Comparative studies of fructose 1,6-diphosphate aldolase fromEscherichia coli 518 andLactobacillus casei var.rhamnosus ATCC 7469

A comparative study has been carried out with FDP aldolases fromEscherichia coli 518 andLactobacillus casei ATCC 7469, which had been purified 17.6- and 65-fold, respectively. The aldolase ofL.casei was stable only in the presence of mercaptoethanol, whereas that ofE.coli was strongly inhibited at low (1.0×10^–4 m) and activated at high concentrations (2.0×10^–1 m) of the same compound.p-Chloromercuric benzoic acid inhibited both aldolases, with 40% inhibition at 2×10^–5 m withE.coli aldolase against at 2×10^–4 m withL.casei aldolase. Significant differences were also observed in pH optima and Km values.E.coli aldolase exhibited a maximal activity at pH 9.0 and gave a Km value of 1.76×10^–3 m FDP with strong substrate inhibition above 7×10^–3 m, against pH 6.8–7.0 and a Km of 7.04×10^–3 m FDP forL.casei aldolase. Strong resistance ofL.casei aldolase against inhibition by EDTA, Ca²⁺ and Mn²⁺ was observed compared with complete inhibition at concentrations of 20mm, 40mm and 20mm, respectively, withE. coli aldolase. Polyacrylamide gel electrophoresis did not reveal any differences between the two enzyme preparations.The differences of the properties of FDP aldolases from different bacterial genera are discussed in relation to other Class II aldolases.     

Inactivation of Brazilian wild type and enterotoxigenicEscherichia coli by chlorine

The kinetic inactivation parameters of four wild strains and two enterotoxigenic strains ofEscherichia coli exposed to commercial calcium hypochlorite were determined. The four wild strains (1A, 3C, 4D and 8H) were isolated from lettuce bought in São Paulo (Brazil), and the two enterotoxigenic strains (TR69 and TR101) were originally isolated from human patients. Decimal reduction time D, for 10 mg L^–1 available chlorine at pH 6.8, varied between 71.4 s for the wild strain 4D and 31.3 s for the toxigenic strain. The D values obtained for wild strain 1A exposed to 5.0 mg L^–1 available chlorine at pH 6.8 varied between 111.1 s and 41.7 s. The D values obtained forE. coli strain TR69 exposed to 10 mg L^–1 available chlorine varied from 15.2 s at pH 5.4 up to 83.3 s at pH 8.2. The use of the most resistant wild strain ofE. coli as a biological standard assures maximal effectiveness in controlling water contamination by chlorination.     

IS8- and Tn2353-mediated cointegration of the plasmids R15 and RP4::Tn1

Summary The plasmids R15 and RP4:: Tn1 form fused structures (85 Md and 92 Md cointegrates). The cointegrates do not resolve practically in recA ^– Escherichia coli cells and have a mean life-time of more than 50 generations in a recA ⁺ background.The 85 Md cointegrates were generated at a frequency of 4×10^–4 per R15 transconjugant during a mating between E. coli [R15; RP4:: Tn1] and E. coli [FColVBtrp:: Tn1755]. These plasmids carry two directly repeated copies of the mobile element IS8 at the junctions between R15 and RP4:: Tn1. The transposition of IS8 from RP4:: Tn1 to the R15 plasmid and the formation of hybrid molecules promoted by this process appear to be induced by the IS8 element of the Tn1755 structure during or after conjugal transfer of FColVBtrp:: Tn1755 into E. coli [R15; RP4:: Tn1] cells.The formation of the 92 Md cointegrates occurs at a frequency of 2×10^–5. The fused molecules of R15 and RP4:: Tn1 carry two direct copies of an 8.65 Md R15 fragment at the junctions between these replicons. The fragment has specific features of a new transposon. This element designated Tn2353 determines resistance to Hg, Sm and Su and contains two sites for each BamHI, BglII and SalI and three sites for both EcoRI and PstI. The physical map and some other characteristics of Tn2353 are presented.Abbreviations Ap ampicillin - EtBr ethidium bromide - Km kanamycin - Md megadaltons - Sm streptomycin - Su sulfanilamide - Tc tetracycline - [] brackets indicate plasmid-carrier state     

