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Biomedical subjects

A Maxwell

Publications and source records attributed to A Maxwell.

At least 19 recordsLinked to original sources
PubMed2000

The additional 165 amino acids in the B protein of Escherichia coli DNA gyrase have an important role in DNA binding.

DNA gyrase is the only enzyme known to negatively supercoil DNA. The enzyme is a heterotetramer of A(2)B(2) subunit composition. Alignment of the primary sequence of gyrase B (GyrB) from various species shows that they can be grouped into two classes. The GyrB of Gram-negative eubacteria has a stretch of about 165 amino acids in the C-terminal half, which is lacking in other GyrB subunits and type II topoisomerases. In Escherichia coli, no function has so far been attributed to this stretch. In this study, we have tried to assess the function of this region both in vivo and in vitro. A deletant (GyrBDelta160) lacking this region is non-functional in vivo. The holoenzyme reconstituted from gyrase A (GyrA) and GyrBDelta160 shows reduced but detectable supercoiling and quinolone-induced cleavage activity in vitro. GyrBDelta160 retains its ability to bind to GyrA and novobiocin. However, when reconstituted with GyrA, the deletant shows greatly impaired DNA binding. The intrinsic ATPase activity of the GyrBDelta160 is comparable to that of wild type GyrB, but this activity is not stimulated by DNA. These studies indicate that the additional stretch present in GyrB is essential for the DNA binding ability of E. coli gyrase.

Adenosine Triphosphatases↗
PubMed2000

Temperature-sensitive suppressor mutations of the Escherichia coli DNA gyrase B protein.

Escherichia coli strain LE316 contains a mutation in gyrB that results in the substitution of Val164 to Gly and confers both chlorobiocin resistance and temperature sensitivity. Selection for suppressors of the ts phenotype yielded second-site mutations in GyrB at His38 and Thr157. The properties of proteins bearing these mutations have been characterized, and a mechanism of suppression is proposed based upon structural considerations.

Antibiotics, Antineoplastic↗
PubMed2000

Tectoridins modulate skeletal and cardiac muscle sarcoplasmic reticulum calcium-release channels.

The isoflavones tectoridin (TTR) and 3'-hydroxy TTR (3'-TTR) were isolated from an Ayurvedic herbal preparation Vacä and evaluated for their affinity and effect on ryanodine receptors (RyR) using junctional sarcoplasmic reticulum vesicles (JSRVs). In [(3)H]ryanodine displacement binding affinity assays, TTR and 3'-TTR exhibited IC(50) values of 17.3 +/- 1.3 microM (K(d) = 6.7 +/- 0.4 microM) and 6.6 +/- 1.4 microM (K(d) = 2.4 +/- 0.2 microM), respectively, for fast skeletal muscle RyR (RyR1) compared with an IC(50) value for ryanodine of 6.2 +/- 0.4 nM (K(d) = 2.4 nM). TTR demonstrated a 3-fold higher affinity for cardiac RyR (RyR2) [IC(50) value of 5.2 +/- 0.6 microM (K(d) = 0.95 +/- 0.3 microM)] than for RyR1. The displacement isotherms for both TTRs paralleled that for ryanodine, consistent with the notion that all three are likely binding to similar site(s) on the receptors. Calcium efflux from and calcium influx into JSRVs were used to measure function effects of TTRs on binding to RyR. In calcium efflux assays, TTR (up to 1 mM) enhanced the release of (45)Ca(2+) from JSRVs in a concentration-dependent manner (EC(50act) of 750 microM). Higher concentrations deactivated (partially closed) RyR1. 3'-TTR had similar effects, but was approximately 2-fold more potent, exhibiting an EC(50act) value of 480 microM. Using passive calcium influx assays, TTR activated and deactivated RyR1 in a time- and concentration-dependent manner. The aglycone tectorigenin also was effective in displacing [(3)H]ryanodine from RyR1 but not from RyR2. These results demonstrate that TTRs are capable of interacting at ryanodine binding sites to differentially modulate fast skeletal and cardiac calcium-release channels.

Animals↗
PubMed1999

The interaction of DNA gyrase with the bacterial toxin CcdB: evidence for the existence of two gyrase-CcdB complexes.

