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T Pineda

Publications and source records attributed to T Pineda.

8 recordsLinked to original sources
PubMed1997

Electrochemical evidence on the molten globule conformation of cytochrome c.

To explore a new approach for characterizing the molten globule conformation, cyclic voltammetric studies of salt induced transitions at acidic pH of cyt c have been carried out. The use of modified electrodes has made the observation of direct electrochemistry in native cyt c possible. However, most of these electrodes do not show reversible responses at acidic pH, due to the fact that, for this system, a deprotonated electrode surface is needed. In these studies, we have used a 6-mercaptopurine and cysteine-modified gold electrodes which are effective for direct rapid electron transfer to cyt c, even in acid solutions. The change in the absorption bands of cyt c are used to monitor the conformational states and, hence, to compare the voltammetric results. Under the experimental conditions where the A state of cyt c is obtained, a reversible voltammetric signal is observed. The midpoint peak potentials are found to be very close to the formal potential of native cyt c. Results are discussed in terms of a cooperative two-state transition between the acid unfolded and the globular acidic states of cyt c. This finding establishes, for the first time, the similarity of both the native and the molten globule-like conformations in terms of its redox properties.

Animals↗
PubMed1996

Spectroscopic studies of myo-inositol monophosphatase with a novel fluorescent substrate.

myo-Inositol monophosphatase catalyzes dephosphorylation of the synthetic substrate anthraniloyl-2'-AMP. Binding of this fluorescent substrate to Tb(III)-monophosphatase was monitored by luminescence spectroscopy. The anthraniloyl chromophore excited at 330 nm sensitizes the long lived luminescence of enzyme bound Tb(III) at 490, 545, 585 and 620 nm. Assuming a mechanism of radiationless energy transfer, the actual distance of separation between the donor anthraniloyl moiety and the acceptor Tb(III) was calculated to be R = 10 angstroms. The binding studies support the earlier observation of Bone et al. (Proc. Natl. Acad. Sci. USA 89 (1992) 10031-10035) that the substrate and the lanthanide Gd(III) interact with a common binding domain of the protein. The catalytic activity of the monophosphatase is completely dependent upon Mg(II) ions which elicit changes in the secondary structure of the protein as revealed by circular dichroism measurements. Binding of Mg(II) ions tend to stabilize the secondary structure of the phosphatase against guanidinium-HCl denaturation.

Adenosine Monophosphate↗
PubMed1995

Characterization of monomeric 4-aminobutyrate aminotransferase at low pH.

4-Aminobutyrate aminotransferase undergoes a reversible process of association/dissociation at low pH. At pH 5.0, monomeric species exist predominantly in solution as revealed by FPLC and time-dependent emission anisotropy measurements. The observed rotational correlation time at pH 5.0, phi obs = 25 ns, corresponds to a compact spherical unit of 52 kDa. An increase in the net charge of the macromolecule at pH 5.0 is responsible for destabilization of the dimeric structure, (WEL approximately 41.84 kJ/mol), but the dissociation of the protein does not perturb the secondary structure as revealed by CD measurements. The fluorescent probe 1-anilinonaphthalene-8-sulfonate (ANS), bound to hydrophobic sites of the enzyme, was used to monitor the kinetics of protein dissociation by stopped-flow spectroscopy. The dissociation of the dimeric structure at pH 5.0 was characterized by a relaxation time of 18 ms. The rate of association of monomeric subunits at pH 7.0 was too fast to be detected in the stopped-flow instrument. These observations have some bearing on the mechanism of reconstitution of dimeric structures of 4-aminobutyrate aminotransferase in the cell.

4-Aminobutyrate Transaminase↗
PubMed1994

Unfolding of 4-aminobutyrate aminotransferase equilibrium and kinetic studies.

The unfolding of pig liver 4-aminobutyrate aminotransferase by urea has been investigated at equilibrium. The overall process was reversible as judged from the recovery of catalytic activity after dilution of urea-treated samples. Unfolding of the enzyme was monitored by circular dichroism and fluorescence spectroscopy. The steepness of the fluorescence and CD changes between 2 and 8 M urea, and the lack of any discernible plateau suggests that unfolding of the protein is a cooperative process. The unfolding of 4-aminobutyrate aminotransferase as a function of urea concentration was monitored by fluorescence measurements of the tryptophanyl residues. The kinetic results indicate that the aminotransferase unfolds in a single kinetic phase. Unfolded 4-aminobutyrate aminotransferase recovers immediately its catalytic activity upon dilution with buffers of neutral pH. Based on equilibrium and kinetic results, a two-state model for the unfolding of the aminotransferase is proposed.

