Effect of selenite and selenate on rat liver nuclear 3,5,3′-triiodothyronine (T3) receptor

The present study was undertaken to investigate the effects of selenite (Se^IV) or selenate (Se^VI) on nuclear T₃ receptors of rat liver. Selenite at 0.1 μM (p<0.01) inhibited the T₃ specific binding to rat liver nuclear receptors. The specific binding of the T₃ receptor was fully restored when even 1.0 μM selenite was separated from the T₃ receptor by gel filtration. No inhibitory effect of selenite (up to 100 μM) on the T₃ binding to nuclear receptor was found in the presence of 1.0 mM dithiothreitol. The rate of dissociation of the T₃-nuclear receptor complex was effectively increased by 0.1 μM selenite. Selenate up to 1 mM as well as sulfite or sulfate up to 0.1 mM did not exert an inhibitory effect on T₃ receptors. The results based on the in vitro experiments suggest that the selenium in the form of selenite may reversibly affect the T₃ binding on the receptor molecule.     

A novel effect of molybdate on the binding of [3H]aldosterone to gel-filtered type I receptors in brain cytosol

Recently we reported that adding molybdate to crude steroid-free cytosol at 0°C results in a dose-dependent reduction in the binding of [³H]aldosterone ([³H]ALDO), to Type I adrenocorticosteroid receptors. In the experiments outlined here, we found that addition of molybdate to steroid-free brain cytosol produces a 30–50% increase in the subsequently measured maximal specific binding capacity (B _MAX) of [³H]ALDO-Type I receptors if the cytosol is subjected to Sephadex G-25 gel filtration prior to steroid addition. These manipulations were found to have no effect on the equilibrium dissociation constant (K _d) of the receptors. In contrast, when gel filtration of steroid-free cytosol was performed in the absence of molybdate, there was a 2-fold increase in the K_d and over a 50% reduction in the subsequently measuredB _MAX of [³H]ALDO-Type I receptors. When molybdate was added to this steroid-free cytosol immediately following gel filtration, there was no reduction (or increase) in Type I receptor [³H]ALDO binding capacity compared with nongel-filtered controls. The addition of as little as 2 mM molybdate to crude steroid-free cytosol was found to stabilize the binding capacity of Type I receptors during exposure to 22°C incubations; however, when gel-filtered steroid-free cytosol was exposed to these conditions at least 10 mM molybdate was required to stabilize Type I receptor binding capacity. Adding the sulfhydryl reducing reagent, dithiothreitol, to the various steroid-free cytosols had little effect on [³H]ALDO-Type I receptor binding. The effects of molybdate, revealed in this study, on Type I receptors in brain cytosol subjected to gel filtration are clearly different from those seen with receptors in crude cytosol preparations, as well as from those reported in the literature for other steroid receptors. Possible mechanisms of action of molybdate on unoccupied Type I receptors in crude and gel-filtered cytosol are discussed.     

Endogenous inhibitor of GABAB and GABAA receptors

An endogenous inhibitor of γ-aminobutyric acid (GABA) receptors was partially purified from bovine brain striatum. It was obtained as a low molecular weight fraction by gel filtration on Biogel P-2 and was adsorbed to Dowex AG 50W-X8, but not to Dowex AG 1-X8. It was ninhydrin-negative, basic, heat-stable substance. It caused dose-dependent inhibition of Na⁺-independent [³H]GABA bindings. Scatchard plot analysis of the [³H]GABA binding to GABA “B” receptor recognition site showed this inhibitor increased the K_d value (24.1 nM to 3.6 nM) without changing the B_max. On the other hand, Scatchard plot analysis of the [³H]GABA binding to GABA “A” receptor recognition site showed that the inhibitor decreased number of binding sites (706 fmol/mg protein to 494 fmol/mg protein) without affecting the K_d value. These results suggest that the endogenous inhibitor functions as a modulator for GABA_B and GABA_A receptors.     

