Dextran-NAD

K. F. Gu and T. M. S. Chang, Conversion of ammonia or urea into L-leucine, L-valine and L-isoleucine using artificial cells containing an immobilized multienzyme system and dextran-NAD+, glucose dehydrogenase for co-factor recycling, A 4/0, 11(1), 24-28 (1988). 

Leca and Marty [26] Cider Wine Whisky Co-immobilisation of alcohol dehydrogenase (ADH), NADH oxidase and high molecular weight NAD (NAD-dextran) in a poly(vinyl alcohol) bearing stryrylpyridinium groups (PVA-SbQ) matrix Pt/+550 vs. Pt ... 

Fig. 8 Synthesis of amino acids by a multienzyme system consisting of leucine dehydrogenase (LeuDH) catalyzing the reductive amination of the corresponding keto acid, L-lactate dehydrogenase (l-LDH), and lactate for the regeneration of NADH and urease for the in situ generation of ammonia. The coenzyme NAD+ was covalently bond to dextran, enzymes and dextran-coupled NAD+ were...

The electrochemical detection utilized the re-oxidation of hexacyano-ferrate(II) on a platinum electrode. For pyruvate determination this assay was extended to a 3-enzyme system by the addition of glutamate p5u-uvate transaminase, which produces alanine from pyruvate. All enz5unes were used in solution in a reaction chamber of approximately 2 pi directly in front of the electrode. The cofactor NAD" " was coupled to dextran with a molecular weight of 40,000 to avoid its replacement for each assay. As the sensor responded to L-alanine and pyruvate again a differential measurement was required when a sample contained both compounds. It was applied to off-line monitoring of a cultivation of S. cerevisiae and data showed good correlation to the photometric assays. 

The mediator NMP has been used for lactate determination with a platinum electrode in front of which LDH and dextran-bound NAD ... 

The cofactor of ADH, NAD+, may not be replaced by other electron acceptors. Malinauskas and Kulys (1978) attempted to construct a reagentless alcohol sensor by coimmobilizing ADH with dextran-bound NAD+ by a dialysis membrane in front of an oxygen electrode. The O2 consumption was indicated via the reoxidation of NADH by NMP+. The system has also been used to measure NAD+ with high sensitivity. In... 

Figure 5a. AA and 1/ data for the system dextran sulfate-HSA-fluoresceine. Albumin concentration ([HSA]), 1 lO M. Fluoresceine concentration, 1 pH = 5.02, acetate buffer (O.IM). ( ) NaDS (M = 4 10 ). AA is the difference in AA of fluoresceine at 500 and 550 nm. Data for the system dextran sulfate-HSA-rhodamine B. Experimental conditions as above. (9) NaDS, M — 4 10 CA NaDS, M = /0 .

In the case of dextran (0.1-1%) and amyiopectin (0.05%) the potentiometric measurements do not indicate any interaction with NaDS, the activity of NaDS solutions is the same with and without these polymers. An amylose solution of 0.025% was turbid and it did not clear up even in the presence of 20 mmoLkg" NaDS. However, the potentiometric measurements showed binding of NaDS according to a Langmuir isotherm with a saturation value of 0.27 mmol/g amylose. The binding of NaDS on amylose is regarded as an adsorption on solid amylose particles and not as an interaction between them in solution. It is concluded that the investigated polysaccharides do not interact with NaDS in aqueous solution. 

Fig. 2. Irreversible modification of the polymerase by dADP-ribose. Ten pi portions of 10% washed HeLa cell lysate were incubated with 10 pi of 25 pM [3 P]NAD (63 G/mmol) and 100 pg/ml DNase I. 5 mM 3-aminobenzamide was included in sample (a). After incubation at 37 C for 5 min, 10% TCA was added to samples a and b. Samples c-e were chilled on ice for 5 min, and centrifuged. The pellets were resuspended in lysis buffer (40 mM Tris-Cl (pH 8), 10 mM Mg(O.CO.CH3)2, 5% Dextran, 0.05% Trition X-1(X) and 0.1 mM CaCl2) plus DNase with (c) no nucleotide, (d) 5 mM unlabeled NAD or (e) 5 mM unlabeled dNAD, and were incubated again for 10 min. The incubation was stopped by the addition of TCA. The precipitates were dissolved in 40 pi of gel buffer containing 1% SDS, 1% 2-mercaptoethanol and were analyzed by 7%-15% polyacrylamide gel electrophoresis. Track (c) controls for losses in the wash. The bands labeled in the presence of 3-aminobenzamide are probably the result of non-enzymatic addition of p PJdADP-ribose that was present in the precursor.

Duplicate T-175 flasks of Cos cells were incubated in the presence or absence of plasmid DNA (25 pg per flask), with DEAE dextran for 3 hr cells were harvested at 48 hr, washed, detached and sonicated in 0.25 M sucrose buffer containing 50 mM Tris pH 8.0, 2 mM MgCl2, 1 mM DTT and 0.1 mM PMSF. Initial velocity (45 sec) assays with [32p]NAD were performed as previously described (20). 

Study of interaction of Blue Dextran 261 with calf spleen NAD glycohydrolase... 

For instance, an NAD" analogue bound to a water-soluble dextran polymer has been applied in the preparation of an enzyme electrode (127) and a model enzyme reactor. Medicinal applications in this field are obvious. One example will illustrate the principle. The oxidoreduction reaction, NAD -I-substrate- NADH-I-H + product, can be coupled to immobilized enzymes. In this model system, the substrate is pumped into a chamber containing the dextran-bound NAD" and two NAD -linked dehydrogenases. At the other end the product of the reaction is removed at the same rate by ultrafiltration. Hence, the process can be recycled. 

Figure 10.15 Effect of the external dextran (Dextran T 500) concentration on the CMC of NaDS. The data were obtained with Sephadex G-10 at 25 C. Cq = concentration of NaDS in the gel phase Cm = concentration in the external solution, (a) In 40 % dextran solution (b) in 20% dextran solution (c) in the absence of dextran (0.1 m NaCl). From Janado et al, [94] with permission.

Poly(vinyl benzoic acid) (HPVB) -PcHj-CH JL 0 CH2C00H Sodium dextran sulfate (NaDS) - 0CH2 HO OSOjNa... 

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