Search PubMed⌕ Search

Biomedical subjects

E Zocchi

Publications and source records attributed to E Zocchi.

At least 37 records · Page 2Linked to original sources
PubMed1997

CD38 and ADP-ribosyl cyclase catalyze the synthesis of a dimeric ADP-ribose that potentiates the calcium-mobilizing activity of cyclic ADP-ribose.

CD38, a lymphocyte differentiation antigen, is also a bifunctional enzyme catalyzing the synthesis of cyclic ADP-ribose (cADPR) from NAD+ and its hydrolysis to ADP-ribose (ADPR). An additional enzymatic activity of CD38 shared by monofunctional ADP-ribosyl cyclase from Aplysia californica is the exchange of the base group of NAD+ (nicotinamide) with various nucleophiles. Both human CD38 (either recombinant or purified from erythrocyte membranes) and Aplysia cyclase were found to catalyze the exchange of ADPR with the nicotinamide group of NAD+ leading to the formation of a dimeric ADPR ((ADPR)2). The dimeric structure of the enzymatic product, which was generated by recombinant CD38 and by CD38(+) Namalwa cells from as low as 10 microM NAD+, was demonstrated using specific enzyme treatments (dinucleotide pyrophosphatase and 5'-nucleotidase) and mass spectrometry analyses of the resulting products. The linkage between the two ADPR units of (ADPR)2 was identified as that between the N1 of the adenine nucleus of one ADPR unit and the anomeric carbon of the terminal ribose of the second ADPR molecule by enzymatic analyses and by comparison with patterns of cADPR cleavage with Me2SO:tert-butoxide. Although (ADPR)2 itself did not release Ca2+ from sea urchin egg microsomal vesicles, it specifically potentiated the Ca2+-releasing activity of subthreshold concentrations of cADPR. Therefore, (ADPR)2 is a new product of CD38 that amplifies the Ca2+-mobilizing activity of cADPR.

ADP-ribosyl Cyclase↗
PubMed1997

The CD38/cyclic ADP-ribose system: a topological paradox.

CD38 was first identified as a lymphocyte differentiation antigen that showed typical properties of an orphan receptor involved in many programs of cell proliferation and activation. However, CD38 proved also to be a bifunctional ectoenzyme that catalyzes the transient formation of cyclic ADP-ribose (cADPR) in a variety of cell types. This property raises many intriguing and so far unanswered questions, since cADPR is a new second messenger molecule directly involved in the control of calcium homeostasis by means of receptor-mediated release of calcium from ryanodine-sensitive intracellular stores. The relationship between receptor-like and enzymatic properties of CD38 is still unknown. The apparent topological paradox of ectocellular synthesis and intracellular activity of cADPR might be explained by: (a) influx of cADPR across the plasma membrane to reach its target stores, as suggested by experiments on cerebellar granule cells; and (b) NAD(+)-induced internalization, following membrane oligomerization, of CD38 with consequent partial import of cADPR metabolism to an intracellular compartment, as recently observed in lymphoid B cells. These two distinct mechanisms and other potential ones (e.g. binding of ectocellularly formed cADPR to cell surface receptors and initiation of signal-transducing pathways across the plasmamembrane) seem to be paradigmatic of processes affecting different types of cells. Although in some biological systems, such as Aplysia and sea urchin egg, cADPR metabolism is restricted to the intracellular environment, in mammalian cells the CD38/cADPR system provides new challenges in terms of subcellular compartmentation and qualifies as an unusual example of "ectobiochemistry" with potential, still unrecognized, properties of cellular regulation.

ADP-ribosyl Cyclase↗
PubMed1996

Ectocellular in vitro and in vivo metabolism of cADP-ribose in cerebellum.

CD38, a type II transmembrane glycoprotein predominantly expressed in blood cells, is a bifunctional ectoenzyme directly involved in the metabolism of cADP-ribose (cADPR). This is a potent Ca2+ mobilizer in several types of cells. The relationship between the ectocellular site of cADPR production and its intracellular calcium-related functions is poorly understood. Cultured rat cerebellar granule cells showed both enzymic activities of CD38, ADP-ribosyl cyclase and cADPR hydrolase, at a ratio of 16 to 1 respectively, and were immunostained by the anti-(human CD38) monoclonal antibody IB4. In these cells externally added cADPR and beta-NAD+ (the precursor of cADPR), but not alpha-NAD+ or ADP-ribose, enhanced the peak of the depolarization-induced rise in intracellular Ca2+ concentration. This effect was inhibited by 1 microM ryanodine, suggesting a potentiation of calcium-induced calcium release by cADPR. CD38 ectoenzyme activities, ADP-ribosyl cyclase and cADPR hydrolase, were also demonstrated in vivo by microdialysis of adult rat cerebellum, where IB4 bound to granule neurons selectively. Trace amounts (11.5 +/- 3.8 nM) of NAD+ were detected by microdialysis sampling and sensitive assays in the basal interstitial fluid of the cerebellum. These results provide a link between ectocellular cADPR turnover and intracellular calcium mobilization in cerebellum.

