A. De Flora

Cyclic GMP-dependent and -independent Effects on the Synthesis of the Calcium Messengers Cyclic ADP-ribose and Nicotinic Acid Adenine Dinucleotide Phosphate

Cyclic ADP-ribose (cADPR) and nicotinic acid adenine dinucleotide phosphate (NAADP) have been shown to mobilize intracellular Ca2+ stores by totally independent mechanisms, which are pharmacologically distinct from that activated by inositol trisphosphate. Although cADPR and NAADP are structurally and functionally different, they can be synthesized by a single enzyme having ADP-ribosyl cyclase activity. In this study, three different assays were used to measure the metabolism of cADPR in sea urchin egg homogenates including a radioimmunoassay, a Ca2+release assay, and a thin layer chromatographic assay. Soluble and membrane-bound ADP-ribosyl cyclases were identified and both cyclized NAD to produce cADPR. The soluble cyclase was half-maximally stimulated by 5.3 μm cGMP, but not by cAMP, while the membrane-bound form was independent of cGMP. The two forms of the cyclase were also different in the pH dependence of utilizing nicotinamide guanine dinucleotide (NGD), a guanine analog of NAD, as substrate, indicating they are two separate enzymes. The stimulatory effect of cGMP required ATP or ATPγS (adenosine 5′-O-(3-thiotriphosphate)) and a cGMP-dependent kinase activity was shown to be present in the soluble fraction. The degradation of cADPR to ADP-ribose was catalyzed by cADPR hydrolase, which was found to be predominantly associated with membranes. Similar to the membrane-bound cyclase, the cADPR hydrolase activity was also independent of cGMP. Both the soluble and membrane fractions also catalyzed the synthesis of NAADP through exchanging the nicotinamide group of NADP with nicotinic acid (NA). The base-exchange activity was independent of cGMP and the half-maximal concentrations of NADP and NA needed were about 0.2 mm and 10 mm, respectively. The exchange reaction showed a preference for acidic pH, contrasting with the neutral pH optimum of the cyclase activities. The complex metabolic pathways characterized in this study indicate that there may be a multitude of regulatory mechanisms for controlling the endogenous concentrations of cADPR and NAADP.

Human erythrocyte glucose 6-phosphate dehydrogenase: electron microscope studies on structure and interconversion of tetramers, dimers and monomers.

Abstract The structure and interconversion of the three discrete forms of human erythrocyte glucose 6-phosphate dehydrogenase, i.e. tetramers, dimers and monomers, have been investigated by electron microscopy; this is the first such study of a human enzyme to be recorded. The monomer has an axial ratio of about 2.0 and dimensions of 68 A × 34 A, which, together with the previously determined molecular weight of 50,000, suggest a roughly cylindrical shape. Association of two monomers depends on NADP and SH groups and yields the dimeric form; this seems to be accompanied by a reduction of monomer axial ratio to about 1.4. Further assembly of two dimers (obtained by lowering the pH and the ionic strength or by addition of Mg2+) gives the tetramer; this last species has a tetrahedral structure with D2 symmetry. Both the pH-dependent dissociation of tetramers to dimers and the association of dimers to tetramers have been found to occur within a few seconds. Dissociation of the dimer, as induced by either glucose 6-phosphate or NADPH2, appears to be less rapid and to yield a larger proportion of monomers than that estimated by the corresponding loss of catalytic activity. This observation and parallel sucrose gradient centrifugation suggest that a variable fraction of the monomers is instantly converted to an apparently active form in the course of assay under standard conditions.

An improved procedure for rapid isolation or glucose 6-phosphate dehydrogenase from human erythrocytes ☆

Abstract Coupling of N6-(aminohexyl)-adenosine 2′,5′-bisphosphate to BrCN-activated agarose was exploited to develop a simple procedure by which homogeneous glucose 6-phosphate dehydrogenase can be isolated in good yield and in a short time (2 days) from human erythrocytes. The method involves three steps, i.e., chromatography on DEAE-Sephadex, chromatography on phosphocellulose and affinity chromatography on the above ligand-matrix complex. This procedure is applicable for the purification of glucose 6-phosphate dehydrogenase from single donors.

Metabolic reduction of chromium by alveolar macrophages and its relationships to cigarette smoke.

Pulmonary alveolar macrophages (PAM), obtained by bronchoalveolar lavage from 47 individuals, reduced hexavalent chromium [Cr(VI)] and decreased its mutagenicity. Their specific activity--mostly mediated by cytosolic, enzyme-catalyzed mechanisms--was significantly higher than in corresponding preparations of mixed-cell populations from human peripheral lung parenchyma or bronchial tree, or from rat lung or liver. At equivalent number of PAM, Cr(VI) reduction, total protein, and some oxidoreductase activities were significantly increased in smokers. No appreciable variation could be detected between lung cancer and noncancer patients. In rats, the Cr(VI)-reducing activity of PAM preparations was induced by Aroclor 1254. Thus, alveolar macrophages provide crucial defense mechanisms not only by phagocytizing metals, but also by metabolically reducing Cr(VI). The epithelial-lining fluid (ELF) also displayed some Cr(VI) reduction. Together with already investigated metabolic processes occurring inside lung cells, these mechanisms are expected to determine thresholds in the pulmonary carcinogenicity of chromium.