Proton-linked sugar transport systems in bacteria

The cell membranes of various bacteria contain proton-linked transport systems ford-xylose,l-arabinose,d-galactose,d-glucose,l-rhamnose,l-fucose, lactose, and melibiose. The melibiose transporter ofE. coli is linked to both Na⁺ and H⁺ translocation. The substrate and inhibitor specificities of the monosaccharide transporters are described. By locating, cloning, and sequencing the genes encoding the sugar/H⁺ transporters inE. coli, the primary sequences of the transport proteins have been deduced. Those for xylose/H⁺, arabinose/H⁺, and galactose/H⁺ transport are homologous to each other. Furthermore, they are just as similar to the primary sequences of the following: glucose transport proteins found in a Cyanobacterium, yeast, alga, rat, mouse, and man; proteins for transport of galactose, lactose, or maltose in species of yeast; and to a developmentally regulated protein of Leishmania for which a function is not yet established. Some of these proteins catalyze facilitated diffusion of the sugar without cation transport. From the alignments of the homologous amino acid sequences, predictions of common structural features can be made: there are likely to be twelve membrane-spanning -helices, possibly in two groups of six, there is a central hydrophilic region, probably comprised largely of -helix; the highly conserved amino acid residues (40–50 out of 472–522 total) form discrete patterns or motifs throughout the proteins that are presumably critical for substrate recognition and the molecular mechanism of transport. Some of these features are found also in other transport proteins for citrate, tetracycline, lactose, or melibiose, the primary sequences of which are not similar to each other or to the homologous series of transporters. The glucose/Na⁺ transporter of rabbit and man is different in primary sequence to all the other sugar transporters characterized, but it is homologous to the proline/Na⁺ transporter ofE. coli, and there is evidence for its structural similarity to glucose/H⁺ transporters in Plants.In vivo andin vitro mutagenesis of the lactose/H⁺ and melibiose/Na⁺ (H⁺) transporters ofE. coli has identified individual amino acid residues alterations of which affect sugar and/or cation recognition and parameters of transport. Most of the bacterial transport proteins have been identified and the lactose/H⁺ transporter has been purified. The directions of future investigations are discussed.     

A suitable method for construction and cloning hybrid plasmids containing EcoRI-fragments of E. coli genome.

Summary A convenient procedure for the isolation of specificEcoRI-fragments ofE. coli genome and their amplification on Km-resistance plasmid vector CK 11 is described. The hybrid molecules were constructedin vitro usingEcoRI-digestion, followed by ligation. Then appropriatedE. coli strain was transformed with ligated DNA mixture and hybrid plasmids CK 11-arg ⁺, CK 11-his ⁺, CK 11-thr ⁺ and CK 11-leu ⁺ containing loci ofE. coli genome were selected by molecular cloning. The hybrid plasmids obtained consisted of oneEcoRI-fragment of initial plasmid CK 11 and one respective specific portion ofE. coli genome.     

Spironolactone antagonism of aldosterone action on Na+ transport and RNA metabolism in toad bladder epithelium

Summary In earlier studies, aldosterone increased the incorporation of precursors into a class of cytoplasmic RNA with the characteristics of messenger RNA (mRNA), in toad bladder epithelium. In the present studies, this effect was analyzed further with a competitive antagonist, spironolactone (SC-9420). Paired hemibladders were labeled with³H-uridine (30 min pulse–140 min chase), with or without aldosterone (3.5×10^–8 m, 7×10^–8 m) in the presence or absence of SC-9420 (7×10^–6 m, 2.5×10^–5 m) at molar ratios of 2001 to 2801. Cytoplasmic RNA, either the total phenol-SDS extract or polyadenylated-RNA (poly(A)(+)-RNA) obtained by oligo-deoxythymidylate-cellulose (oligo(dT)-cellulose) chromatography was analyzed in linear 5–20% sucrose gradients. Eight sets of experiments were completed in which the short-circuit current (scc) was monitored for 180 min and the incorporation of³H-uridine (30 min pulse–150 min chase) was simultaneously determined on pools of epithelia from 5 to 10 hemibladders. The fractional change inscc correlated linearly with the fractional change in³H-uridine of 12S cytoplasmic RNA (r=0.95,p<0.001). The poly(A)(+)-RNA fraction had no detectable rRNA or tRNA and gave a heterogeneous pattern, typical of mRNA, in the sucrose gradients. In the presence of exogenous aldosterone, SC-9420 inhibited the incorporation of³H-uridine into poly(A)(+)-RNA (particularly 12S). These results support the inference that induction of mRNA mediates the action of aldosterone on Na⁺ transport.     

Decolorization kinetics of a recombinant Escherichia coli strain harboring azo-dye-decolorizing determinants from Rhodococcus sp.

