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P Galletti

Publications and source records attributed to P Galletti.

At least 19 recordsLinked to original sources
PubMed1991

Biochemical rationale for the use of CDPcholine in traumatic brain injury: pharmacokinetics of the orally administered drug.

A pharmacokinetic analysis of CDPcholine has been carried out treating either rats or dogs by oral administration with the double labelled molecule. [methyl-14C,5-3H]CDPcholine represents a useful tool to test the structural integrity of this compound during the transmembrane transport and to follow the metabolic fate of cytidine and choline fragments. Furthermore, the identification of the labelled metabolites of the exogenously administered CDPcholine in the various organs allows us to draw inferences about its pharmacological mechanism(s). These studies appear of great interest in view of the extensive therapeutic use of the molecule in the treatment of several CNS pathologies including traumatic brain injury. The results of this work can be summarized as follows. (a) The molecule is rapidly cleaved at the level of the pyrophosphate bridge and a fast uptake of the hydrolytic products occurs. (b) The metabolism of the molecule is characterized by a differential utilization of the two moieties by the various organs. Liver is the most active organ in utilizing CDPcholine with a preferential uptake of the choline fragment. (c) The [3H]cytidine moiety, in all the organs examined, appears to be incorporated into the nucleic acid fraction via the cytidine nucleotide pool. The [14C]choline moiety is in part converted into betaine, which in turn acts as methyl donor to homocysteine, yielding [14C]methionine, subsequently incorporated into proteins. The time-dependent increase in the labelling of phospholipids is indicative of a recycling of choline methyl groups via CDPcholine and/or S-adenosylmethionine. (d) The uptake of CDPcholine by the brain is relatively low; however, a good metabolic utilization of the drug can be observed.(ABSTRACT TRUNCATED AT 250 WORDS)

Administration, Oral↗
PubMed1990

Age-related antihypertensive and haemodynamic effects of verapamil SR: clinical results and effects on atrial natriuretic peptide.

The antihypertensive effect of the calcium-antagonist verapamil was investigated in two groups of patients, adult hypertensives (AH, less than 65 years of age) and elderly hypertensives (EH, greater than 65 years of age), who were treated with 240 mg p.o. sustained-release (SR) verapamil for 4 months. Arterial blood pressure was significantly reduced in both groups: the responders' rate was 65% in the AH group and 82% in the EG group. The heart rate was slightly but not significantly reduced. An improvement in cardiac haemodynamics was observed [cardiac index (CI), from 3.00 +/- 0.51 to 3.25 +/- 0.83 ml min-1 m-2 in AH and from 2.35 +/- 1.08 to 3.04 +/- 0.86 ml min-1 m-2 in EG]. We also evaluated the plasma concentrations of atrial natriuretic peptide (ANP) before and after treatment; ANP levels increased significantly only in the EH group. No serious side effects occurred. In conclusion, verapamil SR provided effective and well-tolerated antihypertensive treatment in both adult and elderly patients.

Age Factors↗
PubMed1989

Enzymatic methyl esterification of a deamidated form of mouse epidermal growth factor.

The enzyme S-adenosylmethionine:protein carboxyl-O-methyl-transferase, type II (EC 2.1.1.77; PCMT) from eukaryotes methyl esterifies peptides containing isoAsp residues, which can arise from spontaneous deamidation of labile Asn residues. We report here a study on in vitro methyl esterification of mouse EGF by bovine brain PCMT. This peptide contains two Asn in the sequences Asn1-Ser2 and Asn16-Gly17. It is known from the literature that the presence of a small residue on the carboxyl side of asparaginyl makes this residue susceptible to deamidation through the spontaneous formation of a succinimide intermediate. Therefore EGF was incubated under deamidating conditions (pH 9.0, 37 degrees for 48 h) and the extent of deamidation monitored by enzymatically measuring the NH3 produced during the alkali treatment: a release of 0.80 mol NH3/mol EGF was calculated. The alkali-treated EGF, analyzed by anion-exchange chromatography, shows two major components identified as native EGF (nEGF) and its deamidated form (dEGF). When incubated in the presence of purified PCMT neither nEGF nor dEGF showed any methyl accepting capability. Since it is known that the three-dimensional structure of a protein may hinder the methyl esterification of a potential ethyl accepting site, dEGF was unfolded by reducing and alkylating the intrachain disulfide bridges. Only a slight increase in the methyl accepting capability could be observed. Conversely, when EGF was deamidated after its unfolding, the resulting protein was stoichiometrically methylated by PCMT, presumably at level of isoAsp16. Our findings strongly suggest that the three-dimensional structure of a protein is a major specificity determinant for both deamidation and methyl esterification processes.

Alkylation↗
PubMed1988

Enzymatic methyl esterification of synthetic tripeptides: structural requirements of the peptide substrate. Detection of the reaction products by fast-atom-bombardment mass spectrometry.

