Expression of the mutant (1735T-DEL) tissue-nonspecific alkaline phosphatase gene from hypophosphatasia patients

Hypophosphatasia (HOPS) is an inherited disorder characterized by defects in skeletal mineralization due to the deficiency of tissue-nonspecific alkaline phosphatase (TNSALP). To date, various mutations in the TNSALP gene have been identified. Especially, a deletion of T at position 1735 (1735T-del) located in exon 12 has been detected in three genetically unrelated Japanese patients, which seems to be one of the hot spots among the causative mutations in Japanese HOPS patients. 1735T-del causes a frame shift downstream from codon 503 (Leu), and consequently the normal termination codon at 508 is eliminated. Since a new inframe termination codon appears at codon 588 in the mutant DNA, the resultant protein is expected to have 80 additional amino acids. Expression of the mutant TNSALP gene using COS-1 cells demonstrated that the protein translated from the mutant 1735T-del had undetectable ALP activity, and its molecule size was larger than normal, as expected. Interestingly, an immunoprecipitation study of patients' sera using antibody against TNSALP revealed an abnormal protein which corresponded in size to the mutated TNSALP expressed by COS-1 cells, suggesting that the abnormal TNSALP is made by HOPS patients. The detection of TNSALP in cells transfected with 1735T-del using an immunofluorescent method exhibited only a faint signal on the cell surface, but an intense intracellular fluorescence after permeabilization.     

Defective intracellular transport of tissue-nonspecific alkaline phosphatase with an Ala162-->Thr mutation associated with lethal hypophosphatasia

We have studied the biosynthesis and intracellular transport of tissue-nonspecific alkaline phosphatase (TNSALP) transiently expressed in COS-1 cells. Mutations were introduced into TNSALP to examine the effects of a single amino acid substitution on the activity and biosynthesis of TNSALP. The cells expressing wild-type TNSALP exhibited more than 200-fold higher alkaline phosphatase activity than untransfected ones. Pulse-chase experiments showed that TNSALP was synthesized as a 66-kDa endoglucosaminidase H (Endo H)-sensitive form and converted to EndoH-resistant forms with heterogenous molecular masses ( approximately 80 kDa), which finally appeared on the cell surface as judged by digestion with phosphatidylinositol-specific phospholipase C (PI-PLC). In contrast, a TNSALP with a Glu218-->Gly mutation exhibited no phosphatase activity at all and the 66-kDa Endo H-sensitive form was the only molecular species throughout the chase in the transfected cells. In accordance with this finding, digestion with PI-PLC and immunofluorescence observation confirmed that this mutant was never expressed on the cell surface. Another mutant with a Ala162-->Thr substitution, which naturally occurs in association with a lethal hypophosphatasia, exhibited a low activity and only a small fraction of the 66-kDa form acquired Endo-H resistance and reached the cell surface. Since the wild-type and the mutant TNSALPs were labeled with [3H]ethanolamine, a component of glycosylphosphatidylinositol (GPI), it is unlikely that the impaired intracellular transport of the two mutants is due to a failure in their modification by GPI. Interestingly, the 66-kDa Endo H-sensitive form of the TNSALP mutants but not that of the wild-type, was found to form an interchain disulfide-bonded high-molecular-mass aggregate within the cells. These results suggest that impaired intracellular transport of the TNSALP (Ala162-->Thr) molecule caused by its aggregation is the molecular basis for the lethal hypophosphatasia carrying this mutation.     

Intracellular retention and degradation of tissue-nonspecific alkaline phosphatase with a Gly317-->Asp substitution associated with lethal hypophosphatasia

One point mutation which converts glycine-317 to aspartate of tissue-nonspecific alkaline phosphatase (TNSALP) was reported to be associated with lethal hypophosphatasia (Greenberg, C. R., et al. Genomics 17, 215-217, 1993). In order to define the molecular defect of TNSALP underlying the pathogenesis of hypophosphatasia, we have examined the biosynthesis of TNSALP with a Gly317-->Asp substitution. When expressed in COS-1 cells, the mutant did not exhibit alkaline phosphatase activity at all, indicating that the replacement of glycine-317 with aspartate abolishes the catalytic activity of TNSALP. Pulse-chase experiments showed that the newly synthesized mutant failed to acquire Endo H-resistance and to reach the cell surface. Interestingly, this TNSALP mutant was found to form a disulfide-bonded high-molecular-mass aggregate and was rapidly degraded within the cell, though the mutant protein was modified by glycosylphosphatidylinositol (GPI). Lactacystin, an inhibitor of the proteasome, obstructed the degradation of the mutant protein, suggesting the involvement of proteasome as a part of quality control of TNSALP.     