A 6.3 kb DNA fragment containing genes responsible for azo-dye decolorization was cloned and expressed in Escherichia coli. The resulting recombinant strain E. coli CY1 decolorized 200 mg azo dye (C.I. Reactive Red 22) l^–1 at 28 °C at 8.2 mg g cell^–1 h^–1, while the host (E. coli DH5) had no color-removal activity. Addition of 0.5 mM isopropyl--d-thiogalacto-pyranoside (IPTG) increased the decolorization rate 3.4-fold. The dependence of the decolorization rate on initial dye concentration essentially followed Monod-type kinetics and the maximal rate occurred with the dye at 600 mg l^–1. The decolorization rate of E. coli CY1 was optimal at 40 °C and pH 11. Aeration (increased dissolved O₂ level) strongly inhibited the decolorization, but decolorization occurred effectively under static incubation conditions (no agitation was employed). The CY1 strain also exhibited excellent stability during repeated-batch operations.     

Investigation of enhancement of two processes, sedimentation and conjugation, when bacteria are concentrated in ultrasonic standing waves

Cells aggregate and can be recovered from suspension when exposed to an ultrasonic standing wave field. The acoustic force on individual cells in a standing wave decreases with particle volume. A plane ultrasonic field generated by a transducer driven at 3.3 MHz was used here to investigate the removal of Escherischia coli, cells with dimensions of the order of 1.0 m, from batch suspension by sedimentation over a range of concentrations (10³ to 10¹⁰ cells ml^–1). Cell removal efficiencies greater than 90% were achieved at initial concentrations of 10¹⁰ cells ml^–1. Removal efficiencies decreased gradually to zero, as initial bacterial concentration was reduced to 10⁷ cells ml^–1. It was found that, when low concentrations of E. coli (10³ to 10⁵ cells ml^–1) were added to suspensions of larger particles (i.e. yeast cells) that were of sufficient concentration to form aggregates in the sound field, E. coli could be harvested to an efficiency of 40%. The results imply that the E. coli became trapped and sediment with aggregates of larger particles. Some strains of bacteria are capable of DNA transfer by conjugation. The transfer rate of E. coli RP4 plasmid is order of magnitudes greater when conjugation occurs on solid medium rather than in liquid suspension. We have investigated whether the conjugation rate would also be higher in ultrasonically induced E. coli clumps than in free suspension. The donor strain was mixed with a recipient strain of E. coli, then sonicated in a capillary at 4.6 MHz in a tubular transducer for 5 min. The bacteria aggregated successfully. Results showed a three-fold increase in the rate of conjugation compared to a liquid mating control.     

Strain improvement ofArthrobacter simplex by protoplast fusion

Anl-tryptophan auxotroph and milky mutants were derived from an inducible cholesterol oxidase-producing bacterium,Arthrobacter simplex USA18, via UV-mutagenesis. Protoplasts of these mutants and a constitutive cholesterol oxidase producer, strain US3011, were prepared by growing cells in the presence of ampicillin (20g ml^–1) followed by digestion with lysozyme. Protoplast fusion between tested strains with complementary characteristics was achieved in the presence of 20–40% polyethylene glycol 6000. The fusion frequency was about 1.5–1.7×10^–3. The cholesterol oxidase activity of four fusants in a cholesterol-containing medium was 20–60% higher than that of parental strains. This study demonstrated that protoplast fusion is applicable to strain improvement ofArthrobacter strains for enzyme production.     

Function of ribonuclease H in initiation of DNA replication in Escherichia coli K-12

Summary Escherichia coli rnh mutants lacking ribonuclease H (RNase H) activity can tolerate deletion of the origin of DNA Replication (oriC) and transposon-insertional inactivation of an initiator gene (dnaA:Tn10). Introduction of the recA200 allele encoding a thermolabile RecA protein intornh ^– dnaA: Tn10 and rnh ^– oriC mutants strains rendered DNA synthesis and colony formation of these mutants temperature sensitive. The temperature sensitivity and the broth sensitivity (Srm^–) of the rnh ^– dnaA: Tn10 recA200 strain was suppressed by the presenceof plasmids (pBR322 derivatives) carrying dnaA ⁺only when the intact oriC site was present on the chromosome. Lack of RNase H activity neither promoted replication of minichromosomes (pOC24 and pasn20) in the absence of required DnaA⁺ protein nor inhibited dnaA ⁺–dependent minichromosome replication. These results led to the conclusion that RNase H is not directly involved in the events leading to initiation of DNA replication at oriC. Rather, it functions as a specificity factor by eliminating certain forms of RNA-DNA hybrids which could otherwise be used to prime DNA replication at sites other than oriC.     

