Biomedical subjects

G Guidotti

Publications and source records attributed to G Guidotti.

At least 37 records · Page 2Linked to original sources

PubMed1998

Golgi localization and functional expression of human uridine diphosphatase.

A full-length E(ecto)-ATPase (Plesner, L. (1995) Int. Rev. Cytol. 158, 141-214) cDNA was cloned from a human brain cDNA library; it encodes a 610-amino acid protein that contains two putative transmembrane domains. Heterologous expression of this protein in COS-7 cells caused a significant increase in intracellular membrane-bound nucleoside phosphatase activity. The activity was highest with UDP as substrate and was stimulated by divalent cations in the following order: Ca2+ >> Mg2+ > Mn2+. The results of immunofluorescence staining indicate that this protein is located in the Golgi apparatus. UDP hydrolysis was increased in the presence of Triton X-100 or alamethicin, an ionophore that facilitates movement of UDP across the membrane, suggesting that the active site of this UDPase is on the luminal side of the Golgi apparatus. This is the first identification of a mammalian Golgi luminal UDPase gene. Computer-aided sequence analysis of the EATPase superfamily indicates that the human UDPase is highly similar to two hypothetical proteins of the nematode Caenorhabditis elegans and to an unidentified 71.9-kDa yeast protein and is less related to the previously identified yeast Golgi GDPase.

Amino Acid Sequence↗

PubMed1998

Widespread expression of ecto-apyrase (CD39) in the central nervous system.

We have shown that ecto-apyrase protein is expressed in primary neurons and astrocytes in cell culture (T.-F. Wang, P.A. Rosenberg, G. Guidotti, 1997. Mol. Brain Res. 1997, 47: 295-302). Here we present immunohistochemical studies showing that ecto-apyrase protein is widely distributed in rat brain, as it is present in neurons of the cerebral cortex, hippocampus and cerebellum as well as in glial cells and endothelial cells. Ecto-apyrase is enriched in brain postsynaptic density membrane fractions and is localized in proximity to synaptophysin, the marker of synaptic vesicles. These results together with the observation that P2 purinergic receptors are present throughout the brain suggest that ecto-apyrase is involved in regulating synaptic transmission mediated by extracellular ATP.

Adenosine Triphosphatases↗

PubMed1997

Biochemical characterization of the subunits of the Na+/K+ ATPase expressed in insect cells.

The Na+/K+ ATPase is composed of two subunits called alpha and beta chains. In insect cells, independently expressed alpha and beta chains are localized to intracellular membranes. Sucrose density gradient sedimentation, crosslinking analysis, and immunoprecipitation of radio-labeled proteins show that the alpha chains expressed alone are in large aggregates of different molecular weights with less than 4% being monomeric. Analysis by non-reducing SDS-PAGE and immunoblotting show that the beta chains expressed alone are in Triton X-100 insoluble, disulfide-linked aggregates. Co-expression of both subunits in insect cells results in only a small fraction (less than 15%) of the alpha chains being assembled as the active recombinant enzyme, with at least 22% of the active recombinant enzyme localized to the plasma membrane as determined by a biochemical assay. The small amount of beta chain at the plasma membrane in cells that express both subunits is beyond the limit of detection by the biochemical assay. Immunoprecipitation of Triton X-100 soluble alpha chains from radio-labeled cells expressing both subunits shows that the alpha chains are mostly in large aggregates containing beta chains. These results suggest that, in insect cells, the availability of correctly folded beta chains is the rate limiting step in the assembly of active Na+/K+ ATPase.

Animals↗

PubMed1997

Intracellular localization of Na,K-ATPase alpha2 subunit mutants.

The Na,K-ATPase is an essential plasma membrane transporter of mammalian cells composed of two subunits, alpha and beta, of which there are several isoforms. We investigated the effect of a substitution, S364P, on the subcellular localization and enzymatic activity of the wild-type alpha2 and alpha2L111R,N122D (alpha2RD) subunits. The substitutions, L111R and N122D, lower the affinity of the alpha2 subunit for the inhibitor ouabain roughly one thousand-fold (E. A. Jewell and J. B. Lingrel, J. Biol. Chem. 266, 16925-16930, 1991) and were introduced into the alpha2 subunit to distinguish its enzymatic activity from that of the endogenous alpha1 subunit of COS-7 cells. The S364P substitution is located in the ATP binding site, only five residues from the aspartyl residue which is phosphorylated during the catalytic cycle of the Na,K-ATPase. This substitution dramatically decreases the amount of enzymatic activity associated with expression of the alpha2RD subunit. Despite the fact that S364P substitution does not block association of the alpha2RD subunit with the endogenous beta1 subunit, it prevents the alpha2 and alpha2RD subunits from accumulating in the plasma membrane and results in their localization in the endoplasmic reticulum.