The C-terminal segment is essential for maintaining the quaternary structure and enzyme activity of the nitric oxide forming nitrite reductase from Achromobacter cycloclastes

We have constructed and expressed a series of mutated nitrite reductase (NIR) mutants based on the sequence of NIR from Achromobacter cycloclastes. Deleting a pentapeptide, an undecapeptide, or a heptadecapeptide from the C-terminus of NIR resulted in a series of C-terminal deletion mutated proteins designated as NIR-5, NIR-11, and NIR-17, respectively. A C-terminally extended mutated protein, NIR+8, was also produced, which contains an extra octapeptide attached to the C-terminus of the wild-type NIR. An SDS-PAGE system using tris-tricine buffer could retain the native NIR in its trimeric form, thus offering a convenient method to check the quaternary structure of NIR analogs. By using this system it was found that NIR-5 was maintained as trimer and retained 72% of wild-type enzyme activity. However, both NIR-11 and NIR-17 behaved as monomers in the SDS-PAGE and lost all their enzyme activity. Although NIR+8 maintained its trimeric structure it was enzymatically inactive. These results clearly indicate that the C-terminal undecapeptide is essential for maintaining the quaternary structure as well as the full enzymatic activity, as expected from the X-ray crystallography studies.     

A homozygous inactivating mutation in the parathyroid hormone/parathyroid hormone-related peptide receptor causing Blomstrand chondrodysplasia

We describe a patient with Blomstrand chondrodysplasia, a lethal genetic disorder characterized by extremely advanced endochondral bone maturation, in whom a homozygous missense mutation is present in the gene coding for the PTH/PTHrP receptor that leads to the substitution of a proline for a leucine in the N-terminal portion of the receptor (P132L). PTH-induced cAMP accumulation was severely reduced in COS-7 cells expressing P132L receptors compared to that of cells expressing wild-type receptors, and PTH-induced inositol phosphate accumulation was not detectable in cells expressing the mutant receptor. Similar results were obtained using PTHrP as an agonist. Maximal specific binding of radioiodinated [Tyr36]PTHrp(1-36) by cells transfected with the P132L receptor was < 10% of that observed for cells transfected with the wild-type receptor. Despite the reduction in radioligand binding to P132L receptors, the intensity and distribution of the fluorescent signal resulting from the expression of receptors fused to GFP were similar for cells transfected with the wild-type and mutant P132L receptors, suggesting a similar degree of cell surface expression. These results firmly establish the role of abnormalities in the PTH/PTHrP receptor in the pathogenesis of Blomstrand chondrodysplasia, and thereby confirm the importance of signaling through the PTH/PTHrP receptor in human fetal skeletal development. Because the amino-acid mutated in the patient described here is otherwise conserved in all mammalian class II G protein-coupled receptors, this abnormality may provide insights into structural features needed for the normal function of this family of receptors.     

Interferon-gamma activates the oxidative killing of Candida albicans by human granulocytes

The sequence proline-proline-glycine-proline is highly conserved in cytochrome P450 families 1 and 2, and similar proline rich sequences are found in other cytochromes P450. Since this sequence immediately follows the NH2-terminal hydrophobic membrane insertion signal, it potentially could function as a signal either for retention of cytochrome P450 in the endoplasmic reticulum or for its correct orientation in the membrane. To test this possibility, DNA sequence coding for this tetrapeptide was deleted from cytochrome P450 2C2 cDNA. Translation of the mutated mRNA in a reticulocyte cell-free system containing canine microsomal membranes resulted in the insertion of the protein into the membrane with a topology indistinguishable from that of normal cytochrome P450 2C2. The mutated protein was expressed in COS1 cells and its distribution, assayed by immunofluorescence, was similar to that of cytochrome P450 2C2. Furthermore, if a short peptide containing a potential glycosylation site was fused to the N-terminus of the mutant protein, the new hybrid protein was glycosylated in COS1 cells and the carbohydrate moiety remained sensitive to cleavage by endoglycosidase H. These results indicate that the protein was inserted and retained in the endoplasmic reticulum membrane. Pulse-chase studies showed that the mutated protein was degraded about four times as fast as cytochrome P450 2C2. In contrast to cytochrome P450 2C2, no (omega-1) hydroxylase activity was detected in COS1 cells expressing the mutated protein at similar steady-state levels as the wild-type protein. These results indicate that, although the conserved PPGP tetrapeptide is not required for cellular localization of cytochrome P450 in the endoplasmic reticulum membrane, its deletion decreases the stability of the protein and abolishes enzymatic activity.     