ADP-ribosyl Cyclase↗
PubMed1996

NAD+-dependent internalization of the transmembrane glycoprotein CD38 in human Namalwa B cells.

CD38 is a transmembrane glycoprotein involved as an orphan receptor in many physiological processes of lymphocytes. It is also a bifunctional enzyme that catalyzes at its ectocellular domain the synthesis from NAD+ (cyclase) and the hydrolysis (hydrolase) of the calcium-mobilizing metabolite cyclic ADP-ribose (cADPR). A still unexplained paradox concerns the relationship between ectocellular localization of CD38 and intracellular calcium-releasing activity of its intermediate product cADPR. Incubation of CD38+ human Namalwa B cells with external NAD+ elicited extensive membrane down-regulation of CD38 and its internalization in non-clathrin-coated vesicles. Since the internalized CD38 was demonstrated to be enzymatically active, this NAD+-dependent process is a hitherto unrecognized means for shifting cADPR metabolism from the cell surface to the intracellular environment.

ADP-ribosyl Cyclase↗
PubMed1995

Structural role of disulfide bridges in the cyclic ADP-ribose related bifunctional ectoenzyme CD38.

Human CD38, a type II cell surface glycoprotein, is a bifunctional ectoenzyme catalyzing both ADP-ribosyl cyclase and cyclic ADP-ribose (cADPR) hydrolase reactions. It shares a high degree of sequence homology with the cyclase from Aplysia species and studies of site-directed mutagenesis have recently demonstrated the importance, but not elucidated the role, of several cysteine residues highly conserved between these proteins. N-Ethylmaleimide, iodoacetamide and thiol-oxidizing reagents failed to affect either the cyclase or the weaker hydrolase activity of the Aplysia californica protein. Likewise, these reagents did not impair the two activities of CD38 purified from human erythrocytes. beta-mercaptoethanol had no effect on the Aplysia enzyme activities, while it inactivated both the cyclase and the cADPR hydrolase of CD38 by inducing its extensive oligomerization. In intact erythrocytes the beta-mercaptoethanol-dependent enzyme inactivation was completely prevented by prior cross-linking of the membrane proteins with glutaraldehyde. These data demonstrate that none of the cysteine residues plays any direct catalytic role in CD38 and Aplysia proteins, and that disulfide bridges are essential for maintaining the monomeric, catalytically active structure of CD38.

ADP-ribosyl Cyclase↗
PubMed1995

Self-aggregation of purified and membrane-bound erythrocyte CD38 induces extensive decrease of its ADP-ribosyl cyclase activity.

The transmembrane glycoprotein CD38 is a bifunctional enzyme that catalyzes at its ectocellular domain both the synthesis and the hydrolysis of cyclic ADP-ribose (cADPR). The complete reaction, converting NAD+ to nicotinamide and ADP-ribose, reproduces an NAD+glycohydrolase (NADase) reaction. CD38 purified from human erythrocyte membranes has been recently shown to undergo stable oligomerization induced by either NAD+ or beta-mercaptoethanol. We demonstrate that oligomerization is also triggered by reduced glutathione (GSH) and that the GSH-induced self-aggregation of purified CD38 is accompanied by extensive and comparable decrease of its ADP-ribosyl cyclase and NADase activities. GSH-induced oligomerization of CD38 and strong enzyme inactivation take place also in situ on erythrocyte membranes.

ADP-ribosyl Cyclase↗
PubMed1994

Self-aggregation of the transmembrane glycoprotein CD38 purified from human erythrocytes.

The 46 kDa human transmembrane glycoprotein CD38 is a multicatalytic enzyme exhibiting ADPribosyl cyclase, cyclic ADPribose hydrolase and NAD(+)-glycohydrolase activities at its extracellular domain. When CD38, purified to homogeneity from human erythrocyte membranes, was incubated with NAD+ or beta-mercaptoethanol, extensive aggregation took place. Addition of both compounds to CD38 led to the formation of still larger aggregates (over 300 nm), which were resistant to TCA precipitation. Extensive and stable CD38 self-aggregation was shown by, i) SDS-PAGE and autoradiography of the [32P]NAD(+)-incubated CD38, ii) SDS-PAGE followed by immunochemical detection of CD38 on the transblots, iii) direct electron microscopy on negatively stained CD38 samples. Self-aggregation of CD38 might be correlated with its putative function as a transducer of activation and proliferation signals in a number of hematopoietic cells.

