Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.2.1.17 (lysozyme)
21,489 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The cDNA, coding for the first metal-binding domain (MBD1) of Menkes protein, was cloned into the T7-system based vector, pCA. The T7 lysozyme-encoding plasmid, pLysS, is shown to be crucial for expression, suggesting that the protein is toxic to the cells. Adding copper to the growth medium did not affect the plasmid stability. MBD1 is purified in two steps with a typical yield of 12 mg.L-1. Menkes protein, a P-type ATPase, contains a sequence GMXCXSC that is repeated six times, at the N-terminus. The paired cysteine residues are involved in metal binding. MBD1 has only two cysteine residues, which can exist as free thiol groups (reduced), as a disulphide bond (oxidized) or bound to a metal ion [e.g. Cu(I)-MBD1]. These three MBD1 forms have been investigated using CD. No major spectral change was seen between the different MBD1 forms, indicating that the folding is not changed upon metal binding. A copper-bound MBD1 was also studied by EPR, and the lack of an EPR signal suggests that the oxidation state of copper bound to MBD1 is Cu(I). Cu(I) binding studies were performed by equilibrium dialysis and revealed a stoichiometry of 1 : 1 and an apparent Kd = 46 microM. Oxidized MBD1, however, is not able to bind copper. Different copper complexes were investigated for their ability to reconstitute apo-MBD1. Given the same total copper concentration CuCl43- was superior to Cu(I)-thiourea (structural analogue of metallothionein) and Cu(I)-glutathione (used at fivefold higher copper concentration) although the latter two were able to partially reconstitute apo-MBD1. Cu(II) was not able to reconstitute apo-MBD1, presumably due to Cu(II)-induced oxidation of the thiol groups. Based on our results, glutathione and/or metallothionein are likely candidates for the in vivo incorporation of copper to Menkes protein.
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PMID:Expression, purification and copper-binding studies of the first metal-binding domain of Menkes protein. 1049 Nov 37

We describe the design and characterization of a set of fusion proteins of the Escherichia coli lactose (lac) permease in which a set of five different soluble "carrier" proteins (cytochrome(b562), flavodoxin, T4 lysozyme, beta-lactamase and 70 kDa heat shock ATPase domain) were systematically inserted into selected loop positions of the transporter. The design goal was to increase the exposed hydrophilic surface area of the permease, while minimizing the internal flexibility of the resulting fusion proteins in order to improve the crystallization properties of the membrane protein. Fusion proteins with insertions into the central hydrophilic loop of the lac permease were active in transport lactose, although only the fusion proteins with E. coli cytochrome(b562), E. coli flavodoxin or T4 lysozyme were expressed at near wild-type lac permease levels. Eight other loop positions were tested with these three carriers, leading to the identification of additional fusion proteins that were active and well-expressed. By combining the results from the single carrier insertions, we have expressed functional "double fusion" proteins containing cytochrome(b562) domains inserted in two different loop positions.
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PMID:Insertion of carrier proteins into hydrophilic loops of the Escherichia coli lactose permease. 1210 Sep 94

At least 16 proteins are thought to be involved in forming the enteropathogenic Escherichia coli (EPEC) type III translocation apparatus which delivers virulence factors into host cells, yet their function and location have not been determined. A biochemical analysis was performed on three components: EscN, a predicted cytoplasmic ATPase; EscV, a predicted inner membrane protein; and EscC, a predicted outer membrane secretin. Wild-type EPEC and mutants constructed in these genes were fractionated by lysozyme treatment, ultracentrifugation, and selective detergent extraction. Fractionation revealed that the type III effectors Tir and EspB required a complete type III apparatus for any degree of export by EPEC, suggesting a continuous channel. Epitope-tagged EscC, EscV, and EscN were localized by fractionation, confirming computer modeling predictions for their location. Transcomplementation experiments revealed that localization of EscV and EscN was unaffected by mutations in other examined type III components. Remarkably, localization of EscC was altered in escV or escN mutants, where EscC accumulated in the periplasm. EscC was correctly localized in the escF needle component mutant, indicating that secretin localization is independent of needle formation. These data indicate that, contrary to previous indications, correct insertion and function of EscC secretin in the outer membrane depends not only on the sec-dependent secretion pathway but also on other type III apparatus components.
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PMID:Secretin of the enteropathogenic Escherichia coli type III secretion system requires components of the type III apparatus for assembly and localization. 1276 Nov 13

