pH-dependent perturbation of Ras-guanine nucleotide interactions and Ras guanine nucleotide exchange

p21Ras (Ras) proteins cycle between active GTP-bound and inactive GDP-bound states to mediate signal transduction pathways that promote cell growth, differentiation, and apoptosis. To better understand how cellular regulatory factors, such as guanine nucleotide exchange factors (GEFs) and nitric oxide (NO), modulate Ras-guanine nucleotide binding interactions, we have conducted NMR and kinetic studies to investigate the pH dependence of Ras-GDP interactions and Ras-guanine nucleotide exchange (GNE). pH-sensitive amide protons were identified and found to be associated with residues in the switch I (Phe28-Asp30) and switch II (Asp57 and Thr58) regions of Ras. Furthermore, most of the residues that interact with Mg2+ exhibit pH-sensitive amide proton chemical shifts which appear to be coupled to pH-dependent Ras Mg2+ binding and guanine nucleotide binding affinity. These results suggest that perturbation of Mg2+ interactions within the Ras-guanine nucleotide complex is critical for pH-dependent dissociation of guanine nucleotide ligands from Ras. Notably, these same regions undergo conformational changes upon association with the Ras GEF, SOS. In addition, although we have recently shown that addition of NO to Ras in the presence of oxygen produces a Ras thiyl radical intermediate that promotes Ras GNE, we have also postulated that another byproduct of this reaction, a H+, may contribute to NO-mediated GNE. However, the results presented herein suggest that the H+ byproduct of the reaction is unlikely to be involved in the NO-mediated Ras GNE.     

Preparation of cyclic nucleotide antisera with thyroglobulin-cyclic nucleotide conjugates

Antisera to cyclic AMP and cyclic GMP were obtained by immunizing rabbits with antigens prepared by conjugating the 2'0-succinyl derivative of the cyclic nucleotides to thyroglobulin. The cyclic nucleotide-thyroglobulin conjugates were injected intradermally into multiple sites on the backs of the animals. This immunization procedure resulted in the production of antiserum in four of five animals capable of binding at a final serum dilution of greater than 1:10,000, 20% of the corresponding iodinated cyclic nucleotide derivative added. The antisera were also highly specific. The antiserum for cyclic AMP had a 2500-fold or greater relative affinity for cyclic AMP than other nucleotides or nucleosides, while that for cyclic GMP had a 5000-fold or greater affinity for 2'0 acetylated nucleotides or nucleosides except for acetylated cyclic IMP. The obstacles to measuring cyclic nucleotides, particularly cyclic GMP, in tissues were also overcome by refining and simplifying the methods for iodination, purification and assay. Furthermore, a "disequilibrium" incubation was developed as an alternative to the acetylation method to increase the sensitivity of the radioimmunoassay. Thus, the levels of both cyclic GMP and cyclic AMP can be determined rapidly and easily in the same tissue sample.     

Evaluation of nucleotide derivatives and sugar nucleotide analogs as inhibitors of glycosyltransferases.

The inhibition of glycosyltransferases was studied using uridine monophosphate derivatives and uridine diphosphate sugar analogs. Modification in the nucleoside portion caused selective inhibition of glycosyltransferases.     

Histochemical localization of nucleotide pyrophosphatase and cyclic nucleotide phosphodiesterase in seeds and shoots of Triticum