ADP-ribosyl Cyclase↗
PubMed1993

A single protein immunologically identified as CD38 displays NAD+ glycohydrolase, ADP-ribosyl cyclase and cyclic ADP-ribose hydrolase activities at the outer surface of human erythrocytes.

The three ectoenzyme activities, NAD+ glycohydrolase, ADP-ribosyl cyclase and cyclic ADP-ribose hydrolase were purified to homogeneity from solubilized human erythrocyte membranes. The purification procedure involved three sequential chromatography steps on hydroxylapatite, immobilized Cu++ and immobilized anti-CD38 monoclonal antibody resins. The final step yielded a single 46 kDa protein displaying all three enzymatic activities. Since the protein bound specifically to the anti-CD38 resin, it was immunologically identified as CD38, a 46 kDa surface antigen involved in activation and proliferation of lymphocyte populations.

ADP-ribosyl Cyclase↗
PubMed1993

Free ADP-ribose in human erythrocytes: pathways of intra-erythrocytic conversion and non-enzymic binding to membrane proteins.

We have previously identified free ADP-ribose (ADPR) as a normal metabolite in mature human erythrocytes. In this study the metabolic transformations of ADPR were investigated in both supernatants from erythrocyte lysates and intact erythrocytes, loaded with ADPR by means of a procedure involving hypotonic haemolysis and isotonic resealing. In both experimental systems, the main pathway was a dinucleotide pyrophosphatase-catalysed hydrolysis to yield AMP, which was readily converted into the adenylic and inosinic nucleotide pools. To a lesser extent, ADPR underwent conversion into a compound that was identified as ADP-ribulose (ADPRu), on the basis of m.s., n.m.r. spectroscopy and enzymic analysis. ADPRu was also susceptible to degradation by the dinucleotide pyrophosphatase, which was partially purified from erythrocyte lysates and characterized with respect to its substrate specificity. Isomerization of ADPR to ADPRu was markedly enhanced by ATP. Incubation of unsealed haemoglobin-free erythrocyte membranes with labelled ADPR did not cause any transformation of this nucleotide and resulted in its trichloroacetic acid- and formic acid-resistant binding to a number of membrane cytoskeletal proteins. These proteins include spectrin, glyceraldehyde 3-phosphate dehydrogenase (Ga3PDH), three proteins of molecular masses 98, 79 and 72 kDa, which apparently comigrate with bands 3, 4.1 and 4.2 respectively, and two additional proteins of molecular masses 58 and 41 kDa. Acid-resistant binding of ADPR, as well as of NAD+, to Ga3PDH was confirmed for the enzyme purified from human erythrocytes.

Adenine Nucleotides↗
PubMed1993

Production and hydrolysis of cyclic ADP-ribose at the outer surface of human erythrocytes.

Hemoglobin-free membranes from human erythrocytes are able to convert beta-NAD+ to cyclic ADP-ribose, a calcium mobilizer as potent as inositol 1,4,5-trisphosphate. Identification of cyclic ADP-ribose was based on HPLC analyses and its Ca(2+)-mobilizing activity on sea urchin egg microsomes. Erythrocyte membranes also hydrolyze cyclic ADP-ribose to ADP-ribose. By comparing the cyclic ADP-ribose-synthesizing and -hydrolyzing activities on unsealed and right-side-out resealed ghosts, it can be concluded that both are localized at the extracellular side of the membrane. This is confirmed by the demonstration of both enzyme activities on the surface of intact human red cells. Identification of the two enzymes involved in cyclic ADP-ribose metabolism might suggest some physiological role of this nucleotide in red cells.

ADP-ribosyl Cyclase↗
PubMed1993

Adenosine diphosphate ribulose in human erythrocytes: a new metabolite with membrane binding properties.

