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Biomedical subjects

A Pande

Publications and source records attributed to A Pande.

At least 37 records · Page 2Linked to original sources
PubMed1990

Prevalence of parainfluenza type 1 virus in Lucknow (India).

A total of 568 children suffering from acute respiratory infections were studied from September, 1986 to June, 1988 using indirect immunofluorescence technique to know the frequency of infections caused by parainfluenza viruses 1 and 3. Though sporadic cases of parainfluenza 1 (2.5%) and parainfluenza 3 (3.2%) were seen round the year, and increase in cases of parainfluenza virus type 1 infection was observed only in October, 1987 in Lucknow.

Age Factors↗
PubMed1989

Purple membrane: color, crystallinity, and the effect of dimethyl sulfoxide.

In an effort to understand the nature of chromophore-protein interactions in bacteriorhodopsin (bR), we have reinvestigated dimethyl sulfoxide (DMSO)-induced changes in bR [Oesterhelt et al. (1973) Eur. J. Biochem. 40, 453-463]. We observe that dark-adapted bR (bR560) in aqueous DMSO undergoes reversible transformation to a species absorbing maximally at 480 nm (bR480). Beginning at 40% DMSO, this change results in complete conversion to bR480 at 60% DMSO. The kinetics of the reaction reveal that this transformation takes place predominantly through the all-trans isomeric form of the pigment. Thermal isomerization of the 13-cis chromophore to the all-trans form is, therefore, the rate-limiting step in the formation of bR480 from the dark-adapted bR. As in native bR, the chromophore in bR480 is linked to the protein via a protonated Schiff base, and its isomeric composition is predominantly all-trans. The formation of bR480 is associated with minor changes in the protein secondary structure, and the membrane retains crystallinity. These changes in the protein structure result in a diminished chromophore-protein interaction near the Schiff base region in bR480. Thus, we attribute the observed spectroscopic changes in bR in DMSO to structural alteration of the protein. The 13-cis chromophoric pigment appears to be resistant to this solvent-induced change. The changes in the protein structure need not be very large; displacement of the protein counterion(s) to the Schiff base, resulting from minor changes in the protein structure, can produce the observed spectral shift.

Bacteriorhodopsins↗
PubMed1989

Effect of lipid-protein interaction on the color of bacteriorhodopsin.

Detergent solubilization and subsequent delipidation of bacteriorhodopsin (bR) results in the formation of a new species absorbing maximally at 480 nm (bR480). Upon lowering the pH, its absorption shifts to 540 nm (bR540). The pK of this equilibrium is 2.6, with the higher pH favoring bR480 (Baribeau, J. and Boucher, F. (1987) Biochim. Biophysica Acta, 890, 275-278). Resonance Raman spectroscopy shows that bR480, like the native bR, contains a protonated Schiff base (PSB) linkage between the chromophore and the protein. However, the Schiff base vibrational frequency in bR480, and its shift upon deuteration, are quite different from these in the native bR, suggesting changes in the Schiff base environment upon delipidation. Infrared absorption and circular-dichroism (CD) spectral studies do not show any net change in the protein secondary structure upon formation of bR480. It is shown that deprotonation of the Schiff base is not the only mechanism of producing hypsochromic shift in the absorption maximum of bR-derived pigments, subtle changes in the protein tertiary structure, affecting the Schiff base environment of the chromophore, may play an equally significant role in the color regulation of bR-derived pigments.

Bacteriorhodopsins↗
PubMed1987

Resonance Raman spectroscopy of octopus rhodopsin and its photoproducts.

We report here the resonance Raman spectra of octopus rhodopsin and its photoproducts, bathorhodopsin and acid metarhodopsin. These studies were undertaken in order to make comparisons with the well-studied bovine pigments, so as to understand the similarities and the differences in pigment structure and photochemical processes between vertebrates and invertebrates. The flow method was used to obtain the Raman spectrum of rhodopsin at 13 degrees C. The bathorhodopsin spectrum was obtained by computer subtraction of the spectra containing different photostationary mixtures of rhodopsin, isorhodopsin, hypsorhodopsin, and bathorhodopsin, obtained at 12 K using the pump-probe technique and from measurements at 80 K. Like their bovine counterparts, the Schiff base vibrational mode appears at approximately 1660 cm-1 in octopus rhodopsin and the photoproducts, bathorhodopsin and acid metarhodopsin, suggesting a protonated Schiff base linkage between the chromophore and the protein. Differences between the Raman spectra of octopus rhodopsin and bathorhodopsin indicate that the formation of bathorhodopsin is associated with chromophore isomerization. This inference is substantiated by the chromophore chemical extraction data which show that, like the bovine system, octopus rhodopsin is an 11-cis pigment, while the photoproducts contain an all-trans pigment, in agreement with previous work. The octopus rhodopsin and bathorhodopsin spectra show marked differences from their bovine counterparts in other respects, however. The differences are most dramatic in the structure-sensitive fingerprint and the HOOP regions. Thus, it appears that although the two species differ in the specific nature of the chromophore-protein interactions, the general process of visual transduction is the same.

