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Biomedical subjects

M R Mazzoni

Publications and source records attributed to M R Mazzoni.

At least 37 records · Page 2Linked to original sources
PubMed1996

Interaction of transducin with light-activated rhodopsin protects It from proteolytic digestion by trypsin.

The tryptic cleavage pattern of transducin (Gt) in solution was compared with that in the presence of phospholipid vesicles, rod outer segment (ROS) membranes kept in the dark, or ROS membranes containing light-activated rhodopsin, metarhodopsin II (Rh*). When Gt was in the high affinity complex with Rh*, the alphat subunit was almost completely protected from proteolysis. The protection of alphat at Arg310 was complete, while Arg204 was substantially protected. The cleavage of alphat at Lys18 was protected in the presence of phospholipid vesicles, ROS membranes kept in the dark, or ROS membranes containing Rh*. The cleavage of betat was slower in the presence of ROS membranes or phospholipid vesicles. When the Rh*. Gt complex was incubated with guanyl-5'-yl thiophosphate, a guanine nucleotide analog known to release the high affinity interaction between Gt and Rh*, the protection at Arg310 and Arg204 was diminished. From our results, we propose that Rh* either physically blocks access of trypsin to Arg204 and Arg310 or maintains the heterotrimer in such a conformation that these cleavage sites are not available. Since Arg204 is involved in the switch interface with betagammat (Lambright, D. G., Sondek, J., Bohm, A., Skiba, N. P., Hamm, H. E., and Sigler, P. B. (1996) Nature 379, 311-319), it may be that betagammat is implicated in protecting this cleavage site in the receptor-bound, stabilized heterotrimer. Arg310 is not near the betagammat subunit, thus we believe that the high affinity binding of Gt to Rh* physically or sterically blocks access of trypsin to this site. Thus, Arg310, only a few angstroms away from the carboxyl terminus of alphat, which is known to directly bind to Rh*, is likely to also be a part of the Rh* binding site. This is in agreement with other studies and has implications for the mechanism by which receptors catalyze GDP release from G proteins. The protection of Lys18 in the presence of phospholipid vesicles suggests that the amino-terminal region is in contact with the membrane, consistent with the crystal structure of the heterotrimer (Lambright, D. G., Sondek, J., Bohm, A., Skiba, N. P., Hamm, H. E., and Sigler, P. B. (1996) Nature 379, 311-319).

Amino Acid Sequence↗
PubMed1993

Regulation of agonist binding to A2A adenosine receptors: effects of guanine nucleotides (GDP[S] and GTP[S]) and Mg2+ ion.

Adenosine acts as a neuromodulator through at least two receptor subtypes, A1 and A2. A2 receptors have been further divided into A2A (high agonist affinity) and A2B (low agonist affinity) receptors. Both A1 and A2 receptors belong to the superfamily of guanine nucleotide-binding regulatory protein (G protein)-coupled receptors. A Gs protein couples the A2A receptor to the activation of adenylyl cyclase. In order to elucidate the mechanism of coupling between the A2A receptor and Gs, we studied the modulation by guanine nucleotides and divalent cations of agonist binding to the A2A receptor in rat striatal membranes, using [3H]CGS 21680 as a selective high-affinity agonist. We demonstrated that in rat striatal membranes agonist binding to A2A receptors was modulated by guanine nucleotides. Both GDP and GTP inhibited [3H]CGS 21680 binding to rat striatal membranes with about equal potency. The nonhydrolyzable analogs, GDP[S] and GTP[S], were equipotent inhibitors and approx. 100-times more potent than GDP and GTP. Data from competition studies with labeled and unlabeled CGS 21680 when analyzed by nonlinear regression demonstrated the presence of two binding sites in rat striatal membranes with mean values for KD of 5.6 and 343 nM and Bmax of 200 and 942 fmol/mg protein. The high-affinity binding site has the characteristics of the A2A receptor. In the presence both of (0.1 mM) GDP[S] and GTP[S], the KD values for the high-affinity site were increased severalfold, whereas the low-affinity site was no longer detected in filtration assays. Dissociation studies revealed monophasic dissociation curves both in the absence and presence of 0.1 mM GDP[S]. However the K-1 value increased in the presence of guanine nucleotide. We also showed that in bovine striatal membranes agonist binding to A2A receptors was modestly modulated by guanine nucleotides, suggesting differences of receptor Gs-protein-coupling a mechanism in different species. Divalent cations often increase agonist binding to different receptors, whereas Mg2+ ions play a role in regulating the initial steps of G-protein activation. We investigated the effects of divalent cations on [3H]CGS 21680 binding to the A2A receptor and determined the requirement of these cations to obtain the modulation of binding by guanine nucleotides. We found that millimolar concentrations of divalent cations were required to obtain an effective interaction between the A2A receptor and Gs. The high-affinity binding of [3H]CGS 21680 to the A2A receptor in rat striatal membranes was dependent on the presence of Mg2+ ions.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1993

Tryptophan207 is involved in the GTP-dependent conformational switch in the alpha subunit of the G protein transducin: chymotryptic digestion patterns of the GTP gamma S and GDP-bound forms.

