Recombinant human DNA (cytosine-5) methyltransferase. II. Steady-state kinetics reveal allosteric activation by methylated dna.
(73/3161)
Initial velocity determinations were conducted with human DNA (cytosine-5) methyltransferase (DNMT1) on unmethylated and hemimethylated DNA templates in order to assess the mechanism of the reaction. Initial velocity data with DNA and S-adenosylmethionine (AdoMet) as variable substrates and product inhibition studies with methylated DNA and S-adenosylhomocysteine (AdoHcy) were obtained and evaluated as double-reciprocal plots. These relationships were linear for plasmid DNA, exon-1 from the imprinted small nuclear ribonucleoprotein-associated polypeptide N, (CGG.CCG)(12), (m(5)CGG. CCG)(12), and (CGG.CCG)(73) but were not linear for (CGG. Cm(5)CG)(12). Inhibition by AdoHcy was apparently competitive versus AdoMet and uncompetitive/noncompetitive versus DNA at +info)
Antigen presentation: TAP dances with ATP.
(74/3161)
Assembly of antigen-presenting complexes between class I MHC molecules and peptide requires formation of a complex between the 'ABC' peptide transporter, TAP, and newly synthesized class I molecules. Recent studies have provided new insights into the role of ATP in peptide binding, transport and release. (+info)
Specificity of novel allosterically trans- and cis-activated connected maxizymes that are designed to suppress BCR-ABL expression.
(75/3161)
Chronic myelogenous leukemia (CML) is associated with the presence of the Philadelphia chromosome, which is generated by the reciprocal translocation of chromosomes 9 and 22. In the case of L6 (b2a2) mRNA, it is difficult to cleave the abnormal mRNA specifically because the mRNA includes no sequences that can be cleaved efficiently by conventional hammerhead ribozymes near the BCR-ABL junction. We recently succeeded in designing a novel maxizyme, which specifically cleaves BCR-ABL fusion mRNA, as a result of the formation of a dimeric structure. As an extension of our molecular engineering of maxizymes, as well as to improve their potential utility, we examined whether an analogous conformational change could be induced within a single molecule when two maxizymes were connected via a linker sequence. An active conformation was achieved by binding of the construct to the BCR-ABL junction in trans, with part of the linker sequence then acting as an antisense modulator in cis (within the complex) to adjust the overall structure. Results of studies in vitro in the presence of cetyltrimethylammonium bromide (CTAB) (but not in its absence) suggested that a certain kind of connected maxizyme (cMzB) might be able to undergo a desired conformational change and, indeed, studies in vivo confirmed this prediction. Therefore, we successfully created a fully functional, connected maxizyme and, moreover, we found that the activity and specificity of catalytic RNAs in vivo might be better estimated if their reactions are monitored in vitro in the presence of CTAB. (+info)
Design of an adenosine phosphorylase by active-site modification of murine purine nucleoside phosphorylase. Enzyme kinetics and molecular dynamics simulation of Asn-243 and Lys-244 substitutions of purine nucleoside phosphorylase.
(76/3161)
Our objective was to alter the substrate specificity of purine nucleoside phosphorylase such that it would catalyse the phosphorolysis of 6-aminopurine nucleosides. We modified both Asn-243 and Lys-244 in order to promote the acceptance of the C6-amino group of adenosine. The Asn-243-Asp substitution resulted in an 8-fold increase in K(m) for inosine from 58 to 484 microM and a 1000-fold decrease in k(cat)/K(m). The Asn-243-Asp construct catalysed the phosphorolysis of adenosine with a K(m) of 45 microM and a k(cat)/K(m) 8-fold that with inosine. The Lys-244-Gln construct showed only marginal reduction in k(cat)/K(m), 83% of wild type, but had no activity with adenosine. The Asn-243-Asp;Lys-244-Gln construct had a 14-fold increase in K(m) with inosine and 7-fold decrease in k(cat)/K(m) as compared to wild type. This double substitution catalysed the phosphorolysis of adenosine with a K(m) of 42 microM and a k(cat)/K(m) twice that of the single Asn-243-Asp substitution. Molecular dynamics simulation of the engineered proteins with adenine as substrate revealed favourable hydrogen bond distances between N7 of the purine ring and the Asp-243 carboxylate at 2.93 and 2.88 A, for Asn-243-Asp and the Asn-243-Asp;Lys-244-Gln constructs respectively. Simulation also supported a favourable hydrogen bond distance between the purine C6-amino group and Asp-243 at 2.83 and 2.88 A for each construct respectively. The Asn-243-Thr substitution did not yield activity with adenosine and simulation gave unfavourable hydrogen bond distances between Thr-243 and both the C6-amino group and N7 of the purine ring. The substitutions were not in the region of phosphate binding and the apparent S(0.5) for phosphate with wild type and the Asn-243-Asp enzymes were 1.35+/-0.01 and 1.84+/-0.06 mM, respectively. Both proteins exhibited positive co-operativity with phosphate giving Hill coefficients of 7.9 and 3.8 respectively. (+info)
RSR13, a synthetic allosteric modifier of hemoglobin, improves myocardial recovery following hypothermic cardiopulmonary bypass.
