The conformation of linear gramicidin is sequence dependent

A comparative monolayer and infrared study of analogues of gramicidin A containing either tyrosines or naphthylalanines instead of tryptophans indicates that the nature of the aromatic residues influences the favoured conformation of the peptides. Polar residues favour the single stranded _DL helix while non polar residues favour the double stranded helix. For partly tryptophan to naphthylalanine substituted analogues the positions of the substitutions orientate the favored conformation. The nature of these substitutions may also modify the peptide-lipid interactions. Correspondence to: F. Heitz Chemical structures of the gramicidin A analogues mentioned in this paper. The differences from gramicidin A are underlined. GM^–: GT:     

Estrogens protect against hydrogen peroxide and arachidonic acid induced DNA damage

The ability of estrogens to protect against DNA damage induced by either hydrogen peroxide or arachidonic acid alone or in combination with Cu²⁺ was investigated. DNA strand breaks were determined by conversion of double stranded supercoiled ØX-174 RFI DNA to double stranded open circular DNA and linear single stranded DNA. Estradiol-17β significantly decreased the formation of single and double strand breaks in DNA induced by H₂O₂ alone or with Cu²⁺. Equilin (an equine estrogen) was more effective than estradiol-17β at the doses tested. Arachidonic acid in the presence of Cu²⁺ caused the formation of high levels of linear DNA which was protected by estrogen with equilen being more effective. These studies suggest that estrogens through this protective effect on DNA damage might contribute to cardioprotection.     

Purification and Characterization of DNA Polymerase Obtained from the Membrane Fraction of E. coli

Purification studies were conducted on DNA polymerase bound to the membrane fraction of E. coli HF 4704. Purified enzyme (Fraction V) required Mg²⁺ and showed an optimun pH of 7.2. Various kinds of salt indicated a stimulative effect at concentrations lower than 0.1 m. Fraction V was unstable at an acidic condition (pH 5.0) but was rather stable at an alkaline condition (pH 9.0). The enzyme activity was lost by incubation at 45°C for 30min but was stabilized by the addition of DNA. The enzyme contained exonuclease activity but no endonuclease activity. The enzyme produced only light density DNA of various sizes. The function of this enzyme as considered to fill single stranded region of the double stranded primer DNA.     

Malic enzyme of Chromatium vinosum

Malic enzyme of the phototrophic bacterium Chromatium vinosum strain D that lacks malate dehydrogenase was partially purified yielding a specific activity of 55 units/mg protein. The constitutive enzyme with a molecular weight of 110,000 and a pH optimum of 8.0 was absolutely dependent on the presence of a monovalent cation (NH ₄ ⁺ , K⁺, Cs⁺, or Rb⁺) as well as a divalent cation (Mn²⁺, or Mg²⁺). The enzyme was inhibited by oxaloacetate, glyoxylate, and NADPH. The K _0.5 value for L-malate and the inhibition constants for oxaloacetate and glyoxylate are dependent on the concentration of the monovalent cation, whereas the K _m value for NADP (18 M) and the K ₁ value for NADPH (42 M) are independent. Throughout all kinetic measurements hyperbolic saturation curves and linear double reciprocal plots were obtained.Abbreviations OAA oxaloacetate - OD optical density     

Ultraviolet reactivation of the single stranded DNA phage phiX 174.

