Affinity partitioning of phosphofructokinase from baker's yeast using polymer-bound Cibacron blue F3G-A

1. Phosphofructokinase from baker's yeast is partitioned between the phases of an aqueous two-phase system, containing dextran (Mr = 500000) and poly(ethyleneglycol) (Mr = 6000), in favour of the dextran-rich phase. By covalent binding of the dye Cibacron blue F3G-A to poly(ethyleneglycol) the enzyme can be extracted to the phase rich in this polymer, i.e. affinity partitioning. 2. The affinity partitioning effect, measured as the logarithmic increase of the partition coefficient by introducing polymer-bound Cibacron blue, depends on several factors. The influence of dye-polymer concentration, polymer concentration, polymer molecular weight, kind of salt and salt concentration, pH and temperature has been studied. 3. The effect of ATP, ADP, AMP, ITP, fructose 1,6-bis-phosphate and fructose 6-phosphate show large differences in the binding strength of these substances to the Cibacron blue binding sites. AMP cannot compete with Cibacron blue while ATP is strongly competing. 4. The use of affinity partitioning for enzyme isolation and determination of ligand binding is discussed, as well as possible mechanisms concerning this type of liquid/liquid extraction.     

Adjustable ‘cross-linking’ of neighboring DNA molecules in liquid-crystalline dispersions through (daunomycin-copper) polymeric chelate complexes

The interaction of daunomycin molecules with double-stranded DNA in the liquid-crystalline state was investigated. It was shown that at a certain extent of daunomycin binding a change of the mechanism of anthracycline orientation with reference to the DNA chain occurs. This is testified by the alteration of the sense of spatial packing of the DNA molecules in liquid-crystalline dispersions formed as a result of phase separation in poly(ethyleneglycol)-containing solutions, as well as by the onset of the reaction of daunomycin with divalent copper ions. Using this reaction, polymeric (daunomycin-copper) chelate cross-links between the DNA molecules fixed in the liquid-crystalline dispersions were formed. The length of such cross-links is adjusted by the distance between the DNA molecules, which, in turn, depends on the concentration of poly(ethyleneglycol) used for phase separation. The above molecular building mechanism may lead to new interesting applications.     

Acetyl-coenzyme-A carboxylase of Candida Lipolytica. 1. Purification and properties of the enzyme.

Acetyl-coenzyme-A carboxylase has been isolated in homogeneous form from Candida lipolytica. The homogeneity of the enzyme preparation is evidenced by analytical ultracentrifugation, dodecyl-sulfate-polyacrylamide gel electrophoresis and Ouchterlony double-diffusion analysis. The purified enzyme exhibits a specific activity of 8.0 U/mg protein at 25 degrees C and contains 1 mol biotin/263000 g protein. The sedimentation coefficient (S20,W) of the enzyme is 18 S. It has been shown by dodecyl-sulfate-polyacrylamide gel electrophoresis that the enzyme possesses only one kind of subunit with a molecular weight of 230000. This finding, together with the biotin content, indicates that the C. lipolytica enzyme has a highly integrated subunit structure. The C. lipolytica enzyme is very labile, but is stabilized by glycerol. The enzyme is markedly activated by poly(ethyleneglycol), the activation being due principally to a decrease in the Km values for substrates. Even in the presence of this activator, the Km value for acetyl-CoA of the C. lipolytica enzyme is much higher than that of the enzyme from Saccharomyces cerevisiae and animal tissues. The C. lipolytica enzyme, unlike the enzyme from animal tissues, is not activated by citrate.     

Structural properties of poly C-scleroglucan complexes

Successive changes of solvent conditions can be used to dissociate and reassociate the triple-helical structure of (1,3)-beta-D-glucans. Ultramicroscopic techniques have revealed a blend of circular and other structures following renaturation. When this solvent exchange process is carried out in the presence of certain polynucleotides, the process creates a novel macromolecular complex. Here, we use size exclusion chromatography (SEC) to study such (1,3)-beta-D-glucan-polynucleotide complexes. Online multi-angle laser-light scattering (MALLS) and refractive index (RI) detectors allowed determination of molecular weight and radius of gyration of the molecules. An ultraviolet (UV) detector allowed specific detection of the polynucleotide. The poly-cytidylic acid (poly C) shifted to coelution with the linear fraction of the scleroglucan following the renaturation of poly C-scleroglucan blends, indicating that poly C is incorporated in linear, but not in circular, structures of scleroglucan. This conclusion was consistent with AFM topographs that revealed a decreased fraction of circular structures upon addition of poly C during the renaturation process. The combined information about radius of gyration (R(g)) and molecular weight (M(w)) allowed us to conclude that the poly C-scleroglucan complexes are more dense and have a higher persistence length than linear scleroglucan triple helixes. The experimentally determined mass per unit length was used as a basis for elucidating possible molecular arrangements within the poly C-scleroglucan complex.     