(77/3161)
BACKGROUND: During hypothermic blood cardioplegia, oxygen delivery to myocytes is minimal with ineffective anaerobic metabolism predominating. RSR13, 2-[4-[[(3,5-dimethylanilino) carbonyl]methyl]phenoxy]-2-methylpropionic acid, a synthetic allosteric modifier of hemoglobin (Hb), increases release of oxygen from Hb, increasing oxygen availability to hypoxic tissues, and reverses the hypothermia-dependent increase in Hb oxygen affinity. We studied recovery of myocardial mechanical and metabolic function and examined myocardial morphology after cardioplegia, comparing RSR13 (1.75 mmol/L)-supplemented blood (RSR13-BC) to standard blood cardioplegia (BC). METHODS AND RESULTS: Twelve dogs underwent 15 minutes of 37 degrees C global ischemia on cardiopulmonary bypass, followed by 75 minutes of hypothermic cardioplegia (13 degrees C) with either BC (n=6) or RSR13-BC (n=6). There were no differences in baseline function between groups. Cardiac function was assessed after 30 minutes of 37 degrees C reperfusion (BC versus RSR13-BC, respectively) by measuring: % return to normal sinus rhythm (0/100%), % of baseline+dP/dt (33.7+/-1.7/76.3+/-1.9), % of baseline-dP/dt (26.6+/-2.0/81.1+/-1.6), stroke volume (3.5+/-0.5/7.1+/-0.9 mL), cardiac output (340+/-20/880+/-40.3 mL/min), and LVEDP (11.3+/-2.2/0. 3+/-2.9 mm Hg). Postischemic oxidative and metabolic parameters including myocardial lactate, pyruvate, ATP content, and percent water content also were determined. Histological analysis demonstrated preservation of endothelial and myocyte morphology in hearts receiving RSR13-BC compared with BC. CONCLUSIONS: These results indicate that in the setting of hypothermic cardiopulmonary bypass, RSR13 improves recovery of myocardial mechanical and metabolic function compared with standard hypothermic BC. Findings from this study suggest that RSR13-BC, by decreasing hemoglobin oxygen affinity, improves oxidative metabolism and preserves cellular morphology, resulting in significantly improved contractile recovery on reperfusion. (+info)
Site-directed mutagenesis reveals two epitopes involved in the subtype selectivity of the allosteric interactions of gallamine at muscarinic acetylcholine receptors.