Summary When UV-irradiated X174 was grown in pre-irradiated host cells of various strains, ultraviolet reactivation (UVR) was observed only in recombination proficient strains such as E. coli C (uvrA ⁺ recA ⁺) and HF4704 (uvrA ^- recA ⁺), but not in the recombination deficient strain HF4712 (uvrA ⁺ recA ^-). By increasing the multiplicity of infection, no rise in the amount of such reactivation was observed. From the study of the neutral and alkaline sucrose gradient sedimentation patterns of DNA samples extracted from unirradiated cells infected with unirradiated phage, it appears that after the conversion of the viral single stranded (SS) DNA to the double stranded form (DS), nicks or scissions were produced on it within all three strains, which were ultimately sealed up in the recA ⁺ but persisted within the recA ^- host cells. When UV-irradiated phage infected unirradiated host cells, such nicking of the DS DNA appeared to be much more extensive in uvrA ⁺ recA ⁺, but slightly reduced in uvrA ⁺ recA ^- and severely suppressed in uvrA ^- recA ⁺ strains. When the host cells were also UV-irradiated, the conversion of the infecting viral SS DNA to DS DNA as well as its subsequent nicking were reduced in all the three strains to a much greater extent. Although nicking of the DS DNA molecule is an essential step even in the normal intracellular replication of X DNA, the production and the sealing up of such nicks appear not to have any positive correlation with UVR of these phages. A drastic reduction in nicking due te pre-irradiation of the host cells might, however, mean slowing down of the replication of the damaged parental RF molecules which would facilitate their repair perhaps through recombination with the homologous parts of the host genome.     

Effect of Glycosylation on the Catalytic and Conformational Stability of Homologous α-Amylases

summary. A thermostable -amylase from B. licheniformis (BLA) and a mesophilic amylase from B. amyloliquefaciens (BAA) were covalently coupled to oxidized synthetic sucrose polymers (OSP400 and OSP70) and polyglutaraldehyde (PGA) by reductive alkylation to study the effect of neoglycosylation on the activity, kinetic and thermodynamic stability. The catalytic efficiency of the modified enzymes was comparable to that of the native enzyme. Covalent coupling decreased the rate of inactivation at all the temperatures studied, both in the presence and absence of added Ca²⁺. The stability of the native enzyme was found to increase upon modification as observed from the increase in t_1/2 in the absence of Ca²⁺ ions by about 1.5–13.7 times (at 85°C) in the case of BLA and 5.7–8.4 times (at 50°C) for BAA. The highest stability was observed for OSP400 modified enzyme with C_m and T_m values of 0.63 M and 7.92°C for BLA and 0.85 M and 5.3°C for BAA, respectively. The order of stability was OSP400 > OSP70 > PGA > Native for both BLA and BAA. The stability of the modified amylases obtained from the present study were superior compared to most of the single and double mutants obtained by site-directed mutagenesis that were constructed so as to enhance the intrinsic stability of these enzymes.This article is dedicated to Dr. P.V. Sundaram.     

Properties of Bound Inorganic Phosphate on Bovine Mitochondrial F1F0-ATP Synthase

Beef-heart mitochondrial F₁F₀-ATP synthase contained six molecules of bound inorganic phosphate (P_i). This phosphate exchanged completely with exogenous ³²P_i when the enzyme was exposed to 30% (v/v) dimethyl sulfoxide (DMSO) and then returned to a DMSO-free buffer (Beharry and Bragg 2001). Only two molecules were replaced by ³²P_i when the enzyme was not pretreated with DMSO. These two molecules of ³²P_i were not displaced from the enzyme by the treatment with 1 mM ATP. Similarly, two molecules of bound ³²P_i remained on the DMSO-pretreated enzyme following addition of ATP, that is, four molecules of ³²P_i were displaced by ATP. The ATP-resistant ³²P_i was removed from the enzyme by pyrophosphate. It is proposed that these molecules of ³²P_i are bound at an unfilled adenine nucleotide-binding noncatalytic site on the enzyme. Brief exposure of the enzyme loaded with two molecules of ³²P_i to DMSO, followed by removal of the DMSO, resulted in the loss of the bound ³²P_i and in the formation of two molecules of bound ATP from exogenous ADP. A third catalytic site on the enzyme was occupied by ATP, which could undergo a P_i ATP exchange reaction with bound P_i The presence of two catalytic sites containing bound P_i is consistent with the X-ray crystallographic structure of F₁ (Bianchet, et al., 1998). Thus, five of the six molecules of bound P_i were accounted for. Three molecules of bound P_i were at catalytic sites and participated in ATP synthesis or P_i ATP exchange. Two other molecules of bound P_i were present at a noncatalytic adenine nucleotide-binding site. The location and role of the remaining molecule of bound P_i remains to be established. We were unable to demonstrate, using chemical modification of sulfhydryl groups by iodoacetic acid, any gross difference in the conformation of F₁F₀ in DMSO-containing compared with DMSO-free buffers.     