Saccharide binding to three Gal/GalNAc specific lectins: Fluorescence,spectroscopic and stopped-flow kinetic studies

Fluorescence and stopped-flow spectrophotometric studies on three plant lectins fromPsophocarpus tetragonolobus (winged bean),Glycine max (soybean) andArtocarpus integrifolia (jack fruit) have been studied usingN-dansylgalactosamine as a fluorescent ligand. The best monosaccharide for the winged bean agglutinin I (WBA I) and soybean (SBA) is Me-GalNAc and for jack fruit agglutinin (JFA) is Me-Gal. Examination of the percentage enhancement and association constants (1.51×10⁶, 6.56×10⁶ and 4.17×10⁵ M^–1 for SBA, WBA I and JFA, respectively) suggests that the combining regions of the lectins SBA and WBA I are apolar whereas that of JFA is polar. Thermodynamic parameters obtained for the binding of several monosaccharides to these lectins are enthalpically favourable. The binding of monosaccharides to these lectins suggests that the-OH groups at C-1, C-2, C-4 and C-6 in thed-galactose configuration are important loci for interaction with these lectins. An important finding is that the JFA binds specifically to Galß1-3GaINAc with much higher affinity than the other disaccharides which are structurally and topographically similar.The results of stopped-flow spectrometry on the binding ofN-dansylgalactosamine to these lectins are consistent with a bimolecular single step mechanism. The association rate constants (2.4×10⁵, 1.3×10⁴, and 11.7×10⁵ M^–1 sec^–1 for SBA, WBA I and JFA, respectively) obtained are several orders of magnitude slower than the ones expected for diffusion controlled reactions. The dissociation rate constants (0.2, 3.2×10^–2, 83.3 sec^–1 for SBA, WBA I and JFA, respectively) obtained for the dissociation ofN-dansylgalactosamine from its lectin complex are slowest for SBA and WBA I when compared with any other lectin-ligand dissociation process.Abbreviations SBA Soybean agglutinin - WBA I Winged bean agglutinin (Basic) - JFA Jack fruit agglutinin - PNA Peanut agglutinin - Con A Concanavalin A - Dansyl (Dns) 5-dimethylaminonaphthalene-I-sulphonyl - 2GaINDns N-dansylgalactosamine - dGal 2-deoxygalactose - l-Ara l-arabinose - d-Fuc d-fucose - l-Rha l-rhamnose - N-acetyllactosamine Galß4GlcNAc - melibiose Gal6Glc     

Effect of ogt expression on mutation induction by methyl-, ethyl- and propylmethanesulphonate in Escherichia coli K12 strains

We have previously reported the isolation of an Escherichia coli K12 mutant that is extremely sensitive to mutagenesis by low doses of ethylating agents. We now show by Southern analysis that the mutation involves a gross deletion covering at least the ogt and fnr genes and that no O⁶-alkylguanine-DNA-alkyltransferase activity is present in cell-free extracts of an ada::Tn10 derivative of these bacteria. Confirmation that sensitisation to ethylation-induced mutagenesis was attributable to ogt and not to any other loci covered by the deletion was obtained by constructing derivatives. Thus an ogt::kan^r disruption mutation was introduced into the parental ogt ⁺ bacteria, and the ogt::kan^r mutation was then eliminated by cotransduction of ogt ⁺ with the closely linked Tet ^r marker (zcj::Tn10). The (ogt-fnr) deletion or ogt::kan^r disruption mutants were highly sensitive to ethyl methanesulphonate-induced mutagenesis, as measured by the induction of forward mutations to l-arabinose resistance (Ara¹). Furthermore, the number of Ara^r mutants increased linearly with dose, unlike the case in ogt ⁺ bacteria, which had a threshold dose below which no mutants accumulated. Differences in mutability were even greater with propyl methanesulphonate. Overproduction of the ogt alkyltransferase from a multicopy plasmid reduced ethylmethanesulphonate-induced mutagenesis in the ogt mutant strains and also methylmethanesulphonate mutagenesis in ada ^– bacteria. A sample of AB 1157 obtained from the E. coli K12 genetic stock centre also had a deletion covering the ogt and fnr genes. Since such deletions greatly influence the mutagenic responses to alkylating agents, a survey of the presence of the ogt gene in the E. coli K12 strain being used is advisable.     