The limited proteolytic pattern of transducin, Gt, and its purified subunits with chymotrypsin were analyzed and the cleavage sites on the alpha t subunit were identified. The alpha t subunit in the GTP gamma S bound form was cleaved into a major 38 kD fragment, whereas alpha t-GDP was progressively digested into 38, 23, 21, and 15 kD fragments. The beta gamma t subunit was not very sensitive to proteolytic digestion with chymotrypsin. The gamma t subunit was not cleaved and only a small portion of beta t was digested into several fragments. In order to determine which proteolytic fragment of alpha t still contained the carboxyl terminal region, chymotrypsinization was carried out using Gt previously 32P-labeled at Cys347 by pertussis toxin-catalyzed ADP-ribosylation. The 32P-label was mainly associated with the alpha t subunit and a 15 kD fragment. The 23 and 21 kD fragments were not 32P-labeled. Analysis of amino terminal sequences of 38, 21, and 15 kD proteolytic bands allowed the identification of the major cleavage sites. Chymotrypsin had two cleavage sites in the amino terminal region of alpha t, at Leu15 and Leu19. Chymotrypsin removed 15-19 amino acid residues from the amino terminus of alpha t, generating two peptides (38 kD) which comigrates in gel electrophoresis. Chymotrypsin also cleaved at Trp207 in a conformation-dependent manner. Trp207 of alpha t-GTP gamma S was resistant to proteolysis but alpha t-GDP and the 38 kD fragments of alpha t-GDP produced the 23 and 21 kD fragments, respectively, and a 15 kD fragment containing the carboxyl terminus.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1991

Structural analysis of rod GTP-binding protein, Gt. Limited proteolytic digestion pattern of Gt with four proteases defines monoclonal antibody epitope.

The epitope of monoclonal antibody (mAb 4A), which recognizes the alpha subunit of the rod G protein, Gt, has been suggested to be both at the carboxyl terminus (Deretic, D., and Hamm, H.E. (1987) J. Biol. Chem. 262, 10839-10847) and the amino terminus (Navon, S.E., and Fung, B.K.-K. (1988) J. Biol. Chem. 263, 489-496) of the molecule. To characterize further the mAb 4A binding site on alpha t and to resolve the discrepancy between these results limited proteolytic digestion of Gt or alpha t using four proteases with different substrate specificities has been performed. Endoproteinase Arg-C, which cleaves the peptide bond at the carboxylic side of arginine residues, cleaved the majority of alpha t into two fragments of 34 and 5 kDa. The alpha t 34-kDa fragment in the holoprotein, but not alpha t-guanosine 5'-O-(3-thiotriphosphate), was converted further to a 23-kDa fragment. A small fraction of alpha t-GDP was cleaved into 23- and 15-kDa fragments. Endoproteinase Lys-C, which selectively cleaves at lysine residues, progressively removed 17 and then 8 residues from the amino terminus, forming 38- and 36-kDa fragments. Staphylococcus aureus V8 protease is known to remove 21 amino acid residues from the amino-terminal region of alpha t, with the formation of a 38-kDa fragment. L-1-Tosylamido-2-phenylethyl chloromethyl ketone-treated trypsin cleaved alpha t progressively into fragments of known amino acid sequences (38, then 32 and 5, then 21 and 12 kDa) and a transient 34 kDa fragment. The binding of mAb 4A to proteolytic fragments was analyzed by Western blot and immunoprecipitation. The major fragments recognized by mAb 4A on Western blots were the 34- and 23-kDa fragments obtained by endoproteinase Arg-C and tryptic digestion. Under conditions that allowed sequencing of the 15- and 5-kDa fragments neither the 34- nor the 23-kDa fragments could be sequenced by Edman degradation, indicating that they contained a blocked amino terminus. The smallest fragment that retained mAb 4A binding was the 23-kDa fragment containing Met1 to Arg204. Thus the main portion of the mAb 4A antigenic site was located within this fragment, indicating that the carboxyl-terminal residues from Lys205 to Phe350 were not required for recognition by the antibody. Additionally, the antibody did not bind the 38- and 36-kDa or other fragments containing the carboxyl terminus, showing that the amino-terminal residues from Met1 to Lys17 were essential for antibody binding to alpha t.

Amino Acid Sequence↗
PubMed1990

Superoxide dismutase-like activity of 1,2,3-triazole derivatives.