Animals↗

PubMed1997

A 0.1-700 Hz current through a voltage-clamped pore: candidate protein for initiator of neural oscillations.

A protein of mass 7643 Da and sequence identical to that of subunit c, the pore part, of the mitochondrial adenosine triphosphate synthase complex, was co-purified with cholesterol in crystals formed from a chloroform/methanol extract of bovine brain gray matter plasma membranes. Reconstitution of the protein-containing crystals in phospholipid bilayers and assay of current by patch-clamp analysis, showed an oscillating cation current at constant voltage, typically of frequency 0.5-200 Hz. The ceroid-lipofuscinoses state in mammals and man (Batten disease), in which subunit c accumulates in lysosomes, affords a rich source of the protein. Pure subunit c from affected sheep liver (in the absence of cholesterol) was also assayed, the current displaying identical sodium oscillations to those of brain crystals. The results suggest that if a protein similar to subunit c resides in the plasma membrane of neural cells, it could be responsible for spontaneous oscillations in brain tissue. The relevance of these results to the pathogenesis of Batten disease is discussed.

Action Potentials↗

PubMed1997

Phosphate transport in yeast vacuoles.

The vacuole of the yeast Saccharomyces cerevisiae is a major storage compartment for phosphate. We have measured phosphate transport across the vacuolar membrane. Isolated intact vacuoles take up large amounts of added [32P]phosphate by counterflow exchange with phosphate present in the vacuoles at the time of their isolation. The bidirectional phosphate transporter has an intrinsic dissociation constant for phosphate of 0.4 mM. Exchange mediated by this carrier is faster than unidirectional efflux of phosphate from the vacuoles. The transporter is highly selective for phosphate; of other anions tested, only arsenate is also a substrate. Transport is strongly pH-dependent with increasing activity at lower pH. Similar phosphate transport behavior was observed in right-side-out vacuolar membrane vesicles.

Biological Transport↗

PubMed1997

The alpha2L111R,N122D isoform of the Na,K-ATPase expressed in HeLa cells does not undergo an adipocyte-like increase in activity in response to insulin.

In the rat adipocyte, insulin increases potassium uptake by a preferential activation of the alpha2 isoform of the Na,K-ATPase. The question under consideration here is whether expression of the alpha2 isoform is sufficient to replicate its differential activation by insulin. Accordingly, we compared the effect of insulin on the activity of the ouabain resistant rat alpha1 and alpha2RD (alpha2L111R,N122D) isoforms in HeLa cells. In HeLa cells, in contrast to the rat adipocyte, insulin produces an increase of equal magnitude in the rate of 86Rb+/K+ uptake by the ouabain resistant rat alpha1 and rat alpha2RD subunits. We conclude that the mechanism of insulin activation of the alpha2RD isoform in HeLa cells differs from that of the wild type alpha2 isoform in the rat adipocyte.

Adipocytes↗

PubMed1997

Sac1p of Saccharomyces cerevisiae is not involved in ATP release to the extracellular fluid.

One activity ascribed to Sac1p is the transport of ATP into the lumen of the endoplasmic reticulum of Saccharomyces cerevisiae; therefore, the question of whether this protein plays a role in ATP efflux from yeast was addressed. Preliminary results suggested that deletion of the SAC1 gene eliminated nigericin-stimulated ATP efflux. However, further experimentation revealed that this result was caused by a pronounced extracellular ATPase activity for sac1delta cells at alkaline pH, conditions required to measure extracellular ATP in wild type cells. At acid pH, sac1delta cells exhibit glucose-dependent, nigericin-stimulated ATP efflux. sac1delta cells express less acid phosphatase activity in the periplasm than do wild type cells, thus increasing the stability of extracellular ATP. At alkaline pH, however, sac1delta cells tend to lose structural integrity and release lactate dehydrogenase as well as an unidentified ATPase activity to the extracellular fluid. Therefore, Sac1p is not involved in ATP efflux from S. cerevisiae.

Adenosine Triphosphate↗

PubMed1997

Ubiquitination of Na,K-ATPase alpha1 and alpha2 subunits.

Na,K-ATPase, an essential transporter of mammalian cells, is an oligomeric transmembrane protein composed of two subunits, alpha and beta, of which there are several isoforms. In this study, we demonstrate that the alpha1 and alpha2 isoforms of the Na,K-ATPase alpha subunit are modified by the covalent attachment of ubiquitin polymers in COS-7 cells. We propose that polyubiquitination of the Na,K-ATPase alpha subunit may play a role in regulating its degradation.

