Optimized assay for purine nucleoside phosphorylase by reversed-phase high-performance liquid chromatography

Halfpenny, Anne P.; Brown, Phyllis R.

doi:10.1016/s0021-9673(01)91379-2

Cited by 19 publications

(3 citation statements)

References 14 publications

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“…For measurement of PNP and ADA activities, the brain was homogenized in four volumes of 0.25 M sucrose and the homogenate was centrifuged for 10 min at 20,000 g. The supernatant portion was assayed. PNP activity was estimated using inosine as substrate and measuring by HPLC the hypoxanthine produced (Halfpenny and Brown, 1980). When the ADA activity was estimated, adenosine was the substrate and the inosine formed was measured by HPLC (Hartwich et al, 1978).…”

Section: Enzyme Assaysmentioning

confidence: 99%

Lesch‐Nyhan Syndrome, Caffeine Model: Increase of Purine and Pyrimidine Enzymes in Rat Brain

Miñana

Portolés

Jordá

et al. 1984

Journal of Neurochemistry

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Rats ingesting high doses of caffeine reproduce the self-destructive behaviour observed in the Lesch-Nyhan syndrome. This syndrome includes a deficit in hypoxanthine-guanine phosphoribosyltransferase. We have observed, however, that the activity of hypoxanthine-guanine phosphoribosyltransferase increases in direct proportion to the concentration of caffeine found in rat brain. It appears, therefore, that the caffeine model is not a true model for the Lesch-Nyhan syndrome, or alternatively, that the deficit in hypoxanthine-guanine phosphoribosyltransferase is coincidental and not a main key to the multifarious aspects of the syndrome, particularly the self-mutilation. The possibility that levels of dopamine are increased in the caffeine model are discussed as a basis for the destructive behaviour. We have found also that ingestion of large amounts of caffeine increases the activities in rat brain of adenosine deaminase, purine nucleoside phosphorylase, aspartate carbamoyl-transferase, dihydroorotase, and dihydroorotate oxidase.

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Section: Enzyme Assaysmentioning

confidence: 99%

Lesch‐Nyhan Syndrome, Caffeine Model: Increase of Purine and Pyrimidine Enzymes in Rat Brain

Miñana

Portolés

Jordá

et al. 1984

Journal of Neurochemistry

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“…RBC lysates were prepared from 5% packed RBCs as described above. This assay measures the hypoxanthine product of the PNP reaction, formazan, which is produced when PNP catalyzes phosphorolysis of inosine in the presence of inorganic orthophosphate to release hypoxanthine . Assay mixtures consisting of 100 m M HEPES (pH 7.0), 1 m M inosine, 50 m M potassium phosphate (pH 7.0), 0.075% Triton X‐100, 1 m M iodonitrotetrazolium chloride, xanthine oxidase (20 mU), and 0–2.2 mU of PNP or cell and RBC lysates were incubated at 25°C.…”

Section: Methodsmentioning

confidence: 99%

Lupus‐Associated Functional Polymorphism in PNP Causes Cell Cycle Abnormalities and Interferon Pathway Activation in Human Immune Cells

Ghodke‐Puranik

Dorschner

Vsetecka

et al. 2017

Arthritis & Rheumatology

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Background Systemic lupus erythematosus (SLE) is frequently characterized by type I interferon (IFN) pathway activation. We previously found a missense SNP in the purine nucleoside phosphorylase (PNP) gene was associated with high IFN in SLE (rs1049564, P=1.24 ×10−7). PNP is a key enzyme in purine metabolism. In this study, we perform functional follow-up of this polymorphism in human cells. Methods Type I IFN was measured in patient sera using a reporter cell assay. Structural modeling of the PNP variant was performed using Pymol software. PNP mRNA, protein levels and type I IFN-induced gene expression were measured in lymphoblastoid cell lines with known PNP rs1049564 genotypes. Cell cycle was assayed using flow cytometry. Results Structural modeling indicated no major disruption in folding related to rs1049564. We found that homozygous rs1049564 TT lymphoblastoid cells had decreased PNP mRNA and protein levels, and TT cells had reduced PNP enzymatic activity even when the amount of PNP was controlled. TT cells had a 2-fold increase in S-phase block as compared to homozygous CC cells. The S-phase block could be pharmacologically reversed with hypoxanthine and adenosine, supporting relative PNP deficiency as the cause of the S-phase block. Type I IFN-induced transcripts were increased in a dose-response fashion related to the rs1049564 T allele both at baseline and after type I IFN stimulation. Conclusions The rs1049564 PNP T allele is a loss-of-function variant, inducing S-phase block and IFN pathway activation in lymphocytes. The S-phase block can be rescued in our in vitro experiments, suggesting a potential for personalized therapeutics.

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“…35 (11) Purine nucleoside phosphorylase (PNP; EC 2.4.2.1) catalyzes the conversion of purine nucleosides such as inosine, guanosine and their 2'-deoxyribonucleosides to purine bases and ribose 1-phosphate or 2-deoxyribose 1-phosphate. Deficiency of PNP is closely associated with immunodeficiency characterized by severely defective T-cell function with normal B-cell immunity.1,2).…”

mentioning

confidence: 99%

Assay of purine nucleoside phosphorylase in erythrocytes by flow-injection analysis with fluorescence detection.

Hayashi

Zaitsu

Ohkura

1987

CHEMICAL & PHARMACEUTICAL BULLETIN

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Optimized assay for purine nucleoside phosphorylase by reversed-phase high-performance liquid chromatography

Cited by 19 publications

References 14 publications

Lesch‐Nyhan Syndrome, Caffeine Model: Increase of Purine and Pyrimidine Enzymes in Rat Brain

Lesch‐Nyhan Syndrome, Caffeine Model: Increase of Purine and Pyrimidine Enzymes in Rat Brain

Lupus‐Associated Functional Polymorphism in PNP Causes Cell Cycle Abnormalities and Interferon Pathway Activation in Human Immune Cells

Assay of purine nucleoside phosphorylase in erythrocytes by flow-injection analysis with fluorescence detection.

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