Analysis of the reduction in expression of tetracycline resistance determined by transposon Tn10 in the multicopy state

The tet genes of transposon Tn10 have been mapped in a 2,200 bp DNA sequence by analysing deletion and Tn5 insertion mutations. When the tet genes were present on multi-copy plasmids the level of resistance expressed was about ten-fold lower than that determined by a single copy of Tn10 in the E. coli chromosome. The 36K tet protein known to be encoded by R100 in E. coli minicells was not detected when they harboured a multicopy tet plasmid. However, normal high levels of resistance were expressed when the tet genes were recombined into the host chromosome as part of a lambda lysogen, showing that the multicopy effect was phenotypic. Most of the Tn5 insertions and deletions in tet which caused Tc^s mutations also prevented expression of high level Tc^r from a chromosomal Tn10 element present in the same cell. Only those insertions in the promoter-proximal 90–130 bp of a 1,275 bp HindII fragment known to carry the gene encoding the 36K tet protein did not reduce the single copy Tn10 resistance level.A gene fusion system that results in the constitutive synthesis of -galactosidase from a tet promoter has been used to assay tet repressor activity. The basal (uninduced) -galactosidase level in cells carrying multicopy tet plasmids was 10–20 fold lower than those carrying a single copy. The tet:: Tn5 mutants defective in the trans-dominant multicopy effect still made normal amounts of tet repressor showing that repressor overproduction was not responsible for this effect. In addition a repressor-defective constitutive mutant did not exhibit a higher resistance level when located on a multicopy plasmid vector. We postulate that a regulatory mechanism recognises the amino-terminus of the tet structural gene product when attempts are being made to overproduce the protein and prevents further translation.     

A protonmotive force as the source of energy for galactoside transport in energy depletedEscherichia Coli

Summary Uracil transport inSaccharomyces cerevisiae is mediated by a specific permease which does not recognize other pyrimidines such as uridine, cytosine, thymine, 2-hydroxypyrimidine or 5-amino-uracil; hypoxanthine and 6-amino-uracil slightly inhibit the uptake of uracil in a strain lacking cytosine permease activity. Wild type cells concentrate extracellular uracil before its transformation into UMP and subsequent incorporation into nucleic acids. A strain lacking UMP pyrophosphorylase and uridine ribohydrolase (strainfur 1–8 rh, in which the endogenous production as well as the utilization of uracil are lacking) is able to concentrate¹⁴C-2 uracil from the medium. At the same time no other¹⁴C-2 labelled compound could be detected in this strain, thus suggesting that the uptake of uracil in yeast occurs by active transport which is not coupled to the UMP pyrophosphorylase. The optimal pH of uracil uptake in standard growth conditions was 4.3. It was deduced from experiments performed on strainfur 1–8 rh with³H-5 and¹⁴C-2 uracil that the intracellular pool of uracil is recycled once the steady-state has been reached. First order kinetics with similar rate constants were observed for uracil efflux in strainfur 1–8 rh (k min^–1=0.75±0.08) as well as in the strain lacking uracil permease,fur 1–8 rh fur 4–6 (k min^–1=0.60±0.08). The intracellular pool of¹⁴C-2 uracil can be chased in strainfur 1–8 rh by addition of³H uracil without inducing a large initial acceleration of the exit rate (the rate constant remained at 0.60). 2-4-dinitrophenol inhibits the uptake of uracil but also reduces the efflux of uracil in strainfur 1–8 rh fur 4–6. These data and the comparison with cytosine transport in the same organism support the hypothesis that, whereas uracil uptake is a permease mediated active transport, the efflux of uracil does not involve the uracil uptake permease. A coefficient of permeability of 7.4×10^–7 cm sec^–1 was calculated for uracil.     

The role of quinate and shikimate in the metabolism of lactobacilli

The metabolism of (–)-quinate and shikimate by one heterofermentative strain,actobacillus pastorianus, and by one homofermentative strain,Lactobacillus plantarum, has been studied using growing and washed cells. Both organisms reduced quinate and shikimate under anaerobic conditions in the presence of suitable hydrogen donors including fructose, glucose andd(–) andl(+)-lactates. The end-product ofL.pastorianus metabolism was dihydroshikimate butL.plantarum carried the reduction a stage further tocis-3,4-dihydroxycyclohexanecarboxylate and formed, simultaneously, catechol. The enzymes involved in these reductions are induced; their importance in the metabolism of lactobacilli is discussed.     

