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Biomedical subjects

J P Rossi

Publications and source records attributed to J P Rossi.

At least 19 recordsLinked to original sources
PubMed2001

Quantitation of plasma membrane calcium pump phosphorylated intermediates by electrophoresis.

P-ATPases are characterized by the formation of acid-stable phosphorylated intermediates (EP) during their reaction cycle. We have developed a microscale method to determine EP that involves the phosphorylation of the enzyme using [gamma-(32)P]ATP and precipitation with TCA; separation of the sample by SDS-PAGE, and measurement of the enzyme protein and (32)P-labeled EP by digital analysis of both the stained gel and its autoradiogram, respectively. The principal advantages of this method over typical procedures (filtration and centrifugation) are the low amount of enzyme required and the substantial decrease in the blank values and data scattering produced by unspecific phosphorylation and nonquantitative recovering of the enzyme. Application of this new method to a purified preparation of the plasma membrane calcium ATPase (PMCA) results in overcoming the difficulties of measuring EP at high ATP concentrations. A biphasic behavior of the substrate curve for EP was observed when the study was extended to ATP levels within the physiological range. Since, in principle, the method does not require the use of highly purified preparations, it could be helpful for the study of phosphorylated intermediates especially under conditions in which small amounts of protein are available, e.g., mutated variants of P-ATPases.

Adenosine Triphosphate↗
PubMed2001

Synthesis and human leukocyte elastase inhibitory evaluation of phosphate triesters and acyl phosphates of penam sulfides and sulfones.

The synthesis of 6,6-dibromo-3alpha-(diphenylphosphate)oxymethyl-2,2-dimethyl penam sulfone (3a), 6alpha-chloro-3alpha-(diphenylphosphate)oxymethyl-2,2-dimethyl penam sulfone (3b), benzyl 6alpha-(diphenyl-phosphate)oxypenicillanate sulfone (4) and 6,6-dibromo-3alpha-(methylphosphate)carbonyl-2,2-dimethylpenam sulfone (12) are reported. When tested as inhibitors of human leukocyte elastase, the compound 4 proved to be the most active.

Esters↗
PubMed2000

Oligomerization of the plasma membrane calcium pump involves two regions with different thermal stability.

Ca(2+) pump dimerization was studied by using a combined approach of thermal denaturation and fluorescence resonance energy transfer. The measurement of calcium pump ability to dimerize after the unfolding of individual functional domains of the enzyme demonstrated the existence of two different regions involved in the self-association process. One of these regions is highly susceptible to thermal unfolding and was identified as the calmodulin (CaM)-binding domain. The other region whose thermal stability is higher than those of the catalytic and CaM-binding domains could be related with the previously found C28W-binding regions.

Binding Sites↗
PubMed2000

Thermal stability of the plasma membrane calcium pump. Quantitative analysis of its dependence on lipid-protein interactions.

Thermal stability of plasma membrane Ca(2+) pump was systematically studied in three micellar systems of different composition, and related with the interactions amphiphile-protein measured by fluorescence resonance energy transfer. Thermal denaturation was characterized as an irreversible process that is well described by a first order kinetic with an activation energy of 222 +/- 12 kJ/mol in the range 33-45 degrees C. Upon increasing the mole fraction of phospholipid in the mixed micelles where the Ca(2+) pump was reconstituted, the kinetic coefficient for the inactivation process diminished until it reached a constant value, different for each phospholipid species. We propose a model in which thermal stability of the pump depends on the composition of the amphiphile monolayer directly in contact with the transmembrane protein surface. Application of this model shows that the maximal pump stability is attained when 80% of this surface is covered by phospholipids. This analysis provides an indirect measure of the relative affinity phospholipid/detergent for the hydrophobic transmembrane surface of the protein (K(LD)) showing that those phospholipids with higher affinity provide greater stability to the Ca(2+) pump. We developed a method for directly measure K(LD) by using fluorescence resonance energy transfer from the membrane protein tryptophan residues to a pyrene-labeled phospholipid. K(LD) values obtained by this procedure agree with those obtained from the model, providing a strong evidence to support its validity.