The effect of poly(ethyleneglycol) esters on the partition of proteins and fragmented membranes in aqueous biphasic systems

The hydroxyl groups of poly(ethyleneglycol) have been esterified (partly) with a number of carboxylic acids. When these esters are included in dextranpoly(ethyleneglycol)-water biphasic systems the partitions of proteins and membranes between the two phases (and the interface) are in some cases strongly affected. The affinity of serum albumin for the poly(ethyleneglycol)-rich phase is strongly increased when the fatty acid group consists of more than 10 carbon atoms. The partition also depends on the number of double bonds in the fatty acid. A corresponding relationship is found for membranes from spinach chloroplasts. The partitions of ovalbumin, lysozyme (EC 3.2.1.17) and ribonuclease (EC 3.1.4.22) are not influenced by the fatty acid esters. Esters of dibasic carboxylic acids show a minute but marked effect on the partition of proteins in general while malate and tartrate esters affect strongly the partition of chloroplast membranes. The partitions of both proteins and membranes are influenced by poly(ethyleneglycol) deoxycholate. Experiments with malate dehydrogenase (EC 1.1.1.37), lactate dehydrogenase (EC 1.1.1.27), fumarase (EC 4.2.1.2), enolase (EC 4.2.1.11) and glutamate-oxaloacetate transaminase (EC 2.6.1.1) show that their partitions, measured on enzymic activity basis, is changed when esters of benzoic, linolenic, tartaric or deoxycholic acid are included in the biphasic system. The mechanism behind the effect of the esterified poly(ethyleneglycol) on the partition of biomaterial, in this type of aqueous biphasic systems, is discussed in terms of a direct binding of the esters to the partitioned material.     

Determinants of liposome partitioning in aqueous two-phase systems: Evaluation by means of a factorial design

This article evaluates the influence of five parameters on liposome partitioning in aqueous two-phase systems (ATPSs), composed of poly(ethyleneglycol) (PEG)/dextran (Dx), using the factorial experimental design together with a multiple regression. Mathematical models to quantify the influence of these parameters, individually and/or jointly, on liposome partitioning in ATPS were developed. The models were statistically tested and verified by experimentation. This approach was then used to define the conditions for the preferential accumulation of liposomes in the top PEG-rich phase. The models predicted a significant effect of liposome surface charge, PEG molecular weight, phase-forming polymer concentration, and phosphate ion concentration on the partition behavior of liposomes. For negatively charged liposomes, it was found that the smaller the molecular weight of PEG and polymer concentration and the larger the phosphate ion concentration, the greater the partition coefficient of the liposomes. No significant effect of pH, at the range of 6-8, on liposome partitioning was noted. This approach has led to the development of an optimal two-phase system where 90% of negatively charged liposomes accumulated in the PEG phase. In addition to the general scientific value of this research, it has a technological importance as ATPSs may be useful for removing the unentrapped drug from liposomes during their preparation for pharmaceutical applications. (c) 1996 John Wiley & Sons, Inc.     

Effect of histone H1, poly(ethyleneglycol) and DNA concentration on intermolecular and intramolecular ligation by T4 DNA ligase

The efficiency of ligation of linear DNA and the relative amounts of intramolecular versus intermolecular ligation may be triggered by a number of additive agents. The results show that it is possible to mimic the effect of poly(ethyleneglycol) 6000 by simply increasing DNA concentration about 15-fold: both the rate and the extent of the reaction are greatly enhanced, and intermolecular ligation is largely favored. However, in this case the stimulation by salts, which occurs in poly(ethyleneglycol) solutions, is not observed; we suggest that salts enhance the hydrophobic interactions between ligase and DNA that take place in the presence of poly(ethyleneglycol). We also show that histone H1, which is involved in the formation of chromatin fibers, is able to stimulate intermolecular ligation by T4 ligase. This effect is more specific than a simple neutralisation of the phosphate groups of the DNA by positive charges of the histone; it still occurs at 125 mM NaCl and in the presence of the four core histones. The implications of the finding concerning the mode of action of histone H1 on DNA are discussed.     