CcdB is a bacterial toxin that targets DNA gyrase. Analysis of the interaction of CcdB with gyrase reveals two distinct complexes. An initial complex (alpha) is formed by direct interaction between GyrA and CcdB; this complex can be detected by affinity column and gel-shift analysis, and has a proteolytic signature which is characterised by a 49 kDa fragment of GyrA. Surface plasmon resonance shows that CcdB binds to the N-terminal domain of GyrA with high affinity. In this mode of binding, CcdB does not affect the ability of gyrase to hydrolyse ATP or promote supercoiling. Incubation of this initial complex with ATP in the presence of GyrB and DNA slowly converts it to a second complex (beta), which has a lower rate of ATP hydrolysis and is unable to catalyse supercoiling. The efficiency of formation of this inactive complex is dependent on the concentrations of ATP and CcdB. We suggest that the conversion between the two complexes proceeds via an intermediate, whose formation is dependent on the rate of ATP hydrolysis.

Adenosine Triphosphatases↗
PubMed1999

Locking the DNA gate of DNA gyrase: investigating the effects on DNA cleavage and ATP hydrolysis.

Supercoiling by DNA gyrase involves the passage of one segment of double-stranded DNA through another. This requires a DNA duplex to be cleaved and the broken ends separated by at least 20 A. This is accomplished by the opening of a dimer interface, termed the DNA gate, which is covalently attached to the broken ends of the DNA. After strand passage, the DNA gate closes allowing the reunion of the broken ends. We have cross-linked the DNA gate of gyrase using cysteine cross-linking to block gate opening. We show that this locked gate mutant can bind quinolone drugs and perform DNA cleavage. However, locking the DNA gate prevents strand passage and the ability of DNA to stimulate ATP hydrolysis. We discuss the mechanistic implications of these results.

Adenosine Triphosphate↗
PubMed1999

Probing the two-gate mechanism of DNA gyrase using cysteine cross-linking.

Cross-linking a pair of novel cysteine residues on either side of the bottom dimer interface of DNA gyrase blocks catalytic supercoiling. Limited strand passage is allowed, but release of the transported DNA segment (T segment) via opening of the bottom dimer interface is prevented. In contrast, ATP-independent relaxation of negatively supercoiled DNA is completely abolished, suggesting that T-segment entry via the bottom gate is blocked. These findings support a two-gate model for supercoiling by DNA gyrase and suggest that relaxation by gyrase is the reverse of supercoiling. Cross-linking a truncated version of gyrase (A64(2)B2), which lacks the DNA wrapping domains, does not block ATP-dependent relaxation. This indicates that passage of DNA through the bottom dimer interface is not essential for this reaction. The mechanistic implications of these results are discussed.

Adenosine Triphosphate↗
PubMed1999

A model for the mechanism of strand passage by DNA gyrase.

The mechanism of type II DNA topoisomerases involves the formation of an enzyme-operated gate in one double-stranded DNA segment and the passage of another segment through this gate. DNA gyrase is the only type II topoisomerase able to introduce negative supercoils into DNA, a feature that requires the enzyme to dictate the directionality of strand passage. Although it is known that this is a consequence of the characteristic wrapping of DNA by gyrase, the detailed mechanism by which the transported DNA segment is captured and directed through the DNA gate is largely unknown. We have addressed this mechanism by probing the topology of the bound DNA segment at distinct steps of the catalytic cycle. We propose a model in which gyrase captures a contiguous DNA segment with high probability, irrespective of the superhelical density of the DNA substrate, setting up an equilibrium of the transported segment across the DNA gate. The overall efficiency of strand passage is determined by the position of this equilibrium, which depends on the superhelical density of the DNA substrate. This mechanism is concerted, in that capture of the transported segment by the ATP-operated clamp induces opening of the DNA gate, which in turn stimulates ATP hydrolysis.

Adenosine Triphosphatases↗
PubMed1999

Thermodynamic studies on anion binding to apotransferrin and to recombinant transferrin N-lobe half molecules.

