Antigenic relatedness and N-terminal sequence homology define two classes of periplasmic flagellar proteins of Treponema pallidum subsp. pallidum and Treponema phagedenis

The periplasmic flagella of many spirochetes contain multiple proteins. In this study, two-dimensional electrophoresis, Western blotting (immunoblotting), immunoperoxidase staining, and N-terminal amino acid sequence analysis were used to characterize the individual periplasmic flagellar proteins of Treponema pallidum subsp. pallidum (Nichols strain) and T. phagedenis Kazan 5. Purified T. pallidum periplasmic flagella contained six proteins (Mrs = 37,000, 34,500, 33,000, 30,000, 29,000, and 27,000), whereas T. phagedenis periplasmic flagella contained a major 39,000-Mr protein and a group of two major and two minor 33,000- to 34,000-Mr polypeptide species; 37,000- and 30,000-Mr proteins were also present in some T. phagedenis preparations. Immunoblotting with monospecific antisera and monoclonal antibodies and N-terminal sequence analysis indicated that the major periplasmic flagellar proteins were divided into two distinct classes, designated class A and class B. Class A proteins consisted of the 37-kilodalton (kDa) protein of T. pallidum and the 39-kDa polypeptide of T. phagedenis; class B included the T. pallidum 34.5-, 33-, and 30-kDa proteins and the four 33- and 34-kDa polypeptide species of T. phagedenis. The proteins within each class were immunologically cross-reactive and possessed similar N-terminal sequences (67 to 95% homology); no cross-reactivity or sequence homology was evident between the two classes. Anti-class A or anti-class B antibodies did not react with the 29- or 27-kDa polypeptides of T. pallidum or the 37- and 30-kDa T. phagedenis proteins, indicating that these proteins are antigenically unrelated to the class A and class B proteins. The lack of complete N-terminal sequence homology among the major periplasmic flagellar proteins of each organism indicates that they are most likely encoded by separate structural genes. Furthermore, the N-terminal sequences of T. phagedenis and T. pallidum periplasmic flagellar proteins are highly conserved, despite the genetic dissimilarity of these two species.     

The periplasmic flagella of Serpulina (Treponema) hyodysenteriae are composed of two sheath proteins and three core proteins.

The major components of the periplasmic flagella of the spirochaete Serpulina (Treponema) hyodysenteriae strain C5 were purified and characterized. We demonstrate that the periplasmic flagella are composed of five major proteins (molecular masses 44, 37, 35, 34 and 32 kDa) and present their location, N-terminal amino acid sequence and immunological relationship. The 44 kDa and the 35 kDa protein are on the sheath of the periplasmic flagellum, whereas the 37, 34 and 32 kDa protein reside in the periplasmic flagellar core. The two sheath flagellar proteins are immunologically related but have different N-terminal amino acid sequences. The N-terminus of the 44 kDa protein shows homology with the sheath flagellins of other spirochaetes, but the 35 kDa protein does not. The three core proteins are immunologically cross-reactive and their N-terminal amino acid sequences are almost, but not completely, identical, indicating that the core proteins are encoded by three distinct genes. The core proteins show extensive N-terminal sequence similarities and an immunological relationship with periplasmic flagellar core proteins of other spirochaetes.     

Evidence that prestin has at least two voltage-dependent steps

Prestin is a voltage-dependent membrane-spanning motor protein that confers electromotility on mammalian cochlear outer hair cells, which is essential for normal hearing of mammals. Voltage-induced charge movement in the prestin molecule is converted into mechanical work; however, little is known about the molecular mechanism of this process. For understanding the electromechanical coupling mechanism of prestin, we simultaneously measured voltage-dependent charge movement and electromotility under conditions in which the magnitudes of both charge movement and electromotility are gradually manipulated by the prestin inhibitor, salicylate. We show that the observed relationships of the charge movement and the physical displacement (q-d relations) are well represented by a three-state Boltzmann model but not by a two-state model or its previously proposed variant. Here, we suggest a molecular mechanism of prestin with at least two voltage-dependent conformational transition steps having distinct electromechanical coupling efficiencies.     

Identification of two iron-repressed periplasmic proteins in Haemophilus influenzae.

