ABSTRACT
Cytidine deaminase (CDA) purified from human placenta revealed the presence of five isoenzymatic forms that differ only in their isoelectric point. Since human cytidine deaminase exists in two variants (CDA 1 and CDA 2) with a non-conservative amino acid substitution at codon 27, in this work we demonstrate that these two variants may combine together in vitro, giving five CDA isoforms as observed in vivo from human placenta. For this purpose, each of the two forms of CDA was purified close to homogeneity and dissociated into monomers in the presence of a small amount of sodium dodecyl sulfate as a dissociating agent. The monomers were mixed together and subjected to anion-exchange chromatography and to chromatofocusing analysis in order to visualize the formation of the five isoforms. Furthermore, for both CDA 1 and CDA 2 some substrates and inhibitors of CDA were assayed, with the aim of demonstrating different kinetic behavior between the two natural variants.
Subject(s)
Cytidine Deaminase/chemistry , Cytidine Deaminase/isolation & purification , Anion Exchange Resins/pharmacology , Chromatography , Chromatography, Gel , Chromatography, Ion Exchange , Cloning, Molecular , Codon , DNA, Complementary/metabolism , Escherichia coli/metabolism , Humans , Isoelectric Focusing , Kinetics , Placenta/enzymology , Protein Engineering/methods , Protein Isoforms , Recombinant Proteins/chemistry , Resins, Synthetic , Sodium Dodecyl Sulfate/chemistryABSTRACT
The first identification and characterization of a catalytic activity associated with NadR protein is reported. A computer-aided search for sequence similarity revealed the presence in NadR of a 29-residue region highly conserved among known nicotinamide mononucleotide adenylyltransferases. The Escherichia coli nadR gene was cloned into a T7-based vector and overexpressed. In addition to functionally specific DNA binding properties, the homogeneous recombinant protein catalyzes NAD synthesis from nicotinamide mononucleotide and ATP.
Subject(s)
Bacterial Proteins , Nicotinamide-Nucleotide Adenylyltransferase/metabolism , Repressor Proteins/metabolism , Amino Acid Sequence , Cloning, Molecular , DNA/metabolism , Escherichia coli , Gene Expression , Molecular Sequence Data , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/isolation & purification , Recombinant Fusion Proteins/metabolism , Repressor Proteins/genetics , Repressor Proteins/isolation & purificationABSTRACT
The efficacy of long-term treatments with the vasodilator antihypertensive pildralazine alone and in combination with propranolol or dihydrochlorothiazide in preventing blood pressure increase and cerebrovascular lesions in saline-drinking spontaneously hypertensive rats (SHR) was evaluated. Groups of 20-40 animals were treated daily for 13 weeks. At the end of the experiments the brains were examined histologically and vascular lesions, ranging from arterial wall damage to cerebral infarction, were checked. In the control groups (n = 60), systolic blood pressure gradually rose to over 200 mmHg, and about 74% of the animals had lesions. Pildralazine at a dose of 1 mg/kg p.o. significantly inhibited the onset of severe hypertension; the combination of pildralazine with moderately effective doses of propranolol (10 mg/kg p.o.) or dihydrochlorothiazide (5 mg/kg p.o.) completely prevented the blood pressure increase. When blood pressure control was achieved, the incidence of cerebrovascular lesions was also significantly reduced.
