Adenosine diphosphate as a regulatory ligand in beef heart cytosol nucleoside diphosphokinase

Colomb, Maruice G.; Chéruy, A.; Vignais, V Pierre; Duplaa, Anne M.; Meyer, Christiane

doi:10.1021/bi00708a005

Cited by 18 publications

(6 citation statements)

References 26 publications

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“…The enzyme has a relative molecular mass close to I00000 and is composed of 17-kDa polypeptide chains. Therefore it probably has a hexameric structure, in agreement with the results of Palmieri et al [50] in the case of the yeast enzyme, Colomb et al [46] for the beef heart cytosol enzyme, and Robinson et al [51] for the beef brain particulate enzyme. This is a discrepancy compared to the relative molecular mass of 80000 suggested by Burns and Islam [52] by SDS gel electrophoresis of the chick brain soluble NDP kinase.…”

Section: Discussionsupporting

confidence: 89%

“…This was done by incubating the semi-purified NDP kinase in the presence of an excess of [y-32P]ATP at 37 "C. The reaction was stopped by adding SDS to the incubation mixture at 37 ' C, as described by Colomb et al [46]. The sample was further analyzed by SDS gel elec- proteins or by counting the radioactivity in gel slices (Fig.…”

Section: Phosphovylution Of the N D P Kinusementioning

confidence: 99%

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Nucleoside diphosphate kinase from brain

Huitorel

Simon

Pantaloni

1984

European Journal of Biochemistry

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Tubulin strictly requires GTP for its polymerization. Nevertheless, microtubule assembly can be observed in the presence of ATP as the only nucleotide triphosphate, due to the nucleoside diphosphate kinase (NDP kinase) present in microtubule preparations, and which phosphorylates the GDP into GTP.We have purified this enzyme from pig brain to homogeneity, and shown that its relative mass is close to 100000 in its native state, and 17000 under denaturing conditions. Therefore it is probably a hexamer, as previously shown for the enzyme from other sources, and also presents a microheterogeneity, with the major isoforms between PI 5.0 and 6.0. The enzyme is transiently phosphorylated during catalysis, as expected within a ping-pong bi-bi mechanism.The effect of the NDP kinase on pure tubulin polymerization was studied : in the presence of NDP kinase, the lag time observed in the kinetics of microtubule assembly was shorter and the final extent of assembly was unchanged. The effect of the enzyme was observed at enzyme concentrations 900-fold lower than tubulin concentration, which shows that the NDP kinase acts catalytically.Kinetic data show that the catalytic effect of the NDP kinase is faster than the rate of nucleotide exchange on tubulin under the same conditons. This result demonstrates that the tubulin-GDP complex itself is a substrate for the enzyme, which may indicate that the G D P bound to tubulin at the E site is exposed on the surface of dimeric tubulin. Tubulin has been shown to bind two molar equivalents of guanine nucleotides, in vivo [14-161 and in vitro [17, 181. One GTP is non-exchangeable at the N site, in vitro [II, 171 and in vivo [16], and is not hydrolyzed [19, 201. One GTP or G D P is exchangeable with free GTP or G D P [I 1,211 at the E site, which is located on the p subunit [22]. GTP is necessary for microtubule assembly [23] and is hydrolyzed at the E site [I 1, 12, 24-26] after addition of the tubulin dimer at microtubule end [27, 281. In the absence of added GTP, microtubule assembly can be observed in vitro in the presence of ATP and NDP kinase when G D P is bound at the E site of tubulin [12,13, 291.The mechanism by which the G D P is phosphorylated before microtubule assembly is still a matter of controversy since some authors presented evidence for a possible direct phosphorylation of the G D P in situ at the E site of tubulin [I31 whereas others suggested that the reaction only occurs on free nucleotides [30]. This is not a trivial point since NDP kinases are known to follow a ping-pong mechanism [31], i.e. they only have one nucleotide binding site per subunit. As previously quoted [30], a phosphorylation of the G D P in situ on the E site of tubulin supposes that this G D P is sufficiently exposed at the tubulin surface to 'mimic the behavior of unbound nucleotide'.On an other hand, it has been shown that the major source of GTP in vivo is the phosphorylation of GDP at the expense of ATP, catalyzed by the NDP kinase [32, 331. Furthermore, the GTP concentration in the cell c...

