Regulation of AMP deaminase from chicken erythrocytes. A kinetic study of the allosteric interactions.
The allosteric properties of AMP deaminase [EC 220.127.116.11] from chicken erythrocytes have been qualitatively and quantitatively accounted for by the concerted transition theory of Monod et al., on the assumption that this enzyme has different numbers of binding sites for each ligand. Theoretical curves yield a satisfactory fit for all experimental saturation functions with respect to activation by alkali metals and inhibition by Pi, assuming that the numbers of binding sites for AMP, alkali metals, and Pi are 4, 2, and 4, respectively. The enzyme was inhibited by concentrations of ATP and GTP below 0.1 and 0.25 mM, respectively, whereas activation of the enzyme was observed at ATP and GTP concentrations above 0.4 and 1.5 mM, respectively. These unusual kinetics with respect to ATP and GTP could be also accounted for by assuming 2 inhibitory and 4 activating sites for each ligand. (+info
Regulation of chicken erythrocyte AMP deaminase by phytic acid.
AMP deaminase [EC 18.104.22.168] purified from chicken erythrocytes was inhibited by phytic acid (inositol hexaphosphate), which is the principal organic phosphate in chicken red cells. Kinetic analysis has indicated that this inhibition is of an allosteric type. The estimated Ki value was within the normal range of phytic acid concentration, suggesting that this compound acts as a physiological effector. Divalent cations such as Ca2+ and Mg2+ were shown to affect AMP deaminase by potentiating inhibition by lower concentrations of phytic acid, and by relieving the inhibition at higher concentrations of phytic acid. These results suggests that Ca2+ and Mg2+ can modify the inhibition of AMP deaminase by phytic acid in chicken red cells. (+info
ATIC-ALK: A novel variant ALK gene fusion in anaplastic large cell lymphoma resulting from the recurrent cryptic chromosomal inversion, inv(2)(p23q35).
The subset of CD30-positive anaplastic large cell lymphomas (ALCL) with the NPM-ALK gene fusion arising from the t(2;5)(p23;q35) forms a distinct clinical and prognostic entity. Recently, various cytogenetic, molecular, and protein studies have provided evidence for the existence of several types of variant ALK fusions in up to 20% of ALK+ ALCL, of which only one, a TPM3-ALK fusion resulting from a t(1;2)(q25;p23), has so far been cloned. A cryptic inv(2)(p23q35) has been described as another recurrent cytogenetic alteration involving ALK and an unidentified fusion partner in some ALCL. In a screen for variant ALK gene fusions, we identified two ALCL that were negative for NPM-ALK by reverse transcriptase-polymerase chain reaction, but were positive for cytoplasmic ALK with both polyclonal and monoclonal antibodies to the ALK tyrosine kinase domain, consistent with ALK deregulation by an alteration other than the t(2;5) Case 1 was a T-lineage nodal and cutaneous ALCL in a 52-year-old woman, and Case 2 was a T-lineage nodal ALCL in a 12-year-old girl. FISH analysis confirmed ALK rearrangement in both cases. An inverse polymerase chain reaction approach was then used to identify the ALK translocation partner in Case 1. We found an in-frame fusion of ALK to ATIC, a gene previously mapped to 2q34-q35. We then confirmed by DNA polymerase chain reaction the localization of ATIC to yeast artificial chromosome (YAC) 914E7 previously reported to span the 2q35 break in the inv(2)(p23q35). FISH analysis in Case 1 confirmed rearrangement of YAC 914E7 and fusion to ALK. The ATIC-ALK fusion was confirmed in Case 1 and also identified in Case 2 by conventional reverse transcriptase-polymerase chain reaction using ATIC forward and ALK reverse primers. ATIC encodes an enzyme involved in purine biosynthesis which, like other fusion partners of ALK, is constitutively expressed and appears to contain a dimerization domain. ATIC-ALK fusion resulting from the inv(2)(p23q35) thus provides a third mechanism of ALK activation in ALK+ ALCL. (+info
A new variant anaplastic lymphoma kinase (ALK)-fusion protein (ATIC-ALK) in a case of ALK-positive anaplastic large cell lymphoma.
