02 May 2003
Defining the minimal catalytic domain of poly(ADP-ribose) glycohydrolase
T. Sizemore, R. Meyer, M. Meyer-Ficca, E. Jacobson, M. JacobsonMed Sci Monit 2003; 9(1): 62-0 :: ID: 15201
Abstract
Poly(ADP-ribosyl)ation alters the function of numerous regulatory proteins within the cell. The posttranslational modifications are mediated by the poly(ADP-ribose) polymerase (PARP) family of enzymes. Only one enzyme, poly(ADP-ribose) glycohydrolase (PARG), is known to catalyze the hydrolysis of the polymer to free ADP-ribose by exo/endo-glycosidic cleavage. Previous research has identified amino acid residues essential for enzymatic activity in a sub region in the catalytic C-terminal region of PARG. Secondary structure predictions indicate that this sub region of PARG may contain a minimal catalytic domain with an ADP-ribosyltransferase fold similar to that observed in PARP-1 and bacterial toxin ADP-ribosyltransferases. In order to identify, purify and characterize the smallest catalytically active fragment of human PARG (hPARG), we designed a PCR strategy that was based on the exon/intron organization of the hPARG gene. Amplified regions of various portions of the PARG catalytic domain were ligated into a bacterial expression vector pBAD102/D-TOPO and the presence of the desired inserts was confirmed by sequence analysis. PARG fragments carrying engineered His-tags were expressed and purified and the presence of catalytic activity was detected by glycohydrolase assays using 32P labeled ADP-ribose polymers. This approach allowed the identification of a minimal catalytic domain of PARG, which should facilitate our understanding of PARG structure and function relationships and potentially allow structural analysis of the PARG catalytic center by crystallography in the future.
Keywords: PARG, PARP, gene cloning, ADP-ribose, ADP-ribose polymers, glycohydrolase assay
Editorial
01 March 2024 : Editorial
Editorial: First Regulatory Approvals for CRISPR-Cas9 Therapeutic Gene Editing for Sickle Cell Disease and Transfusion-Dependent β-ThalassemiaDOI: 10.12659/MSM.944204
Med Sci Monit 2024; 30:e944204
In Press
21 Feb 2024 : Clinical Research
Potential Value of HSP90α in Prognosis of Triple-Negative Breast CancerMed Sci Monit In Press; DOI: 10.12659/MSM.943049
22 Feb 2024 : Review article
Differentiation of Native Vertebral Osteomyelitis: A Comprehensive Review of Imaging Techniques and Future ...Med Sci Monit In Press; DOI: 10.12659/MSM.943168
23 Feb 2024 : Clinical Research
A Study of 60 Patients with Low Back Pain to Compare Outcomes Following Magnetotherapy, Ultrasound, Laser, ...Med Sci Monit In Press; DOI: 10.12659/MSM.943732
26 Feb 2024 : Clinical Research
Predictive Value of Combined HbA1c and Neutrophil-to-Lymphocyte Ratio for Diabetic Peripheral Neuropathy in...Med Sci Monit In Press; DOI: 10.12659/MSM.942509
Most Viewed Current Articles
17 Jan 2024 : Review article
Vaccination Guidelines for Pregnant Women: Addressing COVID-19 and the Omicron VariantDOI :10.12659/MSM.942799
Med Sci Monit 2024; 30:e942799
16 May 2023 : Clinical Research
Electrophysiological Testing for an Auditory Processing Disorder and Reading Performance in 54 School Stude...DOI :10.12659/MSM.940387
Med Sci Monit 2023; 29:e940387
14 Dec 2022 : Clinical Research
Prevalence and Variability of Allergen-Specific Immunoglobulin E in Patients with Elevated Tryptase LevelsDOI :10.12659/MSM.937990
Med Sci Monit 2022; 28:e937990
01 Jan 2022 : Editorial
Editorial: Current Status of Oral Antiviral Drug Treatments for SARS-CoV-2 Infection in Non-Hospitalized Pa...DOI :10.12659/MSM.935952
Med Sci Monit 2022; 28:e935952