Role of tyrosine 129 in the active site of spinach glycolate oxidase

The enzymatic properties and the three-dimensional structure of spinach glycolate oxidase which has the active-site Tyr129 replaced by Phe (Y129F glycolate oxidase) has been studied. The structure of the mutant is unperturbed which facilitates interpretation of the biochemical data. Y129F glycolate oxidase has an absorbance spectrum with maxima at 364 and 450 nm (epsilon max = 11400 M-1 cm-1). The spectrum indicates that the flavin is in its normal protonated form, i.e. the Y129F mutant does not lower the pKa of the N(3) of oxidized flavin as does the wild-type enzyme [Macheroux, P., Massey, V., Thiele, D. J., and Volokita, M. (1991) Biochemistry 30, 4612-4619]. This was confirmed by a pH titration of Y129F glycolate oxidase which showed that the pKa is above pH 9. In contrast to wild-type glycolate oxidase, oxalate does not perturb the absorbance spectrum of Y129F glycolate oxidase. Moreover oxalate does not inhibit the enzymatic activity of the mutant enzyme. Typical features of wild-type glycolate oxidase that are related to a positively charged lysine side chain near the flavin N(1)-C(2 = O), such as stabilization of the anionic flavin semiquinone and formation of tight N(5)-sulfite adducts, are all conserved in the Y129F mutant protein. Y129F glycolate oxidase exhibited about 3.5% of the wild-type activity. The lower turnover number for the mutant of 0.74 s-1 versus 20 s-1 for the wild-type enzyme amounts to an increase of the energy of the transition state of about 7.8 kJ/mol. Steady-state analysis gave Km values of 1.5 mM and 7 microM for glycolate and oxygen, respectively. The Km for glycolate is slightly higher than that found for wild-type glycolate oxidase (1 mM) whereas the Km for oxygen is much lower. As was the case for wild-type glycolate oxidase, reduction was found to be the rate-limiting step in catalysis, with a rate of 0.63 s-1. The kinetic properties of Y129F glycolate oxidase provide evidence that the main function of the hydroxyl group of Tyr129 is the stabilization of the transition state.     

Single point mutations distributed in 10 soluble and membrane regions of the Nicotiana plumbaginifolia plasma membrane PMA2 H+-ATPase activate the enzyme and modify the structure of the C-terminal region

The Nicotiana plumbaginifolia pma2 (plasma membrane H+-ATPase) gene is capable of functionally replacing the H+-ATPase genes of the yeast Saccharomyces cerevisiae, provided that the external pH is kept above 5.0. Single point mutations within the pma2 gene were previously identified that improved H+-ATPase activity and allowed yeast growth at pH 4.0. The aim of the present study was to identify most of the PMA2 positions, the mutation of which would lead to improved growth and to determine whether all these mutations result in similar enzymatic and structural modifications. We selected additional mutants in total 42 distinct point mutations localized in 30 codons. They were distributed in 10 soluble and membrane regions of the enzyme. Most mutant PMA2 H+-ATPases were characterized by a higher specific activity, lower inhibition by ADP, and lower stimulation by lysophosphatidylcholine than wild-type PMA2. The mutants thus seem to be constitutively activated. Partial tryptic digestion and immunodetection showed that the PMA2 mutants had a conformational change making the C-terminal region more accessible. These data therefore support the hypothesis that point mutations in various H+-ATPase parts displace the inhibitory C-terminal region, resulting in enzyme activation. The high density of mutations within the first half of the C-terminal region suggests that this part is involved in the interaction between the inhibitory C-terminal region and the rest of the enzyme.     

