Characterization of a new periplasmic single-domain rhodanese encoded by a sulfur-regulated gene in a hyperthermophilic bacterium Aquifex aeolicus

Rhodaneses (thiosulfate cyanide sulfurtransferases) are enzymes involved in the production of the sulfur in sulfane form, which has been suggested to be the relevant biologically active sulfur species. Rhodanese domains occur in the three major domains of life. We have characterized a new periplasmic single-domain rhodanese from a hyperthermophile bacterium, Aquifex aeolicus, with thiosulfate:cyanide transferase activity, Aq-1599. The oligomeric organization of the enzyme is stabilized by a disulfide bridge. To date this is the first characterization from a hyperthermophilic bacterium of a periplasmic sulfurtransferase with a disulfide bridge. The aq-1599 gene belongs to an operon that also contains a gene for a prepilin peptidase and that is up-regulated when sulfur is used as electron acceptor. Finally, we have observed a sulfur-dependent bacterial adherence linked to an absence of flagellin suggesting a possible role for sulfur detection by A. aeolicus.     

Theoretical studies of cytochrome P-450. Characterization of stable and transient active states, reaction mechanisms and substrate-enzyme interactions

The techniques of theoretical chemistry embodied in ab initio and semiempirical quantum mechanical and empirical energy methods have been used to elucidate the relationship between structure, spectra and function of the oxidative metabolizing heme proteins, the cytochrome P-450s, using a recent X-ray structure of a P-450cam-camphor complex. Specifically, the origin of the spin state changes when substrate binds to the oxidized resting state and the nature of the transient biologically active oxygen transfer state have been described. Mechanisms of hydroxylation and epoxidation, the two fundamental oxidative reactions performed by these enzymes, have been deduced and the role of substrate binding and orientation on product distribution investigated.     

Penetration of various mononuclear ribonucleases into rat experimental granulation-tissue fibroblasts and their intracellular effects

The penetration of five different mononuclear ribonucleases into the subcellular particles of rat experimental granulation-tissue fibroblasts was compared, along with the effects of the enzymes on the fibroblast RNA fractions. Ribonucleases from normal and silica-treated rat peritoneal macrophages have been shown before to regulate the nucleic acid and protein metabolism of rat experimental granulation-tissue fibroblasts. These biologically active enzymes were taken into the fibroblasts in a greater amount than the corresponding human monocyte enzymes and the biologically inactive rat macrophage ribonuclease. The biologically active macrophage enzymes were incorporated mainly into the nuclear fraction. The other three mononuclear ribonucleases were not found particularly in any subcellular compartment. Both biologically active macrophage enzymes degraded the nuclear RNA of fibroblasts and released it to the soluble fraction in contrast to the biologically inactive macrophage enzyme and ribonuclease from normal human monocytes. Instead the ribonuclease from normal human monocytes seemed to degrade RNA in the soluble fraction. There were no marked differences in the subcellular effects of ribonucleases from normal and silica-treated macrophages. However, the treatment of human monocytes with silica changed their ribonuclease so that it split the nuclear RNA of fibroblast and released it to the soluble fraction in the same way as the biologically active macrophage enzymes did.     

Biochemical characteristics of new thermosensitive secretory mutants of yeasts

New thermosensitive mutants of the yeast Saccharomyces cerevisiae which block the secretion of periplasmic enzymes at restriction temperature have been obtained. These mutants accumulate active low molecular weight and mature invertase species in the cell; the buoyant density of the cells in a Percoll gradient is higher than that in the wild strain cells. The mutant cells transferred to permissive temperature (25 degrees C) in the absence of protein synthesis can secrete some amount of accumulated invertase. It was found that the secretory defects of conditional mutants do not affect the activity of cytoplasmic enzymes (e.g., alcohol dehydrogenase) or the level of total protein synthesis and glycosylation and do not induce non-specific disturbances in energy metabolism and plasma membrane functions at restriction temperature. Some strains of new secretory mutants revealed uncoupled defective secretion of periplasmic enzymes and intrinsic membrane proteins (proline permease). The possibility of branching of the secretory pathway for periplasmic enzymes and cytoplasmic membrane proteins is discussed.     

