Phospholipase of rat intestine: mode of action]

Phospholipase activities of rat intestinal mucosa homogenate have been determined from lysophosphatidylcholines [14C] and phosphatidylcholines [-3H-14C]. In the presence of phosphatidylcholines, at pH 6.5, the homogenate has a phospholipase B activity. At pH 8.5, a phospholipase A2 activity was shown. In the presence of lysophospatidylcholines, at pH 6.5, we notice a lysophospholipase A1 activity. A kinetic study of the reactions allows us to separate the activity B into a phospholipase A2 activity and a lysophospholipase A1 activity. Thus, it appears that the total phospholipase activity of rat intestinal mucosa would results from a phospholipase A2 activity and a lysophospholipase A1 activity.     

Crystal structure of a heterodimer of editosome interaction proteins in complex with two copies of a cross-reacting nanobody

The parasite Trypanosoma brucei, the causative agent of sleeping sickness across sub-Saharan Africa, depends on a remarkable U-insertion/deletion RNA editing process in its mitochondrion. A approximately 20 S multi-protein complex, called the editosome, is an essential machinery for editing pre-mRNA molecules encoding the majority of mitochondrial proteins. Editosomes contain a common core of twelve proteins where six OB-fold interaction proteins, called A1-A6, play a crucial role. Here, we report the structure of two single-strand nucleic acid-binding OB-folds from interaction proteins A3 and A6 that surprisingly, form a heterodimer. Crystal growth required the assistance of an anti-A3 nanobody as a crystallization chaperone. Unexpectedly, this anti-A3 nanobody binds to both A3(OB) and A6, despite only ~40% amino acid sequence identity between the OB-folds of A3 and A6. The A3(OB)-A6 heterodimer buries 35% more surface area than the A6 homodimer. This is attributed mainly to the presence of a conserved Pro-rich loop in A3(OB). The implications of the A3(OB)-A6 heterodimer, and of a dimer of heterodimers observed in the crystals, for the architecture of the editosome are profound, resulting in a proposal of a 'five OB-fold center' in the core of the editosome.     

Molecular size and symmetry of the bacterioferritin of Escherichia coli. X-ray crystallographic characterization of four crystal forms

X-ray crystallographic data from four crystal forms of Escherichia coli bacterioferritin show that the molecule has a diameter in the range 119 to 128 A. Molecules are composed of 24 subunits arranged in 432 symmetry. In both size and symmetry the molecule resembles ferritin from eukaryotes. The four crystal forms are monoclinic, space group P2(1) with unit cell dimensions a = 118.7 A, b = 211.6 A, c = 123.3 A and beta = 119.1 degrees; orthorhombic, C222(1), a = 128.7 A, b = 197.1 A, c = 202.8 A; tetragonal, P4(2)2(1)2, a = b = 210.6 A, c = 145.0 A and cubic, I432, a = 146.9 A.     

Study of the electrostatic effects of mutations on the surface of dehaloperoxidase-hemoglobin A

Point mutations of dehaloperoxidase-hemoglobin A (DHP A) that affect the surface charge have been prepared to study the interaction between DHP A with its substrate 2,4,6-trichlorophenol (TCP). Kinetic studies of these surface mutations showed a correlation, in which the more positively charged mutants have increased catalytic efficiency compared with wild type DHP A. As a result, the hypothesis of this study is that there is a global electrostatic interaction between DHP A and TCP. The electrostatic nature of substrate binding was further confirmed by the result that kinetic assays of DHP A were affected by ionic strength. Furthermore, isoelectric focusing (IEF) gel study showed that the pI-6.8 for DHP A, which indicates that DHP A has a slight negative charge pH 7, consistent with the kinetic observations.     

Identification of a functionally relevant signal peptide of mouse ficolin A

Mouse ficolin A is a plasma protein with lectin activity, and plays a role in host defense by binding carbohydrates, especially GlcNAc, on microorganisms. The ficolin A subunit consists of an N-terminal signal peptide, a collagen-like domain, and a C-terminal fibrinogen-like domain. In this study, we show that ficolin A can be synthesized and oligomerized in a cell and secreted into culture medium. We also identify a functionally relevant signal peptide of ficolin A by using MS/MS analysis to determine the N-terminal sequence of secreted ficolin A. When the signal peptide of mouse ficolin A was fused with enhanced green fluorescent protein (EGFP), EGFP was released into HEK 293 cell medium, suggesting that the signal peptide can efficiently direct ficolin A secretion. Moreover, our results suggest that the signal peptide of ficolin A has potential application for the production of useful secretory proteins.     