Protein expression by Haemophilus influenzae under iron-limiting growth conditions was examined. The five type b strains and four nontypeable strains studied all expressed a new protein of about 40 kDa when deprived of iron during growth. Most strains also expressed a protein of about 31 kDa under the same growth conditions. Both the 40- and 31-kDa proteins were not expressed by cells grown in iron-replete medium. The 40- and 31-kDa proteins were not expressed in iron-deficient medium to which an excess of ferric nitrate had been added, and therefore it was concluded that their expression was iron regulated. These iron-repressed proteins were localized to the periplasmic space. The amino-terminal sequences of both proteins were determined. The N-terminal sequence of the 40-kDa protein had 81% similarity to the N terminus of Fbp, the major iron-binding protein of Neisseria gonorrhoeae and N. meningitidis. The 31-kDa protein sequence showed no homology with any known protein sequence. As no plasmids were found in the strains, it was concluded that these proteins were chromosomally encoded.     

VirD2 gene product from the nopaline plasmid pTiC58 has at least two activities required for virulence.

Virulence genes virD1 and virD2 are required for T-DNA processing in Agrobacterium tumefaciens. The regions within virD2 contributing to T-DNA processing and virulence were investigated. Some insertional mutations in virD2 prevented T-DNA border endonucleolytic cleavage and produced an avirulent phenotype. However, a non-polar insertion immediately after bp 684 of the 1344 bp open reading frame of virD2 did not inhibit endonucleolytic cleavage but still caused a loss of virulence. This suggested that in addition to T-DNA border cleaving activity, the VirD2 protein has another virulence function which resides in the C-terminal half of the protein. Comparative nucleotide sequence analyses of virD2 showed that the first 684 bp were 81% homologous to virD2 of an octopine Ti plasmid whereas the remaining 660 bp were only 44% homologous. A plasmid containing the virD region from octopine Ti plasmid could restore both virulence and processing to a nopaline virD2 mutant. No complementation resulted when a nopaline virD2 clone containing a region similar to eukaryotic nuclear envelope transport sequences was deleted from the 3' end. These results suggest that virD1 and only the first half of virD2 are required to encode for the T-DNA processing endonuclease, and that the 3'-half of virD2 encodes a function separate from endonuclease activity that is required for virulence.     

A complex plasma plant sterol locus on mouse chromosome 14 has at least two genes regulating intestinal sterol absorption

We previously identified two inbred mouse strains, C57BL/6J and CASA/Rk, with different plasma plant sterol levels. An intercross between these strains revealed a broad plasma plant sterol locus on chromosome 14, which peaked at 17 centimorgan (cM) with a maximum logarithm of the odds score of 9.9. Studies in a chromosome 14 congenic strain, 14KK, with a 4-60 cM CASA/Rk interval on the C57BL/6J background revealed that males, but not females, had decreased plasma plant sterol levels and intestinal cholesterol absorption. In two subcongenic strains, 14PKK and 14DKK, with 4-19.5 and 19.5-60 cM CASA/Rk intervals, respectively, both males and females had decreased plasma plant sterol levels and decreased intestinal cholesterol absorption. Compatible with the decreased plasma plant sterol phenotype, 14PKK mice had increased biliary plant sterol excretion, whereas 14DKK mice did not. Therefore, gender-dependent interactions of genes at the 14PKK and 14DKK intervals are likely to underlie the 14KK interval effect on plasma plant sterol levels and sterol absorption from the intestine. These studies confirm the plasma plant sterol locus on mouse chromosome 14 and provide evidence that there are at least two sets of genes operating: one set affecting intestinal sterol absorption and biliary excretion, and the other set mainly affecting intestinal sterol absorption.     

