Enzyme activities leading to NAD synthesis in human lymphocytes.

Pyridine nucleotide levels and the activities of enzymes involved in NAD synthesis (nicotinic acid phosphoribosyltransferase, nicotinic acid- and nicotinamide mononucleotide-adenylyltransferase) have been assayed in human normal lymphocytes by an HPLC method using radioactive or nonradioactive substrates. NAD concentration was 46.4 +/- 17.2 pmol 10(-6) cells, and that of NADP was 14.5 +/- 3.9 pmol 10(-6) cells (mean +/- standard deviation). The adenylyltransferase activity using nicotinic acid mononucleotide as substrate was 1.530 +/- 0.216 nmol h(-1) 10(-6) cells, using nicotinamide mononucleotide was 1.466 +/- 0.354 nmol h(-1) 10(-6) cells. The apparent K(M) values were 0.015 mM for the former substrate and 0.167 mM for the latter. The mean activity of nicotinic acid phosphoribosyltransferase was 0.038 +/- 0.014 nmol h(-1) 10(-6) cells, and the apparent K(M) for nicotinic acid was 0.165 mM. The proposed methods, easy and rapid to perform, are reliable and sensitive, avoiding the use of radiolabels except for NAPRT and displaying a very low activity. The reported findings, together with the previous ones in human erythrocytes, can provide an useful base to investigate NAD metabolism in humans through the study of blood cells.

Biochemical and MALDI analysis of the human sperm antigen gp20, homologue of leukocyte CD52.

In previous work we demonstrated that gp20, a sialoglycoprotein of human sperm is homologous to the leukocyte antigen CD52 and that anti-gp20 recognizes an antigen of the same molecular weight as that recognized by CAMPATH-1 (anti CD52) in leukocytes and sperm, but with some differences. In this study we used anti-gp20 to perform immunoblot analysis of many different sperm, seminal plasma and leukocyte samples. The sperm and seminal plasma antigens were similar and appeared to consist of two components, whereas the leukocyte antigen is unique. Evidence of the presence of two components of the sperm antigen, running respectively at about 19 and 21 kDa, was obtained by analyzing the purified antigen stained with Coomassie brilliant blue and by immunoblot analysis of the antigen after two-dimensional electrophoresis. Both components had an isoelectric point (pI) between 3 and 6. MALDI analysis of the purified antigen confirmed the presence of two components and indicated masses (Mr) of 8243 and 10908. The possible relationship between these findings and the presence of two forms of the CD52 gene differing at two aminoacids C-terminal to the GPI-anchor site has been discussed.

Purine and pyridine nucleotide metabolism in the erythrocytes of patients with Rett syndrome.

The possible involvement of purine and pyridine metabolism in Rett syndrome, a neurodegenerative disorder of unknown aetiology affecting females, was investigated. The levels of purine and pyridine nucleotides and their metabolites were determined by HPLC in the erythrocytes and plasma of 31 Rett patients and of 17 age-matched controls. Nucleotide production rate from extracellular precursors was determined in intact cells and enzyme activities were assayed in crude lysates using the same HPLC method. Decreased plasma nicotinamide concentrations and lower erythrocyte activities of hypoxanthine phosphoribosyl transferase, adenine phosphoribosyl transferase and phosphoribosylpyrophosphate synthetase were observed in Rett children compared with age-matched controls, while the production rate of IMP from hypoxanthine and of total pyridine nucleotides from nicotinic acid by intact erythrocytes was significantly increased. No significant difference was found in any of the other parameters examined. These findings give a new contribution to the knowledge of the biochemical alterations in Rett syndrome and encourage further investigations in the nucleotide field.

Nicotinic acid phosphoribosyltransferase activity in human erythrocytes: studies using a new HPLC method.

A non-radiochemical method linked to reverse-phase high-performance liquid chromatography was developed to determine the activity of nicotinic acid phosphoribosyltransferase (EC 2.4.2.11) in crude lysates of human red blood cells. The method is accurate and easily reproducible in different chromatographic systems. The enzyme activity was determined in erythrocytes of healthy subjects and in patients with different purine disorders showing altered NAD levels. Very low enzyme activity was found in a boy hemizygous for phosphoribosylpyrophosphate synthetase superactivity, consistent with the low erythrocyte NAD concentration.

Further insight into NAD synthesis in human erythrocytes: significance and relevance.

The cellular levels of NAD and its rate of synthesis from nicotinic acid (NA) were found to decrease from reticulocyte-rich fractions to mature erythrocytes separated on a density gradient. Decreased activity of NA-phosphoribosyltransferase in crude lysates was also observed from young to mature cells, while the uptake of extracellular NA did not show any difference. Impaired NAD synthesis, more likely than progressive breakdown, is suggested to cause NAD decrease during erythrocyte maturation.

Density increase and ageing of erythrocytes in stored blood.

An increase in the density of erythrocytes was observed after storage of whole blood for 30 days at 4 degrees C in either acid citrate-dextrose or citrate-phosphate-dextrose-adrenaline. Glucose-6-phosphate dehydrogenase activity in unfractionated red blood cell lysates did not vary with the storage time. Enzyme activity in the lighter fraction separated by density gradient centrifugation was higher than that in heavier fractions. The decline in glucose-6-phosphate dehydrogenase activity with density was less marked after storage of whole blood for 30 days. It is suggested that density modifications are not related to the ageing of erythrocytes and additional mechanisms may be involved.

Nucleotide synthesis in human erythrocyte: correlations between purines and pyridines.

The regulation of erythrocyte synthesis of nicotinate and adenine nucleotides has been investigated. Some effectors of the two committed enzymes, nicotinate- and adenine phosphoribosyltransferases, have been identified on crude lysates and on partially purified preparations of the former. Enzyme characteristics have been correlated with the nucleotide synthesis achieved in intact cells incubated in suitable mediums containing (14-C)-nicotinate or adenine. Inorganic phosphate, Mg ions and adenine nucleotides proved to be important effectors of pyridine synthesis, whose products, in turn, do not influence adenine nucleotide production. The production of pyridine nucleotides is little lower than that of adenine nucleotides in intact cells, even if adenine phosphoribosyl-transferase activity appears to be much more limited, inside the cell, than nicotinate phosphoribosyltransferase.