Pyrimidine 5′-Nucleotidase Acquired Deficiency in β-Thalassemia: Involvement of Enzyme-SH Groups in the Inactivation Process

Abstract

Pyrimidine 5′-nucleotidase (P5′N) partial deficiency has been described in several hematological disorders and also in the β-thalassemic trait. To check if the P5’N deficiency in thalassemia was acquired we used thalassemic red cells (from either homo- or heterozygous subjects), whose P5′N activity was lower than in control cells. After separation on a density gradient, activity in lighter cells was similar to controls and less than 50% in denser cells. The most probable mechanism for this faster inactivation involves enzyme -SH groups modification by oxidation and reaction with monofunctional aldehydes produced by membrane lipid peroxidation. In vitro challenge of thiol enzymes as pyruvate kinase (PK), adenylate kinase (AK) and P5′N with increasing concentrations of GSSG, hexanal (HEX) and 4-hydroxynonenal (HNE), showed that HNE is the most powerful among the enzyme inhibitors tested and that P5′N activity is a more sensitive index of -SH groups damage, when compared to PK and AK.

Pyrimidine 5′-Nucleotidase Acquired Deficiency in Beta-Thalassaemia: Involvement of Enzyme-SH Groups in the Inactivation Process

“…In addition, our results supported the hypothesis of a probable in vivo oxidative stress mechanism as the cause of the acquired P5N deficiency observed in P-thalassaemia trait [3]. Although the evidence presented by David et al [1] strongly confirms our results, no mention has been made of our paper, published in Enzyme one year ago [2], In our opinion, the results of David et al [1] stress the fact that P5N-SH groups are more sensi tive to 4-hydroxynonenal than to other aldehydes, but neither the importance of these compounds in p-thal assaemia nor their possible in vivo effect on P5N activity has been elucidated. Conversely, they are in accordance with the different effect of in vitro peroxi dation observed on pyruvate kinase activity when compared to P5N and adenylate kinase activities [2], which showed partial recovery after the addition of P-mercaptoethanol to the RBC incubation medium containing H2O2.…”

Genetic basis of hemolytic anemia caused by pyrimidine 5′ nucleotidase deficiency

“…The 286-amino acid form of the P5ЈN-1 enzyme has a predicted mass of 32.7 kd. It contains 5 cysteine residues, some of which may be implicated in the acquired P5ЈN-1 deficiency associated with lead poisoning 22 and the oxidative stress of ␤-thalassemia 23 ; interestingly, these cysteine residues all occurred within 70 amino acids of the NH 2 -amino end of the protein. The tertiary structure of the protein is not yet known, but predictions of secondary structure suggest that it is a globular protein consisting of approximately 30% ␣ helices and 26% extended strands.…”

A new simple approach for the determination of pyrimidine 5′‐nucleotidase activity in human erythrocytes using an ELISA reader

“…This may be due to the high reticulocyte count (6.5%) or because P5 0 N-1 activity is progressively inhibited until the erythrocyte lead concentration reaches 200 lg/dL RBCs at which point the activity is maximally depressed (Paglia, Valentine & Brockway, 1984; Sakai & Ushio, 1986). P5 0 N-1 partial deficiency has been described in the South European b thalassemia carriers (David et al, 1989). In our finding, ten b thalassemia carriers showed marginally reduced purine/pyrimidine nucleotide ratios, but the P5 0 N-1 activity was normal.…”