Immobilization of <Emphasis Type="Italic">Streptomyces</Emphasis> phospholipase D on a Dowex macroporous resin

The immobilization of phospholipase D produced by Streptomyces sp. YU100 was evaluated to see it would be practical for industrial applications. To accomplish this, the purified enzyme, which contained 53 unit/mg of protein, was subjected to immobilization on various matrices. When immobilization supports including calcium alginate gel, polyacrylamide gel, and macroporous resin were evaluated, the highest enzyme retention ratio (> 42%) was observed on a Dowex MSA-2 macro-porous resin. This may have occurred as a result of the ability of the hydrophobic domain of phospholipase D to interact with the polystyrene backbone of the resin, as well as the ability of the dimethylethanolamine group of the MSA-2 resin to retain the enzyme by forming hydrogen bonds with the acidic residues of the enzyme. Upon the operation of a reactor packed with enzyme that had been immobilized on a Dowex MSA-2 resin, greater than 80% of the initial enzyme activity was retained for 16 days. During the reaction, phosphatidylcholine became bound to the immobilized resin and interfered with the enzyme reaction, therefore, the resin was washed with ethyl ether every 2 h. A process for recovering excessive l-serine from phospholipids using the Dowex MR-3 resin was designed, and the separated l -serine was employed again after replacing the amount that was used.     

Factors affecting the rates at which nucleotides bind to Dowex 1 resin

The relative rates at which adenine nucleotides bind to Dowex 1 anion-exchange resin are determined by diffusion through the immobile fluid film surrounding the resin particles and thus are unaffected by how tightly the nucleotides bind. By using a fine resin and stirring efficiently, the time needed for equilibration can be reduced from 4 h to less than 3 min.     

Procedure for isolation of Thienamycin from fermentation broths

An improved process for the isolation of thienamycin, produced by the actinomycete Streptomyces cattleya, has been developed. The isolation procedure consists of three chromatographic steps, volume reduction by reverse osmosis between the steps, and freezedrying for obtaining the final product. The chromatographic steps are as follows: (1) ion exchange chromatography on Dowex 1 × 2 resin in the bicarbonate cycle, (2) gel chromatography on Dowex 1 × 2 resin in the chloride cycle, (3) reverse phase chromatography on XAD-2 resin. This procedure is useful for processing large volumes of fermentation broth.     

Pictet-spengler reaction using ion-exchange resin as a catalyst and support for 'catch and release' purification

The Pictet-Spengler reaction between tryptamine and aldehydes was catalyzed by Dowex 50W-X4 acidic ion-exchange resin. The products were obtained from the resin in high purity by 'catch and release' without the need for separate chromatographic purification.     

The interaction of cytostatic drugs with adsorbents in aqueous media. The potential implications for liposome preparation

A new method that involves the use of the cation exchange resin Dowex 50W-X4 to remove non-encapsulated drugs from liposome dispersions was investigated. Cytostatic drugs widely varying in their molecular structure can be removed from aqueous solutions by Dowex 50W-X4. The applicability of the resin to separate free from liposome-bound drugs was illustrated for a number of cytostatic drugs (cisplatin, doxorubicin, vincristine). The technique presented allows for a rapid, efficient and convenient procedure for the free drug removal from liposome dispersions without dilution of the liposomal preparation. Studies with liposome-encapsulated drugs will be facilitated by the use of this method, since it avoids many of the problems introduced by conventional methods as dialysis, gel filtration and centrifugation/washing. To elucidate the interaction mechanism of doxorubicin with Dowex 50W-X4, alternative adsorbents were studied for their doxorubicin binding properties. In the adsorption process of doxorubicin onto Dowex 50W-X4 both electrostatic (ion exchange) and hydrophobic effects play a role. The results indicate that hydrophobic contributions to the interaction are responsible for the high resistance offered by the binding forces against desorption of adsorbed doxorubicin. For other adsorbents the interactions are either mainly of an electrostatic or a hydrophobic nature.     

MEASUREMENT OF THICKNESS WITHIN SECTIONS BY QUANTITATIVE ELECTRON MICROSCOPY

To apply the method of quantitative electron microscopy to the measurement of mass in thin sections, the thickness of the section at or very near the structure to be studied must be known. Dowex anion exchange resin AG 1 x 2, stained with phosphotungstic acid (PTA) at pH 6.4, was used as a thickness standard which could be embedded and sectioned. The sectioned PTA-Dowex appeared uniformly stained and exhibited suitable electron opacity. The stoichiometry of the reaction between PTA and the Dowex resin was measured by three independent methods based on gravimetric, colorimetric, and nitrogen determinations whose results showed close agreement. From the PTA uptake, the density of the stained spheres was calculated. Mass of a defined area of PTA-Dowex was measured by quantitative electron microscopy, and from this mass and density, the volume and then the thickness were calculated. The values for thickness were compared to those obtained by interference microscopy on the embedding medium alone in the same sections.     