Calcium-Transporting ATPases↗
PubMed2000

Export and folding of signal-sequenceless Bacillus licheniformis beta-lactamase in Escherichia coli.

Two genetically engineered variants of the Bacillus licheniformis beta-lactamase gene were expressed in Escherichia coli. One variant coded for the exo-small mature enzyme without the signal peptide. The other coded for the exo-large mature enzyme preceded by 10, mostly polar, residues from an incomplete heterologous signal. As observed following the extraction by a lysozyme-EDTA treatment, the signal-less variant was exported to the periplasm with nearly 20% efficiency, whereas the variant with the N-terminal extension was translocated to a lesser degree; interestingly, nearly all of the former and half of the latter were extracted by osmotic shock, which may be of importance for our understanding of cellular compartments. The fact that a signal-less protein is translocated with substantial yields raises questions about the essential role of signal peptides for protein export. As folding and export are related processes, we investigated the folding in vitro of the two variants. No differences were found between them. In the absence of denaturant, they are completely folded, fully active and have a large DeltaG of unfolding. Under partially denaturing conditions they populate several partially folded states. The absence of significant amounts of a non-native state under native conditions makes a thermodynamic partitioning between folding and export less likely. In addition, kinetic measurements indicated that these B. licheniformis lactamases fold much faster than E. coli beta-lactamase. This behavior suggests that they are exported by a kinetically controlled process, mediated by one or more still unidentified interactions that slow folding and allow a folding intermediate to enter the export pathway.

Amino Acid Sequence↗
PubMed1999

Molecular characterization of the glycated plasma membrane calcium pump.

We have previously demonstrated (Diabetes 39:707-711, 1990) that in vitro glycation of the red cell Ca(2+) pump diminishes the Ca(2+)-ATPase activity of the enzyme up to 50%. Such effect is due to the reaction of glucose with lysine residues of the Ca(2+) pump (Biochem. J. 293:369-375, 1993). The aim of this work was to determine whether the effect of glucose is due to a full inactivation of a fraction of the total population of Ca(2+) pump, or to a partial inactivation of all the molecules. Glycation decreased the V(max) for the ATPase activity leaving unaffected the apparent affinities for Ca(2+), calmodulin or ATP. The apparent turnover was identical in both, the glycated and the native enzyme. Glycation decreased the V(max) for the ATP-dependent but not for the calmodulin-activated phosphatase activities. Concomitantly with the inhibition, up to 6.5% of the lysine residues were randomly glycated. The probabilistic analysis of the relation between the enzyme activity and the fraction of nonmodified residues indicates that only one Lys residue is responsible for the inhibition. We suggest that glucose decreases the Ca(2+)-ATPase activity by reacting with one essential Lys residue probably located in the vicinity of the catalytic site, which results in the full inactivation of the enzyme. Thus, Ca(2+)-ATPase activity measured in erythrocyte membranes or purified enzyme preparations preincubated with glucose depends on the remaining enzyme molecules in which the essential Lys residue stays unglycated.

Binding Sites↗
PubMed1999

Identification of Bordetella pertussis virulence-associated outer membrane proteins.

Bordetella pertussis virulence-associated 30-, 32-, 90- and 95-kDa outer membrane proteins were purified and their N-terminal amino acid sequences were determined. The 30- and 32-kDa outer membrane proteins showed identity to the C-terminal region of the precursors of the serum resistance protein (BrkA) and the tracheal colonization factor, respectively. We confirmed the cleavage site of these precursors after N731 for BrkA and after N393 for tracheal colonization factor. Associated with the 32-kDa outer membrane protein, we found a new group of 36-kDa virulence-associated peptides. The 95-kDa outer membrane protein showed identity to Vag8. The 90-kDa outer membrane protein did not show homology with the described proteins. We report the N-termini sequence of Vir-90, a novel potential virulence factor.

