Biochemical and molecular genetic correlation in adenylosuccinate lyase deficiency

An homology model of human adenylosuccinate lyase structure shows that P100A substitution distorts the amino acid chain of domain I in the proximity of His-86, which behaves as general acid in the catalysis, and may expose Cys-98 and Cys-99 to oxidising agents. This model is in line with the observation that the defective protein is strongly inhibited by 4-hydroxy-2-nonenal, an hydroxyalkenal that is known to form thio-ether linkage with proteins

Metabolic distress associated with impaired control by alternative substrate: two examples taken from purine matabolism

The steady-state kinetic analysis, despite being generally regarded as a solved problem, is far from trivial and might give new insight for understanding the role of the enzymes in their physiological context. In this paper, we focus our attention on enzymes with broad specificity that can use alternative substrates in different metabolic pathway

Activation mechanisms of store-operated calcium channels in human neutrophils.

Calcium mobilization plays an important role in the regulation of superoxide anion secretion by neutrophils. Intracellular calcium increase is predominantly a result of calcium influx from extracellular media through the opening of calcium-permeable channels, which is subsequent to the emptying of intracellular stores. This capacitative mechanism, referred as store-operated calcium entry (SOCE), implies that depletion of agonist-sensitive intracellular calcium stores generates a secondary signal that promotes plasma membrane calcium influx. Accumulating evidence indicates that three protein families (TRPC, STIM, and Orai) play obligatory roles in the activation of this pathway by forming a ternary heterologous complex on the plasma membrane. This complex might mediate communication between the endoplasmic reticulum and the plasma membrane, perhaps by facilitating coupling between TRPC and inositol 3,4,5-trisposphate receptors. STIM1 behaves as a sensor of Ca2+ level in the endoplasmic reticulum, while Orai 1, by interacting with TRPC and STIM1, might act as a regulatory subunit that operates the transduction of the signals to the calcium-permeable channels on the plasma membrane. The role of the microtubule network in the regulation of SOCE is still obscure. Experiments performed with microtubule inhibitors gave rise to contradictory results, since these compounds induced partial depletion of Ca2+ stores and influx of bivalent cations from extracellular medium in the cytosol, but at the same time they inhibited SOCE triggered by agonists that are known to deplete Ca2+ from the endoplasmic reticulum

Role of adenylosuccinate lyase in purine biosynthesis and interconversion: molecular biology and genetic defects.

Adenylosuccinate lyase catalyses two steps in the synthesis of purine nucleotides leading to nonhydrolytic cleavage of succinyl groups to yield fumarate: the conversion of 5-aminoimidazole-4-(N-succinylocarboxamide) ribonucleotide (SAICAR) into 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) along the de novo pathway and the formation of adenosine monophosphate (AMP) from adenylosuccinate (S-AMP) in the conversion of inosine monophosphate into adenine nucleotides. In addition to its role in purine biosynthesis, the enzyme is involved, together with AMP deaminase and S-AMP synthetase, in the purine nucleotide cycle. The reaction mechanism involves a general base catalyst, removing a proton from the succinyl group of the substrate, acting in concert with a general acid catalyst that protonates the amino group left on AICAR or AMP. The enzyme shows four active-site clefts, located on the boundaries between three subunits of the tetramer, which contain two highly-conserved histidine residues and a glutamate-histidine relay that are involved in catalysis. Human adenylosuccinate lyase gene is located in chromosome 22 and spans over 23 kb with 13 exons of 146 to 4536 bp. Two adenylosuccinate lyase mRNA isoforms, which are produced by an alternative splicing of exon 12, have been identified in human tissues. Exon 12 deletion results in the translation of a protein missing 59 amino acids, which is completely inactive. The deficiency of adenylosuccinate lyase causes variable degrees of psychomotor retardation, often accompanied by epileptic seizures and/or autistic features. The existence of genetic heterogeneity for the adenylosuccinate lyase deficiency could account for variability of the clinical presentation. The defect is characterised by the appearance of succinylpurines in cerebrospinal fluid and urine. Deficiency of purine nucleotides, toxic effects, and impairment of energy metabolism are potential mechanisms of cerebral damage

Urinary methylxanhine and autistic disorder: absence of previously reported correlation.

We were unable to reveal significant difference in the levels of xanthine and methylxanthines in the urine samples from 59 patients diagnosed with autistic symptoms and 64 age- and sex-matched normal volunteers. Our data suggest that abnormalities in xanthine and methylxanthine excretion (US Patent 20020019406 A1, Feb. 12, 2002) represent distincly uncommon symptoms in autism

Carotenoids and lung cancer: biochemical aspects

Carotenoids are part of the human diet and a regular low-dose intake of these compounds from natural sources is normally preferred. Carotenoid supplementation in various diseases, including cancer, was described to be useful, but evidence has been obtained that high-dose supplementation of beta-carotene may be unsafe, especially to smokers and asbestos-exposed workers, because of a stastically detected increased cancer risk. The negative effect might be mediated by carotenoid breakdown products having a high reactivity towards biomolecules. It has been suggested that these compounds originate from nonenzymatic cleavage of carotenoids by oxidants liberated in large amounts by neutrophils that accumulate in various inflammatory diseases and, in particular, in pulmonary disorders characterized by profound abnormalities in inflammatory pathways, such as those triggered by tobacco smoking. Carotenoid breakdown products, in turn, may affect neutrophil response in different ways that depend on the concentration that is reached by these products in the medium. In vitro studies show that nanomolar and micromolar concentrations of carotenoid derivatives stimulate superoxide production by neutrophils activated by phorbol myristate acetate (PMA), while a slight inhibition is noticed with cells activated by the chemotactic tripeptide N-formyl-Met-Leu-Phe (f-MLP). At higher concentrations, carotenoid breakdown products inhibit superoxide production in the presence of both PMA and f-MLP

A comparative study of the hemolytic effect of polyenic antibiotics and of other cholesterol-binding agents.

Macrocyclic polyenic antibiotics were compared, on the basis of their hemolytic efficiency (expressed as critical occupancy level and as mean intrinsic association constant) with each other and with other cholesterol-specific hemolytic agents, such as digitonin and streptolysin O. In all cases, except for the larger polyenic antibiotics (amphotericin B and nystatin), the experimental results were compatible with the existence in the membrane of a large number of identical binding sites which are independent from each other. Simultaneous addition of two different agents gave either synergistic or antagonistic effects, indicating that digitonin, streptolysin O and filipin have different mechanisms of action from each other and from the mycosamine-containing polyenes

Effect of insulin on lateral diffusion of pyrene in rat liver plasma membrane.

The yield of excimer formation by pyrene molecules inserted in rat liver plasma membranes in sensibly decreased in the presence of 1 nM insulin. This effect can be interpreted as indicating a decrease of the value of the translational diffusion coefficient of the dye within the membrane

POLYENE ANTIBIOTICS INHIBIT SUPEROXIDE-PRODUCING NADPH OXIDASE IN A POLYMORPHONUCLEAR CELL-FREE SYSTEM

We studied the effect of polyene macrolide antibiotics on the NADPH-dependent superoxide production induced by arachidonic acid in a cell-free system consisting of the membrane and cytosolic fractions obtained from bovine polymorphonuclear leukocytes. Preincubation of the membrane fraction with polyenes before addition of the soluble components of the reaction mixture resulted in a dose-dependent inhibition of superoxide production