The mechanism of charge propagation in "ion channel sensors" (ICSs) consisting of gold electrodes modified with a layer of charged proteins and highly charged redox-active marker ions in solution was investigated by electrochemical techniques, QCM and AFM. The study is based on seven proteins (concanavalin A, cytochrome c, glucose oxidase, lysozyme, thyroglobulin, catalase, aldolase, and EF1-ATPase) in combination with seven electroactive marker ions ([Fe(CN)6]3-, [Fe(CN)6]4-, [Ru(NH3)6]3+, mono-, di-, and trimeric viologens), as well as a series of suppressor and enhancer ions leading to the following general statements: (i) electrostatic binding of charged marker ions to the domains of the protein is a prerequisite for an electrochemical current and (ii) charge propagation through the layer consists of electron hopping along surface-confined marker ions into the pores between adsorbed proteins. It is further shown that (iii) marker ions and suppressor ions with identical charge compete for oppositely charged sites on the protein domain, (iv) electrostatically bound multilayers of marker or enhancer ions with alternating charge form on a charged protein domain, and (v) self-exchange and exergonic ET catalysis between adsorbed marker ions and marker ions in solution take place. In addition to fundamental insight into the mechanism of charge propagation, valuable information for the design, optimization, and tailoring of new biosensors based on the ICS concept is demonstrated by the current findings.
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PMID:Charge propagation in "ion channel sensors" based on protein-modified electrodes and redox marker ions. 1608 79

The yvgW gene of Bacillus subtilis has been reported to encode a product which resembles CPx-type ATPase having a function related to Cd2+ and Zn2+ resistance through efflux of this metal. We recently showed that yvgW gene product is also important for sporulation in B. subtilis. The present study was focused on the functional characterization of yvgW in the sporulation process of B. subtilis. The analysis of yvgW expression showed that a significant expression took place during the late stage of sporulation (T5-T8). The deletion of spoIIAC and spoIIGB genes, encoding for sigmaF and sigmaE, respectively, resulted in the complete elimination of yvgW-lacZ expression while the deletion of the spoIIIG coding for sigmaG decreased the yvgW-lacZ expression to only 37% that of the wild type level. In contrast, the deletion of spoIVCB gene coding for sigmaK had no significant effects on the yvgW-lacZ expression. Transcription initiation site of yvgW during sporulation was determined by 5'-RACE-PCR, indicating that -10 and -35 sequences exhibited very good homology with the consensus sequences recognized by RNA polymerase containing sigmaE. Moreover, through the construction of yvgWDelta537-1351::spc, yvgW mutant cells were investigated for their spore properties, such as their resistance profiles against heat, chloroform and lysozyme, pointing out that spores of the mutant cells showed high sensitivity to heat and chloroform, but resistance to lysozyme. The level of dipicolinic acid was also significantly reduced to approximately 63% in yvgW spores as compared to wild type spores. Furthermore, the analyses of the nutrition-specific germination and outgrowth characteristics of the null mutant and the wild type cells revealed no defect in the initiation of yvgW spore germination but they returned to vegetative state more slowly than the wild type spores in minimal medium.
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PMID:Sporulation-specific expression of the yvgW (cadA) gene and the effect of blockage on spore properties in Bacillus subtilis. 1690 59

This study was undertaken to examine physiological responses to acidification of environmental water in the "cobalt" variant of rainbow trout (Oncorhynchus mykiss), which exhibits malformation of the pituitary, by following changes in plasma levels of cortisol and electrolytes, blood pH, gill Na(+), K(+)-ATPase activity, and immune functions after exposure to acid water (pH 4.5). Resting levels of plasma cortisol and lysozyme were significantly lower in the cobalt variant than in the normal trout, whereas plasma ceruloplasmin was significantly higher in the cobalt variant, suggesting that some endocrine factors, lacking or deficient in the cobalt variant, are important for the regulation of its immune functions. Blood pH was slightly but significantly lower in the cobalt variant at rest. After exposure to acid water for 24 h, both the normal trout and cobalt variant showed a significant elevation in plasma cortisol, although the increased level in the cobalt variant was still lower than that in the normal trout transferred to neutral water. No differences were seen in blood pH, plasma electrolytes, and gill Na(+), K(+)-ATPase activity between the normal trout and the cobalt variant, indicating that the cobalt variant regulates ion balance when exposed to acid water, despite malformation of the pituitary. Although the normal trout showed a reduction in plasma lysozyme level after acid exposure, there was no significant change in the cobalt trout. Adverse effects of pituitary malformation on ion balance and immune functions may be compensated by extrapituitary factors in the cobalt variant when it is exposed to acid water.
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PMID:Effects of acid water exposure on plasma cortisol, ion balance, and immune functions in the "cobalt" variant of rainbow trout. 1697 89