Purification and properties of soybean leghemoglobin messenger RNA

Poly(A)-containing leghemoglobin mRNA from soybean root nodules has been purified 84-fold, as judged by its ability to direct the in vitro synthesis of leghemoglobin in a wheat germ system. It has a poly(A) content of 8.6% and a molecular weight, estimated by formamide gel electrophoresis, of 260 000. mRNA with a molecular weight of around 143 000 would be sufficient to code for leghemoglobin. Thus, with respect to both its poly(A) content and its unexpectedly high molecular weight, leghemoglobin mRNA is similar to mRNAs isolated from animal tissues.     

Kinetics of the proton-deuteron exchange at position H8 of adenine and guanine in DNA.

Proton-NMR has been used to determine the activation energies and pre-exponential factors for the deuterium exchange of AH8 in poly(dA-dT).poly(dA-dT), and for GH8 in poly(dG-dC).poly(dG-dC). No simple relationship between the kinetic parameters and molecular conformation was found. By addition of 4.5 M NaCl a transition from the B to the Z conformation was induced for poly(dG-dC).poly(dG-dC), and an increased exchange rate was observed. The exchange rate for poly(dA-dT).poly(dA-dT) also increased below 64 degrees C, and a significant decrease in activation energy on addition of 4.5 M NaCl was observed. The exchange rates at T = 55.8 degrees C were also measured for the AH8 and GH8 in random sequence calf thymus DNA. From the difference in exchange rates, a method of preferential labeling of either the AH8 or the GH8 in high molecular weight DNA is evaluated.     

Isolation and purification of poly(ADP-ribose) glycohydrolase from pig thymus

Poly(ADP-ribose) glycohydrolase has been purified about 12 300-fold from pig thymus with a recovery of 8.5%. The specific activity of the purified enzyme is 13.8 mumol min -1 mg protein -1. The molecular weight was estimated to be 59 000 by gel filtration through Sephadex G-100 in a non-denaturing solvent. Analysis of the final preparation by sodium dodecyl sulphate gel electrophoresis reveals two protein bands of molecular weight, 61 500 and 67 500. The Km value for poly(ADP-ribose) is estimated to be 1.8 microM monomer units. The enzyme preparation is free from phosphodiesterase, NADase and ADP-ribosyltransferase activities. The purified enzyme is inhibited by cyclic AMP, ADP-ribose, naphthylamine, histones H1, H2A, H2B, H3, polylysine, polyarginine, polyornithine and protamine. The inhibition by histone is relieved by an equal mass of DNA. Single-stranded DNA, poly(A), poly(I) and polyvinyl sulphate were inhibitory, but double-stranded DNA was not inhibitory.     

Large-scale preparation of T4 endonuclease VII from over-expressing bacteria

Endonuclease VII is the product of gene 49 of phage T4 and was the first enzyme shown to resolve Holliday structures in vitro [Mizuuchi, K. et al. (1982) Cell 29, 357-365]. Low amounts of the enzyme were originally purified from phage-infected cells [Kemper, B. & Garabett, M. (1981) Eur. J. Biochem. 115, 123-131]. We now report a purification procedure for milligram amounts of cloned endonuclease VII expressed in Escherichia coli with gene 49 under the control of a temperature-inducible promoter on a plasmid system [Tomaschewski, J. (1988) PhD Thesis, University of Bochum, FRG]. The protein was purified 500-fold from crude extracts in five steps with a recovery of 15%. The steps include (a) poly(ethyleneglycol)/dextran two-phase separation; (b) DEAE-cellulose; (c) single-stranded DNA-agarose; (d) Mono-Q and (e) Mono-S chromatography. The final protein was more than 98% pure as estimated from SDS/PAGE analysis. The protein has an apparent molecular mass of 17.8 kDa on SDS-containing polyacrylamide gels and 36 kDa when determined by gel filtration or sedimentation through sucrose gradients in the presence of high salt (600 mM NaCl). In the absence of additional salt, the enzyme has a tendency to aggregate and products of molecular masses differing in steps of about 18 kDa appear on SDS-containing polyacrylamide gels.     