This paper reports the synthesis of some sulfurated 1,2,3-triazole derivatives and the biological evaluation, for the superoxide dismutase-like activity, of these new compounds together with several 1,2,3-triazoles previously described by us. All the compounds possess a SOD-like activity and some of them showed an activity equivalent to or higher than those of penicillamine or thiopronin, which are well known therapeutic drugs in the treatment of rheumatoid arthritis.

Anti-Inflammatory Agents, Non-Steroidal↗
PubMed1989

Effect of monoclonal antibody binding on alpha-beta gamma subunit interactions in the rod outer segment G protein, Gt.

The guanyl nucleotide binding regulatory protein of retinal rod outer segments, called Gt, that couples the photon receptor rhodopsin with the light-activated cGMP phosphodiesterase, can be resolved into two functional components, alpha t and beta gamma t. The effect of monoclonal antibody binding to the alpha t subunit of Gt on subunit association has been investigated in the present study. It was previously shown that this monoclonal antibody, mAb 4A, blocks interactions with rhodopsin and its epitope was located within the region Arg310-Phe350 at the COOH terminus of the alpha t subunit. In this paper, we show that mAb 4A disrupts the Gt complex. Gt migrates in 5-20% linear sucrose density gradients as a monomer, with a sedimentation coefficient of 4.1 +/- 0.07 S, while in the presence of mAb 4A, the alpha t and beta gamma t subunits show sedimentation coefficients of 7.7 +/- 0.2 and 3.7 +/- 0.1 S, respectively. The beta gamma t subunit migrates with the same sedimentation rate as pure beta gamma t. Nonimmune rabbit IgG does not modify the sedimentation behavior of Gt. The Fab fragment of mAb 4A also dissociates the Gt complex, as suggested by the change of the sedimentation rate of alpha t. This effect of mAb 4A on Gt subunit association was also confirmed by immunoprecipitation studies in the presence of detergent. In the presence of detergent, subunit association is not affected, but the formation of Gt oligomers and, therefore, the nonspecific precipitation of beta gamma t subunit are reduced.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Diphosphate↗
PubMed1989

A monoclonal antibody against the rod outer segment guanyl nucleotide-binding protein, transducin, blocks the stimulatory and inhibitory G proteins of adenylate cyclase.

GTP-binding proteins have been implicated as transducers of a variety of biological signaling processes. These proteins share considerable structural as well as functional homology. Due to these similarities, it was thought that a monoclonal antibody that inhibits the light activation of the rod outer segment GTP-binding protein, tranducin (Gt), might exert some functional effect upon the G proteins that regulate the adenylate cyclase system. Antibody 4A, raised against the alpha subunit of Gt, cross-reacted (by hybridization on nitrocellulose) with purified alpha subunits of other G proteins (Gi and Gs, regulatory guanyl nucleotide-binding proteins that mediate inhibition and stimulation of adenylate cyclase, respectively) as long as they were not denatured. This antibody, which interferes with rod outer segment cGMP phosphodiesterase activation by blocking interaction between rhodopsin and Gt, also interfered with actions of both the stimulatory and inhibitory G proteins of adenylate cyclase from rat cerebral cortex membranes. Effects of monoclonal antibody (mAb) 4A were dose-dependent and not reversed by washing. mAb 4A also blocked the Gi-mediated inhibition of adenylate cyclase in the cyc- variant of S49 lymphoma and in doing so raised the level of adenylate cyclase activity in both the cyc- variant and the S49 wild type. There was no effect of mAb 4A on adenylate cyclase activity of the resolved catalytic subunit. These results suggest that the well known sequence homologies among the G proteins involved in cellular signal transduction may extend to the sites that interact with other members of signal-transducing cascades (receptors and effector molecules). Therefore, antibody 4A may serve as a useful tool to probe the similarities and differences among the various systems.

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMed1988

A monoclonal antibody to the alpha subunit of Gk blocks muscarinic activation of atrial K+ channels.

The activated heterotrimeric guanine nucleotide binding (G) protein Gk, at subpicomolar concentrations, mimics muscarinic stimulation of a specific atrial potassium current. Reconstitution studies have implicated the alpha and beta gamma subunits as mediators, but subunit coupling by the endogenous G protein has not been analyzed. To study this process, a monoclonal antibody (4A) that binds to alpha k but not to beta gamma was applied to the solution bathing an inside-out patch of atrial membrane; the antibody blocked carbachol-activated currents irreversibly. The state of the endogenous Gk determined its susceptibility to block by the antibody. When agonist was absent or when activation by muscarinic stimulation was interrupted by withdrawal of guanosine triphosphate (GTP) in the presence or absence of guanosine diphosphate (GDP), the effects of the antibody did not persist. Thus, monoclonal antibody 4A blocked muscarinic activation of potassium channels by binding to the activated G protein in its holomeric form or by binding to the dissociated alpha subunit.

Acetylcholine↗
PubMed1987

Evidence for the existence of a specific binding site for indomethacin on bovine vesicular gland microsomes.