Flowering may be controlled by a quantitative balance between flower-inducing and -inhibiting substances in Pharbitis nil

Phloem exudate prepared from induced cotyledons of Pharbitis nil (SD-PE) showed flower-inducing activity, and the exudate from cotyledons of P. nil grown under continuous illumination (CL-PE) expressed flower-inhibiting activity in apex cultures of P. nil. Following fractionation by ion exchange chromatography, the flower-inducing activity of SD-PE was located in the fraction adsorbed on anion exchange resin (Dowex); the flow-through (FT) fraction from anion and/or cation exchange resins used to separate CL-PE inhibited flowering. The flower-inducing and -inhibiting activities of both fractions from SD-PE and CL-PE were examined in detail. The FT fraction of SD-PE inhibited, and the Dowex fraction of CL-PE induced flowering. The flower-inducing activity of Dowex fraction of SD-PE was about 100 times higher than the same fraction of CL-PE, and the inhibiting activity of FT fraction of CL-PE was about 10 times higher than the same fraction of SD-PE. Therefore, flowering in P. nil may be controlled by a quantitative balance between flower-inducing and -inhibiting substances.     

A modified assay of 3':5'-cyclic-AMP phosphodiesterase.

A modification of the assay of cyclic nucleotide phosphodiesterase involving batch use of Dowex 1 anion exchange resin is described which allows for quantitative recovery of adenosine, guanosine, and their metabolites from the resin slurry. The assay described is suitable for use in crude preparations containing purine catabolizing enzymes. A standardized procedure for determining kinetic parameters of cyclic AMP hydrolysis is also discussed. This procedure was used in the partial characterization of the kinetics of cyclic AMP hydrolysis by rat and rabbit heart supernatant fractions.     

A sensitive method for the assay of guanylate cyclase activity.

A method for the assay of guanylate cyclase is described utilizing alpha-[32P]-GTP as substrate for the enzyme reaction. 100-150 microgram of enzyme protein is incubated in a 15.6 mM Tris-HCl buffer incubation mixture, pH 7.6. The reaction is stopped by the addition of EDTA. The [32P]-cyclic GMP formed is separated by a two-step column chromatography on Dowex 50W-X4 ion-exchange resin and neutral alumina. The recovery for cyclic GMP was about 70%. The blank values ranged from 0.001-0.003% of the added alpha-[32P]-GTP which had been purified by Dowex 50W-X4 column chromatography. This method was employed for the assay of guanylate cyclase activities in different tissues.     

Application of trinitrophenylation for the measurement of alpha-amino residues resulting from peptic digestion.

A sensitive and precise method for the measurement of peptic activity on protein substrate is described. alpha-Amino residues formed by pepsin digestion are photometrically measured by comparing the absorbances of digested and nondigested material which has been trinitrophenylated. The usual problem of high reagent-blank absorbance is eliminated by using an anion exchange resin, Dowex 1-X8. In contrast to Anson's method, the procedure requires only 1/100 the quantity of protein substrate for analysis. It was proved to be particularly useful for the estimation of initial rates of proteolysis.     

Application of nitrous acid deamination of hexosamines to the simultaneous GLC determination of neutral and amino sugars in glycoproteins

A method is described for simultaneous gas chromatographic analysis of neutral sugars and hexosamines in glycoproteins. Sugars are hydrolyzed with the aid of Dowex 50-X2 (H⁺) resin and the resin bound glucosamine and galactosamine are deaminated with NaNO₂ to the coresponding neutral 2,5-anhydrohexoses. Hexoses and 2,5-anhydrohexoses are then quantitated as the corresponding alditol acetates. Application of the procedure to several different glycoproteins is presented.     

Enhancing ethanol fermentability of an artificial acid hydrolyzate with anion exchange resin treatment

To assess the effectiveness of anion exchange resins (Dowex M43 and Dowex monosphere 66) in neutralization and detoxification of an acid hydrolyzate solution, a fermentation medium containing inhibitors was inoculated with Saccharomyces cerevisiae. When treated with resins at a 1:1 ratio (vol:wt) for up to 20 min, 55-67% of furan and more than 95% of phenolic compounds were removed. Ethanol fermentation activity in resin-treated fermentation medium was the same as the control. There was 21-43% of the total sugar loss after one resin treatment, depending on the sugar concentration. Additional treatments increased sugar retention rate to 95%.     

The gill sialic acid content as an index of environmental stress in rainbow trout, Salmo gairdneri, Richardson

The gill sialic acid content in Rainbow Trout exposed to several aqueous concentrations of ammonia NH₃ and/or to a high environmental pH was determined by two different methods. The results showed Warren's method was adequate for the biological material, even without purification of the extracts by Dowex 2.8 resin. A significant increase of the sialic acid concentration occurred in fish exposed to ammonia.     