Determination of two sites of automethylation in bovine erythrocyte protein (d -aspartyl/l -isoaspartyl) carboxyl methyltransferase

Protein (d-aspartyl/l-isoaspartyl) carboxyl methyltransferase (PCM, E.C. 2.1.1.77) was previously shown to be enzymatically methyl esterified in an autocatalytic manner at altered aspartyl residues; methyl esters are observed in a subpopulation of the enzyme termed thePCM fraction [Lindquist and McFadden (1994),J. Protein Chem. 13, 23–30]. The altered aspartyl sites serving as methyl acceptors inPCM have now been localized by using proteolytic enzymes and chemical cleavage techniques in combination with matrix-assisted laser desorption/ionization (MALDI) mass spectrometry to identify fragments of the [³H]automethylated enzyme that contain a [³H]methyl ester. Methylation was positively identified at positions Asn₁₈₈ and Asp₂₁₇ in the enzyme sequence, a consequence of the spontaneous alteration of these sites tol-isoaspartyl ord-aspartyl sites and their methylation by active PCM molecules. The identification of more than one site of automethylation shows thatPCM is not a homogeneous population of damaged PCM molecules, but rather a complex population of molecules with a variety of age-altered damage sites.Abbreviations PCM protein (d-aspartyl/l-isoaspartyl) carboxyl methyltransferase - EDTA disodium ethylenediaminetetraacetate - PMSF phenylmethylsulfonyl fluoride - TEA trifluoroacetic acid - HPLC high-pressure liquid chromatography     

Genetic analysis of functional connectivity between substrate recognition domains ofEscherichia coli glutaminyl-tRNA synthetase

It has previously been shown that the single mutation E222K in glutaminyl-tRNA synthetase (GlnRS) confers a temperature-sensitive phenotype onEscherichia coli. Here we report the isolation of a pseudorevertant of this mutation, E222K/C171G, which was subsequently employed to investigate the role of these residues in substrate discrimination. The three-dimensional structure of the tRNA^Gln: GlnRS:ATP ternary complex revealed that both E222 and C171 are close to regions of the protein involved in interactions with both the acceptor stem and the 3 end of tRNA^Gln. The potential involvement of E222 and C171 in these interactions was confirmed by the observation that GlnRS-E222K was able to mischargesupF tRNA^Tyr considerably more efficiently than the wild-type enzyme, whereas GlnRS-E222K/C171G could not. These differences in substrate specificity also extended to anticodon recognition, with the double mutant able to distinguishsupE tRNA _CUA ^Gln from tRNA ₂ ^Gln considerably more efficiently than GlnRS E222K. Furthermore, GlnRS-E222K was found to have a 15-fold higher K_m for glutamine than the wild-type enzyme, whereas the double mutant only showed a 7-fold increase. These results indicate that the C171G mutation improves both substrate discrimination and recognition at three domains in GlnRS-E222K, confirming recent proposals that there are extensive interactions between the active site and regions of the enzyme involved in tRNA binding.     