Effect of cleavage mutants on syncytium formation directed by the wild-type fusion protein of Newcastle disease virus

The effects of Newcastle disease virus (NDV) fusion (F) glycoprotein cleavage mutants on the cleavage and syncytium-forming activity of the wild-type F protein were examined. F protein cleavage mutants were made by altering amino acids in the furin recognition region (amino acids 112 to 116) in the F protein of a virulent strain of NDV. Four mutants were made: Q114P replaced the glutamine residue with proline; K115G replaced lysine with glycine; double mutant K115G, R113G replaced both a lysine and an arginine with glycine residues; and a triple mutant, R112G, K115G, F117L, replaced three amino acids to mimic the sequence found in avirulent strains of NDV. All mutants except Q114P were cleavage negative and fusion negative. However, addition of exogenous trypsin cleaved all mutant F proteins and activated fusion. As expected for an oligomeric protein, the fusion-negative mutants had a dominant negative phenotype: cotransfection of wild-type and mutant F protein cDNAs resulted in an inhibition of syncytium formation. The presence of the mutant F protein did not inhibit cleavage of the wild-type protein. Furthermore, evidence is presented that suggests that the mutant protein and the wild-type protein formed heterooligomers. By measuring the syncytium-forming activity of the wild-type protein at various ratios of expression of mutant and wild-type protein, results were obtained that are most consistent with the notion that the size of the functionally active NDV F protein in these assays is a single oligomer, likely a trimer. That a larger oligomer, containing a mix of both wild-type and mutant F proteins, has partial activity cannot, however, be ruled out.     

The antibiotic thiostrepton inhibits a functional transition within protein L11 at the ribosomal GTPase centre

A newly identified class of highly thiostrepton-resistant mutants of the archaeon Halobacterium halobium carry a missense mutation at codon 18 within the gene encoding ribosomal protein L11. In the mutant proteins, a proline, conserved in archaea and bacteria, is converted to either serine or threonine. The mutations do not impair either the assembly of the mutant L11 into 70 S ribosomes in vivo or the binding of thiostrepton to ribosomes in vitro. Moreover, the corresponding mutations at proline 22, in a fusion protein of L11 from Escherichia coli with glutathione-S-transferase, did not reduce the binding affinities of the mutated L11 fusion proteins for rRNA of of thiostrepton for the mutant L11-rRNA complexes at rRNA concentrations lower than those prevailing in vivo. Probing the structure of the fusion protein of wild-type L11, from E. coli, using a recently developed protein footprinting technique, demonstrated that a general tightening of the C-terminal domain occurred on rRNA binding, while thiostrepton produced a footprint centred on tyrosine 62 at the junction of the N and C-terminal domains of protein L11 complexed to rRNA. The intensity of this protein footprint was strongly reduced for the mutant L11-rRNA complexes. These results indicate that although, as shown earlier, thiostrepton binds primarily to 23 S rRNA, the drug probably inhibits peptide elongation by impeding a conformational change within protein L11 that is important for the function of the ribosomal GTPase centre. This putative inhibitory mechanism of thiostrepton is critically dependent on proline 18/22. Moreover, the absence of this proline from eukaryotic protein L11 sequences would account for the high thiostrepton resistance of eukaryotic ribosomes.     

Mutagenesis of active-site histidines of Listeria monocytogenes phosphatidylinositol-specific phospholipase C: effects on enzyme activity and biological function

Listeria monocytogenes, a gram-positive facultative intracellular pathogen, produces two distinct phospholipases C. PC-PLC, encoded by plcB, is a broad-range phospholipase, whereas PI-PLC, encoded by plcA, is specific for phosphatidylinositol. It was previously shown that PI-PLC plays a role in efficient escape of L. monocytogenes from the primary phagosome. To further understand the function of PI-PLC in intracellular growth, site-directed mutagenesis of plcA was performed. Two potential active-site histidine residues were mutated independently to alanine, serine, and phenylalanine. With the exception of the activity of the enzyme containing H38F, which was unstable, the PI-PLC enzyme activities of culture supernatants containing each mutant enzyme were <1% of wild-type activity. In addition, the levels of expression of the mutant PI-PLC proteins were equivalent to wild-type expression. Derivatives of L. monocytogenes containing these specific plcA mutations were found to have phenotypes similar to that of the plcA deletion strain in an assay for escape from the primary vacuole, in intracellular growth in a murine macrophage cell line, and in a plaquing assay for cell-to-cell spread. Thus, catalytic activity of PI-PLC is required for all its intracellular functions.     

