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1.
Six strains of Fusobacterium nucleatum were tested for their ability to react with [3H]diisopropylfluorophosphate (DFP), a serine protease inhibitor. Several cytoplasmic proteins were labelled but the strongest labelling was regularly observed in a few outer membrane proteins. The number and the molecular mass of the proteins detected varied according to the strain tested. A 61 kDa protein was labelled in all strains tested, including the two type strains ATCC 10953 and ATCC 25586. A 65 kDa protein was particularly strongly labelled in strains Fev1 and F6.  相似文献   

2.
《The Biochemical journal》1975,148(2):329-333
The synthesis of nitrate reductase and its incorporation into the cytoplasmic membrane of Escherichia coli strain A1004a (5-aminolaevulinic acid auxotroph) does not require synthesis of cytochrome b. The synthesis of the apoprotein(s) of the cytochrome b of the respiratory pathway from NADH to nitrate appears to be inhibited by the absence of haem. No member of the respiratory pathway from NADH to oxygen is capable of reducing nitrate reductase directly. The site on nitrate reductase that oxidizes FMNH2 is located on the cytoplasmic aspect of the cytoplasmic membrane.  相似文献   

3.
The synthesis of membrane protein after infection with bacteriophage T4 was examined. Protein constituents of both the cytoplasmic and outer membrane are made during the infective cycle. In addition, newly synthesized membrane protein is found in material which has a buoyant density greater than that of either of the two host membrane fractions. Polyacrylamide gel analyses and solubilization studies using the detergent Sarkosyl indicate that synthesis of most of the membrane proteins made during the first 5 min of infection is directed by bacterial genes. New membrane proteins synthesized at times greater than 6 min after infection appear to be distinct from those of the host, and new proteins of the outer membrane are different from those of the inner. Proteins in the new dense membrane fraction are similar to those of the outer membrane.  相似文献   

4.
The reconstitution of nitrate reductase activity in mixtures of cytoplasmic fractions from the chlorate-resistant mutants chlA, B, C, and E which are lacking this activity was investigated, and the membrane-like particulate material which formed during this reconstitution was analyzed by polyacrylamide gel electrophoresis. When chlA and chlB extracts are incubated together, the cytoplasmic membrane proteins present in the particles which are formed are contributed by both mutants, and the proteins are essentially the same as the proteins in the cytoplasmic membrane fractions of the two mutants. Identical amounts of protein become particulate when cytoplasmic extracts of any of the mutant strains or wild-type strains are incubated at 32 C either singly or in mixtures, and the formation of particulate material does not appear to be a consequence of nitrate reductase reconstitution. Experiments with wild-type strains indicate that the membrane proteins in the cytoplasmic extract are derived from the cytoplasmic membrane during cell breakage. Reconstitution experiments involving various combinations of preincubated and unincubated extracts of the mutants have allowed a preliminary identification of three types of components which are necessary for the formation of active nitrate reductase: (i) a soluble factor present only in extracts from induced chlB; (ii) a different soluble factor which is missing in chlB but is present in extracts from wild-type, chlA, chlC, and chlE; and (iii) a complex including the nitrate reductase protein which is inactivated by preincubation of the mutant extracts.  相似文献   

5.
The control operative on the nitrate reductase enzyme system of host cyanobacteriumNostoc muscorum was studied after being infected with the cyanophage N-1. Phage infection lifted the host nitrate reductase activity level via accelerating the enzyme synthesis. It was found that the phage-mediated increase in the molybdenum cofactor synthesis was a major contributing factor for apparent elevated nitrate reductase level of the host. This process was inhibited in the presence of erythromycin and tungsten, the inhibitors of protein synthesis and new nitrate reductase synthesis respectively. While the preformed nitrate reductase of healthy cyanobacterium was inhibited by hydrogen peroxide, an oxidizing photosynthetic product, the same enzyme of infected cells remained virtually insensitive to this inhibitor. These data suggest involvement of new nitrate reductase synthesis and its resistance to oxidative inactivation as joint factors controlling the characteristic high enzyme level of host cyanobacterium.  相似文献   