Oestrogen receptor of mammary gland. Inhibition of aggregation and characterization of receptor from lactating gland in the presence of sodium bromide

1. When NaBr, a chaotropic salt, is added, in concentrations ranging from 0.5m to 2m, to low-salt mammary cytosol, (i) age-dependent aggregation of oestrogen receptor is inhibited, (ii) the receptor sediments as a sharp peak at 4.2S on sucrose-gradient centrifugation, with complete disappearance of heavier forms, and (iii) on gel filtration with Sephadex G-200, the receptor is included in the gel matrix. On a calibrated column, the receptor has a Stokes radius of 3.7nm (±6%). 2. Because NaBr inhibits interaction of receptor with other components of cytosol, the values of the sedimentation coefficient, measured by sucrose-gradient sedimentation, and of the Stokes radius, measured by gel filtration, can be accepted with confidence. From these values, it can be computed that the oestrogen-receptor form in NaBr has a mol.wt. of 64000, with a frictional ratio of 1.4. 3. Also, inhibition of aggregation by NaBr allows a 30–90-fold purification of oestrogen receptor. Analysis of this partially purified receptor by sucrose-gradient sedimentation and gel filtration in NaBr gives the same results as for receptor in crude cytosol. On electrofocusing on a pH5–8 gradient, the partially purified oestrogen receptor focuses at pH6.2. On removal of NaBr, receptor aggregates even in this partially purified state. It seems likely that at the protein and ionic concentrations of cytoplasm in vivo, the 64000-mol.wt. receptor form is part of higher states of self- and/or hetero-association with other cytoplasmic components. 4. NaBr up to a concentration of 2m does not inhibit binding of oestrogen by receptor, nor does it decrease the affinity of the interaction (K_D8.9×10⁻¹⁰m). The total number of binding sites in cytosol, however, decreases by approx. 10%, but this decrease may actually be the result of elimination of lower-affinity binding by non-receptor components of cytosol. 5. NaSCN, another chaotropic salt, was also tested but gave less satisfactory results with the mammary cytosol than with uterine cytosol. EDTA was omitted from the buffers because it favours aggregation of mammary oestrogen receptor. KCl (0.4m), sucrose (15%) and ZnSO₄ (3mm) did not prevent aggregation of receptor.     

Functions of calcium in the interaction of progesterone-receptor complex with nuclear components

Chick oviduct cytosol [³H]progesterone-receptor complex treated with 30 mm Ca²⁺ at 0 °C demonstrated a twofold greater binding to isolated chick oviduct nuclei or DNA-cellulose than such complexes activated thermally (25 °C). Divalent ions such as Mg²⁺ and Mn²⁺ were unable to mimic the effect of Ca²⁺ under identical conditions. The capacity of the Ca²⁺-treated progesterone-receptor complex to bind to nuclei or DNA-cellulose reached a peak within 45 min of Ca²⁺ treatment of the complex at 0 °C. This binding gradually declined as a function of incubation time and after 24 h at 0 °C no significant binding was observed. The Ca²⁺- and heat-treated chick oviduct [³H]progesterone-receptor complex was also characterized by DEAE-cellulose and agarose gel nitration chromatography. While heat-activated receptor could be resolved into A and B subunits on DEAE-cellulose, the receptor exposed to Ca²⁺ for 45 min at low temperature yielded the “A” subunit and a broad peak with poor affinity for the anion exchanger. The peak corresponding to “B” subunit was not discernible. The broad peak which eluted before the A peak was subsequently resolved by agarose gel filtration into receptor forms IV and V as described previously by Sherman et al. (M. Sherman, S. Atienza, J. Shansky, and L. Hoffman, 1974, J. Biol. Chem., 249, 5351–5363; M. Sherman, L. Pickering, F. Rollwagen and L. Miller, 1978, Fed. Proc., 37, 167–173). Again DEAE-cellulose chromatography of the progesterone-receptor complex treated as long as 24 h at 0 °C with Ca²⁺ revealed a poorly bound peak which on agarose gel filtration corresponded exclusively to form V. A correlation was apparent between an increase in form V and a gradual decrease in the binding capacity of the Ca²⁺-treated steroid-receptor complex to nuclei, DNA-cellulose, or DEAE-cellulose filters.Based on these findings, I postulate that Ca²⁺ has a functional role in the mechanism of progesterone action in chick oviduct. Firstly, it enhances a low temperature, time dependent binding of the progesterone-receptor complex to chick oviduct nuclear components, and subsequently promotes, by possible activation of endogenous protease(s) the cleavage of the receptor subunits.     

Binding of [3H]heparin to human plasma low density lipoprotein.

