Ribose assay

Step 3. Alkali dissolves most of the RNA-DNA-protein residue and hydrolyzes RNA quantitatively to mononucleotides (91). All four of these contain phosphorus but only the purine nucleotides react in the conventional ribose assay. Alkali also releases phosphoprotein phosphorus as inorganic phosphate (91) (the degree of completeness has not been reported). DNA is alkali resistant as long as the purine-desoxyribose linkages are intact (these bonds are acid labile—see comments under step 1). Once adenine and guanine are removed, alkali lability of DNA appears, due presumably to a cyclization mechanism similar to that which is responsible for RNA lability (14). 

TK activity was determined by a spectrophotometric assay at 340 nm. In 0.5 mL of tris buffer (50 mM) pH 7.6, were added 50 pL of L-erythrulose from a 1 m stock solution in water (0.05 mmol), 25 pL of o-ribose-5-phosphate from a 160 him stock solution in water (4.0 pmol), 5 pC of ThDP from a 21 mM stock solution in water (0.1 pmol), 10 pL of MgCl2 from a 50 mM stock solution in water (0.5 /rmol), 10 pL of NADH from a 14 him solution in water (0.14 /imol), alcohol dehydrogenase (12 units). 

TALDO deficiency can be confirmed in lymphoblasts, fibroblasts and in erythrocytes. These cells are incubated with ribose-5-phosphate, after which formation of transketolase and TALDO products are analysed by gas chromatography with nitrogen phosphorous detection by liquid chromatography tandem mass spectrometry [8, 11]. A similar enzyme assay is available for RPI [2]. Confirmation of the gene defect can be performed by sequence analysis. Disease-causing mutations have been detected in all TALDO-deficient patients and in the RPI-deficient patient. 

As depicted in Figure 6.8 the stability screening was based on DERA activity assay, the retro-aldol reaction of 2-deoxy-D-ribose 5-phosphate to acetaldehyde and D-glyceraldehyde 3-phosphate. D-glyceraldehyde 3-phosphate is further converted by the auxiliary enzymes triose phosphate isomerase and glycerol phosphate dehydrogenase. As the latter reaction consumes NADH it can be measured spectro-pho to metrically by the decrease in absorbance at 340 nm. 

G-protein a-subunits also possess specific residues that can be covalently modified by bacterial toxins. Cholera toxin catalyzes the transfer of ADP-ribose moiety of NAD to a specific arginine residue in certain a-subunits, whereas pertussis toxin ADP-ribosylates those a-subunits that contain a specific cysteine residue near the carboxy-terminus. Modification of the a-subunit by cholera toxin persistently activates these protein by inhibiting their GTPase activity, whereas pertussis toxin inactives Gia protein and thereby results in the uncoupling of receptor from the effector. G-protein a-subunits are regulated by covalent modifications by fatty acids myristate and palmate. These lipid modifications serve to anchor the subunits to the membrane and increase the interaction with other protein and also increase the affinity of the a-subunit for 3y. In this regard, the myristoylation of Gia is required for adenylyl cyclase inhibition in cell-free assay (Taussig et al. 1993). 

J-Lactoglobulin, glycated with ribose, arabinose, galactose, glucose, rhamnose, or lactose, showed no increase in cytotoxicity in the methylthiazoletetrazolium assay against COS-7 and HL-60 cells.163... 

The NAD glycohydrolase (EC 3.2.2.5) in this study catalyzes the hydrolysis of NAD+ to form nicotinamide, adenosine diphosphate ribose (ADPR), and H+. The assay developed for this activity follows the disappearance of the substrate NAD+ and the production of nicotinamide. 

When assayed in their native states, these proteins did not comp>ete with MBP for SecB binding (stabilization of the folded form of the mRBP variant by addition of ribose was required for loss of competitiveness). These results indicate that SecB specifically recognizes nonnative structures and are corroborated by the observation that once folded, neither preMBP nor mMBP is a substrate for SecB (Randall et al., 1990 Weiss and Bassford, 1990). 

