrophoresis. Immunological analysis, however, indicated a high degree of similarity in antigenic determinants between the two forms of the enzyme. Comparison of the rates of incorporation of [35S]methionine into UDP-glucose pyrophosphorylase and into acid-insoluble protein suggests that the maintenance of enzyme protein at a constant level during the transition period from growth to multicellular differentiation is attributable to a rapid turnover of enzyme protein, rather than to a cessation of enzyme synthesis. A decrease in the rate of enzyme degradation as opposed to an induction of enzyme synthesis is suggested as the mechanism of UDP-glucose pyrophosphorylase accumulation during multicellular differentiation.
Detached tomato leaves, supplied with the proteinase inhibitor inducing factor (PIIF) and incubated with water under constant light, exhibited a specificity of intracellular protein turnover directed toward the selective accumulation of heat-stable proteins having disulfide cross-linkages.Approximately 70% of the accumulated proteins could be accounted for in two proteinase.inhibitorsrich in disulfide links.The accumulation of proteins containing disulfides was accompanied by a net loss in total leaf protein, mainly of heat-precipitable proteins having free sulfhydryl residues.Relative rates of synthesis of -S-Sproteins and -SH proteins were assessed by comparing rates of incorporation of isotope into the inhibitor proteins and noninhibitor leaf proteins.Although the inhibitors represented about 12% of total leaf protein after 71 h of induction, only about 2% of total protein synthesis was directed toward inhibitor synthesis during incubation of induced leaves.The marked stability of inhibitors, and other disulfide proteins against degradation in uiuo, appeared to be a major factor providing for their selective accumulation.It was concluded that the state of oxidation of protein-bound half-cystine residues may be a principle parameter influencing the susceptibility of leaf proteins to degradation in uiuo.
The enzyme uridine diphosphate glucose pyrophosphorylase was purified to a final specific activity of approximately 1200 units per mg from etiolated Sorghum vulgare seedlings. The purified enzyme was highly specific for both UTP and UDP-glucose. The divalent cation requirement was most readily satisfied by magnesium, but manganese and cobalt were also effective. The enzyme was characterized kinetically with regard to the mechanism of catalysis. Product inhibition studies indicate an ordered Bi Bi reaction mechanism in which the nucleotide substrate adds first and is released last as product. Evidence was obtained supporting mechanisms in which either free magnesium ion activates catalysis of UDP-glucose synthesis by forming a complex with enzyme in addition to its role in formation of MgUTP2-, or UTP4- inhibits the reaction. In contrast, magnesium ion had no effect on the rate of pyrophosphorolysis beyond its role in formation of MgPPi2-. Michaelis constants for each substrate and dissociation (inhibition) constants for nucleotides combining with enzyme were determined in the presence of excess or limiting concentrations of free magnesium ion. The influence of these mechanisms on the regulation of carbohydrate flow in higher plants is discussed.
