Inner compartment localization of heart mitochondrial nucleoside diphosphokinase

Mitochondria isolated from rat heart contained nucleoside diphosphokinase (EC 2.7.4.6) at a specific activity of 30 mIU/mg protein, or about one half of liver mitochondrial activity, 60 mIU/mg. In contrast to liver mitochondria, no stimulation of O₂ uptake was observed when 150 μM GDP was added to heart mitochondria respiring in post-ADP State 4, and the transphosphorylation of [γ-³²Pi] from ATP into GTP was marginal. However, when heart mitochondria pretreated with oligomycin were solubilized with 0.03% Triton X-100, a five fold increase in the rate of GTP formation was observed. These results show that in heart mitochondria approximately 80% of the nucleoside diphosphokinase activity is localized within the inner compartment.     

Ebselen is a potent non-competitive inhibitor of extracellular nucleoside diphosphokinase

Nucleoside di- and triphosphates and adenosine regulate several components of the mucocilairy clearance process (MCC) that protects the lung against infections, via activation of epithelial purinergic receptors. However, assessing the contribution of individual nucleotides to MCC functions remains difficult due to the complexity of the mechanisms of nucleotide release and metabolism. Enzymatic activities involved in the metabolism of extracellular nucleotides include ecto-ATPases and secreted nucleoside diphosphokinase (NDPK) and adenyl kinase, but potent and selective inhibitors of these activities are sparse. In the present study, we discovered that ebselen markedly reduced NDPK activity while having negligible effect on ecto-ATPase and adenyl kinase activities. Addition of radiotracer [γ ³²P]ATP to human bronchial epithelial (HBE) cells resulted in rapid and robust accumulation of [³²P]-inorganic phosphate (³²Pi). Inclusion of UDP in the incubation medium resulted in conversion of [γ ³²P]ATP to [³²P]UTP, while inclusion of AMP resulted in conversion of [γ ³²P]ATP to [³²P]ADP. Ebselen markedly reduced [³²P]UTP formation but displayed negligible effect on ³²Pi or [³²P]ADP accumulations. Incubation of HBE cells with unlabeled UTP and ADP resulted in robust ebselen-sensitive formation of ATP (IC₅₀ = 6.9 ± 2 μM). This NDPK activity was largely recovered in HBE cell secretions and supernatants from lung epithelial A549 cells. Kinetic analysis of NDPK activity indicated that ebselen reduced the V _max of the reaction (K _i = 7.6 ± 3 μM), having negligible effect on K _M values. Our study demonstrates that ebselen is a potent non-competitive inhibitor of extracellular NDPK.     

Behavior of various ribo- and deoxyribonucleosides, nucleoside monophosphate kinases, and nucleoside diphosphokinase on Blue Sepharose affinity columns.

A rapid and effective separation and purification of cytidylate-deoxycytidylate-uridylate kinase, adenylate kinase, and nucleoside diphosphokinase has been achieved with Blue Sepharose CL6B chromatography. When crude extracts of human crythrocytes or acute myelocytic leukemia cells were applied to the column, adenylate kinase, cytidylate-deoxycytidylate-uridylate kinase, and nucleoside diphosphokinase were retarded while guanylate kinase, cytidine kinase, uridine kinase, and deoxycytidine kinase were unabsorbed. The buffers required to elute the retarded kinases depended on the amount of sample applied to the column.     

The cdc 22 mutation by Schizosaccharomyces pombe is a temperature-sensitive defect in nucleoside diphosphokinase

A number of temperature-sensitive cdc- mutants of Schizosaccharomyces pombe that are affected in DNA replication, were screened for the absence of deoxynucleoside triphosphate(s) when blocked at their restrictive temperature. The preliminary screening simply involved analysis of perchloric acid-soluble cell extracts by two-dimensional thin-layer chromatography on poly(ethyleneimine)-impregnated cellulose. One mutant strain, cdc 22-M45, was found which apparently lacked dTTP. Pulse-labelling of intracellular nucleotides revealed that not only did dTTP become depleted, but that dTDP accumulated when this mutant was blocked by a temperature shift-up, indicating a defective nucleoside diphosphokinase. Nucleoside diphosphokinase from cdc 22-M45 was less active than that from wild-type strain 972 when assayed at high temperatures. The nucleoside diphosphokinase of the mutant also has an altered Km for dTDP at both permissive (25 degrees C), and at the restrictive (36.8 degrees C) temperatures. At the restrictive temperature the Km for dTDP of the mutant enzyme is more than 11-times greater than that of the wild type. Characterisation of the biochemical basis of the defect in this cdc- mutant has shown that in S. pombe, despite its having an apparently complex system of genetic control over progression through S-phase, one factor at least is merely availability of a nucleoside triphosphate precursor to DNA synthesis.