Envelope formation is blocked by mutation of a sequence related to the HKD phospholipid metabolism motif in the vaccinia virus F13L protein

The outer envelope of the extracellular form of vaccinia virus is derived from Golgi membranes that have been modified by the insertion of specific viral proteins, of which the major component is the 37-kDa, palmitylated, nonglycosylated product of the F13L gene. The F13L protein contains a variant of the HKD (His-Lys-Asp) motif, which is conserved in numerous enzymes of phospholipid metabolism. Vaccinia virus mutants with a conservative substitution of either the K (K314R) or the D (D319E) residue of the F13L protein formed only tiny plaques similar to those produced by an F13L deletion mutant, were unable to produce extracellular enveloped virions, and failed to mediate low-pH-induced fusion of infected cells. Membrane-wrapped forms of intracellular virus were rarely detected in electron microscopic images of cells infected with either of the mutants. Western blotting and pulse-chase experiments demonstrated that the D319E protein was less stable than either the K314R or wild-type F13L protein. Most striking, however, was the failure of either of the two mutated proteins to concentrate in the Golgi compartment. Palmitylation, oleation, and partitioning of the F13L protein in Triton X-114 detergent were unaffected by the K314R substitution. These results indicated that the F13L protein must retain the K314 and D319 for it to localize in the Golgi compartment and function in membrane envelopment of vaccinia virus.     

The molecular basis for cross-reacting material-positive hemophilia A due to missense mutations within the A2-domain of factor VIII

Factor VIII (FVIII) is the protein defective in the bleeding disorder hemophilia A. Approximately 5% of hemophilia A patients have normal amounts of a dysfunctional FVIII protein and are termed cross-reacting material (CRM)-positive. The majority of genetic alterations that result in CRM-positive hemophilia A are missense mutations within the A2-domain. To determine the mechanistic basis of the genetic defects within the A2-domain for FVIII function we constructed six mutations within the FVIII cDNA that were previously found in five CRM-positive hemophilia A patients (R527W, S558F, I566T, V634A, and V634M) and one CRM-reduced hemophilia A patient (DeltaF652/3). The specific activity for each mutant secreted into the conditioned medium from transiently transfected COS-1 cells correlated with published data for the patients plasma-derived FVIII, confirming the basis of the genetic defect. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of immunoprecipitated FVIII protein radiolabeled in COS-1 cells showed that all CRM-positive mutant proteins were synthesized and secreted into the medium at rates similar to wild-type FVIII. The majority of the DeltaF652/3 mutant was defective in secretion and was degraded within the cell. All mutant FVIII proteins were susceptible to thrombin cleavage, and the A2-domain fragment from the I566T mutant had a reduced mobility because of use of an introduced potential N-linked glycosylation site that was confirmed by N-glycanase digestion. To evaluate interaction of FVIII with factor IXa, we performed an inhibition assay using a synthetic peptide corresponding to FVIII residues 558 to 565, previously shown to be a factor IXa interaction site. The concentration of peptide required for 50% inhibition of FVIII activity (IC50) was reduced for the I566T (800 mumol/L) and the S558F (960 mumol/L) mutants compared with wild-type FVIII (> 2,000 mumol/L). N-glycanase digestion increased I566T mutant FVIII activity and increased its IC50 for the peptide (1,400 mumol/L). In comparison to S558F, a more conservative mutant (S558A) had a sixfold increased specific activity that also correlated with an increased IC50 for the peptide. These results provided support that the defects in the I566T and S558F FVIII molecules are caused by steric hindrance for interaction with factor IXa.     

Site-directed mutational analysis for the membrane binding of DnaA protein. Identification of amino acids involved in the functional interaction between DnaA protein and acidic phospholipids

DnaA protein, the initiator of chromosomal DNA replication in Escherichia coli, interacts with acidic phospholipids, such as cardiolipin, and its activity seems to be regulated by membrane binding in cells. In this study we introduced site-directed mutations at the positions of hydrophobic or basic amino acids which are conserved among various bacteria species and which are located in the putative membrane-binding region of DnaA protein (from Asp357 to Val374). All mutant DnaA proteins showed much the same ATP and ADP binding activity as that of the wild-type protein. The release of ATP bound to the mutant DnaA protein, in which three hydrophobic amino acids were mutated to hydrophilic ones, was stimulated by cardiolipin, as in the case of the wild-type protein. On the other hand, the release of ATP bound to another mutant DnaA protein, in which three basic amino acids were mutated to acidic ones, was not stimulated by cardiolipin. These results suggest not only that the region is a membrane-binding domain of DnaA protein but also that these basic amino acids are important for the binding and the ionic interaction between the basic amino acids and acidic residues of cardiolipin and is involved in the interaction between DnaA protein and cardiolipin.     