An approach to heterobifunctional poly(ethyleneglycol) bioconjugates

A family of differentially substituted poly(ethyleneglycol) building blocks has been assembled from commercially available material. Their utility is demonstrated by formation of amino acid conjugates, image contrast agents, gold nanoparticles, and functional antibody conjugates. Application in the cellular trafficking of antitumoral agent conjugates is expected.     

Cross-linked polyethylenimine as potential DNA vector for gene delivery with high efficiency and low cytotoxicity

Polyethylenimine (PEI) has been known as an efficient gene carrier with the highest cationiccharge potential.High transfection efficiency of PEI,along with its cytotoxicity,strongly depends on itsmolecular weight.To enhance its gene delivery efficiency and minimize cytotoxicity,we have synthesizedsmall cross-linked PEI with biodegradable linkages and evaluated their transfection efficiencies in vitro.Inthis study,branched PEI with a molecular weight of 800 Da was cross-linked by small diacrylate[1,4-butanediol diacrylate or ethyleneglycol dimethacrylate (EGDMA)] for 2-6 h.The efficiencies of thecross-linked PEI in in vitro transfection of plasmid DNA containing enhanced green fluorescent protein(EGFP) reporter gene were assessed in melanoma B 16F10 cell line and other cell lines.Flow cytometrywas used to quantify the cellular entry efficiency of plasmid and the transgene expression level.Thecytotoxicities of the cross-linked PEI in these cells were evaluated by MTT assay.EGDMA-PEI 800-4h,atypical cross-linked PEI reported here,mediated a more efficient expression of reporter gene than thecommercially available 25-kDa branched PEI control,and resulted in a 9-fold increase in gene deliveryin B16F10 cells and a 16-fold increase in 293T cells,while no cytotoxicity was found at the optimizedcondition for gene delivery.Furthermore,the transfection activity of polyplexes was preserved in thepresence of serum proteins.     

Mesoscopic simulation of self-assembly of linear and dendritic copolymer poly(styrene)-b-poly(ethyleneglycol) in polar and non-polar solvents

In this work, we simulate the microphase separation of aqueous and non-aqueous solutions of diblock copolymer poly(styrene)-b-poly(ethyleneglycol) under different architectures (linear and linear–dendritic) by dissipative particle dynamics. The observed morphologies in water where poly(ethyleneglycol) (PEG) block is soluble are as follows: (1) at low concentrations spherical micelles, cylinders and bicontinuous structures are formed in dendritic structures and spheres, cylinders and perforated lamellas in linear structure. The architectures simulated at low–moderate concentrations show an evolution sphere → cylinder → bicontinuous or perforated lamellas as the concentration is increased. (2) At high concentrated solutions rich defect structures of the sponge type are formed. In a non-aqueous non-polar solution such as cyclohexane, which is a good solvent for the polystyrene block, the formation of well-defined aggregates at low concentrations is not observed; however, irregular structures are achieved in concentrated solutions. We compare these results with a polymeric chimera consisting of a mixture of linear poly(styrene) homopolymer and PEG homopolymer in the linear, G1 or G2 dendritic configurations. Our simulations are in agreement with the experimentally observed structures of these polymers.     

Purification and properties of two ribonucleases and a nuclease from barley seeds

Three enzymes possessing RNAase activity were isolated from barley seeds. These enzymes were further purified by ammonium sulphate precipitation DEAE-cellulose chromatography, gel filtration on Sephadex G-75 and DEAE-Sephadex A-50 chromatography. These enzymes have been characterized and classified as: 1. Plant RNAase I (EC 3.1.27.1). It has a pH optimum at 5.7 and molecular weight of 19 000. 2. Plant RNAase II (EC 3.1.27.1). It has a pH optimum at 6.35 and molecular weight of 19 000. 3. Plant nuclease I (EC 3.1.30.2). It has a pH optimum at 6.8 and molecular weight of 37 000. Two RNAases were purified to homogeneity by means of affinity chromatography on poly(G)-Sepharose 4B, as shown by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate.     

