PubMed Health⌕ Search

Biomedical subjects

D Balasubramanian

Publications and source records attributed to D Balasubramanian.

At least 55 records · Page 3Linked to original sources
PubMed1993

Alteration of dynamic quaternary structure and calcium-binding ability of beta-crystallin by light.

beta-crystallin, one of the three main constituent proteins of the eye lens, exists as an equilibrium population of oligomeric (beta H), trimeric (beta L1) and dimeric (beta L2) species. This equilibrium is dependent on various factors such as the protein concentration, ionic strength and pH of the medium. We have studied the effect of ultraviolet B radiation on the aggregational patterns of beta-crystallin, using size-exclusion chromatography. Irradiation of a solution of beta H-crystallin at 295 nm for about 30 min causes the deaggregation of the hexameric population into dimers. Irradiation for a longer time, however, produces cross-linked high molecular weight products. Irradiation of a beta L2 solution for 30 min does not perturb the elution profile, while irradiation for a longer time increases the content of beta L1 (trimeric) crystallin. Irradiation also causes a decrease in the calcium-binding affinity of the beta-crystallins.

Animals↗
PubMed1992

Hydroxyl radical mediated damage to proteins, with special reference to the crystallins.

Oxidative modification of the eye lens proteins, the crystallins, is known to cause protein cross-links and aggregates which lead to lens opacification or cataracts. We focus attention here on oxidative damage occurring in crystallins and some "control" proteins upon reaction with the hydroxyl radical (.OH) which, in the lens, is generated by photosensitization or by the Fenton reaction. In the present study, we have synthesized and used the bishydroperoxide I as a "photo-Fenton" reagent, in order to photolytically generate pure .OH, free of other oxyradicals. Our findings are the following: (i) Trp residues are oxidized by .OH to N-formylkynurenine and related compounds, but this in itself does not lead to covalent aggregation of the protein. (ii) Tyr residues react with .OH, but apparently do not produce dihydroxyphenylalanine or bityrosine. Nor do protein cross-links occur as a result. (iii) Oxidation of His residues appears to be obligatory for protein cross-linking. Histidine-free proteins do not form high molecular weight products upon reaction with .OH. Protection of His residues by adduct formation in other proteins inhibits cross-linking. (iv) Lys residues seem to participate in the cross-linking reaction. Protection of the Lys residues by maleylation of the protein inhibits cross-linking. (v) The oxidized protein is more acidic in nature than the parent, and it might have altered conformational features.

Animals↗
PubMed1992

Dityrosine formation in the proteins of the eye lens.

The presence of dityrosine crosslinks in the proteins of the lens is a subject of some debate. We have investigated the formation of dityrosine in the lens proteins, the crystallins, through reactions mediated by reactive oxygen species, as well as through direct photolysis of proteins in the UVB region. Multiple methods were used to identify dityrosine. These include amino acid analysis of protein hydrolysates, quenching of the fluorescence at 400-410 nm by borate/boric acid solutions specific for dityrosine, and inhibition of formation of dityrosine in the presence of dithiothreitol. We find that reaction of the crystallins, alpha (alpha), beta (beta) and gamma (gamma), with singlet oxygen (1O2), hydroxyl radicals (.OH) and hydrogen peroxide (H2O2), does not result in the formation of either intermolecular or intramolecular dityrosine. It appears that the formation of dityrosine involves a radical mediated electron transfer reaction. Consistent with this, direct photolysis in the UVB region produces intramolecular dityrosine in all three proteins, with the efficiency varying in the order gamma - > beta - > alpha. Since the levels of electron transfer agents (e.g., Fe ions) in the lens are usually negligible, and since the amount of UVB radiation reaching the gamma--crystallin--rich lens nucleus is normally quite low, the changes of dityrosine formation in the crystallins in vivo appear remote.

Animals↗
PubMed1992

Amphiphilic properties of polysaccharides: dextrin chains as example.