We investigated the effect of ploidy on osmoregulatory, stress and immune responses in non-smolting rainbow trout during saltwater adaptation. Sibling groups of diploid and triploid trout were acclimated in freshwater (FW) and then subjected to abrupt transfer to full strength (35ppt) saltwater (SW) or back to FW. Fish were sampled pre-stress, and 1, 3, 6, 12, 24, 48, 72 and 168h post-stress. Overall mortality in SW was less than 5% in either ploidy, with no mortality in FW. Significant elevations in plasma osmolality and gill ATPase were observed within 1-3h of SW transfer, but retuned to basal levels within 72h indicative of rapid saltwater adaptation and did not differ between ploidy. Furthermore, FW-SW transfer also caused significant and sustained elevations in total blood haemoglobin, plasma IGF-I, cortisol, glucose, total white blood cell counts, increased plasma but decreased mucus lysozyme, and enhanced head kidney macrophage respiratory burst activity. Conversely, FW-FW transfer evoked more transient and less elevated responses, more typical of primary and secondary responses to a single stressor. We conclude that the more elevated levels in these parameters are a function of saltwater adaptation as well as the generic stress response, and that this did not differ between ploidy. Strong positive correlations were found between plasma IGF-I and cortisol, and with osmolality, glucose and WBC, while a negative correlation was found with plasma lysozyme irrespective of ploidy. Overall, the current results suggest that triploidy does not affect the ability of non-smolting trout to adapt to full strength seawater under optimum conditions, and that the osmotic and stress response to such transfer is similar to diploids.
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PMID:The influence of ploidy on saltwater adaptation, acute stress response and immune function following seawater transfer in non-smolting rainbow trout. 1743 69

The chaperonin molecular machine from hyperthermophilic archaeon Pyrococcus furiosus was studied in this paper. The Pyrococcus furiosus chaperonin gene (PfCPN) was amplified by PCR from the Pyrococcus furiosus genomic DNA, and expressed in Escherichia coli BL21-Codonplus(DE)(3)-RIL. The recombinant PfCPN was purified to homogeneity by using ion-exchange and size-exclusion chromatography. It was found that the ATPase activity of the PfCPN was highest at 88 degrees C, and there existed a nested cooperativity of the ATPase activity of the PfCPN. This result suggested that nested allosteric behavior may be common to chaperonin molecular machines from archaea. The half-life (t(1/2)) of the ATPase activity of the PfCPN at 100 degrees C was about 60 min. The PfCPN displayed chaperone activity in preventing lysozyme from thermal inactivation. This chaperone activity was in an ATP-dependent manner.
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PMID:Expression and characterization of the chaperonin molecular machine from the hyperthermophilic archaeon Pyrococcus furiosus. 1744 Sep 15

Escherichia coli HslVU is an ATP-dependent protease consisting of two heat shock proteins, the HslU ATPase and HslV peptidase. In the reconstituted enzyme, HslU stimulates the proteolytic activity of HslV by one to two orders of magnitude, while HslV increases the rate of ATP hydrolysis by HslU several-fold. Here we show that HslV alone can efficiently degrade certain unfolded proteins, such as unfolded lactalbumin and lysozyme prepared by complete reduction of disulfide bonds, but not their native forms. Furthermore, HslV alone cleaved a lactalbumin fragment sandwiched by two thioredoxin molecules, indicating that it can hydrolyze the internal peptide bonds of lactalbumin. Surprisingly, ATP inhibited the degradation of unfolded proteins by HslV. This inhibitory effect of ATP was markedly diminished by substitution of the Arg86 residue located in the apical pore of HslV with Gly, suggesting that interaction of ATP with the Arg residue blocks access of unfolded proteins to the proteolytic chamber of HslV. These results suggest that uncomplexed HslV is inactive under normal conditions, but may can degrade unfolded proteins when the ATP level is low, as it is during carbon starvation.
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PMID:Nucleotide triphosphates inhibit the degradation of unfolded proteins by HslV peptidase. 1746 4

Influence of environmental salinity on expression of distinct corticosteroid receptor (CR) genes, glucocorticoid receptor (GR)-1 and -2, and mineralcorticoid receptor (MR), was examined in osmoregulatory and hemopoietic organs and leucocytes of steelhead trout (Oncorhynchus mykiss). There was no significant difference in plasma cortisol levels between freshwater (FW)- or seawater (SW)-acclimated trout, whereas Na+, K+-ATPase was activated in gill of SW fish. Plasma lysozyme levels also showed a significant increase after acclimation to SW. In SW-acclimated fish, mRNA levels of GR-1, GR-2, and MR were significantly higher in gill and body kidney than those in FW. Head kidney and spleen showed no significant change in these CR mRNA levels after SW-acclimation. On the other hand, leucocytes isolated from head kidney and peripheral blood showed significant decreases in mRNA levels of CR in SW-acclimated fish. These results showed differential regulation of gene expression of CR between osmoregulatory and immune systems.
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PMID:Effects of seawater acclimation on mRNA levels of corticosteroid receptor genes in osmoregulatory and immune systems in trout. 1835 26


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