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Biomedical subjects

D Balasubramanian

Publications and source records attributed to D Balasubramanian.

At least 37 records · Page 2Linked to original sources
PubMed1997

Modified helix-loop-helix motifs of calmodulin--The influence of the exchange of helical regions on calcium-binding affinity.

The four calcium-binding sites, called the helix-loop-helix, or the EF-hand motifs, of calmodulin differ in their ion-binding affinities; this has been thought to arise due to the variations in the sequences of the loop regions where the ion binds. We focus attention here on the role of the flanking helical regions on the calcium-binding affinities. Peptides were synthesized in a manner that simulates the E and F helical flanks of site 4 (the strongest calcium-binding site of the calmodulin) to sandwich the loop sequences of sites 1, 2, 3 and 4 so as to produce peptides named 414, 424, 434 and 444, as well as using the helical flanks of site 1 (the weakest site) to produce peptides 111, 121, 131 and 141. Calcium binding was monitored using the calcium-mimic dye Stains-all (4,4,4',5'-dibenzo-3,3'-diethyl-9-methyl-thiacarbocyanine bromide). Binding abilities were seen to increase several-fold when the E and F helices of site 1 were replaced by those of site 4 (i.e., 111-414). In contrast, the intensity of circular dichroism induced in the absorption bands of the bound achiral dye decreased significantly when the helical flanks of site 4 were replaced with those of site 1 (i.e., 444-141). The helical flanks of site 4 impart greater binding ability to a given loop region, while the helical flanks of site 1 tend to weaken it.

Amino Acid Sequence↗
PubMed1996

Conformation, stability and interactions of corneal keratan sulfate proteoglycan.

We have monitored the molecular conformation, stability, interaction and dynamics of keratan sulfate proteoglycan, the major structural protein component of the cornea, in solution, by studying the fluorescence spectral features of its tryptophan residues as component-specific intrinsic spectral probes (collagen, the other structural component of the cornea, has no tryptophans). Our study suggests that the Trp region of the molecule is in a motionally restricted environment as it exhibits a fluorescence red-edge effect and shows dipole relaxation. The extrinsic spectral probe 8-anilinonaphthalene 1-sulfonate reveals keratan sulfate proteoglycan to possess significant surface hydrophobicity. This dual character of keratan sulfate proteoglycan allows us to label it as an 'ambidextran' proteoglycan. The molecule is stable between pH 5-8 and has a Tm value of 72 degrees C. Disulfide bonds play a role in the stability of the molecule. KSPG is seen to interact with collagen and the model compound, poly(L-proline). Interaction of the proteoglycan with unilamellar vesicles appears to be more interfacial than penetrative. This dual interaction displayed by KSPG with collagen and with lipid assemblages suggests that it plays the role of a 'filler' in the extracellular matrix of the cornea.

Animals↗
PubMed1996

A conformational study of corneal dermatan sulfate proteoglycan using fluorescence spectroscopy.

DSPG is a major proteoglycan of the corneal stroma and is thought to be important for the transparency of the tissue. We have studied its conformation by exploring the microenvironment and dynamics of its lone Tryptophan (Trp) residue using steady state and time resolved fluorescence spectroscopy. DSPG exhibits a doublet Trp fluorescence emission. Such a doublet emission has been observed earlier in the copper protein azurin and in avian lens delta-crystallin. Unlike the above cases where the doublet emission is thought to arise due to vibronic structure or the location of Trp at the interface of interacting subunits, fluorescence quenching, denaturation studies and ANS binding with DSPG indicate the location of Trp at two different environments. Such a situation could arise from the differential glycosylation of the core protein or due to duplexation and aggregation of the glycosaminoglycan chains.

Animals↗
PubMed1996

Fluorescence properties of isolated intact normal human corneas.

We have examined the fluorescence properties of excised intact normal human corneas from over a hundred donors, using synchronous excitation fluorescence spectroscopy. In some of the corneas from the donors, a fluorophore with an excitation band centered at 330 nm was observed. This fluorophore does not seem to correspond to the dityrosine moiety or to any photoproducts of tryptophan. Isolated corneas irradiated with light of 295 nm wavelength do not produce any fluorescent photoproducts, suggesting that the intact tissue has endogenous quenchers, radical scavengers and antioxidants that inhibit its photodamage. The non-tryptophan fluorophores that accumulate in some corneas thus appear to arise largely from the nonenzymatic glycosylation (glycation) of the constituent proteins as similar fluorophores are detected in the corneas of rats in which diabetes is induced.