In the first step, we showed by analytical studies that compound 28 was a donor substrate for transketolase in the presence of D-ribose-5-phosphate as acceptor substrate and that in the second step, the hydroxylated aldehyde released 29 led to the P-elimination of protected L-tyrosine. We showed that the free L-tyrosine can thus be obtained by enzymatic deprotection of N-acetyl-L-tyrosine ethyl ester using acylase and subtilisine. In this conditions, it should be possible to carry out this assay in vivo in the presence of host cells overexpressing transketolase and auxotrophic for L-tyrosin. This strategy should offer the first stereospecific selection test of transketolase mutants. The principle of this assay may be extended to other enzymes that can release aldehydes P-substituted by L-tyrosine. 

The salvage pathway does not involve the formation of new heterocyclic bases but permits variation according to demand of the state of the base (B), i.e. whether at the nucleoside (N), or nucleoside mono- (NMP), di- (NDP) or tri- (NTP) phosphate level. The major enzymes and routes available (Scheme 158) all operate with either ribose or 2-deoxyribose derivatives except for the phosphoribosyl transferases. Several enzymes involved in the biosynthesis of purine nucleotides or in interconversion reactions, e.g. adenosine deaminase, have been assayed using a method which is based on the formation of hydrogen peroxide with xanthine oxidase as a coupling enzyme (81CPB426). 

This assay, which does not require the addition of lipids or detergent, measures the ability of CT or LT to ADP-ribosylate itself in the absence of another ADP-ribose acceptor (except water). ARF added to this assay will also be ADP-ribosylated, but the ADP-ribosylation of it or CT under these conditions does not appear to decrease CT activity or the ability of ARF to stimulate CT (Tsai ef a/., 1991). Reaction products are separated by SDS-PAGE and analyzed by autoradiography as described for Assay 1 (Section 2.3). This assay is the most sensitive of all those listed here and can be used to verify toxin activity observed in other assays. 

This assay measures the ability of CTA or LTA to act as an NADase (first described by Moss ef al. (1976)), catalyzing cleavage of the gly-cosidic bond between the ADP-ribose and the nicotinamide moieties of NAD. Products of the reaction are separated on AG 1-X2 columns and quantified as described for Assay 2 (Section 2.4). 

Like other bacterial ADP-ribosylating toxins (e.g. diphtheria toxin. Pseudomonas aeruginosa exotoxin A, cholera toxin, pertussis toxin, and C. botulinum C2 toxin (Aktories and Just, 1993)), C3 is a mono-ADP-ribosyltransferase (Aktories et ai, 1988b). Treatment of ADP-ribosylated Rho with phosphodiesterase releases 5 -AMP and not phosphoribosyl-AIVtP, a cleavage product of poly(ADP-ribose) (Aktories et ai, 1988b Rubin ef a/., 1988). Accordingly, thymidine, an inhibitor of poly(ADP-ribose)polymerase, does not block C3-like ADP-ribosyltransferases, and can be included in C3 ADP-ribosylation assays to block poly-ADP-ribosylation reactions. 

Assay of cleavage of known intracellular caspases substrates (i.e., Poly ADP Ribose Polymerase)... 

Figure 1 Structure of INS37217 and INS542. INS37217 is a deoxcytidine-uridine dinucleotide [P1-(Uridine 5/)-P4-(2/-deoxycytidine 5 )tetraphosphate, tetrasodium salt] and INS542 is a cytidine-uridine dinucleotide [P1-(Uridine 5 )-P4-(cytidine 5 )tetraphosphate, tetrasodium salt]. The only difference between the two molecules is the absence of the OH-group in the 2 position of the cytidine ribose in INS37217. Both molecules have nearly identical activity in a variety of pharmacological and metabolism assays.

Caspase activation assays and other molecular read-outs for apoptosis Commercially available caspase antibodies, as well as caspase activity assays, allow the rapid measurement of caspase activity in cells. Caspase activity correlates well with the apoptotic program. Examples of other molecular markers used for the indication of apoptosis are Bax, Bcl-2, BCL-XL and PARP (poly (ADP-ribose) polymerase). 

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