Equilibrium constants for the binding of anions to apotransferrin, to the recombinant N-lobe half transferrin molecule (Tf/2N), and to a series of mutants of Tf/2N have been determined by difference UV titrations of samples in 0.1 M Hepes buffer at pH 7.4 and 25 degrees C. The anions included in this study are phosphate, sulfate, bicarbonate, pyrophosphate, methylenediphosphonic acid, and ethylenediphosphonic acid. There are no significant differences between anion binding to Tf/2N and anion binding to the N-lobe of apotransferrin. The binding of simple anions like phosphate appears to be essentially equivalent for the two apotransferrin binding sites. The binding of pyrophosphate and the diphosphonates is inequivalent, and the studies on the recombinant Tf/2N show that the stronger binding is associated with the N-terminal site. Anion binding constants for phosphate, pyrophosphate, and the diphosphonates with the N-lobe mutants K206A, K296A, and R124A have been determined. Anion binding tends to be weakest for the K296A mutant, but the variation in log K values among the three mutants is surprisingly small. It appears that the side chains of K206, K296, and R124 all make comparable contributions to anion binding. There are significant variations in the intensities of the peaks in the difference UV spectra that are generated by the titrations of the mutant apoproteins with these anions. These differences appear to be related more to variations in the molar extinction coefficients of the anion-protein complexes rather than to differences in binding constants.

Anions↗
PubMed1999

Probing the binding of coumarins and cyclothialidines to DNA gyrase.

DNA gyrase is the target of a number of antibacterial agents, including the coumarins and the cyclothialidines. To extend our understanding of the mechanism of action of these compounds, we have examined the previously published crystal structures of the complexes between the 24 kDa fragment of GyrB and coumarin and cyclothialidine drugs and made mutations by site-directed mutagenesis. We used proteolysis as a probe of drug binding to wild-type and mutant proteins. Limited proteolysis of gyrase revealed that binding of these antibiotics is associated with a characteristic proteolytic fingerprint, suggesting a drug-induced conformational change. The ability of the mutants to bind the drugs was studied by testing their ability to induce the coumarin-associated proteolytic signature and to bind to a novobiocin-affinity column. To analyze further the interaction of the drugs with gyrase, we studied the binding using surface plasmon resonance. Mutation of Asn46 to Asp has only a modest effect on the binding of coumarins, while an Asn46 to Leu mutation results in a 10-fold decrease in the affinity. Mutation of Asp73 to Asn completely abolishes binding to both coumarins and cyclothialidines. Mutations at these residues also abolish ATP hydrolysis, explaining the inability of such mutations to occur spontaneously.

Adenosine Triphosphate↗
PubMed1999

Stability of a functional murine satellite DNA-based artificial chromosome across mammalian species.

A 60-Mb murine chromosome consisting of murine pericentric satellite DNA and two bands of integrated marker and reporter genes has been generated de novo in a rodent/human hybrid cell line (mM2C1). This prototype mammalian artificial chromosome platform carries a normal centromere, and the expression of its beta-galactosidase reporter gene has remained stable under selection for over 25 months. The novel chromosome was transferred by a modified microcell fusion method to mouse [L-M(TK-)], bovine (P46) and human (EJ30) cell lines. In all cases, the chromosome remained structurally and functionally intact under selection for periods exceeding 3 months from the time of transfer into the new host. In addition, the chromosome was retained in three first-generation tumours when L-M(TK-) cells containing the chromosome were xenografted in severe combined immunodeficiency mice. These data support that a murine satellite DNA-based artificial chromosome can be used as a functional mammalian artificial chromosome and can be maintained in vivo and in cells of heterologous species in vitro.

Animals↗
PubMed1999

Serum insulin-like growth factor (IGF)-I concentrations are reduced by short-term dietary restriction and restored by refeeding in domestic cats (Felis catus).

