1ehw: Difference between revisions

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==Overview==

We demonstrate here the catalytic activity and subcellular localization of, the Nm23-H4 protein, product of nm23-H4, a new member of the human, nm23/nucleoside diphosphate (NDP) kinase gene family (Milon, L., Rousseau-Merck, M., Munier, A., Erent, M., Lascu, I., Capeau, J., and, Lacombe, M. L. (1997) Hum. Genet. 99, 550-557). Nm3-H4 was synthesized in, escherichia coli as the full-length protein and as a truncated form, missing the N-terminal extension characteristic of mitochondrial, targeting. The truncated form possesses NDP kinase activity, whereas the, full-length protein is inactive, suggesting that the extension prevents, enzyme folding and/or activity. X-ray crystallographic analysis was, performed on active truncated Nm23-H4. Like other eukaryotic NDP kinases, it is a hexamer. Nm23-H4 naturally possesses a serine residue at position, 129, equivalent to the K-pn mutation of the Drosophila NDP kinase. The, x-ray structure shows that the presence of Ser(129) has local structural, effects that weaken subunit interactions. Site-directed mutagenesis shows, that the serine is responsible for the lability of Nm23-H4 to heat and, urea treatment, because the S129P mutant is greatly stabilized., Examination of human embryonic kidney 293 cells transfected with green, fluorescent protein fusions by confocal microscopy shows a specific, mitochondrial localization of Nm23-H4 that was also demonstrated by, Western blot analysis of subcellular fractions of these cells. Import into, mitochondria is accompanied by cleavage of the N-terminal extension that, results in NDP kinase activity. Submitochondrial fractionation indicates, that Nm23-H4 is associated with mitochondrial membranes, possibly to the, contact sites between the outer and inner membranes.

We demonstrate here the catalytic activity and subcellular localization of the Nm23-H4 protein, product of nm23-H4, a new member of the human nm23/nucleoside diphosphate (NDP) kinase gene family (Milon, L., Rousseau-Merck, M., Munier, A., Erent, M., Lascu, I., Capeau, J., and Lacombe, M. L. (1997) Hum. Genet. 99, 550-557). Nm3-H4 was synthesized in escherichia coli as the full-length protein and as a truncated form missing the N-terminal extension characteristic of mitochondrial targeting. The truncated form possesses NDP kinase activity, whereas the full-length protein is inactive, suggesting that the extension prevents enzyme folding and/or activity. X-ray crystallographic analysis was performed on active truncated Nm23-H4. Like other eukaryotic NDP kinases, it is a hexamer. Nm23-H4 naturally possesses a serine residue at position 129, equivalent to the K-pn mutation of the Drosophila NDP kinase. The x-ray structure shows that the presence of Ser(129) has local structural effects that weaken subunit interactions. Site-directed mutagenesis shows that the serine is responsible for the lability of Nm23-H4 to heat and urea treatment, because the S129P mutant is greatly stabilized. Examination of human embryonic kidney 293 cells transfected with green fluorescent protein fusions by confocal microscopy shows a specific mitochondrial localization of Nm23-H4 that was also demonstrated by Western blot analysis of subcellular fractions of these cells. Import into mitochondria is accompanied by cleavage of the N-terminal extension that results in NDP kinase activity. Submitochondrial fractionation indicates that Nm23-H4 is associated with mitochondrial membranes, possibly to the contact sites between the outer and inner membranes.

==About this Structure==

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[[Category: nucleoside diphosphate kinase]]

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@@ Line 4: / Line 4: @@
 ==Overview==
-We demonstrate here the catalytic activity and subcellular localization of, the Nm23-H4 protein, product of nm23-H4, a new member of the human, nm23/nucleoside diphosphate (NDP) kinase gene family (Milon, L., Rousseau-Merck, M., Munier, A., Erent, M., Lascu, I., Capeau, J., and, Lacombe, M. L. (1997) Hum. Genet. 99, 550-557). Nm3-H4 was synthesized in, escherichia coli as the full-length protein and as a truncated form, missing the N-terminal extension characteristic of mitochondrial, targeting. The truncated form possesses NDP kinase activity, whereas the, full-length protein is inactive, suggesting that the extension prevents, enzyme folding and/or activity. X-ray crystallographic analysis was, performed on active truncated Nm23-H4. Like other eukaryotic NDP kinases, it is a hexamer. Nm23-H4 naturally possesses a serine residue at position, 129, equivalent to the K-pn mutation of the Drosophila NDP kinase. The, x-ray structure shows that the presence of Ser(129) has local structural, effects that weaken subunit interactions. Site-directed mutagenesis shows, that the serine is responsible for the lability of Nm23-H4 to heat and, urea treatment, because the S129P mutant is greatly stabilized., Examination of human embryonic kidney 293 cells transfected with green, fluorescent protein fusions by confocal microscopy shows a specific, mitochondrial localization of Nm23-H4 that was also demonstrated by, Western blot analysis of subcellular fractions of these cells. Import into, mitochondria is accompanied by cleavage of the N-terminal extension that, results in NDP kinase activity. Submitochondrial fractionation indicates, that Nm23-H4 is associated with mitochondrial membranes, possibly to the, contact sites between the outer and inner membranes.
+We demonstrate here the catalytic activity and subcellular localization of the Nm23-H4 protein, product of nm23-H4, a new member of the human nm23/nucleoside diphosphate (NDP) kinase gene family (Milon, L., Rousseau-Merck, M., Munier, A., Erent, M., Lascu, I., Capeau, J., and Lacombe, M. L. (1997) Hum. Genet. 99, 550-557). Nm3-H4 was synthesized in escherichia coli as the full-length protein and as a truncated form missing the N-terminal extension characteristic of mitochondrial targeting. The truncated form possesses NDP kinase activity, whereas the full-length protein is inactive, suggesting that the extension prevents enzyme folding and/or activity. X-ray crystallographic analysis was performed on active truncated Nm23-H4. Like other eukaryotic NDP kinases, it is a hexamer. Nm23-H4 naturally possesses a serine residue at position 129, equivalent to the K-pn mutation of the Drosophila NDP kinase. The x-ray structure shows that the presence of Ser(129) has local structural effects that weaken subunit interactions. Site-directed mutagenesis shows that the serine is responsible for the lability of Nm23-H4 to heat and urea treatment, because the S129P mutant is greatly stabilized. Examination of human embryonic kidney 293 cells transfected with green fluorescent protein fusions by confocal microscopy shows a specific mitochondrial localization of Nm23-H4 that was also demonstrated by Western blot analysis of subcellular fractions of these cells. Import into mitochondria is accompanied by cleavage of the N-terminal extension that results in NDP kinase activity. Submitochondrial fractionation indicates that Nm23-H4 is associated with mitochondrial membranes, possibly to the contact sites between the outer and inner membranes.
 ==About this Structure==
@@ Line 30: / Line 30: @@
 [[Category: nucleoside diphosphate kinase]]
-''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Fri Feb 15 15:43:27 2008''
+''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Thu Feb 21 12:27:54 2008''