6.
Dimethyl sulfoxide reductase is a trimeric, membrane-bound, iron-sulfur molybdoenzyme induced in Escherichia coli under anaerobic growth conditions. The enzyme catalyzes the reduction of dimethyl sulfoxide, trimethylamine N-oxide, and a variety of S- and N-oxide compounds. The topology of dimethyl sulfoxide reductase subunits was probed by a combination of techniques. Immunoblot analysis of the periplasmic proteins from the osmotic shock and chloroform wash fluids indicated that the subunits were not free in the periplasm. The reductase was susceptible to proteases in everted membrane vesicles, but the enzyme in outer membrane-permeabilized cells became protease sensitive only after detergent solubilization of the E. coli plasma membrane. Lactoperoxidase catalyzed the iodination of each of the three subunits in an everted membrane vesicle preparation. Antibodies to dimethyl sulfoxide reductase and fumarate reductase specifically agglutinated the everted membrane vesicles. No TnphoA fusions could be found in the dmsA or -B genes, indicating that these subunits were not translocated to the periplasm. Immunogold electron microscopy of everted membrane vesicles and thin sections by using antibodies to the DmsABC, DmsA, DmsB subunits resulted in specific labeling of the cytoplasmic surface of the inner membrane. These results show that the DmsA (catalytic subunit) and DmsB (electron transfer subunit) are membrane-extrinsic subunits facing the cytoplasmic side of the plasma membrane.  相似文献   

7.
In developing a reliable in vitro system for translocating bacterial proteins, we found that the least dense subfraction of the membrane of Escherichia coli was superior to the total inner membrane, both for a secreted protein (alkaline phosphatase) and for an outer membrane protein (OmpA). Compounds that eliminated the proton motive force inhibited translocation, as already observed in cells; since protein synthesis continued, the energy for translocation appears to be derived from the energized membrane and not simply from ATP. Treatment of the vesicles with protease, under conditions that did not interfere with subsequent protein synthesis, also inactivated them for subsequent translocation. We conclude that export of some proteins requires protein-containing machinery in the cytoplasmic membrane that derives energy from the proton motive force.  相似文献   

8.
The five subunits of the membrane-bound adenosine triphosphatase (F1) from Escherichia coli were identified on electrophoretograms of membranes which had been washed with a low-ionic-strength buffer containing the protease inhibitor p-aminobenzamidine. All of the subunits of the membrane-bound F1 appeared to have the same molecular weights and isoelectric points as those of the soluble F1, as judged by two-dimensional electrophoresis. p-Aminobenzamidine inhibited the solubilization of F1 rebound to F1-depleted membranes, and was found to inhibit the membrane-bound adenosine triphosphatase activity to a much greater extent than the solubilized activity. It is therefore unlikely that p-aminobenzamidine inhibits the solubilization of F1 by inhibiting a protease, as suggested previously by Cox et al. (G.B. Cox, J.A. Downie, D.R.H. Fayle, F. Gibson, and J. Radik, J. Bacteriol. 133:287--292, 1978).  相似文献   

9.
Formation of Influenza Virus Proteins   总被引:7,自引:6,他引:1       下载免费PDF全文
Eight virus-specific proteins have been found in chicken embryo fibroblasts infected with fowl plague virus. Among them are two glycoproteins which are the constituents of the hemagglutinin on the virus particle. They are derived from a large precursor glycoprotein by cleavage of a covalent linkage. The reaction can be blocked by the protease inhibitor diisopropylfluorophosphate and the amino acid analogue fluorophenylalanine. This indicates that a peptide bond is cleaved. If infected cells are kept at 25 C, a temperature at which virus maturation is inhibited, the precursor glycoprotein is cleaved at a significantly slower rate than at 37 C. It appears, however, that a reduced synthesis of the carbohydrate-free envelope protein is responsible for the block of virus maturation at 25 C rather than the lower cleavage rate of the precursor.  相似文献   