Free sigma subunit of Bacillus subtilis RNA polymerase binds to DNA

Summary The affinity of Bacillus subtilis RNA polymerase and subunits to DNA was examined by a non-denaturing polyacrylamide slab gel electrophoresis method which made it possible to resolve DNA-bound and free subunits. The results revealed that subunit, but not subunit had a relatively high affinity for double stranded DNA. The subunit was bound maximally to super-coiled pGR1-3 plasmid DNA at a mass ratio of /DNA of 0.7. With B. subtilis double stranded linear DNA one subunit was bound per approximately 1,000 base pairs. The -DNA complex was sufficiently stable for isolation by a molecular gel filtration column. The subunit had much higher affinity for super-coiled than for linear pGR1-3 DNA or for linear double stranded or denatured DNA from B. subtilis, E. coli, and calf thymus. These results indicate that the free B. subtilis subunit, in contrast to the E. coli subunit, can bind by itself to DNA.     

In vivo repair of UV-irradiation damage of single stranded DNA phage phi-X 174

Summary When E. coli C cells, infected with UV irradiated X 174, were allowed to grow in liquid tris-glucose medium at 37° C with aeration, the UV damage of the single stranded (ss) DNA could be repaired to some extent. Such repair was not possible if the irradiated phage were plated immediately on E. coli C in the usual double layer agar method, or if the infected complexes were initially exposed to 0.02 M KCN for 15 min before they were allowed to grow in tris-glucose medium as before. Our results indicate that in order to be repaired, ss DNA containing UV damage must be able to convert itself to a closed circular double stranded replicative form (RF) within the host cells escaping prior scission. The whole process of repair was found to be dependent on protein synthesis in the infected complexes.     

A fluoride-insensitive inorganic pyrophosphatase isolated from Methanothrix soehngenii

An inorganic pyrophosphatase [E.C. 3.6.1.1] was isolated from Methanothrix soehngenii. In three steps the enzyme was purified 400-fold to apparent homogeneity. The molecular mass estimated by gelfiltration was 139±7 kDa. Sodium dodecyl sulfate/polyacrylamide gel electrophoresis indicated that the enzyme is composed of subunits with molecular masses of 35 and 33 kDa in an _{2 2} oligomeric structure. The enzyme catalyzed the hydrolysis of inorganic pyrophosphate, tri-and tetrapolyphosphate, but no activity was observed with a variety of other phosphate esters. The cation Mg²⁺ was required for activity. The pH optimum was 8 at 1 mM PP _i and 5 mM Mg²⁺. The enzyme was heat-stable, insensitive to molecular oxygen and not inhibited by fluoride. Analysis of the kinetic properties revealed an apparent K _m for PP _i of 0.1 mM in the presence of 5 mM Mg²⁺. The V _max was 590 mol of pyrophosphate hydrolyzed per min per mg protein, which corresponds to a K _cat of 1400 per second.The enzyme was found in the soluble enzyme fraction after ultracentrifugation, when cells were disrupted by French Press. Upto 5% of the pyrophosphatase was associated with the membrane fraction, when gentle lysis procedyre were applied.Abbreviation PMSF phenylmethylsulfonyl fluoride     

Pyruvate decarboxylase from Zymomonas mobilis. Isolation and partial characterization

Pyruvate decarboxylase (EC 4.1.1.1) from the ethanol producing bacterium Zymomonas mobilis was purified to homogeneity. This enzyme is an acidic protein with an isoelectric point of 4.87 and has an apparent molecular weight of 200,000±10,000. The enzyme showed a single band in sodium dodecylsulfate gel electrophoresis with a molecular weight of 56,500±4,000 which indicated that the enzyme consists of four probably identical subunits. The dissociation of the cofactors Mg²⁺ and thiamine pyrophosphate at pH 8.9 resulted in a total loss of enzyme activity which could be restored to 99.5% at pH 6.0 in the presence of both cofactors. For the apoenzyme the apparent K _m values for Mg²⁺ and thiamine pyrophosphate were determined to be 24 M and 1.28 M. The apparent K _m value for the substrate pyruvate was 0.4 mM. Antiserum prepared against this purified pyruvate decarboxylase failed to crossreact with cell extracts of the reportedly pyruvate decarboxylase positive bacteria Sarcina ventriculi, Erwinia amylovora, or Gluconobacter oxydans, or with cell extracts of Saccharomyces cerevisiae.Abbreviations Tris-buffer 0,01 M tris-HCl buffer, containing 1 mM MgCl₂ 0.1 mM EDTA, 1.0 mM thiamine pyrophosphate, 2 mM mercaptopropanediol, pH 7.0     