Eukaryotic protein carboxyl methyltransferase catalyzes a two-substrates reaction in which the methyl group of S-adenosylmethionine is transferred to the free carboxyl group of D-aspartyl and L-isoaspartyl-containing peptide or protein substrates. It has been previously shown that at least three binding sites are required for the interaction of adenosylmethionine with the enzyme and/or the protein substrate [Oliva A., Galletti P., Zappia V., Paik W. K. & Kim S. (1980) Eur. J. Biochem. 104, 595-602], while very little is known concerning the structural requirements of the protein substrate. In this study several synthetic tripeptides were selected in order to elucidate the structural requirements of the methyl-accepting substrates. The results obtained with this series of peptides suggested that: (1) three residues appear to be the minimal length, so far identified, required for a productive enzyme-substrate interaction, several dipeptides being ineffective as substrates [McFadden P. N. & Clarke S. (1986) J. Biol. Chem. 261, 11,503-11,511]; (2) the isoaspartyl residue is not recognized unless its alpha-amino group is involved in a carboamide bond; (3) an hydrogen atom on the amide linkage following the isoaspartyl residue is essential for both recognition and catalysis; (4) oligopeptides containing both D-aspartyl and D-isoaspartyl residues are not recognized by this methyltransferase. On the basis of these results, interaction sites between the peptide substrate and the enzyme molecule have been proposed. This paper also reports the first application of fast-atom-bombardment mass spectrometry to the detection of the products of the enzymatic methyl esterification reaction. By this soft ionization technique, the methyl-esterified peptides as well as the corresponding cyclic imides generated during the spontaneous demethylation process have been identified.

Catalysis↗
PubMed1988

Repair of isopeptide bonds by protein carboxyl O-methyltransferase: seminal ribonuclease as a model system.

Previous work has shown that in the peptide segment 62-76 of naturally deamidated alpha subunit of bovine seminal ribonuclease (BS-RNase) the alpha-carboxyl group of iso-Asp67 is selectively methylated by S-adenosylmethionine:protein carboxyl O-methyltransferase [Di Donato, A., Galletti, P., & D'Alessio, G. (1986) Biochemistry 25, 8361-8368]. In the present study this reaction has been characterized, by using the tryptic segment 62-76 of the protein chain (peptide alpha 16). The peptide is stoichiometrically methyl esterified with a Km of 6.17 microM and a Vmax of 19.56 nmol min-1 mg-1, and the product of demethylation has been identified as the cyclic succinimidyl derivative of iso-Asp67-Gly68. The cleavage of the succinimidyl ring yields two isomeric peptides containing an aspartyl residue (peptide alpha 17) and an isoaspartyl residue (peptide alpha 16). On the basis of these results conditions were defined in which repeated cycles of methylation-demethylation led to an effective conversion of peptide alpha 16 into peptide alpha 17, a process that can be interpreted as the repair of an altered isopeptide bond. When the methyl esterification reaction was studied on the native dimeric isoenzymes of seminal RNase and on catalytically active monomeric derivatives, including a stabilized alpha-type subunit, the results of these experiments showed that none of the protein forms were substrates for the methyltransferase. Only the unfolded alpha-type subunit was methylated to a stoichiometric extent. These results indicate that the repair of altered isopeptide bonds is chemically feasible in peptides but is hindered in the case of seminal RNase by its three-dimensional structure.

Amino Acid Sequence↗
PubMed1988

Effects of chronic antihypertensive treatment with ketanserin versus metoprolol on blood pressure and large arteries' compliance in humans: a cross-over double-blind study.

The antihypertensive efficacy of a serotonin-receptor antagonist, ketanserin, was compared with that of a well-established antihypertensive drug, metoprolol, and their cardiac and forearm hemodynamic effects were investigated using echocardiography and bidimensional pulsed Doppler flowmetry, respectively. Twenty hypertensive subjects completed a double-blind, cross-over, randomized study using ketanserin and metoprolol. Two 5-week courses with ketanserin or metoprolol were preceded by a placebo period; the total duration of the study was 15 weeks. Despite a comparable efficacy in reducing systolic and diastolic blood pressure (about 10% of the basal value), the two drugs showed quite different effects on forearm hemodynamics. Ketanserin increased forearm blood flow and induced a significant decrease in forearm vascular resistance (from 141 +/- 16 to 75 +/- 11 mm Hg/mL/sec, P less than .01). Furthermore, this treatment was able to improve brachial artery compliance (from 1.89 +/- .3 to 3.2 +/- .3 cm4/dyne 10(-7), P less than .01). On the contrary, metoprolol did not modify forearm hemodynamics. Both drugs did not significantly modify cardiac performance, as evaluated by left ventricle circumferential fiber shortening. Cardiac output was increased by ketanserin (from 5.9 +/- .3 to 6.6 +/- .5 L/min, P less than .05) and fell during treatment with metoprolol (from 5.9 +/- .4 to 4.9 +/- .3 L/min P less than .01). Thus, the two drugs reduce blood pressure through different hemodynamic mechanisms and the effects of ketanserin on systemic and peripheral circulation seem more favorable.