大肠杆菌周质蛋白提取工艺的研究

研究了精氨酸缓冲液在不同浓度、pH值及提取时间下的周质蛋白提取率,并以溶菌酶法、渗透压休克法作为对比。结果表明浓度0.4 mol/L,pH值8.0,提取时间为45 min时,周质目的蛋白达到0.89 mg/g湿菌,相比其他方法,周质蛋白提取率分别提高93%、187%。实验得到一种高效、方便的大肠杆菌周质蛋白提取工艺,为周质表达的重组蛋白大规模生产奠定了基础。     

Glutathione S-transferase Pi has at least three distinguishable xenobiotic substrate sites close to its glutathione-binding site

Benzyl isothiocyanate (BITC), present in cruciferous vegetables, is an efficient substrate of human glutathione S-transferase P1-1 (hGST P1-1). BITC also acts as an affinity label of hGST P1-1 in the absence of glutathione, yielding an enzyme inactive toward BITC as substrate. As monitored by using BITC as substrate, the dependence of k of inactivation (K(I)) of hGST P1-1 on [BITC] is hyperbolic, with K(I) = 66 +/- 7 microM. The enzyme incorporates 2 mol of BITC/mol of enzyme subunit upon complete inactivation. S-Methylglutathione and 8-anilino-1-naphthalene sulfonate (ANS) each yield partial protection against inactivation and decrease reagent incorporation, whereas S-(N-benzylthiocarbamoyl)glutathione or S-methylglutathione + ANS protects completely. Mapping of proteolytic digests of modified enzyme by using mass spectrometry reveals that Tyr(103) and Cys(47) are modified equally. S-Methylglutathione reduces modification of Cys(47), indicating this residue is at/near the glutathione binding region, whereas ANS decreases modification of Tyr(103), suggesting this residue is at/near the BITC substrate site, which is also near the binding site of ANS. The Y103F and Y103S mutant enzymes were generated, expressed, and purified. Both mutants handle substrate 1-chloro-2,4-dinitrobenzene normally; however, Y103S exhibits a 30-fold increase in K(m) for BITC and binds ANS poorly, whereas Y103F has a normal K(m) for BITC and K(d) for ANS. These results indicate that an aromatic residue at position 103 is essential for the binding of BITC and ANS. This study provides evidence for the existence of a novel xenobiotic substrate site in hGST P1-1, which can be occupied by benzyl isothiocyanate and is distinct from that of monobromobimane and 1-chloro-2,4 dinitrobenzene.     

Evidence for at least two dominant neutralization antigens on human rhinovirus 14.

A collection of 28 mutants of human rhinovirus 14, selected for resistance to 10 individual neutralizing monoclonal antibodies, was used to identify two major neutralization antigens, N-Ag I and N-Ag II. Isoelectric analysis showed that all 16 of the N-Ag I mutants analyzed were charge altered in VP1;8 of 12 N-Ag II mutants were altered in VP3. These results suggest that N-Ag I resides on VP1, whereas N-Ag II lies on VP3. The frequency of charge alterations was much higher than predicted by the genetic code, suggesting that charged amino acids on the antigenic sites play an important role in interaction with neutralizing antibody. Antibodies against N-Ag I and N-Ag II neutralize with widely different efficiencies.     

Testing models for transport systems dependent on periplasmic binding proteins.

A carrier model in which transport across the cytoplasmic membrane is mediated by a periplasmic binding protein (Krupka, R.M. (1992) Biochim. Biophys. Acta 1110, 1-10) is shown to account for many of the properties of these systems: (i) Michaelis-Menten kinetics; (ii) seemingly irreversible uptake; (iii) the absence of exchange transport and counter-transport; (iv) substrate half-saturation constants that in different systems may be lower or higher than the dissociation constant of the binding protein; (v) the high concentration of the binding protein in the periplasm and its weak association with the membrane component. The binding protein appears to function as a valve or rectifier that permits the substrate to enter the cell, but blocks exit in both the energized and de-energized states. The asymmetry depends on both the abruptness and the extent of the conformational change in the binding protein. Characteristically, these systems build up steep gradients across the membrane, circumstances in which such a valve might be important. In agreement with the mechanism, (a) the binding protein is missing in members of the same family of transporters that function in export of the substrate rather than import; and (b) in Gram-positive organisms, which have no periplasmic space, binding proteins function while anchored to the cytoplasmic membrane.     

Evidence that thymocytes require at least two distinct signals to proliferate.

Optimal receptivity of thymocytes to the mitogenic stimulus in macrophage culture fluid (MCF) is limited to the time in culture when these cells are spontaneously proliferating. By providing a brief, nonmitogenic lectin pulse, nonproliferating cells can be "activated" and their ability to respond to MCF totally restored. This procedure provides a model system for evaluating thymocyte proliferation and, in particular, the mechanism of action of MCF.     