The activities of potato nucleotide pyrophosphatase and cyclic nucleotide phosphodiesterase against a common substrate, p-nitrophenyl thymidine 5-phosphate and its histochemical analogue, AS-BI-naphthyl thymidine 5-phosphate, were determined with the aid of relatively specific inhibitors, NAD and 2,3-cAMP, respectively. These inhibitors were utilized to reexamine wheat (Triticum aestivum L. cv. Mironovska 808) seeds and 3–5-d old shoots for the occurrence and histochemical localization of nucleotide pyrophosphatase, and to establish the localization of cyclic nucleotide phosphodiesterase. Nucleotide pyrophosphatase is a cytoplasmic enzyme found to be particularly active in the coleoptile epidermis and hypodermis, leaf mesophyll, as well as in developing fibres and phloem. Cyclic nucleotide phosphodiesterase is also a cytoplasmic enzyme active in the shoot vascular bundles, particularly the xylem, and in the seed. Within the seed it is highly active in the crushed cell layer adjacent to the scutellum and in endosperm cells adjacent to the aleurone layer. Within the embryo, cyclic nucleotide phosphodiesterase is most active in epithelial cells adjacent to the crushed cell layer, the suspensor, radicle and root-cap, as well as in the pro-vascular tissues of the scutellum.     

The pyridine nucleotide and non-pyridine nucleotide dependence of L-glutamate dehydrogenase in the histochemical system

Summary Under histochemical conditions (fresh frozen sections from liver, kidney and cerebellum of the rat) it was shown that the oxidation of L-glutamic acid was carried out by the NAD-dependent L-glutamate dehydrogenase (E.C. 1.4.1.2) and/or the NAD- or NADP-dependent L-glutamate dehydrogenase (E.C. 1.4.1.3) as well as by an enzyme system which is not dependent on externally added NAD, NADP, FAD, FMN or CoQ₁₀ for activity.This non-pyridine dependent activity was related to the L-glutamate dehydrogenases proper, owing to the following: a) the localization of activity noticed corresponds to that obtained with the NAD- or NADP-containing media, b) the incubation time needed for initial formation of red and blue formazans is similar to that with coenzyme-containing media, c) pre-extraction experiments reveal similarity in enzyme diffusion rates, d) the named activity is influenced by the same agents and to the same extent as the activity obtained by the inclusion of NAD or NADP (e.g. dissociation of the dehydrogenase molecule into subunits due to urea, inhibition of activity due to N-ethyl maleimide and 1.10-phenanthroline, activation due to the allosteric effect of ADP and to high substrate concentration, allosteric inhibition caused by GTP and inhibition caused by -ketoglutaric acid, no inhibitory effect of KCN), and e) the named activity was not affected by added PMS (excluding activity due to L-aminoacid oxidase).In the in situ localization of enzyme activity it was found that L-glutamate dehydrogenases E.C. 1.4.1.2 and E.C. 1.4.1.3 co-exist in the cells of kidney and cerebellum, while the L-glutamate dehydrogenase E.C. 1.4.1.3 only was present in liver cells.Finally, it was stated that incubation time should be kept as short as possible in order to avoid Nothing dehydrogenase reaction as well as inhibition due to accumulation of -ketoglutaric acid. Only gel incubation media should be applied.Recipient of a research grant from the Danish Ministry of Education     

Glucocorticoids change the nucleotide and sugar nucleotide pool sizes in cultured human skin fibroblasts

Fibroblasts in culture were treated with the glucocorticoid budesonide. The nucleotide and sugar nucleotide pools were quantitated after separation by isotachophoresis. Steroid treatment induced a 40% increase of the UDP-N-acetylhexosamine pool and a 30% increase of the UDP-glucuronic acid pool whereas the UTP pool was diminished. These effects became apparent after 24 h of incubation and a new steady state was attained after 48 h of incubation. The 3'-phosphoadenosine-5'-phosphosulfate pool was probably not influenced by the glucocorticoid treatment. The half-life of the UDP-N-acetylhexosamine pool was considerably longer in treated than in control cells. The efflux from the UDP-N-acetylhexosamine pool was also lowered in the treated cells. The changed efflux may be due to a decreased glycoconjugate synthesis induced by glucocorticoid treatment. The rate of equilibration of [14C]glucose and [3H]glucosamine with the UDP-N-acetylhexosamine pool was changed by glucocorticoid treatment; especially that of [3H]glucosamine was decreased.