Chemiluminescence associated with phagocytosis of foreign particles in rabbit alveolar macrophages

Rabbit alveolar macrophages exhibit a chemiluminescent response which is associated with phagocytosis of zymosan and polystyrene-butadiene particles. The chemiluminescence reaches a peak in 15 to 25 minutes and then gradually diminishes over the next 1 to 3 hours. During the time of maximal light emission there appears to be no actual uptake of particles, but the response is dependent upon the particle concentration. The metabolic inhibitor, DNP (2,4-dinitrophenol), causes a rapid inhibition of the chemiluminescent response. The addition of ATP to the medium prior to exposure of the cells to particles causes the chemiluminescent response to be greatly diminished, i.e., 0.3mM ATP virtually abolishes the response. These experiments suggest that some metabolic response of the cell to phagocytosis is responsible for the chemiluminescence.     

Induction by L-tryptophan and an analogue, alpha-methyl-DL-tryptophan, of the enzymes catabolizing L-tryptophan in Pseudomonas

An investigation was made of the pattern of induction of the enzymes that metabolize l-tryptophan through kynurenic acid (the quinoline pathway) in Pseudomonas fluorescens. The first four enzymes in the pathway were not induced in the same proportions or in the same time courses. This lack of coordinate induction excludes a mechanism of regulation of these enzymes at a single site as proposed in the operon model. The enzymes were induced in a sequential pattern in the order of their position on the pathway, when they became the limiting reactions. The nonmetabolizable analogue, alpha-methyl-dl-tryptophan, caused a measurable elevation in the levels of the first three enzymes of the same pathway. Evidence is presented that growth of the cells in the presence of alpha-methyl-dl-tryptophan caused the accumulation of endogenous tryptophan, and that induction by the nonmetabolizable analogue is induction by the endogenous tryptophan.     

A study of protein structure changes during hydration by diffuse X-ray scattering: II. Fourier transform analysis of diffuse X-ray scattering data

Radial distribution functions were deduced by Fourier transform analysis of the angular dependences of diffuse X-ray scattering intensities for the following proteins with different hydration degrees: water-soluble α-protein myoglobin, water-soluble (α + β) protein lysozyme, and transmembrane proteins from the photosynthetic reaction centers of purple bacteria Rhodobacter sphaeroides and Blastochlorii (Rhodopseudomonas) viridis. The results of Fourier transform analysis of X-ray scattering intensities give quantitative characteristics of the mechanism underlying the influence of water on the formation of biological macromolecules. On the one hand, water loosens the network of hydrogen bonds, which results in a considerable conformational mobility in the molecules of lysozyme and myoglobin and the reaction centers. On the other hand, water stabilizes and orders the protein globule. A strict correlation was found between the shift of the “first” maximum of the radial distribution function, loosening of the intraglobular hydrogen bonds, increase in the intramolecular mobility, and appearance of pronounced functional activity in macromolecules. The pattern of behavior of the first maximum in the transmembrane proteins of the reaction center was similar to that observed for the water-soluble proteins. However, the first maximum reached the limiting value of 2.9 Å at a considerably lower hydration degree compared with the water-soluble proteins. A quick transition of the protein complex of the reaction center to its native state is due to the fact that the dehydrated conformation of this complex is very close to the native conformation. Comparison of the radial distribution function for water, water-soluble proteins, and transmembrane proteins suggests a quantitative conclusion that water is the least densely packed and ordered system, the water-soluble proteins are more densely packed than water, and the transmembrane proteins are the most densely packed and ordered system.     

Understanding How Noncatalytic Carbohydrate Binding Modules Can Display Specificity for Xyloglucan

Plant biomass is central to the carbon cycle and to environmentally sustainable industries exemplified by the biofuel sector. Plant cell wall degrading enzymes generally contain noncatalytic carbohydrate binding modules (CBMs) that fulfil a targeting function, which enhances catalysis. CBMs that bind β-glucan chains often display broad specificity recognizing β1,4-glucans (cellulose), β1,3-β1,4-mixed linked glucans and xyloglucan, a β1,4-glucan decorated with α1,6-xylose residues, by targeting structures common to the three polysaccharides. Thus, CBMs that recognize xyloglucan target the β1,4-glucan backbone and only accommodate the xylose decorations. Here we show that two closely related CBMs, CBM65A and CBM65B, derived from EcCel5A, a Eubacterium cellulosolvens endoglucanase, bind to a range of β-glucans but, uniquely, display significant preference for xyloglucan. The structures of the two CBMs reveal a β-sandwich fold. The ligand binding site comprises the β-sheet that forms the concave surface of the proteins. Binding to the backbone chains of β-glucans is mediated primarily by five aromatic residues that also make hydrophobic interactions with the xylose side chains of xyloglucan, conferring the distinctive specificity of the CBMs for the decorated polysaccharide. Significantly, and in contrast to other CBMs that recognize β-glucans, CBM65A utilizes different polar residues to bind cellulose and mixed linked glucans. Thus, Gln¹⁰⁶ is central to cellulose recognition, but is not required for binding to mixed linked glucans. This report reveals the mechanism by which β-glucan-specific CBMs can distinguish between linear and mixed linked glucans, and show how these CBMs can exploit an extensive hydrophobic platform to target the side chains of decorated β-glucans.     

Multi‐scale spatial heterogeneity of pectic rhamnogalacturonan I (RG–I) structural features in tobacco seed endosperm cell walls

Plant cell walls are complex configurations of polysaccharides that fulfil a diversity of roles during plant growth and development. They also provide sets of biomaterials that are widely exploited in food, fibre and fuel applications. The pectic polysaccharides, which comprise approximately a third of primary cell walls, form complex supramolecular structures with distinct glycan domains. Rhamnogalacturonan I (RG–I) is a highly structurally heterogeneous branched glycan domain within the pectic supramolecule that contains rhamnogalacturonan, arabinan and galactan as structural elements. Heterogeneous RG–I polymers are implicated in generating the mechanical properties of cell walls during cell development and plant growth, but are poorly understood in architectural, biochemical and functional terms. Using specific monoclonal antibodies to the three major RG–I structural elements (arabinan, galactan and the rhamnogalacturonan backbone) for in situ analyses and chromatographic detection analyses, the relative occurrences of RG–I structures were studied within a single tissue: the tobacco seed endosperm. The analyses indicate that the features of the RG–I polymer display spatial heterogeneity at the level of the tissue and the level of single cell walls, and also heterogeneity at the biochemical level. This work has implications for understanding RG–I glycan complexity in the context of cell‐wall architectures and in relation to cell‐wall functions in cell and tissue development.