Polysaccharide chains are usually considered to be highly hydrophilic, since they contain no obvious apolar moieties. However, it is possible for even these chains to display hydrophobic character, arising out of stereochemical constraints in the chain. We had earlier shown that linear dextrin chains display amphiphilic properties, since all the hydroxyl groups are disposed on one side or face of the chain and the hydrogens disposed on the other. We provide further evidence here for this conclusion that dextrins are amphiphilic chains. In contrast, dextrans and cellulosic chains do not display amphiphilicity. Oligosaccharides that can adopt incipient helical structures might display amphiphilicity. This property might be relevant to intermolecular recognition on cell surfaces, lectin-sugar binding, antigen-antibody interactions and the like, and might be manifested more in a heteromolecular recognition process than as homomolecular self-aggregation.

Buffers↗
PubMed1991

Hydrophobic surfaces in oligosaccharides: linear dextrins are amphiphilic chains.

Polysaccharide chains are usually considered to be highly hydrophilic, since they have no obvious nonpolar moieties in them. Yet, it is possible to realise conformations in these chains wherein all the hydroxy groups are disposed in one side or face of the chain and the hydrogens disposed in the other. We experimentally demonstrate that such an amphiphilic surface is present in linear oligomeric dextrins, i.e., alpha-1,4-linked D-glucosides, but not in alpha-1,6-D-glucosides (dextrans) or in beta-1,4-D-glucosides (cellulose). This amphiphilicity is generated as a consequence of the stereochemical constraints, which vary with the structure of the sugar and with the type of linkage. Oligosaccharide chains that can adopt incipient helical structures might display amphiphilicity. This property might be relevant to intermolecular recognition on cell surfaces, lectin-sugar binding, antigen-antibody interactions and the like, and might be manifested more in heteromolecular recognition process than as homomolecular self-aggregation.

Carbohydrate Conformation↗
PubMed1991

A study of the photodynamic efficiencies of some eye lens constituents.

We have studied the photochemical quantum yields of singlet oxygen production (using the RNO bleaching method) and superoxide production (using the EPR-spin trapping method and the SOD-inhibitable ferricytochrome c reduction spectral assay) of kynurenine (Ky), N-formylkynurenine (NFK), 3-hydroxykynurenine (3HK), kynurenic acid (KUA), and the flavins, riboflavin (RF) and flavin mononucleotide (FMN). Such a study of the photodynamic efficiencies is important since these compounds appear endogenously in the eye. The singlet oxygen quantum yields of the flavins and KUA are high, while Ky and 3HK generate no detectable amounts of singlet oxygen. The superoxide quantum yields of the sensitizers are low compared to their singlet oxygen, and Ky and 3HK produce no detectable amounts of superoxide. The production of the superoxide radical is enhanced in the presence of electron donor molecules such as EDTA and NADH. These results suggest that the production of oxyradicals in the lens may be modulated by the presence of endogenous electron donor molecules such as the coenzymes NADH and NADPH, which are present in significant amounts in some lenses. They also suggest that Ky and 3HK, which are known to be present in aged lenses, might play a protective rather than a deleterious role in the eye.

Animals↗
PubMed1990

The conformational status of a protein influences the aerobic photolysis of its tryptophan residues: melittin, beta-lactoglobulin and the crystallins.

We have studied the aerobic photolysis of the tryptophan residues of the proteins melittin and beta-lactoglobulin when the proteins are in ordered conformations and when they are in randomly coiled states. The results suggest that the conformational status of the protein is a factor that influences the photolysis of the constituent tryptophan residues. This point appears to be of relevance to the photo-oxidation of the tryptophan residues of the eye lens proteins crystallins.

Aerobiosis↗
PubMed1990

The reaction of singlet oxygen with proteins, with special reference to crystallins.