Studies on polyribonucleotides. Synthesis of a polyinosinic: 6-thioinosinic acid copolymer

The enzymatic synthesis of a high molecular weight (s_20,w ～10) copolymer of inosinic and 6-thioinosinic acids [poly(I:s⁶I)] has been achieved. Poly (I:s⁶I) forms a double stranded complex with poly C which has a Tm significantly lower than a poly I · ply C complex of equivalent molecular weight.     

Stimulation of poly(dT) transcription by Bacillussubtilis RNA polymerase in the presence of adenosine monophosphate

The rate of synthesis of poly(A) on a ply(dT) template by $\text{Bacillus}\text{subtilis}$ RNA polymerase is a function of ATP concentration and is expressed as a sigmoidal curve. The addition of millimolar concentration of AMP to low concentrations of ATP stimulates synthesis of poly(A) twenty fold and raises the rate of synthesis to the levels obtained at high ATP concentrations. The reaction is completely dependent upon the presence of poly(dT) and requires the complementary mononucleotide. Stimulation of poly(A) synthesis by AMP is more evident with the holoenzyme. Analysis of poly(A) products by acrylamide gels showed that the poly(A) synthesized in the presence of AMP has an higher molecular weight than poly(A) synthesized in the absence of AMP.     

Poly(ethylene glycol) methacrylate/dimethacrylate hydrogels for controlled release of hydrophobic drugs

Hydrogels have been successfully used to entrap hydrophilic drugs and release them in a controlled fashion; however, the entrapment and release of hydrophobic drugs has not been well studied. We report on the release characteristics of a model hydrophobic drug, the steroid hormone estradiol, entrapped in low (MW 360/MW 550) and high (MW 526/MW 1000) molecular weight poly(ethylene glycol) methacrylate (PEG-MA)/dimethacrylate (PEG-DMA) hydrogels. The cross-linking ratio, temperature, and pH ranged from 10:1 to 10:3, from 33 to 41 degrees C, and from 2 to 12, respectively. The gelation of the PEG-MA/PEG-DMA hydrogel was initiated with UV irradiation. The absence of poly(glutamic acid) in the hydrogel formulation resulted in a loss of pH sensitivity in the acidic range, which was displayed by the hydrogels' similarities in swelling ratios in the pH buffers of pH 2, 4, and 7. Use of high molecular weight polymers resulted in a higher hydrogel swelling (300%) in comparison to the low molecular weight polymers. Drug size was found to be a significant factor. In comparison to 100% estradiol (MW 272) release, the fractional release of insulin (MW 5733) was 12 and 24% in low and high molecular weight gels at pH 2, respectively, and 17% in low molecular weight gels at pH 7. On the release kinetics of the estradiol drug, the hydrogels displayed a non-Fickian diffusion mechanism, which indicated that the media penetration rate is in the same range as the drug diffusion. The synthesis, entrapment, and release of estradiol by the PEG-MA/PEG-DMA hydrogels proved to be successful, but the use of ethanol in the buffers to promote the hydrophobic release of the estradiol in the in vitro environment caused complications, attributed to the process of transesterification.     

Studies on the purification and properties of a 6.8-S DNA polymerase activity found in calf-thymus DNA polymerase-alpha fraction.

The heterogeneity of calf thymus DNA polymerase-alpha has been further investigated. In particular, an enzyme (enzyme D) which exhibits higher activity on poly(dA) . (dT)10 (A:T = 20:1) compared with that on activated DNA, has been further purified and its properties compared with two other activities of the DNA polymerase-alpha fraction (enzymes A1 and C) which do not show a preference for poly(dA) . (dT)10 over activated DNA. As with A1 and C, enzyme D was shown to have many of the characteristic properties of DNA polymerase-alpha in that it is an acidic protein as judged by its binding to DEAE-cellulose, has a molecular weight of about 140000, does not use a poly (A) . (dT)10 template-initiator complex and is inhibited by N-ethylmaleimide. It exhibits anomalous gel filtration behaviour on Sepharose 6B and it binds relatively weakly to DNA-cellulose compared with DNA polymerase-beta. The extreme sensitivity of enzyme D to inhibtion by N-ethylmaleimide distinguishes it from A1 and C, as does its elution position from a DEAE-cellulose column. On the other hand enzymes C and D are readily inactivated by heating at 45 degrees C unlike enzyme A1. The possible interrelationships of the multiple activities of calf thymus DNA polymerase-alpha are discussed.