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P D Dean

Publications and source records attributed to P D Dean.

At least 19 recordsLinked to original sources
PubMed1986

Identification and characterisation of PmaCI an endonuclease of novel specificity from Pseudomonas maltophila.

We report the use of MonoQ FPLC (Fast Protein Liquid Chromatography) for the rapid purification of a novel Type II restriction endonuclease PmaCI, from Pseudomonas maltophila, which recognises the sequence 5'-CAC decreases GTG-3'. The resulting enzyme is free of other nucleases to a level suitable for its characterisation by multiple-substrate digestion and DNA sequencing techniques. This method appears to be widely applicable and we have used it for the isolation of restriction endonucleases of comparable purity from a range of other organisms. Also described is a rapid method for screening a library of small inserted regions in recombinant M13 molecules for the presence and subsequent screening of restriction sites of interest.

Base Sequence↗
PubMed1983

Separation of glycosylated haemoglobins using immobilized phenylboronic acid. Effect of ligand concentration, column operating conditions, and comparison with ion-exchange and isoelectric-focusing.

Haemoglobins from diabetic and non-diabetic individuals have been separated by affinity chromatography using immobilized phenylboronate, which interacts specifically with diol-containing compounds such as glycosylated haemoglobin. The effects of ligand concentration, flow rate, column geometry, preincubation of sample, buffer composition and temperature have been investigated. Significant correlation was found between results from affinity-chromatography and ion-exchange and isoelectric-focusing methods. Isoelectric-focusing of the haemoglobin fractions obtained from affinity chromatography indicate that, in addition to haemoglobin A1c, some haemoglobin A is also bound to immobilized phenylboronic acid. Assays of haemolysates obtained from red blood cells incubated in glucose solutions suggest that unstable pre-(haemoglobin A1c) does not interfere. The assay is not affected by the presence of haemoglobin F.

Boronic Acids↗
PubMed1983

Tandem dye-ligand chromatography and biospecific elution applied to the purification of glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides.

1. A total of 65 immobilized triazine dyes were screened for their ability to purify the dual-nucleotide-specific glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides. From this screen a 'negative' (Matrex Gel Purple A) and a 'positive' (Matrex Gel Orange B) adsorbent were found to be the best in terms of overall purification and yield and were therefore combined to give the best purification. 2. Glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides was purified approx. 56-fold in a two-step tandem chromatographic system using Matrex Gel Purple A followed by Matrex Gel Orange B chromatography to a specific activity of 228 units/mg of protein in a final yield of 73%. 3. A study of the elution characteristics of glucose-6-phosphate dehydrogenase bound to Matrex Gel Orange B by KCl (pulse and gradient) and biospecific eluents (pulse) was carried out. NADP+, NADPH and adenosine 2',5'-bisphosphate were found to be the only effective biospecific eluents. A pulse of 50 microM-NADP+ (1/2 column vol.) was found to give a better purification than a 0-1 M-KCl gradient and therefore was the preferred method of elution. 4. Presaturation of the enzyme with various nucleotides was carried out to determine the effect on the subsequent binding of glucose-6-phosphate dehydrogenase to Matrex Gel Orange B. The results of these and biospecific-elution studies lead us to propose two possible schemes to explain the mechanism of the dye-protein interaction. 5. Reusability, capacity of the adsorbent and effect of varying the ligand concentration were also studied in the purification of glucose-6-phosphate dehydrogenase on Matrex Gel Orange B.

Chromatography, Affinity↗
PubMed1982

Fractionation of membrane proteins on phenylboronic acid-agarose.

Detergent-solubilized plasma-membrane proteins from lymphocytes of patients with chronic lymphocytic leukaemia were applied to phenylboronic acid-agarose. Some of the polypeptides were bound specifically and subsequently eluted with sorbitol. Immunoprecipitation analyses showed that no surface immunoglobulin M was bound, but that most of the histocompatibility antigens HLA-A, HLA-B, HLA-C and HLA-DR were.

Boronic Acids↗
PubMed1981

The effect of ligand presaturation on the interaction of serum albumins with an immobilized Cibacron Blue 3G-A studied by affinity gel electrophoresis.

The interaction of the immobilized triazine dye Cibacron Blue 3G-A with rat, rabbit, sheep, goat, bovine and human serum albumins was studied by affinity gel electrophoresis. Dissociation constants were estimated in each instance and showed human serum albumin to have a significantly higher affinity for the dye than did albumin from any other species. Pretreatment of the defatted proteins with bilirubin (3 mol of bilirubin/mol of protein) did not increase the dissociation constants of the serum albumins, whereas pretreatment with palmitate (7 mol of palmitate/mol of protein) increased the dissociation constant in all cases: 3-fold for human serum albumin, 15-fold for other serum albumins. Increasing the bilirubin/albumin ratio (to 7:1) did not affect the dissociation constant of the albumins studied. Decreasing the palmitate/albumin ratio decreased the dissociation constant for human serum albumin, but did not affect those of bovine and rat albumins. Altering the chain length of the presaturating fatty acid dramatically changed the dissociation constant of both human and bovine serum albumins. Butyrate, hexanoate, octanoate and decanoate did not significantly influence the dissociation constants of bovine and human serum albumins for Cibacron Blue, whereas laurate, myristate and palmitate greatly increased the dissociation constant. These data are discussed in relationship to the behaviour of albumins during dye--agarose column chromatography. In Addendum the effect of nucleotide presaturation on the interaction between Bacillus stearothermophilus 6-phosphogluconate dehydrogenase and the immobilized triazine dyes Cibacron Blue 3G-A and Procion Red HE-3B was examined, and the implications for dye--ligand chromatography are discussed.