Photosensitized oxidation of the eye lens proteins, the crystallins, is thought to lead to protein crosslinks and high molecular weight aggregates. Such protein modifications may be important factors in the formation of lens opacities or cataracts. We focus attention here on type 2 photo-oxidation involving the reaction of singlet oxygen (1O2) with crystallins and some "control" proteins. We find that: (1) trp residues are oxidized to N-formyl kynurenine and related products, but this in itself does not lead to the production of high molecular weight protein aggregates of the protein; (2) tyr residues react with 1O2 but we do not detect dihydroxyphenylalanine or bityrosine nor are protein crosslinks formed as a result; (3) oxidation of his residues appears necessary for high molecular weight protein covalent aggregates to form. Proteins devoid of his, e.g. melittin or bovine pancreatic trypsin inhibitor, do not form high molecular weight products upon reaction with 1O2. Prior reaction and blocking of his inhibits the crosslinking reactions. (4) The oxidized protein is seen to be more acidic than the parent and has an altered tertiary structure. (5) Among the crystallins, reactivity towards 1O2 varies in the order gamma greater than beta greater than alpha and also gamma A/E greater than gamma D greater than gamma B crystallin.

Animals↗
PubMed1989

Binding site conformation dictates the color of the dye stains-all. A study of the binding of this dye to the eye lens proteins crystallins.

The interaction of the cationic carbocyanine dye Stains-all (1-ethyl-2-[3-(1-ethyl-naphthol[1,2-d]thiazolin-2-ylidene)-2- methylpropenyl]naphthol[1,2-d]thiazolium bromide) with the eye lens proteins crystallins has been studied. alpha- and gamma-crystallins do not bind the dye, while beta- and delta-crystallins do, consistent with the fact that the latter two proteins bind the calcium ion. beta-Crystallin resembles parvalbumin in that it induces only the J-band of the bound dye. delta-crystallin, on the other hand, induces only the gamma-band. Analysis of the metachromasia induced in the dye by these and other proteins suggests that Stains-all is responsive to the conformational status of the region to which it binds in a protein. The J-band of the dye is activated when it binds to a globular domain, and the gamma-band is activated when it binds to a helical stretch of the protein.

Animals↗
PubMed1989

Calcium ion binding to delta- and to beta-crystallins. The presence of the "EF-hand" motif in delta-crystallin that aids in calcium ion binding.

Abnormal levels of endogenous calcium ions are known to induce eye lens opacity, and a variety of causative factors has been proposed, including calcium-mediated aggregation and precipitation of the lens proteins crystallins. We have specifically looked in some detail at the interaction of Ca2+ with various crystallins and its consequences. Lenses incubated in solutions containing 10 mM Ca2+ or 5 mM Tb3+ opacified. Fluorescence titration of crystallins with TbCl3 revealed that this ion binds to delta- and beta-crystallins in solution. Equilibrium dialysis showed that four Ca2+ ions bind to one delta-crystallin tetramer with an affinity of 4.3 x 10(3) M-1. Analysis of the amino acid sequence of delta-crystallin reveals the presence of a calmodulin-type "helix-loop-helix" or "EF-hand" calcium ion binding conformational motif in the region comprising residues 300-350. This is a novel feature of the molecule not reported so far. No other crystallins appear to have this motif. beta-Crystallin also binds four Ca2+ ions/aggregate unit of mass 160 kDa, with an affinity of 2.6 x 10(3) M-1, presumably in the midregion of the molecule that is rich in anionic and polar residues. Circular dichroism spectroscopy shows that the binding of calcium ion leads to subtle conformational changes in the molecules, notably in the tertiary structure.

Amino Acid Sequence↗
PubMed1989

Conformational analysis of cyclolinopeptide A, a cyclic nonapeptide: nuclear Overhauser effect and energy minimization studies.