The periplasmic chaperone SurA exploits two features characteristic of integral outer membrane proteins for selective substrate recognition

The Escherichia coli periplasmic chaperone and peptidyl-prolyl isomerase (PPIase) SurA facilitates the maturation of outer membrane porins. Although the PPIase activity exhibited by one of its two parvulin-like domains is dispensable for this function, the chaperone activity residing in the non-PPIase regions of SurA, a sizable N-terminal domain and a short C-terminal tail, is essential. Unlike most cytoplasmic chaperones SurA is selective for particular substrates and recognizes outer membrane porins synthesized in vitro much more efficiently than other proteins. Thus, SurA may be specialized for the maturation of outer membrane proteins. We have characterized the substrate specificity of SurA based on its natural, biologically relevant substrates by screening cellulose-bound peptide libraries representing outer membrane proteins. We show that two features are critical for peptide binding by SurA: specific patterns of aromatic residues and the orientation of their side chains, which are found more frequently in integral outer membrane proteins than in other proteins. For the first time this sufficiently explains the capability of SurA to discriminate between outer membrane protein and non-outer membrane protein folding intermediates. Furthermore, peptide binding by SurA requires neither an active PPIase domain nor the presence of proline, indicating that the observed substrate specificity relates to the chaperone function of SurA. Finally, we show that SurA is capable of associating with the outer membrane. Together, our data support a model in which SurA is specialized to interact with non-native periplasmic outer membrane protein folding intermediates and to assist in their maturation from early to late outer membrane-associated steps.     

Expression and purification of a soluble tissue factor fusion protein with an epitope for an unusual calcium-dependent antibody.

The use of bacterial signal peptides to target recombinant mammalian proteins to the periplasmic space of Escherichia coli (to promote proper disulfide bond formation) has met with variable success. We report the design and use of a bacterial expression vector to direct recombinant fusion proteins to the periplasmic space of E. coli: it contains the signal peptide from the pelB gene of Erwinia carotovora linked to a small peptide epitope for an unusual calcium-dependent antibody (HPC4). HPC4 binds to the epitope in a Ca(2+)-dependent manner, but the epitope itself does not bind Ca2+. We have used this system to express a biologically active, soluble form of tissue factor, the protein responsible for triggering the blood clotting cascade. Soluble tissue factor was secreted into the culture medium at 1-2 mg/liter, from which it could be readily purified using immobilized HPC4 antibody. The HPC4 epitope could be removed by digestion with thrombin or factor Xa, although a free amino terminus was not required for function since soluble tissue factor was equally active with the epitope still in place. This vector/epitope system permits large-scale expression and purification of recombinant soluble tissue factor and should be generally applicable to the isolation of other recombinant proteins. Furthermore, the epitope confers Ca(2+)-dependent binding of the fusion protein to HPC4 antibody while avoiding the creation of a new metal binding site on the fusion protein itself. Tb3+ can bind in this Ca2+ site near Trp, allowing this site to serve as a means of attaching a fluorescent probe to tissue factor.     

Natriuretic peptides--a class of heterologous molecules in plants

Immunological and physiological evidence suggests the presence of biologically active natriuretic peptide hormones (NPs) in plants. Evidence includes specific binding of rat atrial NP, [rANP (99-126)] to plant membranes and the promotion of cyclic guanosine-3',5'-monophosphate (cGMP) mediated stomatal responses. Furthermore, anti-ANP affinity purifies biologically active plant immunoreactants (irPNPs) and a biologically active Arabidopsis thaliana irPNP (AtPNP-A) has been identified. AtPNP-A belongs to a novel class of molecules that share some similarity with the cell wall loosening expansins but do not contain the carbohydrate-binding wall anchor, thus suggesting that irPNPs and ANP are heterologues. We hypothesise that irPNP-like molecules have evolved from primitive glucanase-like molecules that have been recruited to become systemically mobile modulators of homeostasis acting via the plasma membrane. Such a function is compatible with localisation in the conductive tissue and the physiological and cellular modes of action of irPNPs reported to-date.     