10.
The outer membrane fraction from Rhodopseudomonas sphaeroides was isolated by isopycnic density centrifugation. The purity of this fraction was assayed by several methods. When the outer membrane fraction obtained after French press lysis of cells was compared with the outer membrane fragments released during spheroplast formation, the polypeptide profiles were identical. Detergent solubilization of membrane fractions showed that Triton X-100 nonselectively solubilizes both the cytoplasmic membrane and the outer membrane, whereas Deriphat 160 selectively solubilizes the cytoplasmic membrane. Several outer membrane polypeptides, including the major outer membrane protein, exhibited changes in electrophoretic mobility that depended upon the temperature of solubilization in sodium dodecyl sulfate. Solubilization at room temperature in the presence of ions reproduced the effect of thermal denaturation on the major outer membrane polypeptide.  相似文献   

11.
12.
The enzyme transglutaminase has been used to label surface proteins of Escherichia coli cytoplasmic membranes by covalently attaching to them a small fluorescent primary amine, dansyl cadaverine. Spheroplasts lacking outer membrane, osmotically lysed vesicles from the spheroplasts, and vesicles made by breaking cells in a French pressure cell were each labeled with transglutaminase and dansyl cadaverine. When the total cytoplasmic membrane proteins of each were examined on sodium dodecyl sulfate gels, three rather different labeling patterns were obtained. Labeling of the respiratory enzyme, nitrate reductase, in the membranes of each of these preparations was also examined. Membrane-bound nitrate reductase contains three subunits: A, B, and C. Dansyl cadaverine labeling of nitrate reductase in the presence of Triton X-100 indicated that subunits A and C could be labeled. When nitrate reductase was isolated from dansyl cadaverine-labeled spheroplasts, none of the subunits was labeled. When nitrate reductase was isolated from French press vesicles, subunit A was labeled and labeling was enhanced by the presence of nitrate during labeling. When nitrate reductase from osmotic vesicles was examined, subunit A was labeled in the presence of nitrate but no labeled subunits appeared when the vesicles were labeled in the absence of nitrate. It was concluded that (i) nitrate reductase is buried in the membrane with subunit A exposed only on the inner surface of the membrane, (ii) subunit C is sufficiently buried within the membrane so that it is inaccessible to transglutaminase, (iii) subunit B is not labeled under any condition, so its location is not known, and (iv) large osmotic vesicles are probably mosaics in which some protein components have been reoriented.  相似文献   

13.
NADH-Nitrate Reductase Inhibitor from Soybean Leaves   总被引:17,自引:15,他引:2       下载免费PDF全文
A NADH-nitrate reductase inhibitor has been isolated from young soybean (Glycine max L. Merr. Var. Amsoy) leaves that had been in the dark for 54 hours. The presence of the inhibitor was first suggested by the absence of nitrate reductase activity in the homogenate until the inhibitor was removed by diethylaminoethyl (DEAE)-cellulose chromatography. The inhibitor inactivated the enzyme in homogenates of leaves harvested in the light. Nitrate reductases in single whole cells isolated through a sucrose gradient were equally active from leaves grown in light or darkness, but were inhibited by addition of the active inhibitor.

The NADH-nitrate reductase inhibitor was purified 2,500-fold to an electrophoretic homogeneous protein by a procedure involving DEAE- cellulose chromatography, Sephadex G-100 filtration, and ammonium sulfate precipitation followed by dialysis. The assay was based on nitrate reductase inhibition. A rapid partial isolation procedure was also developed to separate nitrate reductase from the inhibitor by DEAE-cellulose chromatography and elution with KNO3. The inhibitor was a heat-labile protein of about 31,000 molecular weight with two identical subunits. After electrophoresis on polyacrylamide gel two adjacent bands of protein were present; an active form and an inactive form that developed on standing. The active factor inhibited leaf NADH-nitrate reductase but not NADPH-nitrate reductase, the bacterial nitrate reductase or other enzymes tested. The site of inhibition was probably at the reduced flavin adenine dinucleotide-NR reaction, since it did not block the partial reaction of NADH-cytochrome c reductase. The inhibitor did not appear to be a protease. Some form of association of the active inhibitor with nitrate reductase was indicated by a change of inhibitor mobility through Sephadex G-75 in the presence of the enzyme. The inhibition of nitrate reductase was noncompetitive with nitrate but caused a decrease in Vmax.