Kinetics of β-glucosidase production by Escherichia coli recombinants harboring heterologous bgl genes

Maximum productivities of -glucosidase by E. coli recombinants, under the control of either lacZ or GALI promoters, were 33 ± 10 and 100 ± 5 IU l^–1 h^–1, respectively.GAL1 promoter of pYES2 enabled the E. colirecombinants to produce 3.1- and 15.1-fold more -glucosidase than that supported by lacZ promoter of pUC18 in E. coli recombinants and donor, respectively.     

Chemotaxis towards sugars inChlamydomonas reinhardtii

Chemotaxis ofChlamydomonas reinhardtii 137c cells towards maltose and sucrose was observed by capillary assay. The threshold concentration was approximately 10^–5 m for maltose and 10^–3 m for sucrose. The peak accumulation of cells occurred at 10^–3 m for maltose and 10^–2 m for sucrose. A selection procedure for chemotaxis mutants was developed. Mutant strain CHE-1 was not attracted by maltose; mutant strain CHE-2 was not attracted by sucrose. Addition of attractant fully inhibited photoaccumulation of cells. After a period of time the photoresponse of cells recovered. Under the conditions of our experiments the period of adaptation lasted 15–20 min in wild-type cells and 4–5 min in mutant cells on the given sugar. Glucose and acetate did not attract cells ofChlamydomonas. Added to the medium, these compounds had no effect on the photoaccumulation of cells.     

A novel purification of ferric citrate receptor (FecA) from Escherichia coli UT5600 and further characterization of its binding activity

In our earlier paper, it was demonstrated that the FecA receptor protein from Escherichia coli UT5600/pBB2 (leu ^–, proC ^–, trpE ^–, entA ^–, rpsl ^–, (ompT-fepA)^–/Ampr, fepA) binds with ferric enterobactin. In order to explore this further the outer membrane receptor protein, FecA, has been isolated from UT5600 (fepA ^–) and purified to homogeneity by DE-52-cellulose anion exchange chromatography followed by MonoPFPLC chromatofocusing. Partially purified FecA and homogeneous FecA show binding activity to [55Fe]ferric enterobactin and the binding is specific. Binding activity of FecA can be enhanced by ferric citrate. Lipopolysaccharide-free FecA as ascertained by silver staining and the endotoxin test still retains the same activity. In vivo uptake studies using different strains of E. coli suggest that FecA in E. coli plays an important role in ferrienterobactin transport.     

Intermolecular and intramolecular transposition and transposition immunity in Tn3 and Tn2660

Summary Intermolecular transposition of Tn2660 into pCR1 was measured at 30°C in recA ^– and recA ⁺ hosts as between 2.6 and 5.5x10^–3, a similar value to that previously found for Tn3. No cointegrate structures were found under conditions where 10⁴ transposition events occurred. Immunity to intermolecular transposition of Tn2660, similar to that found for Tn3 was demonstrated by showing that the above transposition frequency was reduced by a factor of between 10^–3 and 10^–4 when a mutant Tn2660 (resulting in the synthesis of a temperaturesensitive -lactamase) was present in the recipient plasmid. Intramolecular transposition of Tn3 was found to occur under the same conditions as previously demonstrated for Tn2660 giving rise to similar end products, in which the newly introduced Tn3 is oriented inversely to the resident Tn3 and the DNA sequence between the two transposons has been inverted. Thus, in all respects functional identity of the transposition activities of Tn3 and Tn2660 is shown, thereby identifying characteristics of intramolecular transposition that are not readily accommodated by current models of transposition.     

Effects of D-600 on sodium current in squid axons

Summary The effects of the calcium antagonist D-600 (methoxyverapamil) on the excitatory inward sodium current,I _Na, of internally perfused squid giant axons were studied under voltageclamp conditions. We observed little or no effect of the drug when it was added to the external solution at concentrations of 10–200 M. Furthermore, it did not produce a frequency, or use-dependent block ofI _Na when repetitive voltage-clamp pulses were used at rates of 2–5Hz. However, it did produce use-dependent blockade ofI _Na when it was placed internally at a concentration of 200 M. These results in conjunction with other studies suggest that D-600 is a selective blocker of calcium channels in squid axons when the drug is placed in the external solution. Its effects, when placed in the internal solution, are similar to those of permanently charged local anesthetic derivatives, which also produce use-dependent block ofI _Na.