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Section: Discussionsupporting

confidence: 89%

Section: Phosphovylution Of the N D P Kinusementioning

confidence: 99%

Nucleoside diphosphate kinase from brain

Huitorel

Simon

Pantaloni

1984

European Journal of Biochemistry

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“…Such heterogeneity, similar to that reported for other tissues (Cheng et al, 1971(Cheng et al, , 1973, raises the question of whether there is a specific tubulin-binding isoform. However, even mono-isoenzymic preparations of NDP kinase can yield multiple activity peaks, depending on the ionic conditions (Yue et al, 1967), phosphorylation status (Colomb et al, 1974), proteolytic degradation (Robinson et al, 1981), and association with other proteins (Islam & Burns, 1984a). The purified chick brain NDP kinase exhibits the same nucleotide specificity as the activity that copellets with assembled microtubules (Table 1) and the particulate activity.…”

Section: Discussionmentioning

confidence: 99%

Microtubules and nucleoside diphosphate kinase. Nucleoside diphosphate kinase binds to co-purifying contaminants rather than to microtubule proteins

Islam¹,

Burns²

1985

Biochemical Journal

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Nucleoside diphosphate (NDP) kinase has been postulated to generate GTP from the GDP bound to tubulin. The purified chick brain enzyme was studied with respect to its kinetic parameters, and the protein-protein interactions between the NDP kinase and tubulin were examined. No specific interaction is observed between the enzyme and assembled microtubules, tubulin dimers, or tubulin-microtubule-associated protein (MAP) oligomers under a variety of nucleotide conditions. The apparent association is demonstrated to result from NDP kinase binding to a co-purifying contaminant. The absence of detectable NDP kinase-tubulin interactions indicates that NDP kinase does not directly charge up tubulin-GDP.

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“…enzyme solutions contained magnesium acetate (1.5 mmoles/l) in order to obtain the nucleotides in the magnesium form. or ADP (0.8 mmoles/l) were incubated at 3OoC with DPC (1 mmole/l) in triethanolamineacetic acid buffer (40 mmoles/l, pH 7.4) for 10 min, TNBS (2 mmoles/ 1) in sodium hydrogen carbonate (10 mmoles/l) for 30 min or FDNB (20 mmoles/l) in sodium dihydrogen carbonate (40 mmoles/l) for 30 min. activity was compared with that of a control solution in which the modifying agent was omitted.…”

Section: Methodsmentioning

confidence: 99%

“…The fact that the substrate ATP and the product ADP protect, at least partially, the enzyme from inactivation by the reagents used here, indicates that the reactions involve the active site of the enzyme (Table 1). Nucleotides have also been shown to protect some NDP kinases from inactivation by p-chloromercuribenzoat (pCMB) (18,19,20,21). It has been discussed that such a sulfhydryl group may be essential for the qurtenary structure of the enzyme and not a part of the active site of the enzyme (18,211.…”

Section: Solutions Of Dpc In Ethanolmentioning

confidence: 99%

Effect of Chemical Modification of a Histidine and a Lysine Residue of Pea Seed Nucleoside Diphosphate Kinase

Edlund

Heldin

Engström

1982

Upsala Journal of Medical Sciences

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Chemical m o d i f i c a t i o n o f a h i s t i d i n e and l y s i n e r e s i d u e i n a c t i v a t e s pea seed n u c l e o s i d e diphosphate k i n a s e (NDP k i n a s e ) . r e a c t i v e l y s i n e r e s i d u e , a t t h e a c t i v e s i t e o f pea seed NDP kinase, i n a d d it i o n t o t h e h i s t i d i n e r e s i d u e phosphorylated b y t h e s u b s t r a t e ATP as a con-sequence o f t h e enzyme r e a c t i o n . The presence o f a r e a c t i v e l y s i n e a t t h e

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Adenosine diphosphate as a regulatory ligand in beef heart cytosol nucleoside diphosphokinase

Cited by 18 publications

References 26 publications

Nucleoside diphosphate kinase from brain

Nucleoside diphosphate kinase from brain

Microtubules and nucleoside diphosphate kinase. Nucleoside diphosphate kinase binds to co-purifying contaminants rather than to microtubule proteins

Effect of Chemical Modification of a Histidine and a Lysine Residue of Pea Seed Nucleoside Diphosphate Kinase

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