The binding of [³H]heparin to human plasma lipoproteins was measured using a gel filtration assay on columns of Ultrogel AcA 54. [³H]Heparin formed a soluble complex with low density lipoprotein (LDL) as evidenced by the appearance of a new radioactive peak emerging at the void volume where the lipoproteins elute. Free heparin on the other hand was retarded on this column and eluted at a later volume. Heparin binding to LDL could also be demonstrated on columns of Sepharose 4B, in which case two included peaks of ³H were observed to elute in the area of LDL and of heparin. [³H]Heparin did not bind to either high or very low density lipoproteins as determined by the gel filtration assay. The binding of the [³H]heparin to LDL was proportional to both the concentration of LDL and of heparin and both showed saturation kinetics. Cations were not necessary for binding, nor was binding inhibited by EDTA. LDL showed a marked specificity for heparin. Thus, the binding of [³H]heparin to LDL was strongly inhibited by the addition of unlabeled heparin, while other glycosaminoglycans such as chondroitin sulfate, heparan sulfate, keratan sulfate, and dermatan sulfate were not effective inhibitors except at very high concentrations. Salts, especially K₂HPO₄ and (NH₄)₂SO₄, also inhibited binding when added at concentrations of 10 mm or higher suggesting an ionic interaction between heparin and LDL. The pH optimum for binding was between 7.5 and 8.5 but binding fell off markedly above pH 9.0. The [³H]heparin was heterogeneous and could be separated into four fractions on columns of Sephadex G-75. When these fractions were tested for binding to LDL, only the high molecular weight fraction bound to any significant extent. LDL was treated with reagents used to selectively modify basic amino acid residues, and the effect of these treatments on heparin binding was examined. Thus, ethoxyformic anhydride was used for histidine modification, acetic anhydride and succinic anhydride for lysines and cyclohexanedione for arginine residues. In each case there was a significant loss in heparin binding suggesting that various basic amino acids are involved in binding and/or that basic amino acids are necessary to maintain the proper conformation of LDL.     

Purification and characterization of MerR, the regulator of the broad-spectrum mercury resistance genes in Streptomyces lividans 1326

Streptomyces lividans 1326 carries inducible mercury resistance genes on the chromosome, which are arranged in two divergently transcribed operons. Expression of the genes is negatively regulated by the repressor MerR, which binds in the intercistronic region between the two operons. The merR gene was expressed in E. coli using a T7 RNA polymerase/promoter expression system, and MerR was purified to around 95% homogeneity by ammonium sulfate precipitation, gel filtration and affinity chromatography. Gel filtration showed that the native MerR is a dimer with a molecular mass of 31?kDa. Two DNA binding sites were identified in the intercistronic mer promoter region by footprinting experiments. No evidence for cooperativity in the binding of MerR to the adjacent operator sequences was observed in gel mobility shift assays. The dissociation constants (K_D) for binding of MerR were: binding site I, 8.5?×?10^?9?M; binding site II, 1.2?×?10^?8?M; and for the complete promoter/operator region 1?×?10^?8?M. The half-life of the MerR-DNA complex was 19.4?min and 18.8?min for binding site I and binding site II, respectively. The K_D value for binding of mercury(II)chloride to MerR, again determined by mobility shift assay, was 1.1?×?10^?7?M.     

Isolation of the two components of Condylactis toxin.

The two toxic components from the sea anemone Condylactis gigantea have been isolated and purified using gel filtration, ion-exchange chromatography, and preparative isoelectric focusing. The molecular weight of both components is estimated by gel filtration to be 4500. The isoelectric points of the two toxins were determined to be 4.8 and 5.8 with LD₅₀ values of 19 and 58 μg/kg, respectively, when injected into Armadillidium vulgare, a terrestrial crustacean. Low-level labeling with ¹²⁵I using lactoperoxidase was effected for the purpose of estimating homogeneity.     