Amino Acid Sequence↗
PubMed1998

Temperature-induced conformational transition of intestinal fatty acid binding protein enhancing ligand binding: a functional, spectroscopic, and molecular modeling study.

Intestinal fatty acid binding protein (IFABP) undergoes a reversible thermal transition between 35 and 50 degreesC, as revealed by circular dichroism spectroscopy in the near-UV region. For the apoprotein, the molar ellipticity measured at 254 nm (possibly implicating the environment around F17 and/or F55) decreases significantly in this temperature range, while in the holoprotein (bound to oleic acid), this phenomenon is not observed. Concomitantly, an increase in the activity of binding to [14C]oleic acid occurs. Nevertheless, other spectroscopic evidence indicates that the beta-barrel structure, the major motif of this protein, is highly stable up to 70 degreesC. No changes associated with conformation were detected for both structures by fourth-derivative analysis of the UV absorption spectra, circular dichroism in the far-UV region, and intrinsic fluorescence measurements. Further structural information arises from experiments in which binding to the anionic fluorescent probes 1-anilinonaphthalene-8-sulfonic acid (ANS) and its dimer bisANS was examined. The fluorescence intensity of bound ANS diminishes monotonically, whereas that of bisANS increases slightly in the temperature range of 35-50 degreesC. Given the different size of these probes, model building suggests that ANS would be able to sense regions located deeply inside the cavity, while bisANS could also reach the vicinity of the small helical domain of this protein. In light of these results, we believe that this subtle conformational transition of IFABP, which positively influences the binding activity, would involve fluctuations at the peripheral "entry portal" region for the ligand. This interpretation is compatible with the discrete disorder observed in this place in apo-IFABP, as evidenced by NMR spectroscopy [Hodsdon, M. E., and Cistola, D. P. (1997) Biochemistry 36, 1450-1460].

Animals↗
PubMed1998

Chemical modification reveals involvement of different sites for nucleotide analogues in the phosphatase activity of the red cell calcium pump.

The calcium pump of plasma membranes catalyzes the hydrolysis of ATP and phosphoric esters like p-nitrophenyl phosphate (pNPP). The latter activity requires the presence of ATP and/or calmodulin, and Ca2+ [22, 25]. We have studied the effects of nucleotide-analogues and chemical modifications of nucleotide binding sites on Ca2+-pNPPase activity. Treatment with fluorescein isothiocyanate (FITC), abolished Ca2+-ATPase and ATP-dependent pNPPase, but affected only 45% of the calmodulin-dependent pNPPase activity. The nucleotide analogue eosin-Y had an inhibitory effect on calmodulin-dependent pNPPase (Kieosin-Y = 2 microM). FITC treatment increased Kieosin-Y 15 times. Acetylation of lysine residues with N-hydroxysuccinimidyl acetate inactivates Ca2+-ATPase by modifying the catalytic site, and impairs stimulation by modulators by modifying residues outside this site [9]. Acetylation suppressed the ATP-dependent pNPPase with biphasic kinetics. ATP or pNPP during acetylation cancels the fast component of inactivation. Acetylation inhibited only partially the calmodulin-dependent pNPPase, but neither ATP nor pNPP prevented this inactivation. From these results we conclude: (i) ATP-dependent pNPPase depends on binding of ATP to the catalytic site; (ii) the catalytic site plays no role in calmodulin-dependent pNPPase. The decreased affinity for eosin-Y of the FITC-modified enzyme, suggests that the sites for these two molecules are closely related but not overlapped. Acetimidation of the pump inhibited totally the calmodulin-dependent pNPPase, but only partially the ATP-pNPPase. Since calmodulin binds to E1, the E1 conformation or the E2 if E1 transition would be involved during calmodulin-dependent pNPPase activity.