Adult↗
PubMed1996

Calcium binding properties of beta-crystallins.

beta-Crystallins, the oligomeric proteins of the eye lens, were found to bind calcium ions with low affinity, in the millimolar range. Calcium was found to induce conformational changes in the secondary and tertiary structure of beta-crystallins. While these changes in conformation are seen in low ionic strength media, they are masked when the protein is dissolved in an intermediate ionic strength medium, where oligomers of lower molecular weight are formed. The calcium binding takes place in these conditions. The behaviour of this protein with respect to calcium binding is reviewed.

Animals↗
PubMed1996

Effect of UVB radiation on corneal aldehyde dehydrogenase.

PURPOSE: A Class 3 aldehyde dehydrogenase happens to be a major soluble protein constituent of the cornea. Its role is conjectured to be manifold: to protect the tissue from oxidative damage by eliminating the toxic aldehydes produced upon lipid peroxidation under oxidative stress, to act as an UV-absorber, and to maintain the level of the coenzyme NADH in the cornea. We have studied the effect of UVB on the structure and enzyme activity of corneal aldehyde dehydrogenase. METHODS: Aldehyde dehydrogenase was irradiated at 295 nm for varying periods of time and change in its enzyme activity assayed. The structural changes in the molecule accompanying irradiation were monitored using fluorescence and circular dichroism spectroscopy, and its hydrodynamic behavior and surface hydrophobicity studied using gel filtration chromatography and binding of the hydrophobic fluorophore ANS. The protective ability of aldehyde dehydrogenase in preventing aggregation of photolabile proteins, such as Gamma-crystallin of the eye lens, was studied by monitoring the scattering value of the test protein with irradiation by UVB. RESULTS: Aldehyde dehydrogenase is seen to undergo photodamage with alterations in its quaternary structure, though no significant change is noticed in the peptide chain conformation. Under such conditions the molecule continues to act as a protectant by preventing aggregation of photolabile proteins such as the eye lens Gamma-crystallin. CONCLUSIONS: Our earlier studies have shown that the free sulfhydryl groups are important for the antioxidant abilities of aldehyde dehydrogenase. Its protective ability towards photoaggregation of Gamma-crystallin seen here might arise both due to: (i) oxyradical quenching and (ii) the increased surface hydrophobicity of the molecule upon irradiation, which allows it to bind to, and thus inhibit the aggregation of interacting proteins.

Aldehyde Dehydrogenase↗
PubMed1994

Racemization of tyrosine in the insoluble protein fraction of brunescent aging human lenses.

With increase in age, proteins of the eye lens undergo covalent modifications associated with interprotein cross-linking, loss of protein solubility, generation of protein-associated pigments, and racemization of some optically active residues. Precipitated protein aggregates localized in the nucleus of brunescent lenses are modified to such an extent that they become resistant to the commonly used methods of extraction by reducing agents or detergents. Thus, little information is available on the types of protein modifications that occur in the nuclear region of the lens. Here we report the simultaneous solubilization and digestion of the entire nuclear pellet of brunescent human cataract lenses into constituent amino acids and their derivatives through protease action at neutral pH. Fractionation of the resulting hydrolyzate by reverse phase chromatography, followed by spectroscopic and biochemical analysis of one of the purified fractions, showed the presence of racemic tyrosine in the amino acid mixture. Racemization of L-amino acid residues in protein chains is associated with the aging process. This is the first report of racemization of tyrosine in any system studied so far.

Aging↗
PubMed1994

Protein-associated pigments that accumulate in the brunescent eye lens. Identification of a quinoline derivative.