Adult↗
PubMed1986

Selective deamidation and enzymatic methylation of seminal ribonuclease.

Isoenzymatic forms alpha 2, alpha beta, and beta 2 of bovine seminal ribonuclease are generated by the transformation of beta-type into alpha-type subunit through deamidation of a single amide group [Di Donato, A., & D'Alessio, G. (1981) Biochemistry 20, 7232-7237]. The residue involved in this selective deamidation has been identified as Asn67. Deamidation occurs by formation of a cyclic imide intermediate involving the Gly at position 68. Opening of the cyclic imide may occur on either side of the nitrogen, generating both the normal alpha-aspartyl and an isoaspartyl residue at position 67. The alpha-carboxyl of the isoaspartyl residue is effectively methylated by bovine brain protein carboxylmethyltransferase.

Amino Acid Sequence↗
PubMed1986

Enzymatic basis for the calcium-induced decrease of membrane protein methyl esterification in intact erythrocytes. Evidence for an impairment of S-adenosylmethionine synthesis.

The effect of Ca2+ loading, induced by the ionophore A23187, on methyl esterification of membrane proteins (i.e. bands 2.1, 3, 4.1 and 4.5) has been investigated in intact human erythrocytes. When the cells were incubated with L-[methyl-3H]methionine, 40 microM CaCl2 and 10 microM A23187 induce a 50% inhibition of membrane protein methyl esterification. This effect is selectively due to the increased intracellular Ca2+ concentration, as it is antagonized by 10 mM EGTA, and other divalent cations such as Mn2+ do not exert any inhibition. In order to clarify the mechanism(s) of the reported inhibition, the various events involved in the methyl esterification process in vivo were analyzed. L-Methionine uptake as well as protein methylase II activity are not directly affected by altered intracellular Ca2+ concentrations. Conversely in the Ca2+-loaded erythrocytes the conversion of [3H]methionine into [3H]AdoMet, catalyzed by AdoMet synthetase, decreases up to 25%. When the undialyzed erythrocyte cytosolic fraction is assayed in vitro for AdoMet synthetase the activity of the enzyme from the CaCl2/A23187-treated erythrocytes is significantly lower than the control, up to 5 mM ATP. This result suggests that in the Ca2+-loaded erythrocytes the ATP intracellular concentration is significantly lowered. The direct evaluation of ATP intracellular concentration, by HPLC, confirms a significant drop of ATP level, as a consequence of the Ca2+ loading. The removal of Ca2+ from the cells quantitatively restores both the AdoMet synthesis and the methyl esterification levels. The possible role of altered ATP intracellular concentrations as a regulatory factor in the AdoMet-dependent reactions as well as in post-translational protein methylation related to the ageing process is also discussed.

Adenosine Triphosphate↗
PubMed1985

Transport and metabolism of double-labelled CDPcholine in mammalian tissues.

Double-labelled [methyl-14C,5-3H]CDPcholine has been synthesized and subjected to a pharmacokinetic analysis in several biological systems. In transport experiments with intact human erythrocytes no incorporation of radioactivity is observable. On the other hand the results obtained with perfused rat liver suggest a rapid cleavage of the pyrophosphate bridge of the molecule, followed by a rapid uptake of the hydrolytic products. The plasma half-lives of intravenously injected CDPcholine and of its metabolites have been evaluated within 60 sec range. Renal and fecal excretion of the injected radioactivity is negligible: only 2.5% of administered 14C- and 6.5% of the 3H- is excreted up to 48 hr after administration. Liver and kidney are the major CDPcholine metabolizing organs, characterized by a fast and extensive uptake of choline metabolites, followed by a slow release; conversely the rate of uptake of both 3H and 14C-labelled moieties by rat brain is significantly slower, reaching a steady-state level after 10 hr. The characterization of the labelled compounds detectable in the investigated organs provides some insights on the metabolism of the drug: the 3H-cytidine moiety in all the examined organs appears to be incorporated into the nucleic acid fraction via the cytidine nucleotide pool; the [14C]choline moiety of the molecule is in part converted, at the mitochondrial level, into betaine which accounts for about 60% of the total 14C-radioactivity associated with liver and kidney 30 min after administration; [14C]betaine in turn acts as methyl donor to homocysteine yielding [14C]methionine subsequently incorporated into proteins; the time dependent increase in labelled phospholipids is indicative of a recycling of the choline methyl-groups in this lipid fraction via CDPcholine and/or S-adenosylmethionine; the rather extensive amount of labelled methionine detectable in brain probably arises from its uptake from the blood stream, since the enzyme catalyzing the conversion of betaine into methionine is lacking in brain.