人甲状旁腺激素的研究

人甲状旁腺激素(hPTH)是甲状旁腺分泌的多肽激素。它能与骨基质和肾细胞膜上专一性的受体相结合,将调节细胞中钙磷浓度的信号传导到膜内。hPTH活性片段在N端,其N端氨基酸序列与牛、猪PTH高度同源。hPTH及其活性片段在治疗骨及肌肉疾病方面有重要作用,重组hPTH已获成功。     

Atomic structures of periplasmic binding proteins and the high-affinity active transport systems in bacteria

We have determined and refined the X-ray crystal structures of six periplasmic binding proteins that serve as initial receptors for the osmotic-shock sensitive, active transport of L-arabinose, D-galactose/D-glucose, maltose, sulphate, leucine/isoleucine/valine and leucine. The tertiary structures and atomic interactions between proteins and ligands show common features that are important for understanding the function of the binding proteins. All six structures are ellipsoidal, consisting of two similar, globular domains. The ligand-binding site is located deep in the cleft between the two domains. Irrespective of the nature of the ligand (e.g. saccharide, sulphate dianion or leucine zwitterion), the specificities and affinities of the binding sites are achieved mainly through hydrogen-bonding interactions. Binding of ligands induces a large protein conformational change. Three different structures have been observed among the binding proteins: unliganded 'open cleft', liganded 'open cleft', and liganded 'closed cleft'. Here we discuss the functions of binding proteins in the light of numerous crystallographic and ligand-binding studies and propose a mechanism for the binding protein-dependent, high-affinity active transport.     

Lateral diffusion of proteins in the periplasm of Escherichia coli.

We have introduced biologically active, fluorescently labeled maltose-binding protein into the periplasmic space of Escherichia coli and measured its lateral diffusion coefficient by the fluorescence photobleaching recovery method. Diffusion of this protein in the periplasm was found to be surprisingly low (lateral diffusion coefficient, 0.9 X 10(-10) cm2 s-1), about 1,000-fold lower than would be expected for diffusion in aqueous medium and almost 100-fold lower than for an equivalent-size protein in the cytoplasm. Galactose-binding protein, myoglobin, and cytochrome c were also introduced into the periplasm and had diffusion coefficients identical to that determined for the maltose-binding protein. For all proteins nearly 100% recovery of fluorescence was obtained after photobleaching, indicating that the periplasm is a single contiguous compartment surrounding the cell. These data have considerable implications for periplasmic structure and for the role of periplasmic proteins in transport and chemotaxis.     

Bacterial zinc uptake and regulators

Many bacteria use an ABC transporter for high-affinity uptake of zinc with a cluster 9 solute-binding protein. Other members of this protein family transport manganese. At present, it is not always possible to distinguish zinc-specific and manganese-specific transporters on the basis of sequence analysis. Low-affinity ZIP-type zinc transporters in bacteria have also been identified. Most high-affinity zinc uptake systems are regulated by Zur proteins, which form at least three unrelated subgroups of the Fur protein family (regulators of iron transport). High-affinity transport of zinc out of the periplasmic space poses a problem to the cell because zinc is a cofactor of several periplasmic enzymes. Certain zinc-binding proteins in the periplasm might function as chaperones to supply these enzymes with zinc.     

Periplasmic binding protein-dependent transport systems: the membrane-associated components

Periplasmic binding protein-dependent transport systems are multicomponent, consisting of several inner membrane-associated proteins and a periplasmic component. The membrane-associated components of different systems are related in organization and function suggesting that, despite different substrate specificities, each transport system functions by a common mechanism. Current understanding of these components is reviewed. The nature of energy coupling to periplasmic transport systems has long been debated. Recent data now demonstrate that ATP hydrolysis is the primary source of energy for transport. The ATP-binding transport components are the best characterized of a family of closely related ATP-binding proteins believed to couple ATP hydrolysis to a variety of different biological processes. Intriguingly, systems closely related to periplasmic binding protein-dependent transport systems have recently been identified in several Gram-positive organisms (which lack a periplasm) and in eukaryotic cells. This class of transport system appears to be widespread in nature, serving a variety of important and diverse functions.     