4-Nitrophenylphosphatase↗
PubMed1998

The pitch of short-duration fundamental frequency glissandos.

Pitch perception for short-duration fundamental frequency (F0) glissandos was studied. In the first part, new measurements using the method of adjustment are reported. Stimuli were F0 glissandos centered at 220 Hz. The parameters under study were: F0 glissando extents (0, 0.8, 1.5, 3, 6, and 12 semitones, i.e., 0, 10.17, 18.74, 38.17, 76.63, and 155.56 Hz), F0 glissando durations (50, 100, 200, and 300 ms), F0 glissando directions (rising or falling), and the extremity of F0 glissandos matched (beginning or end). In the second part, the main results are discussed: (1) perception seems to correspond to an average of the frequencies present in the vicinity of the extremity matched; (2) the higher extremities of the glissando seem more important; (3) adjustments at the end are closer to the extremities than adjustments at the beginning. In the third part, numerical models accounting for the experimental data are proposed: a time-average model and a weighted time-average model. Optimal parameters for these models are derived. The weighted time-average model achieves a 94% accurate prediction rate for the experimental data. The numerical model is successful in predicting the pitch of short-duration F0 glissandos.

Adult↗
PubMed1998

Effect of 2-hydroxyoestradiol on insulin secretion in normal rat pancreatic islets.

The possible action of 2-hydroxyoestradiol (2-OHE2) on glucose-induced insulin secretion was evaluated in pancreatic islets isolated from normal rats by collagenase digestion and incubated in KRB buffer. Insulin output in response to either 3.3 or 16.6 mM glucose was measured by radioimmunoassay in the absence or presence of different concentrations of 2-OHE2, norepinephrine (NE), or oestradiol. Islets were also incubated with 2-OHE2, NE, or oestradiol plus a fixed concentration (1 microM) of the alpha 2-adrenergic-receptor blocking agent yohimbine. The results showed that 2-OHE2, oestradiol and NE within a range of 0.1 to 20 microM inhibited glucose-induced insulin secretion in a dose-dependent manner: Ki (microM): 0.04 +/- 0.0001, 0.04 +/- 0.0002, and 0.01 +/- 9.1 E-6 respectively. This suppression was significantly reversed by yohimbine. Contrary to NE and 2-OHE2, oestradiol at lower concentrations (increasing within a range of 0.001 to 0.05 microM) in incubation medium in the same experimental conditions had a significant stimulatory effect on insulin secretion. Thus, it would appear that catecholoestrogens suppress islet insulin release via alpha 2-adrenergic receptors, which suggests that oestrogens may exert a dual modulatory effect on insulin secretion by enhancing release via direct interaction with the cytosolic-oestrogen receptor and inhibiting release after their local hydroxylation and the interaction of their new catechol moiety with alpha 2-adrenergic receptors. Our results suggest that these compounds may play a complementary role to CAs as negative modulators, and they also provide a broader scope for understanding the effect of oestrogens and/or their metabolites in the control of endocrine functions other than those related to reproduction.

Adrenergic alpha-Antagonists↗
PubMed1997

The membrane topology of the amino-terminal domain of the red cell calcium pump.