Spectrum Analysis of Esterase Isozymes of culti-species and wild Species in Arachis

The electrophoresis analysis of isozymes of Arachis show a very close relationship among four types of cultispecies (A. hypogaea) and A. monticola, the tetraploid wild species is a related species. Among the five diploid wild species of Arachis Section, both A. cardenasii with A genome and A. batizocoi with B genome are found to be relatively nearer to cultis'pecies than A. correntina, A. stenosperma and A. villosa, while A. rigonii of Erectoides Section and A. pusilla of Triseminala Section are the distant species.     

Mapping of the discontinuous H-kininogen binding site of plasma prekallikrein. Evidence for a critical role of apple domain-2.

Plasma prekallikrein, a zymogen of the contact phase system, circulates in plasma as heterodimeric complex with H-kininogen. The binding is mediated by the prekallikrein heavy chain consisting of four apple domains, A1 to A4, to which H-kininogen binds with high specificity and affinity (K(D) = 1.2 x 10(-8) M). Previous work had demonstrated that a discontinuous kininogen-binding site is formed by a proximal part located in A1, a distal part exposed by A4, and other yet unidentified portion(s) of the kallikrein heavy chain. To detect relevant binding segment(s) we recombinantly expressed single apple domains and found a rank order of binding affinity for kininogen of A2 > A4 approximately A1 > A3. Removal of single apple domains in prekallikrein deletion mutants reduced kininogen binding by 21 (A1), 64 (A2), and 24% (A4), respectively, whereas deletion of A3 was without effect. Transposition of homologous A2 domain from prekallikrein to factor XI conferred high-affinity kininogen binding from the former to the latter. The principal role of A2 for H-kininogen docking to the prekallikrein heavy chain was further substantiated by the finding that cleavage of a single peptide bond in A2 drastically diminished the H-kininogen binding affinity. Furthermore, the epitope of monoclonal antibody PKH6 which blocks kallikrein-kininogen complex formation with an IC(50) of 8 nM mapped to the center portion of domain A2. Our data indicate that domain A2 and two flanking sequence segments of A1 and A4 form a discontinuous binding platform for H-kininogen on the prekallikrein heavy chain. Domain-specific antibodies directed to these critical sites efficiently interfered with contact phase-induced bradykinin release from H-kininogen.     

Effect of Protein A on Adsorption of Bacteriophages to Staphylococcus aureus

Experiments were performed to determine if protein A influenced the association of bacteriophages with Staphylococcus aureus. Bacteriophage adsorption was compared in a S. aureus strain rich in protein A and mutants of this strain with very little protein A, in a strain with little protein A, and in mutants of this strain with increased protein A. In addition, the effect of growth in mannitol-salt broth and trypsin digestion (known to reduce protein A) on bacteriophage absorption was measured. There was an inverse relationship between protein A content of strains and the quantity of bacteriophage absorbed. However, no inhibition of staphylococcal phages was obtained with purified soluble protein A. Protein A as a surface component rendered the bacteria more resistant to adsorption of staphylococcal typing phages presumably by masking the phage receptor sites. When protein A-deficient mutants were incubated with bacteriophages, there was survival of staphylococci with increased protein A content probably due to a selective action.     

Purification and properties of five different forms of human procarboxypeptidases

Three different procarboxypeptidases A and two different procarboxypeptidases B have been isolated for the first time, in a pure and native state, from human pancreatic extracts. These proteins were purified in one or two quick steps by anion-exchange HPLC. All these forms have been biochemically characterized. Two of the procarboxypeptidases A, the A1 and A2 forms, are obtained in a monomeric state while the other, the A3 form, is obtained as a binary complex of a procarboxypeptidase A with a proproteinase E. This complex is stable in aqueous buffers at various ionic strengths and develops carboxypeptidase A and proteinase E activities in the presence of trypsin. The A1 and A2 forms show clear differences in electrophoretic mobility in SDS/polyacrylamide gels, isoelectric point, proteolytic activation process with trypsin and susceptibility to thermal denaturation. In contrast, these properties are similar in the A1 and A3 (binary complex) forms. On the other hand, with respect to the properties listed above, the B1 and B2 forms differ from each other mainly in isoelectric point. An overall comparison of the above properties reveals the unusual character of the A2 form, midway between the other A and B forms. N-terminal extended sequence analysis carried out on these proenzymes confirm that they constitute different isologous forms.     