Incubation of ADPribose with yeast phosphoriboisomerase resulted in the formation of an adenylic nucleotide that was identified with ADPribulose by mass spectrometry. Synthesis of [32P]ADPribulose from [32P]NAD+ by the combined activities of commercial NAD+ glycohydrolase and phosphoriboisomerase allowed us to use it as a labeled internal standard throughout the procedure of purification from trichloroacetic acid extracts of human red blood cells. ADPribulose was purified by means of three sequential reverse phase HPLC separations and its concentration in human erythrocytes was estimated to be 0.11 +/- 0.1 microM. Unsealed erythrocyte ghosts did not transform ADPribulose, which bound to specific membrane proteins with a trichloroacetic and formic acid-resistant binding. The labeled proteins were identified as spectrin, bands 3, 4.1, 4.2 and Glyceraldehyde 3-phosphate dehydrogenase on the basis of their relative mobilities on SDS-PAGE.

Adenosine Diphosphate Ribose↗
PubMed1992

Presence and turnover of adenosine diphosphate ribose in human erythrocytes.

ADP-ribose was detected in human red blood cells (RBC) at 0.45 +/- 0.1 microM concentrations. These levels could be estimated after purification of ADP-ribose by means of three sequential HPLC fractionations of RBC extracts. Extraction was performed by sonication of RBC either in trichloroacetic acid, followed by centrifugation, or in carbonate-bicarbonate buffer, pH 10.0, followed by rapid ultrafiltration. Neither procedure of extraction caused artefactual formation of ADP-ribose. Prolonged incubation of intact RBC in isotonic buffer containing labeled orthophosphate resulted in the slow incorporation of radioactivity into ADP-ribose. Identification of the labeled ADP-ribose was confirmed upon incubation of the purified metabolite with nucleotide pyrophosphatase, yielding radioactive 5'-AMP and ribose 5-phosphate, while its exposure to a nonspecific deaminase resulted in the quantitative formation of labeled inosine diphosphate ribose.

Adenosine Diphosphate↗
PubMed1990

Glyoxylic acid prevents NAD+ and NADH depletion in K562 cells cultured at limiting dilution.

K562 erythroleukemic cells cultured at low population density in the absence of serum die within 12-24 hours, unless 0.1 mM glyoxylic acid is added to the culture medium. Earlier events, preceding cell death and occurring within 2 hours culture, are: a) a marked drop of both the NAD+/NADH ratio and the NAD+ concentration, which is prevented by 10mM benzamide, b) an increased biosynthesis of NAD+, leading to extensive depletion of cellular ATP. In the presence of 0.1 mM glyoxylic acid the NAD+/NADH ratio as well as their absolute concentrations remain unchanged, while NAD+ biosynthesis is absent. A NAD+/NADH glycohydrolase activity is present in the cell extract, inhibited by 10 mM benzamide and with a higher affinity for NADH than for NAD+. Preservation of a high NAD+/NADH ratio by glyoxylic acid apparently prevents enzyme activity and the related loss of pyridine nucleotides.

Adenosine↗
PubMed1990

Multiple small molecular weight guanine nucleotide-binding proteins in human erythrocyte membranes.

Native membranes from human erythrocytes contain the following G proteins which are ADP-ribosylated by a number of bacterial toxins: Gi alpha and Go alpha (pertussis toxin), Gs alpha (cholera toxin), and three proteins of 27, 26 and 22 kDa (exoenzyme C3 from Clostridium botulinum). Three additional C3 substrates (18.5, 16.5 and 14.5 kDa) appeared in conditions of unrestrained proteolysis during hemolysis. SDS-PAGE separation of erythrocyte membrane proteins followed by electroblotting and incubation of nitrocellulose sheets with radiolabeled GTP revealed consistently four GTP-binding proteins with Mr values of 27, 26, 22 and 21 kDa. Although a 22 kDa protein was immunochemically identified as ras p21, the C3 substrate of 22 kDa is a different protein probably identifiable with a rho gene product. Accordingly, at least five distinct small molecular weight guanine nucleotide-binding proteins, whose functions are so far undetermined, are present in native human erythrocyte membranes.

ADP Ribose Transferases↗
PubMed1990

Glyoxylic acid promotes poly(ADP-ribosyl)ation of nuclear proteins in K562 cells cultured at limiting dilution.

Addition of glyoxylic acid to the culture medium allows survival and proliferation of K562 human erythroleukemic cells cultured at low population density in the absence of serum. Concomitantly, glyoxylic acid induces a remarkable increase in nuclear poly(ADP-ribose) content, as compared to control cells cultured without addition of glyoxylate. The latter effect is reversed by addition of micromolar concentrations of benzamide to the cultures. As glyoxylic acid is metabolized through NADH-dependent reduction to glycolic acid only, the observed effects on cell growth and on nuclear poly(ADP-ribose) content seem to be mediated by an increased cellular ability to oxidize NADH.

Benzamides↗