The isolated inhibitor was inactivated in the light, but after 24 hours in the dark full inhibitory activity returned. Equal amounts of inhibitor were present in leaves harvested from light or darkness, except that the inhibitor was at first inactive when rapidly isolated from leaves in light. Photoinactivation of yellow impure inhibitor required no additional components, but inactivation of the purified colorless inhibitor required the addition of flavin.

Preliminary evidence and a procedure are given for partial isolation of a component by DEAE-cellulose chromatography that stimulated nitrate reductase. The data suggest that light-dark changes in nitrate reductase activity are regulated by specific protein inhibitors and stimulators.

  相似文献   

14.
Brain hexokinase (ATP:D-hexose-6-phosphotransferase, EC 2.7.1.1) binds selectively to the outer membrane of rat liver mitochondria but not to inner mitochondrial or microsomal membranes nor to the plasma membrane of human erythrocytes. A protein having subunit molecular weight of 31,000, determined by sodium dodecyl sulfate-gel electrophoresis, has been highly purified from the outer mitochondrial membrane by repetitive solubilization with octyl-beta-D-glucopyranoside followed by reconstitution into membranous vesicles when the detergent is removed by dialysis. When incorporated into lipid vesicles, the protein confers the ability to bind brain hexokinase in a Glc-6-P-sensitive manner as is seen with the intact outer mitochondrial membrane. Hexokinase binding ability and the 31,000 subunit molecular weight protein co-sediment during sucrose density gradient centrifugation. Both hexokinase binding ability and the 31,000 subunit molecular weight protein are resistant to protease treatment of the intact outer mitochondrial membrane while other membrane proteins are extensively degraded. It is concluded that this protein, designated the hexokinase-binding protein (HBP), is an integral membrane protein responsible for the selective binding of hexokinase by the outer mitochondrial membrane.  相似文献   

15.
The proteolytic activity of E.coli measured using 125I-labelled αS1 casein as substrate, is mainly localised in the outer membrane and is due to an intrinsic outer membrane protein which can be solubilized by deoxycholate. This enzyme exhibits maximum activity at pH 7,5 in Tris-HCl buffer, is resistant to thermal denaturation with a half-life of 28 min. at 90°C in deoxycholate-NaCl buffer and is inhibited by ethylene-diamine tetraacetate, high concentrations of p-aminobenzamidine, tosyl-L-lysine chloromethyl ketone, tosyl-L-phenylalaninechloromethyl ketone and by two inhibitors of the processing of the secreted protein precursors, procaine and phenehylalcohol. Whole cells do not exhibit proteolytic activity, nevertheless, some is unmasked when the outer membrane is permeabilized by Tris or ethylenediamine tetraacetate or when vesicles are sonicated. This suggests that the protease is on the inner side of the outer membrane. Because the protease is different from the soluble proteases described in E.coli, and especially from proteases I,II and III, it has been called protease IV.  相似文献   