4-Aminobutyrate Transaminase↗
PubMed1993

Reversible unfolding of pyridoxal kinase.

The unfolding of brain pyridoxal kinase by guanidinium HCl has been investigated at equilibrium. The overall process was reversible as judged from the complete recovery of catalytic activity after removal of guanidinium HCl. Unfolding of pyridoxal kinase was monitored by circular dichroism and fluorescence spectroscopy. The steepness of the spectroscopic changes between 0.2 and 1.5 M guanidinium HCl, and the lack of any discernible plateau suggests that unfolding of the monomer is a cooperative process. A compact intermediate on the unfolding pathway of pyridoxal kinase could not be detected by the method of denaturant gel filtration. The fluorescent analogs of the substrates ATP and pyridoxal were used to assess differences in stability among the domains of the protein. Based on fluorescence and steady emission anisotropy results, it is postulated that the nucleotide domain is more stable than the pyridoxal domain of the kinase.

Animals↗
PubMed1993

Interaction of ATP analogs with yeast 3-phosphoglycerate kinase. Affinity labeling of the hinge region.

Yeast 3-phosphoglycerate kinase is inactivated by incubation with pyridoxal 5'-diphospho-5'-adenosine (AdoP2Pxy) [Tamura, J. K., Rakov, R. D. & Gross, R. L. (1986) J. Biol. Chem. 261, 4126-4133). Incorporation of 1 mol affinity label/mol enzyme was sufficient for complete inactivation of 3-phosphoglycerate kinase. The substrate ATP affords substantial protection against inactivation. Partial protection is afforded by the substrate glycerate 3-phosphate. When AdoP2Pxy-modified phosphoglycerate kinase was reduced with [3H]NaBH4 and subjected to trypsin hydrolysis, only one radioactive peptide was isolated by reverse-phase high-performance liquid chromatography. The amino acid composition and sequence analysis of the purified radioactive peptide revealed that it spans residues 379-403 of the enzyme and Lys385 specifically reacted with the affinity label. This peptide represents the hinge region between the two domains of the protein, where the active site is also located. The fluorescence intensity of enzyme-bound AdoP2Pxy is enhanced when glycerate 3-phosphate is added, suggesting exposure of the fluorescent probe to a more hydrophobic environment. Another fluorescent analog, anthraniloyl-dATP (ant-dATP), which carries the fluorescent reporter group on the ribose ring, binds to the enzyme at two distinct sites with Kd values of 6 +/- 2 microM and 25 +/- 3 microM, as determined by steady-state anisotropy measurements. Bound ant-dATP was displaced from the enzyme by glycerate 3-phosphate and ATP, as monitored by the fluorescence anisotropy. These results suggest that both fluorescent ATP analogs bind to the active site, which is at the hinge region of the enzyme. Model-building studies showed that when AdoP2Pxy is built into the open form of the enzyme, as described in X-ray studies, the pyridoxyl group of AdoP2Pxy cannot reach Lys385 for Schiff-base formation. Labeled Lys385 is on a beta-turn immediately following helix XII, which was suggested to interact with the nucleotide and become ordered at the active site of 3-phosphoglycerate kinase [Watson, H. C., Walker, N. P. C., Shaw, P. J., Bryant, T. N., Wendell, P. L., Fothergill, L. A., Perkins, R. E., Conroy, S. C., Dobson, M. J., Tuite, M. F., Kinesman, A. J. & Kinesman, S. M. (1982) EMBO J. 1, 1635-1640]. The results presented here suggest that binding of substrates cause significant structural changes in the enzyme.

Adenosine Diphosphate↗
PubMed1985

Emergency surgery after coronary disruption complicating PTCA. Report of four cases.

Four out of 150 patients having a PTCA at our institution sustained a coronary artery disruption and underwent emergency surgery. All of them presented a severe stenosis of the LAD, in 2 of them there was also a stenosis at the origin of a diagonal branch. In each case the injured vessel was a diagonal branch. Three patients had a rupture of subepicardial arteries and developed cardiac tamponade. In another patient the rupture was located intramyocardially. Severe hemodynamic derangement occurred in all 4 cases, one resulting in cardiac arrest. The latter was the only patient who died postoperatively, due to neurological damage. Two of the surviving patients sustained a non-transmural myocardial infarction in the area of the injured vessel. However, all 3 survivors recovered easily and are doing well 6 months after surgery.

Angioplasty, Balloon↗