The conformation of cyclolinopeptide A [cyclo(Pro-Pro-Phe-Phe-Leu-Ile-Ile-Leu-Val)], a naturally occurring cyclic nonapeptide has been investigated in dimethylsulfoxide solution by 270 MHz 1H-nmr. A complete assignment of all C alpha H and NH resonances has been accomplished using two-dimensional correlated spectroscopy and nuclear Overhauser effects (NOEs). Analysis of interresidue NOEs and JHNC alpha H values permit construction of a molecular model for the cyclic peptide backbone. The crude model derived from nmr has been used as a starting point for energy minimization, which yields a refined structure largely compatible with nmr observations. The major features of the conformation of cyclolinopeptide A are a Type VI beta-turn centered at Pro(1)-Pro(2), with a cis peptide bond between these residues and a gamma-turn (C7 structure) centered at Ile(6). Two intramolecular hydrogen bonds Val(9) CO--Phe(3)NH (4----1) and Leu(5) CO--Ile(7)NH (3----1) are observed in the low-energy conformation. The limited solvent accessibility observed for the Val(9) and Leu(5) NH groups in the nmr studies are rationalized in terms of steric shielding.

Magnetic Resonance Spectroscopy↗
PubMed1989

Use of micelles in studying drug-binding sites: simulation of the tubulin-bound fluorescence of colchicine.

Enhancement of the fluorescence intensity of colchicine occurs in media of low polarity and appreciable viscosity; this is suggested to be the basis of the intensification of its fluorescence when it is bound to and immobilized in tubulin. We show here that the tubulin-bound fluorescence features of colchicine are largely reconstructed upon solubilizing it in chosen micellar aggregates that offer optimal polarities and microviscosities. Triton X-100 and bile salt micelles intensify the colchicine emission but the maximal effects are obtained with tetrameric aggregates of the peptide melittin. Estimates of the polarity, microviscosity and binding-site dimensions of colchicine are obtained using this mimetic approach. Our results suggest that well chosen micellar systems act as good models to reconstruct and analyze the spectral properties of molecules immobilized in their binding sites.

Binding Sites↗
PubMed1988

In situ optical spectroscopy of some systems of biological interest.

Monitoring the optical absorption or emission spectrum of a condensed phase sample offers information about the supramolecular assembly, packing effects and other features characteristic of the phase that would be missed when one studies solution-state spectra. We have used the technique of photoacoustic spectroscopy to study intact biological specimens, such as algae, parasite cells and the eye lens. Such a study has offered information about the status of endogenous hemin in Plasmodium cells and the mode of interaction of antimalarial drugs of the chloroquine class therein. We have also attempted to do in situ fluorescence spectroscopy on isolated intact eye lenses, which has enabled us to follow the photochemistry and the status of the photoproduct of the oxidation of the trp residues of the crystallins of the lens.

Acoustics↗
PubMed1985

The molten globular intermediate form in the folding pathway of human carbonic anhydrase B.

The acid-induced and guanidinium chloride-induced conformational transitions in human carbonic anhydrase B have been analyzed. A structural form was detected at pH 3, which has a higher secondary structural order than the native enzyme but little tertiary structure. The enzyme dissolved in an intermediate concentration of the denaturant guanidinium chloride (1 M at pH 7.5) also adopts a similar conformational state. This form, denoted as the intermediate form I, possesses most of the characteristics defined for the molten globular state of globular proteins and might serve as the embryonic structural intermediate during the self-organization of the protein into its functional native form.

Animals↗
PubMed1984

The malaria parasite monitored by photoacoustic spectroscopy.

Noninvasive photoacoustic spectroscopy was used to study the malaria parasites Plasmodium chabaudi and Plasmodium berghei, their pigment, and ferriprotoporphyrin IX, which is a by-product of the hemoglobin that the parasite ingests. The results indicate that the pigment consists of ferriprotophorphyrin self-aggregates and a noncovalent complex of ferriprotoporphyrin and protein. Spectra of chloroquine-treated parasites reveal in situ interaction between the drug and ferriprotoporphyrin. Chloroquine-resistant parasites, readily distinguishable by this method, appear to degrade hemoglobin only partially.

Animals↗