Nutritional modulation of insulin-like growth factors (IGF) and their binding proteins (IGFBP) is well established. The effect of nutritional restriction on the serum IGF/IGFBP system of adult cats was investigated to evaluate serum IGF-I as a biochemical marker of nutritional status. Assays for measuring feline serum IGF and IGFBP were validated and normal ranges established in a study population of 46 healthy nonobese adult cats. Serum concentrations of IGF-I and IGF-II correlated significantly with body weight (r = 0.75, P < 0. 0001 and r = 0.34, P < 0.03, respectively). Serum IGFBP profiles were similar to other species, including humans, dogs and guinea pigs. IGFBP-3 was the predominant binding protein reflecting IGF-I concentrations and body size. Serum IGFBP-2 concentrations were high relative to the normal human serum pool (NHS) control. Food withdrawal for 18 h followed by refeeding did not alter circulating IGF or IGFBP concentrations, including IGFBP-1, in nine cats. Short-term dietary restriction of nine adult cats to supply initially 56% (56%M) and then 42.5% (42.5%M) of calculated maintenance energy requirements for 14 d resulted in a significant weight loss (P < 0.01). However, serum IGF-I concentrations fell significantly (-51%, P < 0.01) only with 42.5%M restriction. Serum IGF-II, IGFBP, insulin and albumin concentrations were not altered during the study. We conclude that nutrition does modulate the adult feline IGF/IGFBP system, but to a lesser extent than in other species. Further evaluation is required before serum IGF-I can be used for the assessment of nutritional status in adult cats.

Animals↗
PubMed1998

The N-terminal domain of human topoisomerase IIalpha is a DNA-dependent ATPase.

We have constructed clones encoding N-terminal fragments of human DNA topoisomerase IIalpha. We show that the N-terminal domain (approximately 50 kDa) has an intrinsic ATPase activity that can be stimulated by DNA. The enzyme obeys Michaelis-Menten kinetics showing a approximately 6-fold increase in kcat in the presence of DNA. Cross-linking studies indicate that the N-terminal domain is a dimer in the absence and presence of nucleotides. Using site-directed mutagenesis, we have identified the catalytic residue for ATP hydrolysis as Glu86. Phosphorylation of the N-terminal domain with protein kinase C does not affect the ATPase activity. The ATPase domain of human topoisomerase IIalpha shows significant differences from its counterpart in DNA gyrase and we discuss the mechanistic implications of these data.

Adenosine Triphosphatases↗
PubMed1998

Hydrolysis of ATP at only one GyrB subunit is sufficient to promote supercoiling by DNA gyrase.

Mutation of Glu42 to Ala in the B subunit of DNA gyrase abolishes ATP hydrolysis but not nucleotide binding. Gyrase complexes that contain one wild-type and one Ala42 mutant B protein were formed, and the ability of such complexes to hydrolyze ATP was investigated. We found that ATP hydrolysis was able to proceed independently only in the wild-type subunit, albeit at a lower rate. With only one ATP molecule hydrolyzed at a time, gyrase could still perform supercoiling, but the limit of this reaction was lower than that observed when both subunits can hydrolyze the nucleotide.

Adenosine Triphosphate↗
PubMed1998

Kinetic studies on the removal of iron and aluminum from recombinant and site-directed mutant N-lobe half transferrins.

Kinetic studies have been conducted in pH 7.4 Hepes buffer at 25 degreesC on the removal of Fe(III) and Al(III) from the recombinant N-lobe half molecule of human serum transferrin (Tf/2N) and from the R124A, K206A, and K296A mutants of this protein. The rates of iron removal from Tf/2N by 3-hydroxypyridin-4-one (deferiprone) and nitrilotriacetic acid (NTA) are essentially identical with previous results on N-terminal monoferric transferrin (Tf-FeN). For both Tf/2N and Tf-FeN, iron removal by deferiprone follows simple saturation kinetics, while iron removal by NTA follows simple first-order kinetics. There is some discrepancy between the two proteins with respect to iron removal by PPi, but this may be due to differences in the chloride concentrations among different studies. The addition of Fe(NTA)2 to R124A at ambient bicarbonate concentrations forms the Fe-NTA-Tf ternary complex, but the usual Fe-CO3-Tf complex can be formed by adding ferrous ion in the presence of a larger excess of bicarbonate. This complex releases its iron very rapidly by a mechanism that is first-order with respect to the ligand. This suggests that the first-order component of metal release from transferrin involves the displacement of the synergistic carbonate anion. Since iron removal from K206A and K296A at pH 7.4 is extremely slow, studies have been conducted on the more labile Al3+ complexes of Tf/2N, K206A, and K296A. The removal of Al3+ from Tf/2N by PPi follows the same complex kinetic order with respect to the ligand concentration that is observed for iron removal, while the removal of Al3+ from both K206A and K296A reverts to a simple saturation process. The addition of perchlorate retards the removal of Al3+ from both K206A and K296A, suggesting that these lysine residues are not associated with the allosteric effects of inorganic anions on the rates of metal removal.