Ferrichrome transport in Escherichia coli K-12: altered substrate specificity of mutated periplasmic FhuD and interaction of FhuD with the integral membrane protein FhuB

FhuD is the periplasmic binding protein of the ferric hydroxamate transport system of Escherichia coli. FhuD was isolated and purified as a His-tag-labeled derivative on a Ni-chelate resin. The dissociation constants for ferric hydroxamates were estimated from the concentration-dependent decrease in the intrinsic fluorescence intensity of His-tag-FhuD and were found to be 0.4 microM for ferric aerobactin, 1.0 microM for ferrichrome, 0.3 microM for ferric coprogen, and 5.4 microM for the antibiotic albomycin. Ferrichrome A, ferrioxamine B, and ferrioxamine E, which are poorly taken up via the Fhu system, displayed dissociation constants of 79, 36, and 42 microM, respectively. These are the first estimated dissociation constants reported for a binding protein of a microbial iron transport system. Mutants impaired in the interaction of ferric hydroxamates with FhuD were isolated. One mutated FhuD, with a W-to-L mutation at position 68 [FhuD(W68L)], differed from wild-type FhuD in transport activity in that ferric coprogen supported promotion of growth of the mutant on iron-limited medium, while ferrichrome was nearly inactive. The dissociation constants of ferric hydroxamates were higher for FhuD(W68L) than for wild-type FhuD and lower for ferric coprogen (2.2 microM) than for ferrichrome (156 microM). Another mutated FhuD, FhuD(A150S, P175L), showed a weak response to ferrichrome and albomycin and exhibited dissociation constants two- to threefold higher than that of wild-type FhuD. Interaction of FhuD with the cytoplasmic membrane transport protein FhuB was studied by determining protection of FhuB degradation by trypsin and proteinase K and by cross-linking experiments. His-tag-FhuD and His-tag-FhuD loaded with aerobactin specifically prevented degradation of FhuB and were cross-linked to FhuB. FhuD loaded with substrate and also FhuD free of substrate were able to interact with FhuB.     

Role of carboxyl-terminal charges on S-modulin membrane affinity and inhibition of rhodopsin phosphorylation

S-Modulin shows a higher affinity for urea-stripped frog rod outer segment membranes than s26 (a cone homologue of S-modulin). NaCl at a concentration of several hundred millimolar reduced the membrane affinity of S-modulin to the s26 level. Chimeric S-modulin and s26 whose respective 23 and 29 amino acids at the carboxyl terminus were swapped showed membrane affinites similar to those of s26 and S-modulin, respectively. The membrane affinity of an S-modulin mutant lacking C-terminal positive charges was reduced to the s26 level, while another S-modulin mutant lacking C-terminal negative charges has a higher membrane affinity than wild-type S-modulin. When the molar ratio of recombinant S-modulins to rhodopsin is 0.5, there was no large difference in the inhibition efficiency. However, S-modulin and mutants with high membrane affinities inhibit rhodopsin phosphorylation more efficiently than s26 and mutants with low membrane affinities at the molar ratio of 0.1. These results indicate that the C-terminal positive charges of these Ca2+-binding proteins enhance the membrane affinity and the inhibitory effect on rhodopsin phosphorylation by increasing the concentration of S-modulin on the membrane.     

A case of lung adenocarcinoma associated with remarkably high levels of CA 19-9 and lymphangitis carcinomatosa

A mutant of Methanobacterium thermoautotrophicum with a lesion in membrane Na+-translocating ATPase (synthase) was isolated. The total ATPase activity in permeabilized cells of this mutant was elevated three-fold as compared with the wild-type strain. In contrast to wild-type cells, mutant ATPase was neither inhibited by DCCD nor stimulated by Na+ ions. The methane formation orate of the mutant cells at pH 7.5 under non-growing conditions was nearly twice that of the wild-type strain and was stimulated by sodium ions. On the other hand, the ATP synthesis driven by methanogenesis under the same conditions was lower that of the wild-type under the same conditions, and contrary to the wild-type was not stimulated by Na+ ions. ATP synthesis driven by a potassium diffusion potential in the presence of sodium ions was markedly diminished in the mutant cells. The membrane potential values of the wild-type and the mutant cells in the presence of 10 mM NaCl at pH 7.0 were comparable at energized conditions (-223 mV and -230 mV respectively). The Mg2+-dependent ATPase activity of the 10(5) x g supernatant of broken cells from the mutant cells was 30% higher than in the wild-type. On the other hand, two bands with Mg2+-dependent ATPase activity were identified by native PAGE in this fraction in both wild-type as well as in mutant. These data suggest that the binding of Na+-translocating ATPase (synthase) to the membrane spanning part is changed in the mutant strain.     