Using bovine vesicular gland microsomes and [14C]indomethacin we demonstrated the presence of a specific binding site for nonsteroidal anti-inflammatory drugs. Specific binding of [14C]indomethacin to microsomes was rapid, with most of the ligand bound by 2 min at 4 degrees C. In routine binding assays the incubation temperature was maintained at 4 degrees C, because the maximal specific binding was obtained. Specific [14C]indomethacin binding appeared to increase linearly with increasing protein concentration over the range of 0.1-1.0 mg of microsomal protein. Specific binding was saturable and Scatchard analysis of binding data showed a single class of binding sites with a dissociation constant (Kd) of 3.8 microM and a maximal number of binding sites (Bmax) of about 1272 pmol/mg of protein. When these binding data were plotted according to the Hill equation, a straight line was obtained with a Hill coefficient of 1.0. Structural specificity of the nonsteroidal anti-inflammatory drug site was studied with diclofenac, arylpropionic acids (ketoprofen and indoprofen), and aspirin. Diclofenac and arylpropionic derivatives were able to compete with [14C]indomethacin for binding to microsomes, while aspirin was a weak inhibitor.

Animals↗
PubMed1986

N-benzyl-4-phenyl-1,2,3-triazole derivatives as in vitro inhibitors of the prostaglandin synthesis.

Numerous N-benzyl-4-phenyl-1,2,3-triazole derivatives were synthesized and tested. The compounds were prepared by nucleophilic substitution, 1,3-dipolar cycloaddition and usual functional group conversion reactions. Several derivatives were evaluated in vitro for their ability to inhibit prostaglandin synthesis and to displace labelled [14C] indomethacin from bovine vesicular gland microsomes. Some compounds showed biological activity.

Animals↗
PubMed1985

4-(4-Phenylsubstituted)-1,2,3-triazolacetic acid derivatives in vitro inhibitors of prostaglandin synthesis.

New 4-(4-phenylsubstituted)-1,2,3-triazolacetic acid derivatives of general formula (I) were prepared by nucleophilic substitution, 1,3-dipolar cycloaddition and functional group interconversion reactions. These compounds were evaluated as in vitro prostaglandin synthesis inhibitors. Only the isomeric compounds (II c) and (III e), with a 4-aminophenyl substituent on the triazole ring, inhibit arachidonic acid-induced malondialdehyde formation in human platelets; (II c) and (III e) are as effective as aspirin.

Anti-Inflammatory Agents↗
PubMed1984

Uric acid metabolism in two patients with coexistent Down's syndrome and gout.

Two patients with coexistent Down's syndrome and gout are described. Although increased serum urate levels are frequently reported in Down's syndrome, only a few such patients have been described with concomitant gout. In our patients no significant alterations of the purine salvage pathway were found, and the turnover parameters of uric acid, determined by means of a 14C-labeled uric acid study, were consistent with the metabolic findings observed in normoexcretor gouty patients.

Adult↗
PubMed1982

Controlled multicenter trial of tiopronin and d-penicillamine for rheumatoid arthritis.

Fifty-seven patients took part in a controlled double-blind trial between tiopronin and D-penicillamine as basic treatment for rheumatoid arthritis. Thirty-nine (19 receiving tiopronin, 20 receiving D-penicillamine) completed the trial after 1 year. Both drugs resulted in a decrease of the erythrocyte sedimentation rate, Ritchie index, and Lee index and in a sparing effect on symptomatic antiinflammatory therapy. Improvement in these variables was statistically highly significant at any interval with tiopronin, but was sometimes less or not at all significant with D-penicillamine. Nevertheless, the difference in effects between the 2 drugs never reached statistical significance. Six patients receiving tiopronin and 6 receiving D-penicillamine were taken out of the experiment because of side effects.

Aged↗
PubMed1980

[Tiopronine: new basic treatment for rheumatoid arthritis. Open study of 158 cases].

A study of patients with rheumatoid arthritis permitted us to note the interest of tiopronine (sulfhydryl compound of the d-penicillamine type) in the basic treatment of rheumatoid arthritis. The dose of the drug was on average, 1 gram daily, which, in one series of patients, was administered at the start of treatment and in another series, with an increase of dosage of 250 mg every 20 days. The results, judged by the reduction of the Ritchie and Lee index, of the E.R.S. and the steroid and non-steroid anti-inflammatory requirements, are good (very good and good) in 47% of cases. These patients were in an advanced stage of the disease and there had been numerous therapeutic failures. Side effects similar to those observed during treatment with D-penicillamine were present in 38% of cases, and in 31% required stopping treatment. It is likely that the frequency of stopping treatment for side effects was definitely exaggerated. The place of this new drug in the basic treatment of rheumatoid arthritis requires further study by a double blind test with D-penicillamine, and this is at present in progress.

Adult↗