5′-Nucleotidase in Dictyostelium: protein purification, cloning, and developmental expression

Chanchao, Chanpen; Eristi, Can M.; Favis, Reyna; Rutherford, Charles L.

doi:10.1016/s0304-4165(99)00206-8

Cited by 5 publications

(7 citation statements)

References 40 publications

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“…3A, right). The faint bands at 17s and 26s in the knockout lanes represent the nonspecific hybridization of the 5nt probe to ribosomal RNA, as we have observed previously (Chanchao et al, 1999).…”

Section: Northern and Western Analysis Of The 5nt Null Mutantssupporting

confidence: 80%

“…After transfer, the membrane was subjected to hybridization with labeled 5nt cDNA. The wt produced a band located between the two ribosomal bands corresponding to the known location of the authentic 5nt mRNA (Chanchao et al, 1999). This band was not present in any of the five 5nt null mutant clones (Fig.…”

Section: Northern and Western Analysis Of The 5nt Null Mutantsmentioning

confidence: 87%

“…By utilizing amino acid sequence from the purified 5NT, Chanchao et al (1999) were able to clone the gene encoding this protein. Sequence comparison between the 5nt gene and that of classical alp from other organisms revealed little similarity.…”

mentioning

confidence: 99%

“…Sequence comparison between the 5nt gene and that of classical alp from other organisms revealed little similarity. However, a search of the databases of the Dictyostelium developmental cDNA project at the institute of Biological Science, University of Tsukuba, Japan, using conserved sequences of alps from several different organisms, revealed the presence of several clones of a Dictyostelium sequence with high sequence similarity to alp (Chanchao et al, 1999).…”

mentioning

confidence: 99%

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Analysis of 5′ nucleotidase and alkaline phosphatase by gene disruption in Dictyostelium

Rutherford

Overall

Ubeidat

et al. 2003

Genesis

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In Dictyostelium discoideum a phosphatase with a high pH optimum is known to increase in activity during cell differentiation and become localized to a narrow band of cells at the interface of prespore and prestalk cells. However, it was not clear if this activity is due to a classical "alkaline phosphatase" with broad range substrate specificity or to a "5'nucleotidase" with high substrate preference for 5'AMP. We attempted to disrupt the genes encoding these two phosphatase activities in order to determine if the activity that is localized to the interface region resides in either of these two proteins. During aggregation of 5nt null mutants, multiple tips formed rather than the normal single tip for each aggregate. In situ phosphatase activity assays showed that the wt and the 5nt gene disruption clones had normal phosphatase activity in the area between prestalk and prespore cell types, while the alp null mutants did not have activity in this cellular region. Thus, the phosphatase activity that becomes localized to the interface of the prestalk and prespore cells is alkaline phosphatase.

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Section: Northern and Western Analysis Of The 5nt Null Mutantssupporting

confidence: 80%

Section: Northern and Western Analysis Of The 5nt Null Mutantsmentioning

confidence: 87%

mentioning

confidence: 99%

mentioning

confidence: 99%

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Analysis of 5′ nucleotidase and alkaline phosphatase by gene disruption in Dictyostelium

Rutherford

Overall

Ubeidat

et al. 2003

Genesis

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“…Equilibrative transporters in the plasma membrane facilitate the diffusion of adenosine down its concentration gradient and the accumulation of adenosine outside the cell (8). Nitric oxide, which readily crosses cell membranes (9), also stimulates adenosine accumulation, by acting as a metabolic inhibitor and depleting intracellular ATP (10). Extracellular adenosine acts in a negative feedback loop to inhibit transmitter release from presynaptic terminals (11).…”

Section: Western Blot Analysis Of Pp1 and Pp2a In Neuronal Culturesmentioning

confidence: 99%

NMDA receptor-mediated extracellular adenosine accumulation is blocked by phosphatase 1/2A inhibitors

Rosenberg

2007

Brain Research

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We have previously demonstrated that NMDA receptor-mediated extracellular adenosine accumulation in neuronal cultures is receptor-mediated and requires calcium influx. Because protein kinase C (PKC) is a calcium-dependent enzyme, we hypothesized that activation of PKC might be involved in NMDA-mediated adenosine accumulation. PKC inhibitors however, did not block NMDA-evoked adenosine accumulation, but rather, stimulated basal adenosine accumulation. These data suggested the possibility that NMDA receptor mediated adenosine accumulation involves net dephosphorylation rather than phosphorylation of one or more substrates. Thus, inhibition of kinases would be expected to increase adenosine accumulation and inhibition of phosphatases would be expected to block adenosine accumulation. To test this hypothesis, we used the phosphatase 1/2A inhibitors calyculin A and okadaic acid. Both inhibitors significantly reduced NMDA-evoked adenosine accumulation. In contrast phosphatase 2B inhibitors did not block NMDA-evoked adenosine accumulation. These data suggest that NMDAevoked adenosine accumulation is mediated by activation of phosphatase 1/2A. We have established previously that NMDA-mediated adenosine accumulation is associated with adenosine kinase inhibition. However, adenosine kinase is not a direct substrate for phosphatase 1/2A because inhibition of phosphatase 1/2A did not abolish NMDA-evoked adenosine kinase inhibition. Okadaic acid also had no effect on NO donor-evoked adenosine accumulation, which previously has been shown to be associated with adenosine kinase inhibition. Dephosphorylation of one or more proteins other than adenosine kinase as a consequence of NMDA receptor activation might play an important role in extracellular adenosine regulation, with important consequences for the regulation of excitatory synaptic transmission, plasticity, epileptogenesis, and excitotoxicity.

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Expression pattern of 5′-nucleotidase in Dictyostelium

Ubeidat

Eristi

Rutherford

2002

Mechanisms of Development

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In order to analyze the expression pattern of the 5'-nucleotidase (5nt) gene in Dictyostelium, we made a fusion construct in which the 5nt promoter directed the expression of beta-galactosidase gene. The reporter gene was not active in vegetative amoebae but was expressed during the aggregation stage. At the slug stage, 5nt was highly expressed in pstAB cells. As the slug moved along the substratum, high activity of beta-galactosidase was detected in cells that were left behind in the slime trail. In the completed fruiting body, 5nt was expressed in the lower cup, the anterior like cells (ALC) and the basal disc.

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5′-Nucleotidase in Dictyostelium: protein purification, cloning, and developmental expression

Cited by 5 publications

References 40 publications

Analysis of 5′ nucleotidase and alkaline phosphatase by gene disruption in Dictyostelium

Analysis of 5′ nucleotidase and alkaline phosphatase by gene disruption in Dictyostelium

NMDA receptor-mediated extracellular adenosine accumulation is blocked by phosphatase 1/2A inhibitors

Expression pattern of 5′-nucleotidase in Dictyostelium

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