A systematic study of the membrane-associated regions in the plasma membrane Ca2+ pump of erythrocytes has been performed by hydrophobic photolabeling. Purified Ca2+ pump was labeled with 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)-diazirine ([125I]TID), a generic photoactivatable hydrophobic probe. These results were compared with the enzyme labeled with a strictly membrane-bound probe, [3H]bis-phosphatidylethanolamine (trifluoromethyl) phenyldiazirine. A significant light-dependent labeling of an M(r) 135,000-140,000 peptide, corresponding to the full Ca2+ pump, was observed with both probes. After proteolysis of the pump labeled with each probe and isolation of fragments by SDS-PAGE, a common pattern of labeled peptides was observed. Similarly, labeling of the Ca2+ pump with [125I]TID, either in isolated red blood cell membranes or after the enzyme was purified, yields a similar pattern of labeled peptides. Taken together, these results validate the use of either probe to study the lipid interface of the membrane-embedded region of this protein, and sustain the notion that the conformation of the pump is maintained throughout the procedures of solubilization, affinity purification, and reconstitution into proteoliposomes. In this work, we put special emphasis on a detailed analysis of the N-terminal domain of the Ca2+ pump. A labeled peptide of M(r) 40,000 belonging to this region was purified and further digested with V8 protease. The specific incorporation of [125I]TID to proteolytic fragments pertaining to the amino-terminal region indicates the existence of two transmembrane stretches in this domain. A theoretical analysis based on the amino acid sequence 1-322 predicts two segments with high probability of membrane insertion, in agreement with the experimental data. Each segment shows a periodicity pattern of hydrophobicity and variability compatible with alpha-helical structure. These results strongly suggest the existence of a transmembrane helical hairpin motif near the N-terminus of the Ca2+ pump.

Calcium-Transporting ATPases↗
PubMed1995

Interaction of unsaturated fatty acids with the red blood cell Ca(2+)-ATPase. Studies with a novel photoactivatable probe.

Unsaturated fatty acids, such as oleic acid, increase both the affinity for Ca2+ and the maximum effect of the Ca(2+)-ATPase of red blood cells [Niggli et al. (1981) J. Biol. Chem. 256, 8588-8592]. With the aim of examining the structural and kinetic details of the interaction between unsaturated fatty acids and the enzyme, we designed and synthesized 8-(5'-azido-O-hexanoylsalicylamido)octanoic acid (AS86), a photoactivatable analogue of unsaturated fatty acids. AS86, interacting noncovalently with the enzyme, shares with oleic acid the following properties: (i) it binds reversibly to the plasma membrane Ca(2+)-ATPase; (ii) in the absence of calmodulin, AS86 shows a biphasic behavior; i.e., at low concentrations it increases the affinity for Ca2+ and the maximum velocity of the enzyme, while at higher concentrations it decreases the maximum velocity; (iii) in the presence of calmodulin, AS86 increases slightly the affinity for Ca2+ and decreases the maximum velocity of the Ca2+ pump; and (iv) AS86 inhibits the activity of the enzyme devoid of its calmodulin-binding domain after proteolysis. When the reagent is covalently bound to the native enzyme, and then activated by calmodulin, increasing amounts of AS86 decrease the maximum velocity along a hyperbolic curve without modifying the apparent affinity for Ca2+. These results could be explained by the eventual existence of two different kind of sites recognizing the reagent: one influencing the affinity for Ca2+ and the other inhibitory of the calmodulin effects. When covalently bound, AS86 exerts its inhibitory effects upon the enzyme lacking the calmodulin-binding domain, thus reflecting that this action is promoted by interaction with a site lying outside this region. The purified enzyme is susceptible to be tagged with 125I-AS86. Both the inhibitory effect on the calmodulin-dependent enzymic activity after covalent binding of AS86 and the photoadduct formation between the enzyme and 125I-AS86 are impaired by the presence of oleic acid in a concentration-dependent fashion. Recognition of photoreactive fatty acid analogues by the purified enzyme could be useful to provide further insight on the location of the interacting sites.

Adult↗
PubMed1995

Effect of pH upon Ca(2+)-ATPase activity of rat pancreatic islets: its possible contribution to the inhibitory effect of different insulin secretagogues.