Alteration of quaternary structure and biological activity of concanavalin A

A major molecular species of concanavalin A (Con A), a mitogenic lectin from jack bean seeds, has a quaternary structure composed of four homologous subunits and a tetravalent sugar-binding ability. We show that the tetrameric Con A can be converted into a monovalent monomeric form by either photochemical alkylation or hydrogen peroxide/dioxane oxidation of about two tryptophan residues. A divalent dimeric derivative of Con A is also prepared by sulfomethylamidation of about four carboxyl groups. Chemical properties and mitogenic and hemagglutinating activities of these new Con A derivates are compared with those of the tetravalent Con A, as well as of the Con A derivatives that have appeared in the literature on cell biological studies. The significance of the lectin valences in lymphocyte activation and hemagglutination is also discussed.     

The relevance of N-linked glycosylation to the binding of a ligand to guanylate cyclase C.

The role of carbohydrate moieties at the N-linked glycosylation sites of guanylate cyclase C (GC-C), a receptor protein for guanylin, uroguanylin and heat-stable enterotoxin, in ligand binding and structural stability was examined using site-directed mutagenesis of the putative N-linked glycosylation sites in the extracellular domain (ECD) of porcine GC-C. For this purpose, eight mutant proteins of ECD (N9A, N20A, N56A, N172A, N261A, N284A, N334A and N379A) and six mutant proteins of the complete GC-C (N9A, S11A, N172A, T174A, N379A and T381A) were prepared, in which Ala replaced Asn, Ser and Thr at the N-linked glycosylation consensus sites. All the mutant proteins showed a ligand-binding affinity (K(d)) similar to those of the wild-type proteins, although the deletion of a carbohydrate moiety at each of the N-linked glycosylation sites affected the ligand-binding ability of ECD or GC-C to some degree. However, the mutant proteins of ECD (N379A) and GC-C (N379A and T381A) showed considerably decreased binding ability in the context of maximum capacity (B(max)) to a ligand, despite the fact that the expression levels of these mutant proteins were nearly the same as the wild-type proteins. Moreover, the mutant protein of ECD (N379A) was considerably less stable to a denaturant. These results clearly indicate a crucial role for the carbohydrate moiety at N379, which is located near the transmembrane region, in structural stability, the ability to bind to a ligand and the cyclase catalytic activity of GC-C, and provide a route for the elucidation of the mechanism of the interaction between GC-C and a ligand.     

Purification of limited tryptic fragments of Ca2+,Mg2+-adenosine triphosphatase of the sarcoplasmic reticulum and identification of conformation-sensitive cleavage sites

Sarcoplasmic reticulum membranes were treated with trypsin, and samples enriched with A1a, A1b, and C fragments (Saito, K. et al. (1984) J. Biochem. 95, 1297-1304), respectively, were prepared. A1b and C fragments were purified to apparent homogeneity, and an approximately equimolar mixture of A1(Met1-Arg198), A1a, and A1b fragments free from other contaminants was also obtained through gel permeation and hydroxylapatite chromatography in the presence of sodium dodecyl sulfate. N- and C-terminal amino acid sequence analyses of these peptides were carried out in order to identify the tryptic cleavage sites responsible for the formation of these fragments. Both A1a and A1b fragments had the same C-terminal sequence as A1 fragment. Single cleavage of A1 at T3a (Lys218-Ala219) yielded A1a, while a cleavage between either Lys234-Ile235 or Arg236-Asp237 (collectively designated as T3b) resulted in A1b fragment. Thus, A1a and A1b fragments differed from A1 fragment only by their loss of short stretches corresponding to the N-terminal region of the latter. On the other hand, C fragment represented the C-terminal half of B fragment (Ala506-Gly994). It had the same C-terminal sequence as B fragment and was produced by cleavage at T4 (Lys728-Thr729). Cleavages at T3a and T3b profoundly affected the catalytic properties of SR-ATPase (Imamura, Y. and Kawakita, M. (1986) J. Biochem. 100, 133-141), and it was suggested that the segment of the ATPase molecule including the region between Ala199 and Arg236 is important in mediating the coupling between ATP splitting and Ca2+-transport.     

Characterization of a novel acidic protein of 38 kDa, A0, in yeast ribosomes which immunologically cross-reacts with the 13 kDa acidic ribosomal proteins, A1/A2

A new ribosomal protein of 38 kDa, named A0, was detected in yeast ribosomes on immunoblotting. The antibody used here was that against A1/A2, 13 kDa acidic ribosomal proteins which cross-reacted with A0. Although A0 and A1/A2 share common antigenic determinants, they differ in the following biochemical properties. While A1/A2 could be extracted from ribosomes with ethanol and ammonium sulfate, A0 could not. A0 gave two protein spots in a less acidic region than for A1/A2 on two-dimensional gel electrophoresis. The heterogeneity observed for A0 was ascribable to phosphorylation because one spot disappeared after treatment of the ribosomes with phosphatase. The syntheses of A0 and A1/A2 are directed by different mRNA species, as judged with a cell-free translation system, ruling out the possibility that A0 is a precursor of A1/A2. Although a mammalian ribosomal protein equivalent to A0 has been shown to be associated with 13 kDa acidic proteins in the cytoplasm, essentially no A0 was detected on immunoblotting in the yeast cytosol, while a small but detectable amount of A1/A2 was present. The possibility that A0 is a eukaryotic equivalent of L10 of Escherichia coli is discussed.     