The membrane destabilising action of the antibacterial agent chlorhexidine

Abstract The antibacterial agent chlorhexidine has long been used as an agent for medical antisepsis. This compound is a membrane active agent which probably has its major antibacterial action by interference with the function of cellular membranes. The results demonstrated an inhibition of oxygen utilisation by bacteria which was related to falls in cellular ATP levels. There was an effect on the outer membranes of Gram-negative bacteria which allowed the release of periplasmic enzymes. The inner membrane was not ruptured but its functionality was breached and there was an inhibition of active uptake of small molecules which did not appear to be related to cellular ATP levels.     

The stilbene derivatives,nucleosides, and nucleosides modified by stilbene derivatives

In this short review, including 187 references, the issues of biological activity of stilbene derivatives and nucleosides and the biological and medicinal potential of fusion of these two classes are discussed. The stilbenes, especially the stilbenoids, and nucleosides are both biologically active. Hybrids formed from binding of these compounds have not yet been broadly studied. However, those that have been investigated exhibit desirable medicinal properties. The review is divided in such parts: I. Derivative of stilbene (biomedical investigations, biological activities in cells, enzymes and hazard), parts II. naturally occurred nucleoside and its derivatives: uridine, thymidine and 5-methyluridine, cytidine, adenosine, guanosine and part III. hybrid molecules- drugs and hybrid molecules- nucleoside - stilbene and its derivative.     

Ca2+ is a cofactor required for membrane transport and maturation and is a yield-determining factor in high cell density penicillin amidase production

Penicillin amidases (PAs) from E. coli and A. faecalis are periplasmic enzymes that contain one tightly bound Ca(2+) per molecule that does not directly participate in the enzymatic function. This ion may, however, be required for the maturation of the pre-pro-enzyme. The pro-enzyme of homologous PAs are translocated through the Tat- (E. coli PA(EC)) and Sec- (A. faecalis PA(AF)) transport systems, respectively. Cell fractionation, electrophoresis, immunoblotting, and activity staining demonstrated that Ca(2+) binding is required for the membrane transport and maturation of the pro-enzyme to active enzyme. Pro-enzyme without Ca(2+) was targeted to the membrane but not translocated. Influence of Ca(2+) in medium and feed was studied for high cell density cultivations of E. coli expressing these enzymes. Without Ca(2+) in the feed the synthesis of the pre-pro-enzyme was hardly influenced. At optimal Ca(2+) content in the feed the active enzyme amount could be increased by 2 orders of magnitude up to 0.9 g/L (PA(EC)) and 2.3 g/L (PA(AF)) or 4% (PA(EC)) and 8% (PA(AF)) of the cell dry weight. The corresponding specific activities are 1700 U (PA(EC)) and 14000 U (PA(AF)) per gram cell dry weight, respectively. These values are higher than those published previously. Thus, for optimal yields of the studied and other extra- and periplasmic enzymes that require Ca(2+) or other ions as cofactors for membrane transport and maturation, sufficient cofactor must be added in the feed.     

Ralstonia eutropha TF93 is blocked in tat-mediated protein export

Ralstonia eutropha (formerly Alcaligenes eutrophus) TF93 is pleiotropically affected in the translocation of redox enzymes synthesized with an N-terminal signal peptide bearing a twin arginine (S/T-R-R-X-F-L-K) motif. Immunoblot analyses showed that the catalytic subunits of the membrane-bound [NiFe] hydrogenase (MBH) and the molybdenum cofactor-binding periplasmic nitrate reductase (Nap) are mislocalized to the cytoplasm and to the inner membrane, respectively. Moreover, physiological studies showed that the copper-containing nitrous oxide reductase (NosZ) was also not translocated to the periplasm in strain TF93. The cellular localization of enzymes exported by the general secretion system was unaffected. The translocation-arrested MBH and Nap proteins were enzymatically active, suggesting that twin-arginine signal peptide-dependent redox enzymes may have their cofactors inserted prior to transmembrane export. The periplasmic destination of MBH, Nap, and NosZ was restored by heterologous expression of Azotobacter chroococcum tatA mobilized into TF93. tatA encodes a bacterial Hcf106-like protein, a component of a novel protein transport system that has been characterized in thylakoids and shown to translocate folded proteins across the membrane.     