Animals↗
PubMed1984

Spectroscopic characterization of nitrated purple membranes.

Light-adapted purple membranes were modified with tetranitromethane by a new light-dependent procedure at pH 5.5 which results in a blue-shifted chromophore absorbing at 530nm. This modification affects two aromatic residues. The modified bacteriorhodopsin's ground state chromophore structure is probed by circular dichroism and resonance raman spectroscopy while its photocycle is studied by laser-flash photolysis in the picosecond, microsecond and millisecond time scale. After nitration, the main findings are 1) Interactions between neighboring chromophores are lost, 2) Modified bacteriorhodopsin contains a conformationally changed chromophore but retains a protonated Schiff's base as evidenced by a resonance raman band at 1652 cm-1, 3) A red-shifted intermediate is formed in less than 10 ps after laser excitation, 4) The decay of the M-intermediate is not significantly affected whereas the rise time of the intermediate is enhanced about two fold. These observations are relevant to the role of aromatic acid residues of the apoprotein in the determination of the chromophoric characteristics in bacteriorhodopsin.

Bacteriorhodopsins↗
PubMed1982

Acid-base equilibrium of the Schiff base in bacteriorhodopsin.

Aqueous suspensions of dark-adapted bacteriorhodopsin (bR560) in the purple membrane of Halobacterium halobium are exposed to rapid jumps to high pH. Optical and resonance Raman measurements are carried out by using flow and stationary methods. Above pH congruent to 11.5 bR560 starts to be reversibly converted to a species absorbing at 460 nm (bR460) characterized by an unprotonated Schiff base chromophore. Above pH congruent to 13.0 bleaching takes place, first reversibly and subsequently irreversibly, to a species absorbing around 365 nm (bR365). This process competes with the formation of bR460. The pKa corresponding to the equilibrium (equation in text) is determined as 13.3 +/- 0.3. The value of the corresponding association rate constant determined from the reverse jumps (from pH 12.67 to pH 10 and 9.2) is ka = (3.5 +/- 0.5) X 10(11) M-1 s-1. Thus, starting with bR at pH 12.67 the reprotonation process is diffusion controlled as observed for homogeneous acid-base equilibria. The observed rate of dissociation when jumping from pH 6.5 to 12-13 is slower than that predicted by including the equilibrium (equation in text) The results imply that the Schiff base is titratable in the dark, but its accessibility to external OH- ions is limited. The limitations in the significance of the "apparent" value of pKa = 13.3 observed for the Schiff base titration are discussed in light of possible alterations in the structure of bR resulting from the parallel titration of other protein groups. It is suggested that a light-induced pKa change of at least nine units takes place during the photocycle of light-adapted bR.

Acid-Base Equilibrium↗
PubMed1981

Kinetics of unfolding and folding of horse heart ferricytochrome c with urea.

Kinetic data for the reversible folding and unfolding by urea of horse heart ferricytochrome c, in 0.05 M phosphate + 0.25 M Na2SO4 buffer, pH 7.0, in the region of the main denaturation transition, 4-9 M, are reported. Stopped flow technique and absorptivity at 695, 528, and 361 nm as the monitoring probes were used. The decay profiles in the region of the transition 6-7.2 M urea are adequately described by a rate law with two exponential decay terms, but a rate law with only a single term is found to be applicable at the lower and higher limits of the transition. The apparent rate constant for the fast phase exhibits urea dependence with a minimum value at about 6.5 M urea, while the apparent rate constant of the slow phase is found to be independent of urea and has a value of 0.04 +/- 0.02 s-1. The assignment of the two apparent rate constants to the respective steps and the characterization of the processes involved were carried out through correlation of the kinetic data to the results from equilibrium studies for urea denaturation ( Myer, Y. P., MacDonald, L. H., Verma, B. C., and Pande, A. (1980) Biochemistry 19, 199-207). A mechanism X1 in equilibrium X2 in equilibrium D, where the first step is the urea-dependent unfolding and folding, and the second, an apparent urea-independent process involving possibly reorganization of the unfolded form X2, has been proposed to account for the above findings. The X2 in equilibrium D process is further considered in light of various possibilities: the incorrect folding of the unfolded form, the alteration of heme iron coordination, and the cis-trans isomerization of proline.

Animals↗
PubMed1981

On the mechanism of hydrogen-deuterium exchange in bacteriorhodopsin.