The assessment of child sexual abuse allegations: using research to guide clinical decision making

Escherichia coli DNA gyrase B subunit (GyrB) is composed of a 43-kDa N-terminal domain containing an ATP-binding site and a 47-kDa C-terminal domain involved in the interaction with the gyrase A subunit (GyrA). Site-directed mutagenesis was used to substitute, in both the entire GyrB subunit and its 43-kDa N-terminal fragment, the amino acid Y5 by either a serine (Y5S) or a phenylalanine residue (Y5F). Under standard conditions, cells bearing Y5S or Y5F mutant GyrB expression plasmids produced significantly less recombinant proteins than cells transformed with the wild-type plasmid. This dramatic decrease in expression of mutant GyrB proteins was not observed when the corresponding N-terminal 43 kDa mutant plasmids were used. Examination of the plasmid content of the transformed cells after induction showed that the Y5F and Y5S GyrB protein level was correlated with the plasmid copy number. By repressing tightly the promoter activity encoded by these expression vectors during cell growth, it was possible to restore the normal level of the mutant GyrB encoding plasmids in the transformed bacteria. Treatment with chloramphenicol before protein induction enabled large overexpression of the GyrB mutant Y5F and Y5S proteins. In addition, the decrease in plasmid copy number was also observed when the 47-kDa C-terminal fragment of the GyrB subunit was expressed in bacteria grown under standard culture conditions. Analysis of DNA supercoiling and relaxation activities in the presence of GyrA demonstrated that purified Y5-mutant GyrB proteins were deficient for ATP-dependent gyrase activities. Taken together, these results show that Y5F and Y5S mutant GyrB proteins, but not the corresponding 43-kDa N-terminal fragments, compete in vivo with the bacterial endogenous GyrB subunit of DNA gyrase, thereby reducing the plasmid copy number in the transformed bacteria by probably acting on the level of negative DNA supercoiling in vivo. This competition could be mediated by the presence of the intact 47-kDa C-terminal domain in the Y5F and Y5S mutant GyrB subunits. This study demonstrates also that the amino acid Y5 is a crucial residue for the expression of the gyrase B activity in vivo. Thus, our in vivo approach may also be useful for detecting other important amino acids for DNA gyrase activity, as mutations affecting the ATPase activity or GyrB/GyrB, or GyrB/GyrA protein interactions.     

The oligomycin sensitivity conferring protein of rat liver mitochondrial ATP synthase: arginine 94 is important for the binding of OSCP to F1

The oligomycin sensitivity conferring protein (OSCP) is an essential subunit of the mitochondrial ATP synthase (F0F1) long regarded as being directly involved in the energetic coupling of proton transport to ATP synthesis. To gain insight into the function of OSCP, mutations were made in a highly conserved central region of the subunit, and the recombinant proteins were studied using several biochemical assays. Rat liver OSCP was expressed to high levels in Escherichia coli, solubilized from inclusion bodies, renatured, and purified to homogeneity. The recombinant protein was able to reconstitute oligomycin-sensitive ATPase activity to inner membrane vesicles depleted of F1 and OSCP, and bound to F1 with a stoichiometry of 1:1. A novel fluorescence anisotropy assay was developed to study the affinity of binding of F1 to OSCP, providing a Kd value of 51 +/- 11 nM. Two highly conserved, charged residues (E91 and R94) which lie within the central region of OSCP were mutated, and the recombinant proteins (E91Q, R94Q, and R94A) were purified to homogeneity and judged by CD spectroscopy to have structures similar to that of the wild-type protein. Both R94 mutants demonstrated little or no binding to F1, while the E91Q bound in a manner identical to that of wild-type OSCP. Significantly, all three mutant proteins were able to reconstitute F1 with membranes and to confer oligomycin sensitivity to the same extent as wild-type OSCP. These results demonstrate that a single tight binding site exists on isolated rat liver F1 for OSCP, and implicate arginine 94 as playing a critical role in this site. In addition, these results indicate that this tight binding site is not required for conferral of oligomycin sensitivity to the reconstituted F0F1 complex.