Immunological relationships of glutamine synthetases from marine and terrestrial enterobacteria

Glutamine synthetase (GS) fromBeneckea alginolytica strain 90 was purified to homogeneity as indicated by sodium dodecyl sulface polyacrylamide gel electrophoresis. The purified enzyme was injected into rabbits, and the antiserum, as well as previously prepared antiserum against the GS fromEscherichia coli, was used in Ouchterlony double diffusion experiments. From the results of these studies, the marine and terrestrial enterobacteria could be separated into five major groups consisting of (i)Beneckea and related organisms, (ii)Photobacterium phosphoreum, P. leiognathi, andP. angustum, (iii) species ofAeromonas, (iv)Xenorhabdus luminescens, and (v) species of terrestrial enterobacteria. Evidence is presented that inB. alginolytica, as in other Gramnegative eubacteria, GS activity is regulated by adenylylation and deadenylylation.     

Structure-function relationships amongLathyrus lectins

The structure-function relationship of eight structurally very similarLathyrus lectins, which are two-chain lectins from theVicieae tribe, has been studied. Hemagglutination activity, inhibition of hemagglutination, glycoprotein reactivity and mitogenic activity were determined for each of the lectins. Despite the close structural resemblance, marked differences in the carbohydrate binding activity of theLathyrus lectins were recorded. These functional differences were related to small conformational changes due to minor differences in amino acid sequence of eachLathyrus lectin.     

Structure-function relationships in heme-proteins

Biological systems rely on heme-proteins to carry out a number of basic functions essential for their survival. Hemes, or iron-porphyrin complexes, are the versatile and ubiquitous active centers of these proteins. In the past decade, discovery of new heme-proteins, together with functional and structural research, provided a wealth of information on these diverse and biologically important molecules. Structure determination work has shown that nature has used a variety of different scaffolds and architectures to bind heme and modulate functions such as redox properties. Structural data have also provided insights into the heme-linked protein conformational changes required in many regulatory heme-proteins. Remarkable efforts have been made towards the understanding of factors governing redox potentials. Site-directed mutagenesis studies and theoretical calculations on heme environments investigated the roles of hydrophobic and electrostatic residues, and analyzed the effect of heme solvent accessibility. This review focuses on the structure-function relationships underlying the association of heme in signaling and iron metabolism proteins. In addition, an account is given about molecular features affecting heme's redox properties; this briefly revisits previous conclusions in the light of some more recent reports.     

Structure-function relationships in insulin

A new interpretation of structure-function relationships in the insulin molecule is presented. Negative cooperativity is postulated to arise from a dimerization event occurring between two receptor-bound molecules. The receptor-binding surface of insulin can necessarily not involve residues involved in dimerization as has been generally accepted. Support for this interpretation is based on published data.     

Structure-function relationships of somatostatin analogs

Objective of peptide chemistry has always been the production of analogues for clinical application. Advantages sought over natural peptides are (a) reduced molecular size; (b) prolonged biological half-life, and (c) enhanced specificity. After elucidation of the active core of somatostatin a number of analogues have been synthetized. Among them SMS 201-995, an octapeptide, was selected for further development because of its high potency and prolonged plasma clearance. Procedures extending the duration of action of somatostatin derivatives such as enhancement of lipophilicity and amino acid substitution are described, and factors influencing the specificity of such substances are succinctly analyzed.     

Inhibition of plant glutamine synthetases by substituted phosphinothricins

Glutamine synthetase (GS) utilizes various substituted glutamic acids as substrates. We have used this information to design herbicidal α- and γ-substituted analogs of phosphinothricin (l-2-amino-4-(hydroxymethylphosphinyl)butanoic acid, PPT), a naturally occurring GS inhibitor and a potent herbicide. The substituted phosphinothricins inhibit cytosolic sorghum GS₁ and chloroplastic GS₂ competitively versusl-glutamate, with K_i values in the low micromolar range. At higher concentrations, these inhibitors inactivate glutamine synthetase, while dilution restores activity through enzyme-inhibitor dissociation. Herbicidal phosphinothricins exhibit low K_i values and slow enzyme turnover, as described by reactivation characteristics. Both the GS₁ and GS₂ isoforms of plant glutamine synthetase are similarly inhibited by the phosphinothricins, consistent with the broad-spectrum herbicidal activity observed for PPT itself as well as other active compounds in this series.     

Structure-function relationships of hormone-sensitive lipase.

Research into the structure-function relationships of lipases and esterases has increased significantly during the past decade. Of particular importance has been the deduction of several crystal structures, providing a new basis for understanding these enzymes. The generated insights have, together with cloning and expression, aided studies on structure-function relationships of hormone-sensitive lipase (HSL). Novel phosphorylation sites have been identified in HSL, which are probably important for activation of HSL and lipolysis. Functional and structural analyses have revealed features in HSL common to lipases and esterases. In particular, the catalytic core with a catalytic triad has been unveiled. Furthermore, the investigations have given clear suggestions with regard to the identity of functional and structural domains of HSL. In the present paper, these studies on HSL structure-function relationships and short-term regulation are reviewed, and the results presented in relation to other discoveries in regulated lipolysis.     