Anaplastic lymphoma kinase (ALK)-positive lymphomas ("ALKomas") constitute a distinct molecular and clinicopathological entity within the heterogeneous group of CD30-positive large cell lymphomas. In 80-85% of cases tumor cells express a Mr 80,000 hybrid protein comprising the nucleolar phosphoprotein nucleophosmin (NPM) and the ALK. We report here the cloning and expression of a novel ALK-fusion protein from an ALK-positive lymphoma. This case was selected for molecular investigation because of (a) the absence of NPM-ALK transcripts; (b) the atypical staining patterns for ALK (cytoplasm-restricted) and for NPM (nucleus-restricted); and (c) the presence of a Mr 96,000 ALK-protein differing in size from NPM-ALK. Nucleotide sequence analysis of ALK transcripts isolated by 5'-rapid amplification of cDNA ends revealed a chimeric mRNA corresponding to an ATIC-ALK in-frame fusion. ATIC is a bifunctional enzyme (5-aminoimidazole-4-carboxamide ribonucleotide transformylase and IMP cyclohydrolase enzymatic activities) that catalyzes the penultimate and final enzymatic activities of the purine nucleotide synthesis pathway. Expression of full-length ATIC-ALK cDNA in mouse fibroblasts revealed that the fusion protein (a) possesses constitutive tyrosine kinase activity; (b) forms stable complexes with the signaling proteins Grb2 and Shc; (c) induces tyrosine-phosphorylation of Shc; and (d) provokes oncogenic transformation. These findings point to fusion with ATIC as an alternative mechanism of ALK activation. (+info
Inv(2)(p23q35) in anaplastic large-cell lymphoma induces constitutive anaplastic lymphoma kinase (ALK) tyrosine kinase activation by fusion to ATIC, an enzyme involved in purine nucleotide biosynthesis.
The non-Hodgkin lymphoma (NHL) subtype anaplastic large-cell lymphoma (ALCL) is frequently associated with a t(2;5)(p23;q35) that results in the fusion of the ubiquitously expressed nucleophosmin (NPM) gene at 5q35 to the anaplastic lymphoma kinase (ALK) gene at 2p23, which is not normally expressed in hematopoietic tissues. Approximately 20% of ALCLs that express ALK do not contain the t(2;5), suggesting that other genetic abnormalities can result in aberrant ALK expression. Here we report the molecular characterization of an alternative genetic means of ALK activation, the inv(2)(p23q35). This recurrent abnormality produces a fusion of the amino-terminus of 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase/IMP cyclohydrolase (ATIC), a bifunctional homodimeric enzyme that catalyzes the penultimate and final steps of de novo purine nucleotide biosynthesis, with the intracellular portion of the ALK receptor tyrosine kinase. RT-PCR analysis of 5 ALCL tumors that contained the inv(2) revealed identical ATIC-ALK fusion cDNA junctions in all of the cases. Transient expression studies show that the ATIC-ALK fusion transcript directs the synthesis of an approximately 87-kd chimeric protein that is localized to the cytoplasm, in contrast to NPM-ALK, which typically exhibits a cytoplasmic and nuclear subcellular distribution. ATIC-ALK was constitutively tyrosine phosphorylated and could convert the IL-3-dependent murine hematopoietic cell line BaF3 to cytokine-independent growth. Our studies demonstrate an alternative mechanism for ALK involvement in the genesis of NHL and suggest that ATIC-ALK activation results from ATIC-mediated homodimerization. In addition, expected decreases in ATIC enzymatic function in ATIC-ALK-containing lymphomas may render these tumors more sensitive to antifolate drugs such as methotrexate. (Blood. 2000;95:2144-2149) (+info
Binding of PurH to a muscle-specific splicing enhancer functionally correlates with exon inclusion in vivo.