Metabolism of human erythrocyte glucose-6-phosphate dehydrogenase: VI. Interconversion of multiple molecular forms

Abstract Human erythrocyte glucose-6-phosphate dehydrogenase exists in two catalytically active forms, i.e., tetramers and dimers, and also monomers which are intrinsically devoid of activity. Several factors are involved in the interconversion of the three species. High pH and ionic strength favor dissociation of the tetramer to dimers, while low values of pH and ionic strength and in addition some divalent cations shift the equilibrium toward the tetramers. NADPH2, formed during the operation of glucose-6-P dehydrogenase, inactivates the enzyme through a specific disaggregation which affects only the dimeric form. Dissociation of the tetramer to inactive monomers can, however, occur through a compulsory mechanism involving the previous conversion to dimers which are themselves disaggregated by NADPH2. Therefore, when the enzyme is working in a system where NADPH2 is not immediately reoxidized (i.e., in the absence of glutathione reductase), a sequential tetramer ⇀ dimer ⇀ monomer conversion may occur as a result of the enzymatic activity.

Isolation and partial characterization of an NADP- and NADPH-binding protein from human erythrocytes

Abstract A simple procedure, exploiting an affinity chromatography step on agarose-linked adenosine 2′,5′-bisphosphate, allows the concurrent purification from human red cell lysates of glucose 6-phosphate dehydrogenase (G6PD) and of another protein (FX). The latter, which has a higher electrophoretic mobility than G6PD, is not identifiable with any of a number of erythrocyte enzymes. It is a holoprotein, composed in its native form of two polypeptide chains of 33,000 M r each and of one NADP equivalent. Native FX binds NADPH and this binding is competitive with NADP: The corresponding stoichiometry is 0.5 NADPH equivalents per 33,000 M r (“half-site reactivity”), with a dissociation constant of 1 × 10 −8 m . On the basis of competition experiments, the dissociation constant for NADP is estimated to be 1.8 × 10 −7 m .

Distinctive patterns of NADP binding to dimeric and tetrameric glucose 6-phosphate dehydrogenase from human red cells

Abstract The opposite effects displayed by NADP and NADPH2 on red cell G6PD (i.e., protection and inactivation) were elucidated in terms of a competitive mechanism between the oxidized and the reduced forms of the coenzyme. Dialysis equilibrium experiments showed that the dependence of the NADP binding on the initial NADP concentration followed a simple hyperbola when the enzyme exists as “pure” tetramer and conversely was sigmoid-shaped upon dissociation of the tetramers to dimers. The experimental data were consistent for six and three NADP equivalents bound per mole of tetramer and dimer, respectively.

Human erythrocyte glucose 6-phosphate dehydrogenase. Physical properties.

Summary A number of physical properties of human erythrocyte glucose 6-phosphate dehydrogenase have been investigated under different experimental conditions yielding tetramers, (m.w. = 204,800), dimers (m.w. = 101,400) and monomers (m.w. = 51,300), respectively.

A methemoglobin-dependent and plasma-stimulated experimental model of oxidative hemolysis

Abstract Incubation of human red blood cells with ter-butyl hydroperoxide (tBHP) causes depletion of GSH and the production of highly reactive oxygen derivatives, notably hydroxyl (OH • ) radicals, followed by lysis of the cells. These effects are related to the formation of methemoglobin (MetHb), which catalyzes the homolytic cleavage of tBHP to form OH • radicals. Lysis of red blood cells is the result of lipid peroxidation of membrane components and formation of protein aggregates and is enhanced if the tBHP-treated cells are resuspended in autologous plasma or serum. The tBHP-treated cells provide a useful model for analysis of the sequence of events in oxidative hemolysis.

Mechanisms of perturbation of erythrocyte calcium homeostasis in favism.

Favism is an acute hemolytic anemia triggered by ingestion of fava beans in genetically susceptible subjects with severe deficiency of glucose-6-phosphate dehydrogenase (G6PD) activity. Erythrocytes from 10 favic patients had constantly and markedly increased calcium levels, as compared with values detected in 4 asymptomatic G6PD-deficient controls. Correspondingly, the calcium permeability of erythrocytes, estimated as the fraction of intracellular calcium exchangeable with externally added 45Ca2+, was invariably enhanced in favism and returned to normal patterns after several months from the acute hemolytic crisis. In favic patients, the levels of erythrocyte calcium ATPase activities showed wide variability, ranging from 2.0-12.9 mumol Pi/ml RBC/h, while control values in asymptomatic G6PD-deficient subjects were 10.62 +/- 2.03 mumol Pi/ml RBC/h. Analysis of the calcium ATPase in situ in erythrocyte membranes from favic patients showed the same molecular mass of 134 kD as observed in the control subjects. Exposure of G6PD-deficient erythrocytes in vitro to autoxidizing divicine, a pyrimidine aglycone strongly implicated in the pathogenesis of favism which leads to late accumulation of intracellular calcium, caused: (i) a marked inactivation of calcium ATPase, without changes in the molecular mass of 134 kD; and (ii) the concomitant loss of spectrin, band 3 and band 4.1, all known substrates of the calcium activated procalpain-calpain proteolytic system. Thus, the increased intraerythrocytic calcium apparently results in the degradation of calcium ATPase observed in some favic patients. It is proposed that both enhanced calcium permeability and a calcium-stimulated degradation of the calcium pump are the mechanisms responsible for the perturbation of erythrocyte calcium homeostasis in favism.