Isopentenyl pyrophosphate isomerase and prenyltransferase from tomato fruit plastids

Isopentenyl pyrophosphate isomerase has been isolated from an extract of tomato fruit plastids and purified 245-fold by fractionation with ammonium sulfate, gel filtration on Bio-Gel A 1.5m, ion-exchange chromatography on DEAE-cellulose, gel filtration on Sephadex G-100, and chromatofocusing. Gel filtration on Sephadex G-100 separated the isopentenyl pyrophosphate isomerase from a prenyltransferase fraction that catalyzed the conversion of isopentenyl pyrophosphate to acid-labile compounds in the presence of dimethylallyl, geranyl, or farnesyl pyrophosphates. The molecular weights of the isopentenyl pyrophosphate isomerase and prenyltransferase were determined to be 34,000 and 64,000, respectively, by gel filtration on Sephadex G-100. The only cofactor required by either the isomerase or the prenyltransferase was a divalent cation, either Mg²⁺ or Mn²⁺. Isopentenyl pyrophosphate isomerase could also be totally inactivated by 1 × 10^?3m iodoacetamide, and this property was utilized in the assay of prenyltransferase activity in the presence of contaminating isomerase. The inactivation of isomerase by iodoacetamide is consistent with the stabilization of isopentenyl pyrophosphate isomerase by dithiothreitol. The K_m of isopentenyl pyrophosphate isomerase for isopentenyl pyrophosphate was found to be 5.7 × 10^?6.     

The complex kinetics of auxin-binding to a particulate fraction from tobacco-pith callus

The kinetics of binding of 1-naphthylacetic acid to particulate fractions from tobacco-pith callus were studied. This binding site does not bind auxin at 0° C. Binding experiments performed at 25° C demonstrated an apparent K _a of approx. 6.5·10⁶ M^-1. A filtration method was developed in order to study non-equilibrium kinetics of this binding. Dissociation of the complex of auxin and binding site indicates the presence of at least two binding components with dissociation rate constants (k _off) of 6.1·10^-3 min^-1 and 6.0·10^-2 min^-1. This binding behaviour was not independent, indicating that the binding of auxin to the particulate fractions was more complex than binding of one hormone molecule to one binding site. This complexity was further confirmed by experiments in which the initial velocity of complex formation was measured. A model was worked out into which our data fit without contradictions. It involves the binding of four hormone molecules to one receptor molecule.     

Threonine-sensitive aspartate kinase and homoserine dehydrogenase from Pisum sativum

Aspartate kinase and two homoserine dehydrogenases were partially purified from 4-day-old pea seedlings. A sensitive method for measuring aspartate kinase activity is described. Aspartate kinase activity was dependent upon ATP, Mg²⁺ or Mn²⁺, and aspartate. The aspartate kinase was inhibited in a sigmoidal manner by threonine and K_i for threonine was 0·57 mM. The enzyme could be desensitized to the inhibitor and threonine protected the enzyme against thermal inactivation. Aspartate kinase activity was enhanced by isoleucine, valine and alanine. Homoserine, methionine and lysine were without effect. The homoserine dehydrogenase activity which was associated with aspartate kinase during purification could be resolved into two peaks by gel filtration. The activity of both peaks was inhibited by aspartate and cysteine and one was inhibited by threonine.     

Isolation of a 110 000 molecular weight protein in the purification of the nuclear chick intestinal 1,25-dihydroxyvitamin D-3 receptor

A single protein band of molecular weight 110 000 has been obtained after sodium dodecyl sulfate polyacrylamide gel electrophoresis of purified 1,25-dihydroxyvitamin D-3 (1,25-(OH)₂D-3) receptor from crude nuclear extracts of chick intestinal mucosa, prepared in the presence of the protease inhibitors phenylmethylsulfonyl fluoride and ?-aminocaproic acid. The nuclear extract was subjected to a six-step purification scheme, involving polymin P and ammonium sulfate fractionation, DNA-cellulose affinity chromatography, Sephacryl S-200 gel filtration, blue dextran-Sepharose and a final DNA-cellulose chromatographic step. The receptor was obtained in about 1% yield and was purified approx. 3700-fold from the nuclear extract, as assessed by specific activity. Single peaks were observed with ³H-1,25-(OH)₂D-3-labeled crude nuclear extracts on Sephacryl S-200 gel filtration ($\text{Strokes′ radius}\text{= 35.5}\text{A}\text{?}$) and sucrose density gradient centrifugation (3.5 S). Although the identity of the M_r 110 000 protein will remain inconclusive until methods for further characterization are available, it may represent evidence for a higher molecular weight form of the 1,25-(OH)₂D-3 receptor than that observed previously.     