Regulated alternative splicing of avian cardiac troponin T (cTNT) pre-mRNA requires multiple intronic elements called muscle-specific splicing enhancers (MSEs) that flank the alternative exon 5 and promote muscle-specific exon inclusion. To understand the function of the MSEs in muscle-specific splicing, we sought to identify trans-acting factors that bind to these elements. MSE3, which is located 66-81 nucleotides downstream of exon 5, assembles a complex that is both sequence- and muscle-specific. Purification and characterization of the MSE3 complex identified one component as 5-aminoimidazole-4-carboxamide ribonucleotideformyltransferase/IMP cyclohydrolase (PurH), an enzyme involved in de novo purine synthesis. Recombinant human PurH protein directly binds MSE3 RNA and PurH is the primary determinant of sequence-specific binding in the native complex. Furthermore, we show a direct correlation between the in vitro binding affinity of both the MSE3 complex and recombinant PurH with functional activation of exon inclusion in vivo. Together, these results strongly suggest that PurH performs a second function as a component of a complex that regulates MSE3-dependent exon inclusion. (+info
Characterization of two 5-aminoimidazole-4-carboxamide ribonucleotide transformylase/inosine monophosphate cyclohydrolase isozymes from Saccharomyces cerevisiae.
The Saccharomyces cerevisiae ADE16 and ADE17 genes encode 5-aminoimidazole-4-carboxamide ribonucleotide transformylase isozymes that catalyze the penultimate step of the de novo purine biosynthesis pathway. Disruption of these two chromosomal genes results in adenine auxotrophy, whereas expression of either gene alone is sufficient to support growth without adenine. In this work, we show that an ade16 ade17 double disruption also leads to histidine auxotrophy, similar to the adenine/histidine auxotrophy of ade3 mutant yeast strains. We also report the purification and characterization of the ADE16 and ADE17 gene products (Ade16p and Ade17p). Like their counterparts in other organisms, the yeast isozymes are bifunctional, containing both 5-aminoimidazole-4-carboxamide ribonucleotide transformylase and inosine monophosphate cyclohydrolase activities, and exist as homodimers based on cross-linking studies. Both isozymes are localized to the cytosol, as shown by subcellular fractionation experiments and immunofluorescent staining. Epitope-tagged constructs were used to study expression of the two isozymes. The expression of Ade17p is repressed by the addition of adenine to the media, whereas Ade16p expression is not affected by adenine. Ade16p was observed to be more abundant in cells grown on nonfermentable carbon sources than in glucose-grown cells, suggesting a role for this isozyme in respiration or sporulation. (+info
Human 5-aminoimidazole-4-carboxamide ribonucleotide transformylase/inosine 5'-monophosphate cyclohydrolase. A bifunctional protein requiring dimerization for transformylase activity but not for cyclohydrolase activity.
The bifunctional enzyme aminoimidazole carboxamide ribonucleotide transformylase/inosine monophosphate cyclohydrolase (ATIC) is responsible for catalysis of the last two steps in the de novo purine pathway. Gel filtration studies performed on human enzyme suggested that this enzyme is monomeric in solution. However, cross-linking studies performed on both yeast and avian ATIC indicated that this enzyme might be dimeric. To determine the oligomeric state of this protein in solution, we carried out sedimentation equilibrium analysis of ATIC over a broad concentration range. We find that ATIC participates in a monomer/dimer equilibrium with a dissociation constant of 240 +/- 50 nM at 4 degrees C. To determine whether the presence of substrates affects the monomer/dimer equilibrium, further ultracentrifugation studies were performed. These showed that the equilibrium is only significantly shifted in the presence of both AICAR and a folate analog, resulting in a 10-fold reduction in the dissociation constant. The enzyme concentration dependence on each of the catalytic activities was studied in steady state kinetic experiments. These indicated that the transformylase activity requires dimerization whereas the cyclohydrolase activity only slightly prefers the dimeric form over the monomeric form. (+info