Animals↗

PubMed1997

Effect of ATP binding cassette/multidrug resistance proteins on ATP efflux of Saccharomyces cerevisiae.

Multidrug resistance (MDR) in mammalian tumors or tissues is often associated with the overexpression of the putative drug efflux pump P-glycoprotein (Pgp). One theory concerning the mechanism of Pgp activity is that efflux of ATP is coupled to drug efflux. Evidence in support of this theory has been observed in mammalian cells. Recently, the STS1 gene, which is a multidrug resistance gene related to the mammalian Pgp's, has been characterized in S. cerevisiae. Also, the mouse mdr3 Pgp has been functionally expressed in yeast cells. Therefore, it was of interest to determine whether the expression of these proteins affected ATP efflux from yeast. Although both genes were shown to confer MDR, thus confirming functional expression, the endogenous glucose-dependent, drug-stimulated ATP efflux activity of yeast was not affected by expression of STS1, and was decreased by the expression of mouse mdr3.

ATP Binding Cassette Transporter, Subfamily B↗

PubMed1997

Characterization of brain ecto-apyrase: evidence for only one ecto-apyrase (CD39) gene.

A rat brain cDNA coding for ecto-(Ca,Mg)-apyrase activity was isolated using human CD39 cDNA and functionally expressed in COS-7 cells. The gene codes for a protein with high similarity to human (75% identity) and murine (90% identity) CD39. It is expressed in primary neurons and astrocytes in cell culture as well as in kidney, liver, muscle and spleen. Southern analysis of the mouse genome suggests that there may be a single copy of the ecto-apyrase gene. Interestingly, the human CD39 gene cytologically co-localizes with the susceptibility gene involved in human partial epilepsy with audiogenic symptoms; such a coincidence is consistent with reports on the deficiency of ecto-apyrase activity in the brains of humans with temporal lobe epilepsy and in those of mice with audiogenic seizures.

Adenosine Triphosphatases↗

PubMed1997

Expression of a single gene produces both forms of skeletal muscle cyclic nucleotide-gated channels.

Cyclic nucleotide-gated cation channels in skeletal muscle are responsible for insulin-activated sodium entry into this tissue (J. E. M. McGeoch and G. Guidotti. J. Biol. Chem. 267: 832-841, 1992). These channels have previously been isolated from rabbit skeletal muscle by 8-bromoguanosine 3',5'-cyclic monophosphate (8-BrcGMP) affinity chromatography, which separates them into two populations differing in nucleotide affinity [L. C. Santy and G. Guidotti. Am. J. Physiol. 271 (Endocrinol. Metab. 34): E1051-E1060, 1996]. In this study, a polymerase chain reaction approach was used to identify skeletal muscle cyclic nucleotide-gated channel cDNAs. Rabbit skeletal muscle expresses the same cyclic nucleotide-gated channel as rabbit aorta (M. Biel, W. Altenhofen, R. Hullin, J. Ludwig, M. Freichel, V. Flockerzi, N. Dascal, U. B. Kaupp, and F. Hofmann. FEBS Lett. 329: 134-138, 1993). The entire cDNA for this gene was cloned from rabbit skeletal muscle and an antiserum to this protein produced. Expression of this cDNA produces a 63-kDa protein with cyclic nucleotide-gated channel activity. A similarly sized immunoreactive protein is present in sarcolemma. Purification of the expressed channels reveals that this single gene produces both native skeletal muscle channel populations.

Animals↗

PubMed1996

Characterization of ATP transport into chromaffin granule ghosts. Synergy of ATP and serotonin accumulation in chromaffin granule ghosts.

ATP is an excitatory neurotransmitter that is stored and cosecreted with catecholamines from cells of the adrenal medulla. While the transport of catecholamines into chromaffin granule ghosts has been extensively characterized, there is little information on the mechanism of ATP transport into these structures. Here we show that ATP transport is driven by the electrical component of the electrochemical proton gradient created by the chromaffin granule membrane H+-ATPase, and that the accumulated nucleotide is released from the vesicles by inhibition of the H+-ATPase. GTP and UTP are also substrates for this transporter, distinguishing it from the mitochondrial ADP/ATP exchanger. Accumulation of ADP and ATP (rather than exchange with intravesicular ATP) is demonstrated by high pressure liquid chromatography measurements. The anion transport inhibitor 4,4-diisothiocyanatostilbene-2,2-disulfonic acid (Ki = 27 microM) inhibits ATP transport, while atractyloside, the inhibitor of the mitochondrial ATP/ADP exchanger, is a very poor inhibitor. Finally, we have demonstrated a synergy between the accumulation of ATP and that of serotonin (i.e. more of each solute accumulates when the two are accumulated together), supporting the view that there is an interaction between serotonin and ATP that reduces their effective concentration within the ghosts.