Enzymatic biosynthesis of ergotamine and investigation on some aminoacyl-tRNA synthetases in Claviceps

An enzyme system from Claviceps purpurea (Fr.) Tul. catalyzing the incorporation of l-phenylalanine into ergotamine - ergotamine synthetase - was purified 172-fold. This was done by a combination of ammonium sulfate precipitation, gel filtration, ion-exchange chromatography on DEAE-Sepharose CL-6B, and hydroxyapatite chromatography. The activation of ergotamine specific amino acids as well as d-lysergic acid and dihydrolysergic acid via adenylates, as determined by the ATP-³²PP_i exchange, was investigated. Phenylalanyl-tRNA synthetase, catalyzing the same type of activation reaction, could not be separated from ergotamine synthetase by the purification procedure applied. Therefore, at the present stage of enzyme purification, phenylalanine-dependent ATP-³²PP_i exchange cannot be used to measure ergotamine synthetase activity specifically.Phenylalanyl-tRNA synthetase and leucyl-tRNA synthetase were separated into mitochondrial and cytoplasmic isoenzymes by hydroxyapatite chromatography. Their charging activities of procaryotic versus eucaryotic tRNA and their molecular masses were determined.     

Purification and molecular properties of the acetylcholine receptor from Torpedo electroplax

The acetylcholine receptor of Torpedo electroplax is purified by affinity adsorption using cobra toxin (Naja naja siamensis) covalently attached to Sepharose 4B. Desorption by 10 mm benzoquinonium produces a protein that binds α-[¹²⁵I]bungarotoxin but not [³H]acetylcholine or other reversible cholinergic ligands. On the other hand, desorption by 1 m carbamylcholine produces an acetylcholine receptor protein that binds [³H]acetylcholine, [³H]decamethonium, [³H]nicotine, [¹⁴C]dimethyl-d-tubocurarine, and α-[¹²⁵I]bungarotoxin. The batch method of affinity adsorption employed gives recoveries of acetylcholine receptor (as measured by acetylcholine binding) averaging 69.2 ± 14.6%. The purity of the isolated acetylcholine receptor protein is estimated to be at best 87% as judged by disc gel electrophoresis and electrofocusing.The purified acetylcholine receptor binds 7.8 nmoles acetylcholine/mg protein based on estimation of protein concentration by a spectrophotometric method. Of these, 2.7 nmoles exhibit high affinity (K_D = 0.02 μM) and 5.1 nmoles a lower affinity (K_D = 1.97 μM. If the protein concentration used is that obtained by amino acid analysis, the total specific activity would be 10.4 nmoles acetylcholine bound per milligram protein. The subunit carrying one acetylcholine binding site is estimated to range between 83,000 and 112,000 daltons. In contrast to the membrane-bound or Lubrol-solubilized acetylcholine receptor, the purified acetylcholine receptor shows no autoinhibition with acetylcholine concentrations up to 10 μm. Binding of acetylcholine was totally inhibited by α-bungarotoxin or cobra toxin and was partially blocked by four nicotinic drugs, but not by two muscarinic ones. The amino acids of the acetylcholine receptor are analyzed and compared to those of acetylcholinesterase.     

Nonactivated and activated glucocorticoid receptor complexes from human salivary gland adenocarcinoma cell line

[³H]Triamcinolone acetonide glucocorticoid receptor complexes from human salivary gland adenocarcinoma cells (HSG cells) were shown to be activated with an accompanying decrease in molecular weight in intact cells, as analyzed by gel filtration, DEAE chromatography, the mini-column method and glycerol gradient centrifugation. Glucocorticoid receptor complexes consist of steroid-binding protein (or glucocorticoid receptor) and non-steroid-binding factors such as the heat-shock protein of molecular weight 90 000. To determine whether the steroid-binding protein decreases in molecular weight upon activation, affinity labeling of glucocorticoid receptor in intact cells by incubation with [³H]dexamethasone 21-mesylate, which forms a covalent complex with glucocorticoid receptor, was performed. Analysis by gel filtration and a mini-column method indicated that [³H]dexamethasone 21-mesylate-labeled receptor complexes can be activated under culture conditions at 37°C. SDS-polyacrylamide gel electrophoresis of [³H]dexamethasone 21-mesylate-labeled steroid-binding protein resolved only one specific 92 kDa form. Furthermore, only one specific band at 92 kDa was detected in the nuclear fraction which was extracted from the cells incubated at 37°C. These results suggest that there is no change in the molecular weight of steroid-binding protein of HSG cell glucocorticoid receptor complexes upon activation and that the molecular weight of nuclear-binding receptor does not change, although the molecular weight of activated glucocorticoid receptor complexes does decrease. Triamcinolone acetonide induced an inhibitory effect on DNA synthesis in HSG cells. Dexamethasone 21-mesylate exerted no such effect and blocked the action of triamcinolone acetonide on DNA synthesis. These results suggests that dexamethasone 21-mesylate acts as antagonist of glucocorticoid in HSG cells. The fact that dexamethasone 21-mesylate-labeled receptor complexes could be activated and could bind to DNA or nuclei aas well as triamcinolone acetonide-labeled complexes suggests that dexamethasone 21-mesylate-labeled complexes can not induce specific gene expression after their binding to DNA.     