Intracrinology of estrogens and androgens in breast carcinoma

Intratumoral metabolism and synthesis of biologically active steroids such as estradiol and 5-dihydrotestosterone as a result of interactions of various enzymes are considered to play very important roles in the pathogenesis and development of hormone-dependent breast carcinoma. Among these enzymes involved in estrogen metabolism, intratumoral aromatase play an important role in converting androgens to estrogens in situ from serum and serving as the source of estrogens, especially in postmenopausal patients with breast carcinoma. However, other enzymes such as 17β-hydroxysteroid dehydrogenase (17β-HSD) isozymes, estrogen sulfatase (STS), and estrogen sulfotransferase, which contribute to in situ availability of biologically active estrogens, also play pivotal roles in this intratumoral estrogen production above. Androgen action on human breast carcinoma has not been well-studied but are considered important not only in hormonal regulation but also other biological features of carcinoma cells. Intracrine mechanisms also play important roles in androgen actions on human breast carcinoma cells. Among the enzymes involved in biologically active androgen metabolism and/or synthesis, both 17β-hydroxysteroid dehydrogenase type 5 (17βHSD5; conversion from circulating androstenedione to testosterone) and 5-reductase (5Red; reduction of testosterone to DHT (5-dihydrotestosterone) were expressed in breast carcinoma tissues, and in situ production of DHT has been proposed in human breast cancer tissues. However, intracrine mechanisms of androgens as well as their biological or clinical significance in the patients with breast cancer have not been fully elucidated in contrast to those in estrogens.     

Phosphorylation in vivo and in vitro of the arginine-ornithine periplasmic transport protein of Escherichia coli

The arginine-ornithine periplasmic binding protein, an essential component of the arginine-ornithine transport system of Escherichia coli, was isolated in a phosphorylated form and in a non-phosphorylated form from the periplasmic fluid, after incubation of intact cells with (32P)orthophosphate under conditions similar to those used for arginine transport studies. The binding protein could also be labeled with 32Pi by incubation in vitro of the periplasmic fluid with [gamma-32P]ATP, or by incubation in vitro of the purified binding protein with radioactive ATP, Mg2+ and a phosphokinase enzyme released by osmotic-shock treatment. The two forms of the protein were separated by DEAE-Sephacel chromatography. By several different criteria, which included binding studies, analyses of the amino acid composition of the two forms of the protein, analysis by sodium dodecyl sulfate/polyacrylamide gel electrophoresis and testing for other components of the periplasmic space with affinities for inorganic phosphate, it was concluded that the 32P-labeled protein corresponds to a phosphorylated form of the arginine-ornithine-binding protein. The phosphorylation reaction required Mg2+ and a phosphokinase from the periplasmic fluid. The dissociation constant of the phosphorylated protein for arginine was 5.0 microM (dissociation constant of the unmodified protein equals 0.1 microM), suggesting that the chemically modified protein is the active form of the molecule which releases the ligand for its translocation through the cytoplasmic membrane. The pH-stability profile of the phosphoprotein has a 'U'-shape characteristic of acyl phosphates. Reaction of the phosphorylated binding protein with hydroxylamine at pH 5.4, also released Pi from the phosphoprotein. These properties suggest that the phosphoryl group of the phosphoprotein is linked covalently to a carboxyl function of the protein. This information indicates that ATP is a direct energy donor for the active transport of arginine and ornithine in E. coli, and a step of phosphorylation of the arginine-ornithine-binding protein appears to be involved in the utilization of the phosphate bond energy by the arginine-ornithine transport system.     

Expression of ricin B chain in Escherichia coli

DNA encoding ricin B chain was fused to that encoding the E. coli OmpA signal peptide using the expression secretion vector pIN-111-ompA. When induced, E. coli cells transformed with the recombinant plasmid express ricin B chain. The recombinant product accumulates in the periplasmic space in a soluble, biologically active form.