Animals↗
PubMed1985

Studies on enzyme-substrate interactions of cholinephosphotransferase from rat liver.

In order to elucidate the reaction mechanism and the substrate-binding sites, CDPcholine:1,2-diacylglycerol cholinephosphotransferase (EC 2.7.8.2), prepared from rat liver microsomal fraction, has been subjected to kinetic analysis and substrate specificity studies. Kinetic evidence supports the hypothesis of a Bi-Bi sequential mechanism, involving a direct nucleophilic attack of diacylglycerol on CDPcholine during the reaction. To investigate the substrate requirements for recognition and catalysis, several CDPcholine analogs, modified in the nitrogen base or in the sugar or in the pyrophosphate bridge, have been synthesized, characterized and assayed as substrates and/or inhibitors of the reaction. The amino group on the pyrimidine ring, the 2'-alcoholic function of the ribose moiety as well as the pyrophosphate bridge have been identified as critical sites for enzyme-substrates interactions.

Animals↗
PubMed1979

Methyl acceptors for protein methylase II from human-erythrocyte membrane.

Membrane proteins from human erythrocytes were methylated with purified protein methylase II (S-adenosylmethionine:protein-carboxyl O-methyltransferase, EC.2.1.1.24). The methylated proteins were analyzed by dodecyl sulfate/polyacrylamide gel electrophoresis. Monomeric and dimeric glycophorin A (NaIO4/Schiff-2 and NaIO4/Schiff-1 positive bands) and 'band 4.5' were identified as two major classes of methyl-acceptor polypeptides for protein methylase II. In rabbit erythrocyte membrane where glycophorin A is absent, 'band 4.5' was the only major methyl-acceptor protein component. Extracted and purified glycophorin A from human erythrocytes was also found to be an excellent substrate for protein methylase II with a Km of 35.7 microM. The role of erythrocyte membrane protein methylation is discussed with regard to membrane function.

Animals↗
PubMed1978

Substrate specificity of 5'-methylthioadenosine phosphorylase from human prostate.

5'-Methylthioadenosine phosphorylase was purified approx. 340-fold from human prostate by using affinity chromatography by Hg-coupled Sepharose. The enzyme, responsible for the breakdown of 5'-methylthioadenosine into adenine and methylthioribose 1-phosphate, was partially characterized. The apparent Km for 5'-methylthioadenosine is 25 microM. It is activated by thiols and shows an absolute requirement for phosphate ions. New analogues of 5'-methylthioadenosine were prepared and their activity as substrates or inhibitors of the reaction was investigated. The replacement of the 6-amino group of the adenine moiety by a hydroxy group, as well as the replacement of N-7 by a methinic radical, resulted in an almost complete loss of activity. Otherwise the replacement of sulphur by selenium, as well as that of the methyl group by an ethyl one, is compatible with the activity as substrate. The positively charged sulphonium group also prevents catalytic interaction with the enzyme. The inhibitory effect of 5'-methylthiotubercidin (competitive) and 5'-dimethylthioadenosine sulphonium salt (non-competitive) was also demonstrated. The reported results suggest three binding sites between the substrate and the enzyme.

Adenosine↗
PubMed1978

[5'-methylthioadenosine phosphorylase from the human prostate. 1. Purification and partial characterization].

5'-Methylthioadenosine phosphorylase has been purified approximately 340-fold in 20% yield from human prostate: the use of affinity chromatography by Sepharose-Hg has been found particularly advantageous. The enzyme has been partially characterized and an apparent Km of 2.5 x 10(-5) M has been calculated for 5'-methylthioadenosine. The reaction is activated by thiols and shows an absolute requirement for phosphate ions.

Adenosine↗
PubMed1978

Selective methyl esterification of erythrocyte membrane proteins by protein methylase II.

Methyl esterification of erythrocyte membrane proteins have been demonstrated by incubating the isolated membrane with purified protein methylase II (S-adenosyl-methionine:protein-carboxyl O-methyltransferase, EC 2.1.1.24) and S-adenosyl-L-[methyl-14C]methionine. Methyl esterification of membrane-bound proteins occurred selectively to proteins corresponding to bands 3 (mol wt 97 000), 4 (mol wt 75 000), and 4.5 (mol wt 48 000) [designated according to Steck, T. L. (1974), J. Cell Biol. 62, 1] as identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Mild alkali treated depleted vesicles which lacked bands 1, 2, 5, and 6 had a higher methyl accepting capacity; 500 pmol of methyl groups/mg of depleted vesicle proteins vs. 200 pmol of methyl groups/mg of intact membrane proteins. Alkali-extractable membrane components were not methylated.

Animals↗