Application of epidermal characters to the taxonomy of European species of Antirrhinum (Schrophulariaceae)

Epidermal characters provide additional features in the assessment of taxonomic relationships among the European species of Antirrhinum. Much of this study confirms the classification derived by Webb but important differences have been observed, viz. A. mollissimum may be considered as identical to A. molle whilst A. boissieri, A. ambiguum and A. rupestre may be assigned as distinct species. Evidence from epidermal characters supports Rothmaler's view that A. majus spp. litigiosum is a subspecies of A. majus and should not be amalgamated with A. barrelieri as proposed by Webb. A. × huteri is proposed to be a hybrid between A. hispanicum and A. boissieri. Thus it is proposed that the section Antirrhinum comprises 20 species with A. majus divided into five subspecies.     

Visualization of Subunit Interactions and Ternary Complexes of Protein Phosphatase 2A in Mammalian Cells

Protein phosphatase 2A (PP2A) is a ubiquitous phospho-serine/threonine phosphatase that controls many diverse cellular functions. The predominant form of PP2A is a heterotrimeric holoenzyme consisting of a scaffolding A subunit, a variable regulatory B subunit, and a catalytic C subunit. The C subunit also associates with other interacting partners, such as α4, to form non-canonical PP2A complexes. We report visualization of PP2A complexes in mammalian cells. Bimolecular fluorescence complementation (BiFC) analysis of PP2A subunit interactions demonstrates that the B subunit plays a key role in directing the subcellular localization of PP2A, and confirms that the A subunit functions as a scaffold in recruiting the B and C subunits to form a heterotrimeric holoenzyme. BiFC analysis also reveals that α4 promotes formation of the AC core dimer. Furthermore, we demonstrate visualization of specific ABC holoenzymes in cells by combining BiFC and fluorescence resonance energy transfer (BiFC-FRET). Our studies not only provide direct imaging data to support previous biochemical observations on PP2A complexes, but also offer a promising approach for studying the spatiotemporal distribution of individual PP2A complexes in cells.     

Occurrence of indole alkaloids among secondary metabolites of soil Aspergillus

The occurrence of indole alkaloids among secondary fungal metabolites was studied in species of the genus Aspergillus, isolated from soils that were sampled in various regions of Russia (a total of 102 isolates of the species A. niger, A. phoenicis, A. fumigatus, A. flavus, A. versicolor, A. ustus, A. clavatus, and A. ochraceus). Clavine alkaloids were represented by fumigaclavine, which was formed by A. fumigatus. alpha-Cyclopiazonic acid was formed by isolates of A. fumigatus, A. flavus, A. versicolor, A. phoenicis, and A. clavatus. The occurrence of indole-containing diketopiperazine alkaloids was documented for isolates of A. flavus, A. fumigatus, A. clavatus, and A. ochraceus. No indole-containing metabolites were found among the metabolites of A. ustus or A. niger.     

Antigenic profile of some typical and septate phialide-strains of Aspergillus fumigatus

Antigens from typical and septate phialide-strains of Aspergillus fumigatus, A. fumigatus var. ellipticus, A. fischeri, A. flavus and A. niger were analysed by fused rocket and two-dimensional immunoelectrophoresis. Depsite strain-to-strain variations, isolates of the two morphological forms of A. fumigatus showed a close similarity in their antigenic profile; the percentage of qualitative sharing ranged between 80% and 100%. The number of antigens shared by the isolates of A. fumigatus var. ellipticus and A. fischeri as compared with a typical A. fumigatus isolate was relatively high. By contrast isolates of A. flavus and A. niger, which are taxonomically not so closely related to A. fumigatus, revealed a qualitative sharing of only 16% and 8%, respectively. The number of common antigenically active components identified in culture filtrate antigens from the typical and septate phialide-strains of A. fumigatus was around 29. Two-dimensional electrophoresis with tandem and intermediate gel proved further that the two types of A. fumigatus isolates are antigenically homologous. Present findings also support the view that the antigenically active components of this pathogen are predominantly protein-glycoprotein in nature.