Some properties of adenylate kinase from chemolithotrophically grown Thiobacillus novellus

Adenylate kinase (ATP:AMP phosphotransferase, EC 2.7.4.3) from Thiobacillus novellus was purified 54-fold. The preparation was, on the basis of densitometer scans of polyacrylamide gels, at least 85% pure. The optimum pH in the forward direction (2 ADP ATP+AMP) was about 8.7, and in the reverse 8.2 The enzyme was specific for AMP, ADP and ATP with apparent K _m values of 0.04, 0.34 and 0.09 mM respectively. A double reciprocal plot of specific activity vs. (ADP)² was linear. Both AMP and ATP inhibited the forward reaction with AMP the more inhibitory of the two. AMP inhibition was competitive with respect to ADP with a K _i of 0.125 mM. Although Mg²⁺ was necessary for maximal activity, about 20% of this was obtained in its absence. Co²⁺ and Mn²⁺ at similar concentrations gave 46% and 26% respectively of the activity found with Mg²⁺. Apparent K _m for Mg²⁺ was about 0.054 mM in the forward and 0.15 mM in the reverse direction. pHMB and HgCl₂ were potent inhibitors. Inhibition by pHMB but not HgCl₂ was reversible by GSH or cysteine. Arrhenius plots gave an E _a of 3.25 kcal/mole/degree C in the forward direction without discontinuity. In the reverse, there was a break at 26.7°C with an E _a of 3.62 kcal/mole/degree C for lower temperatures and 3.92 kcal/mole/degree C for higher temperatures.Molecular weights of the enzyme were 46,300±300 by SDS PAGE and 47,800±200 by SDS PAGE after treatment with 5 M urea and about 40,000 by sucrose density gradient centrifugation.Abbreviations APS adenosine-5-phosphosulfate - DEAE diethylaminoethyl - DTT dithiothreitol - E_a energy of activation - ECTEOLA epichlorohydrin triethanolamine - G6P glucose-6-phosphate - GSH glutathione (reduced) - PAGE Polyacrylamide gel electrophoresis - pHMB parahydroxymercuribenzoate - PEP phosphoenolpyruvate - PPi inorganic pyrophosphate - SDS sodium dodecyl sulfate - TEAE triethylaminoethyl Supported by an operating grant to A.M.C. from NSERCSummer student research trainee     

The influence of polyamine-nucleic acid complexes on Fe2+ autoxidation

Summary Polyamines are able to affect Fe²⁺ autoxidation in the presence of suitable low molecular weight phosphorus-containing compounds; the inhibitory effect exerted by polyamines is directly related to their ability to bind phosphorus-containing compounds [1].It is well known that polyamines, as polycations at physiological pH, bind strongly to nucleic acids. In this paper it is shown that polyamines, also in the presence of nucleic acids, inhibit Fe²⁺ autoxidation and thus depress the generation of free oxygen radicals. Most of the nucleic acids tested inhibited Fe²⁺ autoxidation although the concentration which causes half maximal effect differs. Polyamine effect on Fe²⁺ autoxidation varies greatly depending on the single or double stranded nature of the nucleic acid. In the present of single stranded nucleic acids, spermine and spermidine potentiate the inhibition of Fez+ autoxidation by these nucleic acids. A relationship exists between the ability of spermine to interact with single stranded nucleic acids and to inhibit Fe²⁺ autoxidation in their presence. When double stranded nucleic acids are present, polyamines reverse the inhibition of Fee+ autoxidation exerted by these nucleic acids. Molecular mechanisms are proposed to explain these experimental results. The hypothesis that polyamines may inhibit oxidative damage caused to nucleic acids by Fe²⁺ autoxidation, is also discussed.Abbreviations poly [A] polyadenylic acid (5) - poly [C] polycytidylic acid (5) - poly [1] polyinosinic acid (5) - poly [G] polyguanylic acid (5) - poly [A. U] polyadenylic-uridylic acid - poly [A] poly [U] polyadenylic-polyuridylic acid     