Alanine↗
PubMed1998

Conformational changes in DNA gyrase revealed by limited proteolysis.

We have used limited proteolysis to identify conformational changes in DNA gyrase. Gyrase exhibits a proteolytic fingerprint dominated by two fragments, one of approximately 62 kDa, deriving from the A protein, and another of approximately 25 kDa from the B protein. Quinolone binding to the enzyme-DNA complex induces a conformational change which is reflected in the protection of the C-terminal 47-kDa domain of the B protein. An active site mutant (Tyr122 to Ser in the A protein) that binds quinolones but cannot cleave DNA still gives the quinolone proteolytic pattern, while stabilization of a cleaved-DNA intermediate by calcium ions does not reveal any protection, suggesting that the quinolone-induced conformational change is different from an "open-gate" state of the enzyme. A quinolone-resistant mutant of gyrase fails to give the characteristic quinolone-associated proteolytic signature. The ATP-induced dimerization of the B subunits is a key step of the gyrase mechanism. The proteolytic fingerprint of this conformation (stabilized by the non-hydrolyzable ATP analog 5'-adenylyl-beta, gamma-imidodiphosphate (ADPNP) shows a protection of the 43-kDa N-terminal domain of the B subunit. The presence of quinolones does not prevent dimerization since incubation of the enzyme-DNA complex with both ADPNP and quinolones gives rise to a complex whose proteolytic pattern retains the characteristic signature of dimerization but has lost the quinolone-induced protection. As a result, the quinolone-gyrase complex can still hydrolyze ATP, albeit with different kinetic characteristics. We interpret the proteolytic signatures observed in terms of four complexes of gyrase, each representing a particular conformational state.

Adenosine Triphosphate↗
PubMed1998

The DNA gyrase-quinolone complex. ATP hydrolysis and the mechanism of DNA cleavage.

Quinolone binding to the gyrase-DNA complex induces a conformational change that results in the blocking of supercoiling. Under these conditions gyrase is still capable of ATP hydrolysis which now proceeds through an alternative pathway involving two different conformations of the enzyme (Kampranis, S. C., and Maxwell, A. (1998) J. Biol. Chem. 269, 22606-22614). The kinetics of ATP hydrolysis via this pathway have been studied and found to differ from those of the reaction of the drug-free enzyme. The quinolone-characteristic ATPase rate is DNA-dependent and can be induced in the presence of DNA fragments as small as 20 base pairs. By observing the conversion of the ATPase rate to the quinolone characteristic rate, the formation and dissociation of the gyrase-DNA-quinolone complex can be monitored. Comparison of the time dependence of the conversion of the gyrase ATPase with that of DNA cleavage reveals that formation of the gyrase-DNA-quinolone complex does not correspond to the formation of cleaved DNA. Quinolone-induced DNA cleavage proceeds via a mechanism consisting of two cleavage events that is modulated in the presence of a nucleotide cofactor. We demonstrate that quinolone binding and drug-induced DNA cleavage are separate processes constituting two sequential steps in the mechanism of action of quinolones on DNA gyrase.

Adenosine Triphosphate↗
PubMed1998

Insulin-like growth factor axis of gluten-sensitive dogs during a gluten challenge.

Eight gluten-sensitive Irish setters underwent a gluten challenge to investigate changes in the insulin-like growth factor (IGF) axis. In the first study, they were challenged with an acute intraduodenal administration of tryptic-peptic gluten digest and then maintained on dietary gluten for three months. In the second study, the challenge came solely from dietary gluten fed for three months. After the acute intraduodenal administration of gluten, serum IGF-I levels decreased significantly by 21.8 per cent, (P = 0.01) on day 3 after challenge and then returned to normal. There was also a decrease (52.5 per cent, P < 0.03) in the levels of serum IGF-binding protein-3 (IGFBP-3) until day 14 after challenge but they had returned to normal by day 28. In two dogs IGFBP-3 levels decreased through specific serum protease activity. There were no changes in serum IGF-I or IGFBP levels during the second study after the dietary gluten challenge alone, or in four non-gluten-sensitive beagles studied as controls during the acute intraduodenal/dietary gluten challenge.

Animals↗