Adenosine Triphosphate↗

PubMed1996

CD39 is an ecto-(Ca2+,Mg2+)-apyrase.

CD39, a 70- to 100-kDa molecule expressed primarily on activated lymphoid cells, was previously identified as a surface marker of Epstein Barr virus (EBV)-transformed B cells. In this report, we show that an ecto-(Ca2+,Mg2+)-apyrase activity is present on EBV-transformed B cells, but not on B or T lymphomas. The coincidence between CD39 expression and ecto-apyrase activity on immune cells suggests that CD39 may be an ecto-apyrase. This supposition is supported by the observation that the amino acid sequence of CD39 is significantly homologous to those of several newly identified nucleotide triphosphatases. Finally, we show that CD39 indeed has ecto-apyrase activity by expression in COS-7 cells.

Adenosine Triphosphatases↗

PubMed1996

Purification and cloning of a soluble ATP-diphosphohydrolase (apyrase) from potato tubers (Solanum tuberosum).

A soluble ATP-diphosphohydrolase (apyrase, EC 3.6.1.5) has been purified from potato tubers. Solanum tuberosum, to a specific activity of 10,000 mumol P(i)/mg/min. The cDNA corresponding to the potato apyrase has been isolated and termed RROP1. The deduced amino acid sequence contains a putative signal sequence, two hydrophobic regions at the carboxy terminus, two potential Asn-linked glycosylation sites, and four regions in the amino-terminal half that we term ACR (apyrase conserved regions) 1-4 that are highly conserved in known apyrases and related enzymes; garden pea nucleoside triphosphatase, Toxoplasma gondii nucleoside triphosphate hydrolases, and Saccharomyces cerevisiae golgi guanosine diphosphatase. A yeast 71.9-kDa hypothetical protein on chromosome V, a Caenorhabditis elegans hypothetical 61.3-kDa protein on chromosome III, and human CD39, a lymphoid cell activation antigen, also share the conserved ACR regions, but their ability to hydrolyze nucleotides has not been assessed.

Amino Acid Sequence↗

PubMed1996

ATP transport and ABC proteins.

ATP can be exported into the extracellular space, where it has important biological effects. Recent evidence shows that direct ATP export across the plasma membrane is associated with the presence of ABC proteins. Do the ABC proteins pump ATP as well as their other substrates, and if so, why?

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1996

Identification of the cysteine residues involved in the class I disulfide bonds of the human insulin receptor: properties of insulin receptor monomers.

The cysteine residues involved in the class I disulfide bonds between the alpha subunits in the (alpha beta)2 dimer of the human insulin receptor have been identified by labeling with N-ethylmaleimide and by site-directed mutagenesis. Both cysteine 524 and cysteine 682 form interchain disulfide bonds; their conversion to serine residues results in the absence of receptor dimers and the presence of alpha beta monomers. The receptor monomers have a slightly lower affinity for insulin than the native receptor dimers. Insulin binding to the receptor monomers promotes their dimerization in the plasma membrane; at nanomolar concentrations of receptor, both unliganded and liganded receptors are monomers. Receptor monomers are stimulated by insulin to autophosphorylate and to phosphorylate exogenous subtrates with the same efficiency as the receptor dimers. The conclusion is that receptor dimerization is not required to activate the tyrosine kinase activity of the insulin receptor.

Amino Acid Sequence↗

PubMed1996

Reconstitution and characterization of two forms of cyclic nucleotide-gated channels from skeletal muscle.

A cyclic nucleotide-gated channel present in skeletal muscle plasma membrane has previously been identified as being responsible for insulin-activated sodium entry into muscle cells (J. E. M. McGeoch and G. Guidotti. J. Biol. Chem. 267:832-841, 1992). We have isolated this channel activity to further study and characterize it. The channel was solubilized from rabbit skeletal muscle sarcolemma and functionally reconstituted into phospholipid vesicles, as assayed by patch-clamp analysis of the reconstituted proteins. Channel activity was isolated by 8-bromo-guanosine 3',5'-cyclic monophosphate affinity chromatography, producing two distinct peaks of cyclic nucleotide-gated channel activity. These two types of channel activity differ in guanosine 3',5'-cyclic monophosphate affinity and in the ability to be opened by adenosine 3',5'-cyclic monophosphate. The cyclic nucleotide-gated channel from rod outer segments also forms two peaks of activity when purified in this manner. The presence of two forms of channel activity could have implications for the mechanism of insulin-activated sodium entry.

Animals↗