16.
The iron(III) reductase activity of Geobacter sulfurreducens was determined with the electron donor NADH and the artificial electron donor horse heart cytochrome c. The highest reduction rates were obtained with Fe(III) complexed by nitrilotriacetic acid as an electron acceptor. Fractionation experiments indicated that no iron(III) reductase activity was present in the cytoplasm, that approximately one-third was found in the periplasmic fraction, and that two-thirds were associated with the membrane fraction. Sucrose gradient separation of the outer and cytoplasmic membranes showed that about 80% of the iron(III) reductase was present in the outer membrane. The iron(III) reductase could be solubilized from the membrane fraction with 0.5 M KCl showing that the iron(III) reductase was weakly bound to the membranes. In addition, solubilization of the iron(III) reductase from whole cells with 0.5 M KCl, without disruption of cells, indicated that the iron(III) reductase is a peripheral protein on the outside of the outer membrane. Redox difference spectra of KCl extracts showed the presence of c-type cytochromes which could be oxidized by ferrihydrite. Only one activity band was observed in native polyacrylamide gels stained for the iron(III) reductase activity. Excision of the active band from a preparative gel followed by extraction of the proteins and sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed the presence of high-molecular-mass, cytochrome-containing proteins in this iron(III) reductase activity band. From these experimental data it can be hypothesized that the iron(III) reductase of G. sulfurreducens is a peripheral outer membrane protein that might contain a c-type cytochrome.  相似文献   

17.
The disappearance of nitrate reductase activity in leaves of Hordeum vulgare L. during darkness was inhibited by cycloheximide, actinomycin D, and low temperature. Thus, protein synthesis was probably required for the disappearance of nitrate reductase in the dark. Since chloramphenicol did not affect the rate of loss of activity, the degradation or inactivation apparently required protein synthesis by the cytoplasmic ribosomal system. Consistent with this observation, nitrate reductase is also reportedly located in the cytoplasm. Thus, the amount of nitrate reductase activity present in leaves of barley may be controlled by a balance between activating and inactivating systems.  相似文献   

18.
Chlorate-resistant mutants corresponding to each known genetic locus (chlA, chlB, chlC, chlD, chlE) were isolated from Escherichia coli K-12. All these mutants showed decreased amounts of membrane-bound nitrate reductase, cytochrome b, and formic dehydrogenase, but all had normal succinic dehydrogenase activity. Proteins from the cytoplasmic membranes of these mutants were compared to those of the wild type-on polyacrylamide gels. The addition of nitrate to wild-type anaerobic cultures caused increased formation of three membrane proteins. These same proteins, along with one other, were missing in varying patterns in mutants altered at the different genetic loci. One of the missing proteins was found to be the enzyme nitrate reductase, although this protein was present in some mutants lacking nitrate reductase activity. None of the others has been identified.  相似文献   

19.
Effects of protease inhibitors on composition of newly synthesized protein were studied by pulse-labeling E. coli cells with [3H]leucine and analyzing the labeled proteins by sodium dodecylsulfate gel electrophoresis. In addition to tosyl-lysine chloromethylketone that had been studied previously, antipain, leupeptin and diisopropyl fluorophosphate all inhibited production of a major outer membrane protein, protein I. Synthesis of protein I was specifically inhibited by antipain or leupeptin in strain K12, whereas several other proteins were also affected in strain B. Protein synthesis in strain B was generally more sensitive to inhibition by antipain than that in strain K12.  相似文献   

20.
A number of polyacrylamide gel systems and solubilization procedures were studied to define the number and nature of "major" polypeptide bands in the outer membrane of Pseudomonas aeruginosa. It was shown that five of the eight major outer membrane proteins were "heat modifiable" in that their mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis was determined by the solubilization temperature. Four of these heat-modifiable proteins had characteristics similar to protein II of the Escherichia coli outer membrane. Addition of lipopolysaccharide subsequent to solubilization caused reversal of the heat modification. The other heat-modifiable protein, the porin protein F, was unusually stable to sodium dodecyl sulfate. Long periods of boiling in sodium dodecyl sulfate were required to cause conversion to the heat-modified form. This was demonstrated both with outer membrane-associated and purified lipopolysaccharide-depleted protein F. Furthermore, lipopolysaccharide treatment had no effect on the mobility of heat-modified protein F. Thus it is concluded that protein F represents a new class of heat-modifiable protein. It was further demonstrated that the electrophoretic mobility of protein F was modified by 2-mercaptoethanol and that the 2-mercaptoethanol and heat modification of mobility were independent of one another. The optimal conditions for the examination of the outer membrane proteins of P. aeruginosa by one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis are discussed.  相似文献   

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