Folate Receptors in Malignant and Benign Tissues of Human Female Genital Tract

We have characterized the folate receptor in malignant and benign tissues of human female genital tract (Fallopian tube and benign and malignant tissues of uterus). Radioligand binding displayed characteristics similar to those of other folate binding proteins. Those include a high-affinity type of binding (K=10¹⁰M^–1), apparent positive cooperativity, a slow dissociation at pH 7.4 becoming rapid at pH 3.5, and inhibition of binding by folate analogues. The gel filtration profile of Triton X-100 solubilized tissue contained two large peaks of ³H-folate labelled protein (>=130 and 100kDa) as well as a 25 kDa peak. Only a single band of 70 kDa was seen on SDS-PAGE immunoblotting. The large molecular size forms on gel filtration appear to represent folate receptors having a hydrophobic membrane anchor inserted into Triton X-100 micelles. The folate receptor of female genital tract showed cross-reactivity in ELISA and positive immunostaining with rabbit antibodies against human milk folate binding protein. Variations in the ratio of immunoresponse to total high affinity folic acid binding suggests the presence of multiple isoforms of the receptor in different types of malignant and benign tissues.     

A calorimetric study of the binding of 2'-deoxyuridine-5'-phosphate and 5-fluoro-2'-deoxyuridine-5'-phosphate to thymidylate synthetase.

The thermodynamic parameters, ΔH′, ΔG′, and ΔS′, and the stoichiometry for the binding of the substrate 2′-deoxyuridine-5′-phosphate (dUMP) and the inhibitor 5-fluoro-2′-deoxyuridine-5′-phosphate (FdUMP) to Lactobacillus casei thymidylate synthetase (TSase) have been investigated using both direct calorimetric methods and gel filtration methods. The data obtained show that two ligand binding sites are available but that the binding of the second mole of dUMP is extremely weak. Binding of the first mole of dUMP can best be illustrated by dUMP + TSase + H⁺?(dUMP-TSase-H⁺). [1] The enthalpy, ΔH₁′, for reaction [1] was measured directly on a flow modification of a Beckman Model 190B microcalorimeter. Experiments in two different buffers (I = 0.10 m) show that ΔH₁′ = ?28 kJ mol^?1 and that 0.87 mol of protons enters into the reaction. Analysis of thermal titrations for reaction [1] indicates a free energy change of ΔG₁′ = ?30 kJ mol^?1 (K₁ = 1.7 × 10⁵ m^?1). From these parameters, ΔS₁′ was calculated to be +5 J mol^?1 degree^?1, showing that the reaction is almost totally driven by enthalpy changes. Gel filtration experiments show that at very high substrate concentrations, binding to a second site can be observed. Gel filtration experiments performed at low ionic strength (I = 0.05 m) reveal a stronger binding, with ΔG₁′ = ?35 kJ mol^?1 (K₁ = 1.2 × 10⁶ m^?1), suggesting that the forces driving the interaction are, in part, electrostatic. Addition of 2-mercaptoethanol (0.10 m) had the effect of slightly increasing the dUMP binding constant. Binding of FdUMP to TSase is best illustrated by 2FdUMP + TSase + n_HH⁺?FdUMP₂ ? TSase ? (H⁺)_nH. [2] The enthalpy for this reaction, ΔH₂, was also measured calorimetrically and found to be ?30 kJ mol^?1 with n_H = 1.24 at pH 7.4 Assuming two FdUMP binding sites per dimer as established by Galivan et al. [Biochemistry15, 356–362 (1976)] our calorimetric results indicate different binding energies for each site. Based on the binding data, a thermodynamic model is presented which serves to rationalize much of the confusing physical and chemical data characterizing thymidylate synthetase.     