Protein-sequence studies on Rh-related polypeptides suggest the presence of at least two groups of proteins which associate in the human red-cell membrane.

The Rh D blood-group antigen forms part of a complex, involving several other polypeptides, that is deficient in the red cells of individuals who lack all the antigens of the Rh blood-group system (Rhnull red cells). These include components recognized by anti-(Rh D) antibodies and the murine monoclonal antibodies R6A and BRIC 125. We have carried out protein-sequence studies on the components immunoprecipitated by these antibodies. Anti-(Rh D) antibodies immunoprecipitate an Mr-30,000-32,000 polypeptide (the D30 polypeptide) and an Mr-45,000-100,000 glycoprotein (D50 polypeptide). Antibody R6A immunoprecipitates two glycoproteins of Mr 31,000-34,000 (R6A32 polypeptide) and Mr 35,000-52,000 (R6A45 polypeptide). The D30 and R6A32 polypeptides were found to have the same N-terminal amino acid sequences, showing that they are closely related proteins. The D50 polypeptide and the R6A45 polypeptide also had indistinguishable N-terminal amino acid sequences that differed from that of the D30 and R6A32 polypeptides. The putative N-terminal membrane-spanning segments of the two groups of proteins showed homology in their amino acid sequence, which may account for the association of each of the pairs of proteins during co-precipitation by the antibodies. Supplementary data related to the protein sequence have been deposited as Supplementary Publication SUP 50417 (6 pages) at the British Library Document Supply Centre, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1988) 249, 5.     

Proper interaction between at least two components is required for efficient export of proteins to the Escherichia coli cell envelope.

An Escherichia coli mutant carrying delta malE12-18, a 21-base pair deletion confined to the coding DNA of the maltose-binding protein signal peptide, is unable to export maltose-binding protein to the periplasm efficiently. Consequently, such a strain is defective for the utilization of maltose as a sole carbon source. We obtained 16 mutants harboring extragenic delta malE12-18 suppressor mutations that exhibit partial restoration of export to the mutant maltose-binding protein. A genetic analysis of these extragenic suppressor mutations demonstrated that 15 map at prlA, at 72 min on the standard E. coli linkage map, and that 1 maps at a new locus, prlD, at 2.5 min on the linkage map. Our evidence indicates that the prlA and prlD gene products play an important role in the normal pathway for export of proteins to the cell envelope. Efficient execution of the secretory process requires that these prl gene products interact properly with each other so that a productive interaction of these gene products with the signal peptide also can occur. Our data suggest that proper assembly of a complex is required for efficient export of E. coli envelope proteins to their various extracytoplasmic compartments.     

Concanavalin A receptor(s) possibly interacts with at least two kinds of GTP-binding proteins in murine thymocytes

A part of the GTP gamma S-binding activity in murine thymocyte membranes was found to have affinity to a concanavalin A (Con A)-Sepharose column. The material was identified as Gi (inhibitory GTP-binding protein) on the basis of the molecular weight and by islet activating protein-dependent ADP-ribosylation and anti-alpha i (alpha subunit of Gi) immunoblotting. However, when the membranes prepared from Con A-stimulated thymocytes were used, no GTP gamma S-binding activity was detected in the Con A-bound fraction, suggesting that Gi physically and specifically associated with Con A acceptors dissociates upon Con A stimulation. Furthermore, another GTP gamma S-binding protein (25 kDa), which is quite similar to a novel phosphoinositide-specific phospholipase C (PI-PLC)-associated G protein in calf thymocytes (Wang, P., Toyoshima, S., & Osawa, T. (1988) J. Biochem. 103, 137-142), was detected among the Con A-Sepharose-bound proteins with the chemical cross-linking technique. When the 40 kDa and 25 kDa G proteins associated with Con A receptor(s) were isolated and their direct effects on the activity of partially purified PI-PLC as to phosphatidylinositol 4,5-bisphosphate hydrolysis were examined, the 25 kDa G protein was found to enhance the PI-PLC activity more effectively. On the other hand, pretreatment of cells with islet-activating protein completely abolished the inhibitory effect of Con A on the prostaglandin E1 and isoproterenol-induced increases of cellular cAMP.(ABSTRACT TRUNCATED AT 250 WORDS)