Structure-function relationships in plant phenylpropanoid biosynthesis

Plants, as sessile organisms, evolve and exploit metabolic systems to create a rich repertoire of complex natural products that hold adaptive significance for their survival in challenging ecological niches on earth. As an experimental tool set, structural biology provides a high-resolution means to uncover detailed information about the structure-function relationships of metabolic enzymes at the atomic level. Together with genomic and biochemical approaches and an appreciation of molecular evolution, structural enzymology holds great promise for addressing a number of questions relating to secondary or, more appropriately, specialized metabolism. Why is secondary metabolism so adaptable? How are reactivity, regio-chemistry and stereo-chemistry steered during the multi-step conversion of substrates into products? What are the vestigial structural and mechanistic traits that remain in biosynthetic enzymes during the diversification of substrate and product selectivity? What does the catalytic landscape look like as an enzyme family traverses all possible lineages en route to the acquisition of new substrate and/or product specificities? And how can one rationally engineer biosynthesis using the unique perspectives of evolution and structural biology to create novel chemicals for human use?     

Structure-function relationships in an anion-translocating ATPase

The ArsAB ATPase is an efflux pump located in the inner membrane of Escherichia coli. This transport ATPase confers resistance to arsenite and antimonite by their extrusion from the cells. The pump is composed of two subunits, the catalytic ArsA subunit and the membrane subunit ArsB. The complex is similar in many ways to ATP-binding cassette ('ABC') transporters, which typically have two groups of six transmembrane-spanning helical segments and two nucleotide-binding domains (NBDs). The 45 kDa ArsB protein has 12 transmembrane-spanning segments. ArsB contains the substrate translocation pathway and is capable of functioning as an anion uniporter. The 63 kDa ArsA protein is a substrate-activated ATPase. It has two homologous halves, A1 and A2, which are clearly the result of an ancestral gene duplication and fusion. Each half has a consensus NBD. The mechanism of allosteric activation of the ArsA ATPase has been elucidated by a combination of molecular genetics and biochemical, structural and kinetic analyses. Conformational changes produced by binding of substrates, activator and/or products could be revealed by stopped-flow fluorescence measurements with single-tryptophan derivatives of ArsA. The results demonstrate that the rate-limiting step in the overall reaction is a slow isomerization between two conformations of the enzyme. Allosteric activation increases the rate of this isomerization such that product release becomes rate-limiting, thus accelerating catalysis. ABC transporters, which exhibit similar substrate activation of ATPase activity, can undergo similar conformational changes to overcome a rate-limiting step. Thus the ArsAB pump is a useful model for elucidating mechanistic aspects of the ABC superfamily of transport ATPases.     

Immunochemical comparison of glutamine synthetases from some solanaceae plants

The presence of different glutamine synthetase isoenzymes in different Solanaceae plants and their relative antigenicities against antiglutamine synthetase from tomato leaf serum were studied. All the plants tested showed one glutamine synthetase isoenzyme except for Mandragora autumnalis, which showed two, after discontinuous polyacrylamide gel electrophoresis and specific in situ assay. Antigenicities were compared by the double immunodiffusion technique. The Nicotiana glauca enzyme showed equal reactivity to that of Lycopersicon esculentum, but its antigenicity was higher than Withania frutescens, Datura stramonium, and Hyoscyamus niger. The study of relative antigenicities permitted differentiation of the glutamine synthetase enzymes from uncultivated species of Solanaceae.     

Structure-function relationships in immobilized chymotrypsin catalysis

Specific activities and the amounts of active immobilized enzyme were determined for several different preparations of alpha-chymotrypsin immobilized on CNBr-activated Sepharose 4B. Electron paramagnetic resonance (EPR) spectroscopy of free and immobilized enzyme with a spin label coupled to the active site was used to probe the effects of different immobilization conditions on the immobilized enzyme active site configuration. Specific activity of active enzyme decreased and rotational correlation time of the spin label increased with increasing immobilized enzyme loading. Enzyme immobilized using an intermediate six-carbon spacer arm exhibited greater specific activity and spin label mobility than directly coupled enzyme. The observed activity changes due to immobilization were completely consistent with corresponding active site structure alterations revealed by EPR spectroscopy.     

Structure-function relationships of heterodimeric amino acid transporters

Heterodimeric amino acid transporters mediate the transfer of amino acids between organs and between different cell types. Members of this particular family of amino acid transporters are constituted by a heavy chain and an associated light chain. The heavy chain is a type II membrane protein with an intracellular amino terminus, a single transmembrane helix, and a large extracellular domain. The light chain, in contrast, is a typical helix-bundle protein with 12 putative transmembrane helices. Two different heavy chains, designated 4F2hc and rbAT, and seven different light chains have been identified to date. Deletion studies indicate that the extracellular domain of the heavy chain has two subdomains. The carboxy-terminal tip of 4F2hc is critical for recognition of certain light chains, whereas the carboxy-terminal tip of rbAT is involved in substrate transport. Sequence alignments suggest that the major part of the extracellular domain forms an α/β domain similar to bacterial α-amylases. A structural model of the rbAT extracellular domain is presented that is in agreement with experimental observations from several mutations and that aligns well with the α-amylase domain.     