(78/3161)
Gallamine allosterically modulates the binding of classical muscarinic ligands with a potency order of M(2) > M(1),M(4) > M(3), M(5). We have suggested previously that the M(2)/M(5) and M(2)/M(3) selectivities are attributable to an epitope in the sixth transmembrane region or third outer loop (o3) region of the receptor. In this study, analysis of numerous point mutations in this region of the M(5) receptor found that a mutation of V --> N resulted in an increased affinity toward gallamine, suggesting that the asparagine residue at M(2)(419) is responsible for gallamine's M(2)/M(5) selectivity. Mutations in the other subtypes indicated that the acidic residues found at this position in M(1) and M(4) are associated with slightly higher affinity toward gallamine, whereas the valine and lysine residues of M(5) and M(3), respectively, are associated with significantly lower affinity. In the o2 region, replacement of an acidic sequence of M(2) (EDGE) by the corresponding neutral sequence of M(1) (LAGQ) reduced the affinity toward gallamine, as reported previously by others; the converse substitution of the acidic sequence into M(1) significantly increased affinity for gallamine. Substitution of the M(1) sequence into this region of M(5) markedly reduced affinity toward gallamine, whereas substitution into M(4) had no effect. All of the above mutations are consistent with gallamine binding with a similar orientation at each subtype, such that it interacts with acidic residues in the o2 region of M(3) and M(5) and with acidic residues in the o3 region of M(1) and M(4); gallamine appears to interact with both regions of the M(2) subtype. (+info)
Mammalian TOR controls one of two kinase pathways acting upon nPKCdelta and nPKCepsilon.
(79/3161)
There are three conserved phosphorylation sites in protein kinase C (PKC) isotypes that have been termed priming sites and play an important role in PKC function. The requirements and pathways involved in novel (nPKC) phosphorylation have been investigated here. The evidence presented for nPKCdelta shows that there are two independent kinase pathways that act upon the activation loop (Thr-505) and a C-terminal hydrophobic site (Ser-662) and that the phosphorylation of the Ser-662 site is protected from dephosphorylation by the Thr-505 phosphorylation. Both phosphorylations require C1 domain-dependent allosteric activation of PKC. The third site (Ser-643) appears to be an autophosphorylation site. The serum-dependent phosphorylation of the Thr-505 and Ser-662 sites increases nPKCdelta activity up to 80-fold. Phosphorylation at the Ser-662 site is independently controlled by a pathway involving mammalian TOR (mTOR) because the rapamycin-induced block of its phosphorylation is overcome by co-expression of a rapamycin-resistant mutant of mTOR. Consistent with this role of mTOR, amino acid deprivation selectively inhibits the serum-induced phosphorylation of the Ser-662 site in nPKCdelta. It is established that nPKCepsilon behaves in a manner similar to nPKCdelta with respect to phosphorylation at its C-terminal hydrophobic site, Ser-729. The results define the regulatory inputs to nPKCdelta and nPKCepsilon and establish these PKC isotypes downstream of mTOR and on an amino acid sensing pathway. The multiple signals integrated in PKC are discussed. (+info)
Identification of transduction elements for benzodiazepine modulation of the GABA(A) receptor: three residues are required for allosteric coupling.
(80/3161)
Modulation of GABA(A) receptors by benzodiazepines (BZDs) is believed to involve two distinct steps: a recognition step in which BZDs bind and a conformational transition step in which the affinity of the receptor for GABA changes. Previously, using gamma(2)/alpha(1) chimeric subunits (chi), we demonstrated that although the N-terminal 167 gamma(2) amino acid residues confer high-affinity BZD binding, other gamma(2) domains couple BZD binding to potentiation of the GABA-mediated Cl(-) current (I(GABA)). To determine which gamma(2) regions couple binding to potentiation, we generated chis with longer N-terminal gamma(2) segments for voltage-clamp experiments in Xenopus oocytes. Chimeras containing greater than the N-terminal 167 gamma(2) residues showed incremental gains in maximal potentiation for diazepam enhancement of I(GABA). Residues in gamma(2)199-236, gamma(2)224-236 (pre-M1), and particularly gamma(2)257-297 (M2 and surrounding loops) are important for BZD potentiation. For several positive BZD modulators tested, the same regions restored potentiation of I(GABA). In contrast, beta-carboline inverse-agonism was unaltered in chimeric receptors, suggesting that structural determinants for positive and negative BZD allosteric modulation are different. Dissection of the gamma(2)257-297 domain revealed that three residues in concert, gamma(2)T281, gamma(2)I282 (M2 channel vestibule), and gamma(2)S291 (M2-M3 loop) are necessary to impart full BZD potentiation to chimeric receptors. Thus, these residues participate in coupling distant BZD-binding events to conformational changes in the GABA(A) receptor. The location of these novel residues provides insight into the mechanisms underlying allosteric coupling for other members of the ligand-gated ion channel superfamily. (+info)