Two forms of the nucleoside diphosphate kinase of Pseudomonas aeruginosa 8830: altered specificity of nucleoside triphosphate synthesis by the cell membrane-associated form of the truncated enzyme

Abstract

Nucleoside diphosphate kinase (EC 2.7.4.6) (Ndk) is a ubiquitous enzyme functioning in the intracellular distribution of terminal phosphate bond energy among the various nucleotides used in synthetic and regulatory functions in cells. We have previously reported that in Pseudomonas aeruginosa, this important enzyme is transcriptionally regulated by the gene algR2 and posttranslationally regulated by a phosphoprotein phosphatase for the phosphorylated form of Ndk. We report here that an intracellular protease cleaves the 16-kDa form of Ndk to a 12-kDa form that undergoes autophosphorylation with an efficiency almost identical to that of the 16-kDa form. The 12-kDa form was found to be predominantly associated with the P. aeruginosa cell membrane fraction, whereas the 16-kDa form was predominantly cytoplasmic. In the membrane-associated state, the 12-kDa form of Ndk was found to synthesize GTP in preference to other nucleoside triphosphates. The specificity toward GTP synthesis could be abolished by the addition of Tween 20 or Triton X-100. The activity itself could be abolished by the addition of anti-Ndk antibody to the assay mixture. The formation of the 12-kDa form of Ndk and its association with the cell membrane were found to be related to the growth stage of P. aeruginosa, with less than 1% of the 12-kDa Ndk detectable in the membrane fraction at early log phase in comparison with the levels present at late stationary phase.

wt p53 dependent expression of a membrane-associated isoform of adenylate kinase

“…It is in fact known that AK1 can complement nucleoside diphosphate kinase (NDPK) de®ciency in bacteria (Lu and Inouye, 1996), and that mammalian AK1 has full NDPK activity and can e ciently phosphorylate all four ribo-or deoxyribo-nucleosides diphosphates (Lu and Inouye, 1996). In Pseudomonas aeruginosa, under certain growth conditions, cytoplasmic NDPK is proteolytically converted to a membrane-associated form which acquires a strong speci®city for synthesizing GTP in preference to other NTPs (Shankar et al, 1996). Similarly, AK1b could have a more selective substrate speci®city than AK1.…”

Complex Formation of the Elongation Factor Tu from Pseudomonas aeruginosa with Nucleoside Diphosphate Kinase Modulates Ribosomal GTP Synthesis and Peptide Chain Elongation

“…Unlike Pk and Pra that are membrane-associated proteins, EF-Tu is primarily cytoplasmic and can therefore form complexes with the 16-kDa Ndk, which is known to be soluble (4). It is important to emphasize that Pk and Pra can form complexes with only 12-kDa Ndk, which is known to be membrane-associated, and not with the cytoplasmic 16-kDa Ndk (5,8).…”

“…The reaction mixture was incubated for 1 min, followed by addition of 2 l of 10 ϫ SDS buffer. 1 l of the reaction mixture was spotted on to a polyethyleneimine TLC plate and the reaction products were separated in 0.75 M KH 2 PO 4 buffer, pH 3.75, as described previously (4). The TLC plates were air dried, covered with plastic wrap, and autoradiographed.…”

“…Effect of Purified EF-Tu on NTP Synthesizing Activity of Ndk-NTP synthesizing activity was measured in 20-l reaction volumes containing 250 M each of CDP, GDP, and UDP as described previously (4,5). The reaction was initiated by adding different amounts of purified protein and 10.0 Ci of [␥-32 P]ATP (3000 Ci/mmol) along with 0.125 mM nonradioactive ATP.…”

“…Salt Washing of the Ribosome and Its Dissociation-70 S ribosomes from P. aeruginosa were isolated and extracted with 100 mM, 200 mM, 500 mM, and 1.0 M KCl solution in TMNM buffer (10 mM Tris, pH 7.5, 6 mM MgCl 2 , 30 mM NH 4 Cl, and 4 mM 2-mercaptoethanol) sequentially, and at each step the pellet was resuspended while the supernatant was assayed for NTP synthesizing activity. In a separate experiment, 70 S ribosomes were separated into 30 and 50 S subunits by sucrose density gradient centrifugation as described for E. coli ribosomes (10).…”