Recombination-dependent recircularization of linearized pBR322 plasmid DNA following transformation of Escherichia coli

Summary Monomeric pBR322 DNA that had been linearized at its unique SalI site transformed wild-type Escherichia coli with 10² to 10³ times less efficiency than CCC plasmid DNA. Dose-response experiments indicated that a single linear plasmid molecule was sufficient to produce a transformant. Transformation with linearized pBR322 DNA was reduced 10 to 40 fold in recA ^–, recBC ^– or recF ^– backgrounds. In contrast, transformation with CCC DNA was unaffected by the rec status of the host. Transformation with linear pBR322 DNA was increased 3-fold in a DNA ligase-overproducing (lop11) mutant and decreased to a similar degree by transient inactivation of ligase in a ligts7 mutant.A proportion (ranging from about 9% in the wild-type to 42% in a recBC, lop11 mutant) of the transformants obtained with SalI-linearized pBR322 monomeric DNA contained deleted plasmids. Deletion rates were generally higher in rec ^– strains. Dephosphorylation of the termini on linear DNA or the creation of blunt-ended pBR322 molecules (by end-filling the SalI 5 protrusions or by cleavage with PvuII) decreased the transformation frequencywhilst increasing the deletion rate.Linear pBR322 dimeric DNA gave transformation frequencies in recA ⁺ and recA ^– strains that were reduced only 3 to 7 fold respectively relative to frequencies obtained with dimeric CCC DNA. Furthermore, in contrast to transformation with linear monomeric DNA, deletions were not observed.We propose that the majority of transformants arise, not by simple intracellular reannealing and ligation of the two cohesive SelI-termini of a linear molecule, but by intramolecular recombination. Deleted plasmids could be generated therefore during recyclization caused by recombination between short directly repeated sequences within a pBR322 monomer. We suggest that perfectly recircularized monomeric pBR322 molecules, which are found in the majority of transformants, arise primarily by intramolecular recombinational resolution of head-to-tail linear pBR322 dimers. Such linear oligomeric forms are created during preparation of linearized plasmid DNA by annealing of the SalI cohesive termini and constitute a variable proportion of the total molecules present.     

Purification and properties of oxaloacetate decarboxylase fromCorynebacterium glutamicum

Oxaloacetate (OAA) decarboxylase (E.C. 4.1.1.3) was isolated fromCorynebacterium glutamicum. In five steps the enzyme was purified 300-fold to apparent homogeneity. The molecular mass estimated by gel filtration was 118 ± 6 kDa. SDS-PAGE showed a single subunit of 31.7 KDa, indicating an ₄ subunit structure for the native enzyme. The enzyme catalyzed the decarboxylation of OAA to pyruvate and CO₂, but no other -ketoacids were used as substrate. The cation Mn²⁺ was required for full activity, but could be substituted by Mg²⁺, Co²⁺, Ni²⁺ and Ca²⁺. Monovalent ions like Na⁺, K⁺ or NH ₄ ⁺ were not required for activity. The enzyme was inhibited by Cu²⁺, Zn²⁺, ADP, coenzyme A and succinate. Avidin did not inhibit the enzyme activity, indicating that biotin is not involved in decarboxylation of OAA. Analysis of the kinetic properties revealed a K _m for OAA of 2.1 mM and a K _m of 1.2 mM for Mn²⁺. The V _max was 158 µmol of OAA converted per min per mg of protein, which corresponds to an apparent k _cat of 311 s^–1.Abbreviations OAA oxaloacetate - LDH lactate dehydrogenase