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Cloning and characterisation of hAps1 and hAps2, human diadenosine polyphosphate-metabolising Nudix hydrolases.

Leslie NR, McLennan AG, Safrany ST - BMC Biochem. (2002)

Bottom Line: Recent gene duplication has generated the two Nudix genes, NUDT11 and NUDT10.We have characterised their gene products as the closely related Nudix hydrolases, hAps1 and hAps2.These two gene products complement the activity of previously described members of the DIPP family, and reinforce the concept that Ap5A and Ap6A act as intracellular messengers.

View Article: PubMed Central - HTML - PubMed

Affiliation: Division of Cell Signalling, School of Life Sciences, The University of Dundee, Dundee, DD1 5EH, UK. n.r.leslie@dundee.ac.uk

ABSTRACT

Background: The human genome contains at least 18 genes for Nudix hydrolase enzymes. Many have similar functions to one another. In order to understand their roles in cell physiology, these proteins must be characterised.

Results: We have characterised two novel human gene products, hAps1, encoded by the NUDT11 gene, and hAps2, encoded by the NUDT10 gene. These cytoplasmic proteins are members of the DIPP subfamily of Nudix hydrolases, and differ from each other by a single amino acid. Both metabolise diadenosine-polyphosphates and, weakly, diphosphoinositol polyphosphates. An apparent polymorphism of hAps1 has also been identified, which leads to the point mutation S39N. This has also been characterised. The favoured nucleotides were diadenosine 5',5"'-pentaphosphate (kcat/Km = 11, 8 and 16 x 10(3) M(-1) x s(-1) respectively for hAps1, hAps1-39N and hAps2) and diadenosine 5',5"'-hexaphosphate (kcat/Km = 13, 14 and 11 x 10(3) M(-1) x s(-1) respectively for hAps1, hAps1-39N and hAps2). Both hAps1 and hAps2 had pH optima of 8.5 and an absolute requirement for divalent cations, with manganese (II) being favoured. Magnesium was not able to activate the enzymes. Therefore, these enzymes could be acutely regulated by manganese fluxes within the cell.

Conclusions: Recent gene duplication has generated the two Nudix genes, NUDT11 and NUDT10. We have characterised their gene products as the closely related Nudix hydrolases, hAps1 and hAps2. These two gene products complement the activity of previously described members of the DIPP family, and reinforce the concept that Ap5A and Ap6A act as intracellular messengers.

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SDS-PAGE of purified hAps1, hAps1-39N and hAps2. GST-tagged hAps proteins were prepared as described under "Materials and methods". Approximately 10 μg of (a) hAps1, (b) hAps1-39N and (c) hAps2 were analysed by SDS-PAGE. Purity was determined by Coomassie-blue staining a 4–12% Bis-Tris NuPage gel (Novex). Molecular weight standards (MultiMark multi-colored standards) were from Novex.
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Figure 4: SDS-PAGE of purified hAps1, hAps1-39N and hAps2. GST-tagged hAps proteins were prepared as described under "Materials and methods". Approximately 10 μg of (a) hAps1, (b) hAps1-39N and (c) hAps2 were analysed by SDS-PAGE. Purity was determined by Coomassie-blue staining a 4–12% Bis-Tris NuPage gel (Novex). Molecular weight standards (MultiMark multi-colored standards) were from Novex.

Mentions: The ORFs for hAps1, hAps1-39N and hAps2 were cloned into the pGEX6P-1 expression vector and expressed in Escherichia coli (E. coli). After incubation with isopropyl β-D-thiogalactoside (IPTG) (100 μM) at 26°C for 8 hours, a major protein of the expected size was induced and expressed in a soluble form in each case. The glutathione S-transferase (GST)-tagged recombinant proteins were purified by chromatography on Glutathione Sepharose 4 Fast Flow resin (Fig. 4).


Cloning and characterisation of hAps1 and hAps2, human diadenosine polyphosphate-metabolising Nudix hydrolases.

Leslie NR, McLennan AG, Safrany ST - BMC Biochem. (2002)

SDS-PAGE of purified hAps1, hAps1-39N and hAps2. GST-tagged hAps proteins were prepared as described under "Materials and methods". Approximately 10 μg of (a) hAps1, (b) hAps1-39N and (c) hAps2 were analysed by SDS-PAGE. Purity was determined by Coomassie-blue staining a 4–12% Bis-Tris NuPage gel (Novex). Molecular weight standards (MultiMark multi-colored standards) were from Novex.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC117780&req=5

Figure 4: SDS-PAGE of purified hAps1, hAps1-39N and hAps2. GST-tagged hAps proteins were prepared as described under "Materials and methods". Approximately 10 μg of (a) hAps1, (b) hAps1-39N and (c) hAps2 were analysed by SDS-PAGE. Purity was determined by Coomassie-blue staining a 4–12% Bis-Tris NuPage gel (Novex). Molecular weight standards (MultiMark multi-colored standards) were from Novex.
Mentions: The ORFs for hAps1, hAps1-39N and hAps2 were cloned into the pGEX6P-1 expression vector and expressed in Escherichia coli (E. coli). After incubation with isopropyl β-D-thiogalactoside (IPTG) (100 μM) at 26°C for 8 hours, a major protein of the expected size was induced and expressed in a soluble form in each case. The glutathione S-transferase (GST)-tagged recombinant proteins were purified by chromatography on Glutathione Sepharose 4 Fast Flow resin (Fig. 4).

Bottom Line: Recent gene duplication has generated the two Nudix genes, NUDT11 and NUDT10.We have characterised their gene products as the closely related Nudix hydrolases, hAps1 and hAps2.These two gene products complement the activity of previously described members of the DIPP family, and reinforce the concept that Ap5A and Ap6A act as intracellular messengers.

View Article: PubMed Central - HTML - PubMed

Affiliation: Division of Cell Signalling, School of Life Sciences, The University of Dundee, Dundee, DD1 5EH, UK. n.r.leslie@dundee.ac.uk

ABSTRACT

Background: The human genome contains at least 18 genes for Nudix hydrolase enzymes. Many have similar functions to one another. In order to understand their roles in cell physiology, these proteins must be characterised.

Results: We have characterised two novel human gene products, hAps1, encoded by the NUDT11 gene, and hAps2, encoded by the NUDT10 gene. These cytoplasmic proteins are members of the DIPP subfamily of Nudix hydrolases, and differ from each other by a single amino acid. Both metabolise diadenosine-polyphosphates and, weakly, diphosphoinositol polyphosphates. An apparent polymorphism of hAps1 has also been identified, which leads to the point mutation S39N. This has also been characterised. The favoured nucleotides were diadenosine 5',5"'-pentaphosphate (kcat/Km = 11, 8 and 16 x 10(3) M(-1) x s(-1) respectively for hAps1, hAps1-39N and hAps2) and diadenosine 5',5"'-hexaphosphate (kcat/Km = 13, 14 and 11 x 10(3) M(-1) x s(-1) respectively for hAps1, hAps1-39N and hAps2). Both hAps1 and hAps2 had pH optima of 8.5 and an absolute requirement for divalent cations, with manganese (II) being favoured. Magnesium was not able to activate the enzymes. Therefore, these enzymes could be acutely regulated by manganese fluxes within the cell.

Conclusions: Recent gene duplication has generated the two Nudix genes, NUDT11 and NUDT10. We have characterised their gene products as the closely related Nudix hydrolases, hAps1 and hAps2. These two gene products complement the activity of previously described members of the DIPP family, and reinforce the concept that Ap5A and Ap6A act as intracellular messengers.

Show MeSH