Structure-function relationships in eukaryotic nuclei.

It may be that eukaryotic nuclei contain a collection of operationally independent units (genes), each controlled through its interactions with soluble protein factors which diffuse at random throughout the nucleoplasmic space. Alternatively, nuclei might be organized in such a sophisticated fashion that specific genes occupy distinct sites and that spatially ordered RNA synthesis, processing and transport delivers mature RNAs to predestined sites in the cytoplasm. Different fields of research support each of these extreme views. Molecular biologists inspecting the precise details of specific interactions, usually in vitro, inevitably favour the former, while cell biologists working with far more complicated systems generally assume that more elaborate arrangements exist. In considering the importance of nuclear architecture, I have attempted to relate a collection of experiments each of which intimates some close relationship between structural aspects of chromatin organization and the precise mechanisms underlying nuclear function. I will argue that higher-order structures are crucial for achieving the observed efficiency and coordination of many nuclear processes.     

Structure-function relationships in unusual nonvertebrate globins

Based on the literature and our own results, this review summarizes the most recent state of nonvertebrate myoglobin (Mb) and hemoglobin (Hb) research, not as a general survey of the subject but as a case study. For this purpose, we have selected here four typical globins to discuss their unique structures and properties in detail. These include Aplysia myoglobin, which served as a prototype for the unusual globins lacking the distal histidine residue; midge larval hemoglobin showing a high degree of polymorphism; Tetrahymena hemoglobin evolved with a truncated structure; and yeast flavohemoglobin carrying an enigmatic two-domain structure. These proteins are not grouped by any common features other than the fact they have globin domains and heme groups. As a matter of course, various biochemical functions other than the conventional oxygen transport or storage have been proposed so far to these primitive or ancient hemoglobins or myoglobins, but the precise in vivo activity is still unclear. In this review, special emphasis is placed on the stability properties of the heme-bound O2. Whatever the possible roles of nonvertebrate myoglobins and hemoglobins may be (or might have been), the binding of molecular oxygen to iron(II) must be the primary event to manifest their physiological functions in vivo. However, the reversible and stable binding of O2 to iron(II) is not a simple process, since the oxygenated form of Mb or Hb is oxidized easily to its ferric met-form with the generation of superoxide anion. The metmyoglobin or methemoglobin thus produced cannot bind molecular oxygen and is therefore physiologically inactive. In this respect, protozoan ciliate myoglobin and yeast flavohemoglobin are of particular interest in their very unique structures. Indeed, both proteins have been found to have completely different strategies for overcoming many difficulties in the reversible and stable binding of molecular oxygen, as opposed to the irreversible oxidation of heme iron(II). Such comparative studies of the stability of MbO2 or HbO2 are of primary importance, not only for a full understanding of the globin evolution, but also for planning new molecular designs for synthetic oxygen carriers that may be able to function in aqueous solution and at physiological temperature.     

Structure-function relationships in cardiac troponin T

Regions of rabbit and bovine cardiac troponin T that are involved in binding tropomyosin, troponin C and troponin I have been identified. Two sites of contact for tropomyosin have been located, situated between residues 92-178 and 180-284 of troponin T. A cardiac-specific binding site for troponin I has been identified between residues 1-68 of cardiac troponin T, within a region of the protein that has previously been shown to be encoded by a series of exons that are expressed in a tissue-specific and developmentally regulated manner. The binding site for troponin C is located between residues 180-284 of cardiac troponin T. When isolated from fresh bovine hearts, cardiac troponin T contained 0.21 +/- 0.11 mol phosphate per mol; incubation with phosphorylase kinase increased the phosphate content to approx. 1 mol phosphate per mol. One site of phosphorylation was identified as serine-1; a second site of phosphorylation was located within peptide CB3 (residues 93-178) and has been tentatively identified as serine-176. Addition of troponin C to cardiac troponin T does not inhibit the phosphorylation of this latter protein that is catalysed by phosphorylase b kinase.     

Structure-function relationships in nicotinic acetylcholine receptors

1. A combination of molecular, biochemical, electrophysiological and immunological approaches has begun to resolve some of the questions about structure-function relationships of nicotinic acetylcholine receptors (AchRs). 2. Current structural studies suggest that models of the subunits which propose four transmembrane domains are correct. 3. It is also probable that the carboxy termini of the subunits are extracellular, while the putative amphpathic helix is intracellular. 4. Electrophysiological and ligand-binding experiments suggest that the M2 region forms the wall of the ion channel. 5. We have isolated clones from PC12 and rat brain cDNA libraries which we have shown, by functional expression, code for members of a gene family of nicotinic acetylcholine receptor subunits. 6. In situ hybridization studies have shown that the neuronal receptor subunit mRNAs are expressed in the mammalian central nervous system. 7. The muscle and neuronal nicotinic AchR subtypes we have expressed show differences in their pharmacological properties. 8. The isolation and identification of clones which code for receptors and voltage-activated ion channels will help in the understanding of a variety of disease states and assist in the design of drugs which are specific for unique molecular targets.