This work was undertaken in an attempt to elucidate the possible mechanism by which insulin secretagogues produce a fast and transient drop in the Ca(2+)-ATPase activity of the pancreatic islet membrane. For this purpose, the enzyme activity was measured in either homogenates or partially purified membranes of islets previously incubated under different experimental conditions. Ca(2+)-ATPase activity measured in homogenates of islets preincubated with 8 mM glucose decreased significantly compared to control islets incubated with 2.8 mM glucose. The inhibition was also observed when the enzyme activity was measured in homogenates of islets preincubated with 2.8 mM glucose plus 20 mM propionic acid as well as with glucose 2.8 mM in a buffer equilibrated with a gas mixture of O2 and either 12% or 30% CO2. Ca(2+)-ATPase activity decreased significantly in partially purified islet membranes preincubated for 3 min with glucose (2 and 8 mM), 15 mM KCl and 2 mM tolbutamide. These substances did not affect the Ca(2+)-ATPase activity when added directly to the enzyme assay medium. The enzyme activity also decreased when measured in membranes preincubated at pH 6.5. The addition of 1 mM ATP to the preincubation medium protected the Ca(2+)-ATPase activity from the inhibition induced by glucose, KCl and tolbutamide as well as from the one produced by acidic pH in the medium. On account of these results, we suggest that insulin secretagogues, as well as either acidification of B-cell cytosol or islet membrane incubation medium, produce changes at the islet membrane level which promote a decrease in the Ca(2+)-ATPase activity. A shift of the E1-E2 equilibrium of the phosphoenzyme towards E1 may account for such decreased activity. Changes in Ca(2+)-ATPase activity could either favour the decrease or the increase in the cytosolic concentration of Ca2+ in B-cells. Therefore, negative and positive modulation of its activity might allow Ca(2+)-ATPase to play a role in the switch-on and -off mechanism for intracellular Ca2+ signal regulation of B-cell secretion of insulin.

Adenosine Triphosphate↗
PubMed1995

[Circadian variations of performances and basic rhythms].

Difficulties with chronopsychology studies include a masking effect of variables, the combination of different rhythms and variations of strategies. An experiment is conducted to analyze the role of circadian variations of elementary processes in the variations of performance for a complex task. Twenty-four subjects solved anagrams and tried to find the rule of anagram construction, during two sessions, at 10 am and 5 pm. Responses were classified in three groups: (a) discovery of the anagram construction rule (R2 responses); (b) resolution of anagram without discovery of rule (R1 responses); (c) failure, no resolution of anagram (R0 responses). During the second session, R2 performances were better at 10 am than at 5 pm. In contrast, R1 performances were better at 5 pm than at 10 am. Rule application was faster at 10 am than at 5 pm. Results are discussed in terms of variations of short-term memory capacity (Folkard and Monk, 1980). Using chronopsychology to analyze the role of elementary processes in a complex task is discussed.

Adult↗
PubMed1994

Acetylation with succinimidyl acetate affects both the catalytic site and the regulation of the erythrocyte Ca2+ pump.

Acetylation of lysine residues of the erythrocyte Ca2+ pump using succinimidyl acetate (SA) led to its complete inactivation. In the absence of any of the major activators of the pump (namely calmodulin and acidic phospholipids), ATP fully protected the pump from inactivation by SA, with a K0.5 of 13 microM. This value is very close to the Km of the high-affinity site for ATP, thus suggesting that the residue(s) involved is(are) near or at the catalytic site of the Ca(2+)-ATPase. Furthermore, the presence of 500 microM ATP prevented the acetylation of about two residues per molecule of enzyme. Acetylation by SA also prevented the activation of the Ca2+ pump by calmodulin, acidic phospholipids or controlled trypsin proteolysis. This effect of SA treatment was not avoided by the presence of ATP in the preincubation medium, indicating a second set of modified residues. The fact that the three modes of activation were cancelled in a similar fashion by SA suggests that, although acting via different mechanisms, they share at least a common step in which SA-sensitive lysine residues may participate. Moreover, modification of the pump by SA plus ATP decreased the KCa when the activity was measured in both the absence and presence of calmodulin, suggesting that the residue(s) modified in this case is(are) involved directly in the regulation of the affinity for Ca2+.

Acetylation↗