Continuous-flow resonance Raman experiments carried out in bacteriorhodopsin show that the exchange of a deuteron on the Schiff base with a proton takes place in times shorter than 3 ms. Exchange mechanisms based on a base-catalyzed deprotonation followed by reprotonation of the Schiff base are excluded. A mechanism is suggested in which a water molecule interacts directly with the Schiff base deuteron in a concerted exchange mechanism. It appears that in the dark, the binding site is more accessible to neutral water molecules than to charged protons.

Bacteriorhodopsins↗
PubMed1980

Kinetics of the reduction of horse heart ferricytochrome c. Ascorbate reduction in the presence and absence of urea.

The reduction of horse heart cytochrome c with ascorbate in the absence of urea and in its presence, 0 to 8 M, pH 7.0, has been investigated using a stopped flow technique and the absorptivity at 550 nm as the monitoring probes, and by using the rate of oxidizability with molecular oxygen. Reduction is found to be consistent with a mechanism involving (i) a urea-dependent equilibrium step between an ascorbate-reducible and an irreducible form, with a [urea]1/2 of 7.5 M and a reversion rate constant of 0.05 +/- 0.02 s-1, (ii) the binding of ascorbate to cytochrome c, with a binding constant of 5.9 M-1 in the absence of urea which decreases to a value of 2.7 M-1 above 5.5 M urea, and (iii) a reduction step, with a urea-independent rate constant of 2.9 +/- 0.3 s-1. This scheme is interpreted in terms of an electron-transfer pathway involving neither the classical "adjacent" attack nor attack at the exposed heme edge, i.e. "remote" attack, but rather, through an alternate pathway involving binding at some site other than the heme crevice opening and a migration path of rather low electron-transfer efficiency. The urea-linked ascorbate reduction step is th X2 in equilibrium D step of the urea denaturation mechanism (Myer, Y. P., MacDonald L. H., Verma, B. C., and Pande, A. J. (1980) Biochemistry 19, 199-207), and the 9 M urea form, D, is the irreducible form. Form X2 and the other intermediate form, X1, are found to be reducible directly by ascorbate, and not through reversion to the native form of the protein. both the integrity of the heme crevice and the polypeptide-organized structures are of little importance as far as ascorbate reducibility is concerned, but the integrity of the structural and protein functional changes reflecting the X2 in equilibrium D step of the mechanism directly or indirectly determines the reducibility of the protein.

Animals↗
PubMed1979

Further investigation of the role of calcium in human lens protein aggregation.

High-molecular-weight (HMW) protein from human cataractous lenses, isolated by differential centrifugation, was deaggregated in 7M urea and then reaggregated in either the presence or absence of 10 mM CaCl2. Over 90% of the material reaggregated in the presence of calcium appears to have a size greater than 50 X 10(6) daltons. By contrast, only 20% to 25% of the material reaggregated in the absence of calcium has molecular weight greater than 50 X 10(6) daltons. Disulfide formation during reaggregation is unlikely in the latter experiment, since the addition of 50 mM mercaptoethanol caused no change in results. About 60% to 70% of the low-molecular-weight (LMW) protein fraction deaggregated in 7M urea buffer can be converted to HMW species in the presence of 10 mM CaCl2, when the deaggregating agent is removed. However, only 5% to 10% of this protein is converted to HMW species if the deaggregation step is eliminated. Experiments with 45 Ca indicate that whereas calcium is necessary for the formation of the HMW aggregates, only one calcium per approximately 5 X 10(5) daltons remains bound in the reaggregated material. The data suggest that although calcium may be required to induce aggregation to HMW species, it is not required to stabilize such macromolecules. SDS-polyacrylamide gel electrophoresis of the HMW species formed upon reaggregation of the dissociated HMW species with calcium indicates the presence of all the major polypeptide subunits of the original HMW species present in the lens; however, reaggregation in the absence of calcium yields HMW species lacking in the 9600 dalton component.

Calcium Chloride↗
PubMedDate not supplied

Acetylator status, drug metabolism and disease.

Acetylation polymorphism, although discovered 40 years ago, still holds interest not only because many drugs and carcinogens are metabolized by acetylation in the liver but also because advances have been made in the understanding of the molecular genetics of acetylation. It is this genetic variation of drug metabolism that is one of the causes of inter-individual variation of the effect of a drug. Acetylation polymorphism relates to the metabolism of a number of arylamine and hydrazine drugs and carcinogens by cytosolic N-acetyltransferase--NAT2. In humans, 2 genes--NAT1 and NAT2--are responsible for the N-acetyltransferase activity. Studies have revealed several allelic variants of both NAT1 and NAT2. It has been suggested that some of these variants modify the individual susceptibility to disease.

Acetylation↗