Occurrence of 2-Aminobutanoic Acid in the Incubation Medium of Mixed Rumen Ciliate Protozoa1

An unknown, ninhydrin-positive substance detected on paper chromatograms of the endogenous metabolites of mixed rumen ciliate protozoa was isolated and purified by column chromatography with ‘Dowex’ 50-X8 resin and identified as 2-aminobutanoic acid (α-amino-n-butyric acid) on the basis of elementary analysis, mass spectrometry, paper chromatography, infrared spectrometry, and melting point.     

An assay for glucose 6-phosphatase based on the formation of glucose

A rapid, convenient method for the assay of glucose 6-phosphatase dependent on the removal of radioactive substrate from radioactive product by Dowex 2 fluoride is described. The enzymatic reaction is stopped by the addition of an ethanolic slurry of the resin. After the tubes are shaken, the radioactivity of glucose in the clarified supernatant layer is measured. The release of glucose is directly proportional to time and enzyme concentration. Detergents do not interfere with the method.     

Cytokinin biosynthesis in roots of corn

Removal of the quiescent center (QC) from the root apex of maize (Zea mays L., cv. Kelvedon 33) initiates a set of events which culiminate in the regeneration of an intact apex with a newly formed QC. Concomitant with the formation of a new QC is a marked reduction in extractable cytokinins in the tissue of the proximal meristem. Replacing the excised QC with a Dowex (acidic cation-exchange resin) bead affects both root growth and QC regeneration. Root growth is inhibited by plain Dowex beads and Dowex beads treated with zeatin; this inhibition is reversed if the beads have been treated with CaCl₂ (±zeatin). Dowex beads treated with zeatin delay the formation of a new QC; this effect is the same whether or not the beads also contain CaCl₂. The results of this investigation support the notions that cytokinin biosynthesis in roots is a result of activities of both the QC and the proximal meristem, and that cytokinins, at least if supplied exogenously, can play a role in root morphogenesis by delaying the regeneration of the QC.Abbreviations used throughout the text PM proximal meristem - QC quiescent center - RC root cap     

Studies on Cytokinins in Watermelon Seeds

Extracts of immature seeds of watermelon contain three cytokinins which are adsorbed on Dowex 50W ion exchange resin. The Rf of the fastest moving factor is similar to that of zeatin. The slowest moving factor is insoluble in n-butanol and shows some resemblance to zeatin ribotide. The chromatographic properties of the second factor are, however, different from those of any of the known cytokinins and is believed to be new to literature.     

New and general synthesis of beta-C-glycosylformaldehydes from easily available beta-C-glycosylpropanones

A highly effective method for the introduction of a formyl group at the anomeric position of pyranosides was developed via enolisation of beta-C-D-glycopyranosylpropan-2-one using thermodynamic conditions then oxidative cleavage of the more substituted double bond. This sequence affords the desired aldehydes that are conveniently protected as aminals for purification and storage and easily regenerated using Dowex resin H+. In this paper, the syntheses of nine differently protected aldehydes derived from d-glucose, d-galactose, lactose and N-acetyl-d-glucosamine are presented. Our strategy proved to be very efficient in most cases excepted in the D-mannose series.     

A simple procedure for purification of N-carbamoylputrescine: Application to assays of putrescine transcarbamoylase and agmatine iminohydrolase activities

The observation that N-carbamoylputrescine is quantitatively excluded on O-(carboxymethyl)-cellulose columns with simultaneous retention of putrescine and agmatine has been utilized to develop a sensitive radiometric assay for putrescine transcarbamoylase and a colorimetric assay for agmatine iminohydrolase. A simple procedure for obtaining bulk amounts of pure synthetic N-carbamoylputrescine by separation from putrescine and dicarbamoylputrescine on Dowex 50 (Na⁺) resin is described.     

A method for enzymic extraction and the measurement of chloroplast RNA

A method is described for measuring RNA associated with chloroplast thylakoid membranes. Washed thylakoids are incubated with ribonucleases A and T(1), under low Mg(2+) conditions, to release hydrolyzed RNA into solution. After removing the membranes by centrifugation, the mono- and oligonucleotides are adsorbed by Dowex 1-X2 in miniature columns made from Pasteur pipettes, and then eluted with 2 n HCl. RNA is estimated from the absorbance of the eluate at 260 nanometers, with corresponding values obtained by the orcinol reaction for pentose. Polyacrylamide gel electrophoresis patterns of extracted RNA indicate that our current procedures for preparation of thylakoids results in material containing variable and often significant levels of RNA from 80S ribosomes. Thus values for total RNA cannot be used as a valid estimate for the level of 70S ribosomes associated with these membranes, unless an additional procedure is used to estimate the per cent contamination by 80S ribosomes.Recoveries of digested RNA from the Dowex resin of 94 to 98% were obtained with 2 milliliters of HCl eluant, making possible the analysis of thylakoid samples with as little as 4 micrograms of RNA. The procedure involves small columns and only one centrifugation, so that it is useful for obtaining reliable measurements from multiple samples.