Antimicrobial Activity of a Novel Vascular Access Film Dressing Containing Chlorhexidine Gluconate

Background

Covering insertion sites with chlorhexidine impregnated dressings has been proven to be clinically effective in reducing catheter related blood stream infections (CR-BSI). Two chlorhexidine gluconate (CHG)-impregnated dressings are commercially available, a polyurethane foam disk and a film dressing containing a chlorhexidine gluconate-impregnated gel pad. While both have demonstrated efficacy in clinical settings, the major drawback of high cost and impaired IV insertion site visibility limits their usage. A new, simple film dressing containing CHG within its adhesive layer is now available. The objective of this study was to test the in vitro antimicrobial efficacy of the new dressing in comparison to the CHG-impregnated gel dressing.

Methods

Quantitative aliquots of suspensions (concentration of 1.0x10⁶ to 5.0x10⁶ cfu/sample) of clinically relevant challenge organisms (Staphylococcus species, gram-negative bacilli, Candida albicans) were incubated in contact with the new CHG-containing film dressing, a placebo version of the same (negative control) and the commercially available CHG-impregnated gel dressing (positive control). Serial dilutions of the surviving organisms were quantified using the pour plate after 1, 3, 5, and 7 days of incubation in order to calculate an antimicrobial log₁₀ reduction for each organism/dressing combination at each point in time.

Results

The new CHG-containing film dressing delivered greater than 5.0 log₁₀ reduction throughout the 7 days on all aerobic gram-negative bacilli and Staphylococcus species tested. As of day 1 the CHG-containing film dressing provided greater than 5.0 log₁₀ reduction on Candida albicans. There were no statistically significant differences in the log₁₀ reduction between the two dressings tested.

Conclusion

The new CHG-containing film dressing was found to be as effective as the chlorhexidine gluconate-impregnated gel dressing on clinically relevant microbes.     

The relationship of purity to photosynthetic activity in preparations ofCodium fragile chloroplasts

Summary The purity of the chloroplasts isolated from the algaCodium fragile by 1. a conventional centrifugation method, and 2. a method involving a gel filtration stage through Sephadex G 50 coarse, was compared using enzymatic analysis in conjunction with electron microscopy. The findings show that the purer chloroplasts, obtained by gel filtration, could carboxylate more¹⁴CO₂ and evolve more O₂ than the crude preparation from which they originated.     

Effects of electron donors and acceptors on the kinetics of the photoelectric responses in Rhodospirillum rubrum and Rhodopseudomonas sphaeroides chromatophores

Chromatophores of Rhodospirillum rubrum and Rhodopseudomonas sphaeroides were adhered to one side of a collodion film impregnated with a phospholipid solution in decane and 20 ns laser flashes were delivered to produce an electrical potential difference generated across the collodion film in less than 0.2 μs (resolution time of the apparatus). The kinetics of Δψ decay in the dark was studied. In the absence of additions there occurs a ‘rapid’ decay of photoelectric potential (τ ≈ 70 ms) corresponding to charge recombination within the primary dipole P-870⁺-Q^-_A. The rapid decay of Δψ is prevented by ascorbate in the presence of permeable redox dyes which can reduce the photooxidized P-870⁺ rapidly. Under these conditions, Δψ dissipates with τ > 0.5 s typical of a passive discharge of the chromatophore membrane. Prevention of the rapid decay of Δψ by 70–75% can be observed upon addition of excess ubiquinone-10 to the solution of phospholipids used to impregnate the collodion film, and to a lesser extent by addition of some other quinones. The effect of quinones is inhibited by o-phenanthroline. The data obtained show that upon association of chromatophores with the collodion film, the secondary quinone acceptor is extracted from its binding site into a hydrophobic volume of the macroscopic membrane, and this effect can be reversed by exogenous ubiquinone. About 4-times less Q-10 is required to reconstitute Q_B function in chromatophores from Rps. sphaeroides than in those from R. rubrum, which points to a tighter binding of the secondary acceptor in the former. No evidence for electrogenic nature of Q⁻_A → Q_B electron transfer could be obtained in experiments with Q_B-replenished chromatophores.