Animals↗
PubMed1981

Affinity chromatography of yeast alpha-glucosidase using ligand-mediated chromatography on immobilized phenylboronic acids.

The synthesis of 3-nitro-4-(6-aminohexylamido)phenylboronic acid is described. The properties of two novel forms of immobilized phenylboronate agarose adsorbents [m-aminophenylboronic acid-Matrex Gel and 3-nitro-4-(6-aminohexylamido)phenylboronic acid-Sepharose CL-6B] were investigated. Both gels bind and selectively retard the glycoprotein alpha-glucosidase from yeast. The retardation is affected by following parameters: (i) pH, (ii) presence of sugar, (iii) concentration of sugar and (iv) buffer species (especially triethanolamine). Five sugars were studied, namely sorbitol, fructose, ribose, glucose and maltose. The concentration of sugar required to produce significant retardation increased in the above order, whereas the ability of sugar to form a complex with boron decreases in the same order. These effects were observed with crude as well as pure enzyme. Since alpha-glucosidase is a glycoprotein, it is proposed that this protein is mainly bound to these immobilized phenylboronates via sugar (glyco) residues. Displacement of the enzyme from the column is effected by the sugar in the buffer (or in a preincubation mixture). However, the marked pH-dependence (this retardation effect could only be observed at pH 7.4) suggests that these results are not due solely to hydrophobic or ionic mechanisms and are more complex than simple sugar-phenylboronic acid interactions.

Boronic Acids↗
PubMed1981

Use of triazine dyes in the affinity chromatographic purification of alkaline phosphatase from calf intestine.

Dye-ligand chromatography was examined as a method for the purification of alkaline phosphatase (EC.3.1.3.1). Forty six dye-Matrex Gels were assessed for their ability to bind alkaline phosphatase. Most dye adsorbents bound significant quantities of the enzyme. Three dye columns were examined in more detail for their selectivity using gradients of potassium chloride to desorb enzyme protein. Purification of alkaline phosphatase using Cibacron blue 3GA-Sepharose 6B chromatography was enhanced by using affinity elution. The best purifications (290-fold) were obtained using pulsed elution with the substrate alpha-naphthyl phosphate although the inhibitor, inorganic phosphate, was also useful (128-fold purification).

Alkaline Phosphatase↗
PubMed1980

The use of various immobilized-triazine affinity dyes for the purification of 6-phosphogluconate dehydrogenase from Bacillus stearothermophilus.

1. 6-Phosphogluconate dehydrogenase from Bacillus stearothermophilus was purified approximately 260-fold on triazine-immobilized dye columns to a final specific activity of 54 mumol of NADP+ reduced/min per mg of protein and an overall yield of 62%. 2. An investigation of the capacities of different triazine dyes that inhibit 6-phosphogluconate dehydrogenase was carried out. Cibacron Blue F3G-A and Procion Red HE-3B strongly inhibited the enzyme in free solution and were therefore chosen as the ligands in the purification scheme. 3. KCl was found to be the most suitable agent for eluting 6-phosphogluconate dehydrogenase from Procion Red HE-3B-Sepharose 6B. NADP+ could specifically elute 6-phosphogluconate dehydrogenase from Cibacron Blue F3G-A-Sepharose 6B. 4. A study of the effect of temperature on the binding of pure 6-phosphogluconate dehydrogenase to both Cibacron Blue-Sepharose and Procion Red-Sepharose showed that the binding increased with an increase in temperature.

Affinity Labels↗
PubMed1980

Studies on the mechanism of binding of serum albumins to immobilized cibacron blue F3G A.

The interaction of Cibacron Blue F3G A-Sepharose 4B with several serum albumins was studied. Although all albumins used were fond to bind to this adsorbent, human serum albumin was bound to a far greater extent than were the others. From the results of competition experiments and n.m.r. studies of Cibacron Blue and/or bilirubin binding to human serum albumin it is proposed that the mechanism of the interaction between human serum albumin and cibacron Blue is consistent wit Cibacron Blue binding to bilirubin-binding sites. In contrast with these findings with human serum albumin, there is little or no interaction of Cibacron Blue and the bilirubin-binding sites of albumins from rabbit, horse, bovine or sheep sera, although some interaction occurs between Cibacron Blue and the fatty acid-binding sites of these proteins. Structural analogues of Cibacron Blue have been used to investigate the binding of albumins to these ligands.

Animals↗
PubMed1980

Serum equilin, oestrone, and oestradiol levels in postmenopausal women receiving conjugated equine oestrogens ('Premarin').

Two groups of postmenopausal women were seen at monthly intervals during a 6-month trial of cyclic therapy with conjugated equine oestrogens ('Premarin'). the seven women in the first group were taking premarin alone and the six women in the second group were taking premarin plus a progestagen, norethisterone acetate. On each visit, serum unconjugated oestrogens were measured by radioimmunoassay. Mean concentrations for the first group were 393 (+/- 203, SD) pmol/l for 17 beta-oestradiol, 599 (+/- 180) pmol/l for oestrone, and 6840 (+/- 5130) pmol/l for equilin. Corresponding levels for the second group were 342 (+/- 112) pmol/l, 564 (+/- 279) pmol/l, and 8840 (+/- 4020) pmol/l. 3 months after completion of therapy, the oestrone and 17 beta-oestradiol concentrations had returned to pre-treatment levels in both groups, but equilin was detected in 3 out of 3 women in the first group at a mean level of 532 (+/- 267) pmol/l and in 2 out of 4 women in the second group at 1170 (+/- 870) pmol/l. In view of the prolonged presence of equilin and the possible association between treatment with conjugated equine oestrogens and endometrial cancer, it is suggested that equilin-containing compounds should not be given for more than 12 months.

17-Ketosteroids↗