Brunescent (dark brown) cataract is particularly prevalent in the tropics. Enzymatic digestion of the insoluble protein fraction of brunescent cataractous eye lenses from India, followed by high performance liquid chromatographic separation of the pigments and spectroscopic investigations, have led to the identification of one of the pigments as 4-hydroxyquinoline-3-[alpha-aminoacetic acid] (compound A). The 4-hydroxyquinoline moiety is shown to be a photodynamic agent that generates O2.- and leads to protein crosslinking. This suggests that the compound A may play a long-term deleterious role in situ in the lens.

Cataract↗
PubMed1994

Sugar-DNA molecular recognition: specific interaction of alpha-1,4-glucopyranose chains with DNA in the minor groove.

alpha(1,4) glucopyranose chains (dextrins) are shown to interact with DNA via hydrophobic interactions from the minor groove side, while alpha(1,6) dextrans do not bind DNA. The observed specific alpha(1,4) linked sugar-DNA interactions may have importance in recognition of DNA by ene-diyne class of antibiotics via their saccharide units which have mostly alpha(1,4) linkages.

Base Sequence↗
PubMed1994

In situ fluorescence spectroscopic studies on bovine cornea.

The cornea is a transparent ocular tissue and its transparency is thought to be a result of intramolecular interactions and the supramolecular organization of its protein constituents. We have studied the intrinsic fluorescence properties of intact bovine corneas and compared these with that of the opaque sclera. It was observed that with increasing excitation wavelengths the emission maxima shifted toward the red edge exhibiting the phenomenon of red edge excitation shift, which is indicative of immobilization of the constituent fluorophores. The magnitude of the shift increased after photodamage by irradiation at 295 nm. Many of the spectral characteristics of the cornea are shown to be due to its proteoglycans, which show surprisingly significant red edge excitation shift in solution.

Animals↗
PubMed1994

Oxidative damage to the eye lens caused by cigarette smoke and fuel smoke condensates.

Cigarette smoking is reported to increase the risk of cataract. Likewise, the use of smoky cooking fuel is implicated in the etiology of cataract. In an effort to understand the cellular and molecular basis, the in vitro and in vivo cataractogenetic effects of these smoke condensates have been studied using isolated rat lenses and pigmented rats. Isolated capsulated rat lenses are incubated with cigarette smoke condensate (CSC) and firewood smoke condensate (FSC) for varying periods, with and without antioxidants, in the presence and absence of light. CSC and FSC permeate the lens capsule, impart colour and opacify the lens in a light- and dose-dependent manner. Antioxidants offer partial inhibition against the above damage. The condensates contain polycyclic aromatics which generate reactive oxygen species such as O2 photodynamically, and ppb levels of Fenton metal ions which induce oxidative reactions through .OH. Smoke induced damage possibly occurs through systemic absorption and transport of toxic components to several tissues, and specially into the lens, wherein the turnover is slow, leading to chronic accumulation causing oxidative damage to the constituent molecules and to consequent lenticular opacity.

Animals↗
PubMed1993

Nonenzymatic glycation alters protein structure and stability. A study of two eye lens crystallins.

We have investigated the effect of nonenzymatic glycation (fructation) in vitro on the structure and stability of two proteins that are glycated in vivo as a consequence of high endogenous levels of sugar. We find that whereas fructation leads to the structural destabilization of the monomeric gamma-crystallin from the core of the eye lens, it leads to an increase in stability in the multimeric alpha-crystallin of the lens cortex. Thus, while glycated gamma-crystallin shows (a) a longer wavelength of fluorescent emission, indicating a greater exposure of its aromatic side chains to the medium; (b) a reduced secondary structural content; and (c) a more facile denaturation by thermodynamic and chemical means, alpha-crystallin displays the opposite behavior. Furthermore, alpha-crystallin shows an increased tendency toward multimeric aggregation upon fructation. We interpret these differences in the broad context of the effects of neutralization of positive charges on protein structure and stability. Fructation tends to destabilize gamma-crystallin, by effecting a significant reversal in the balance of charges in the protein, at physiological pH. alpha-Crystallin is a multimeric protein whose pI is lower than its pH of optimum stability. Fructation in this case effectively neutralizes the cationic charges and promotes conformational order. This study indicates that although glycation brings about similar changes in the covalent chemical structures of proteins, its influence on the three-dimensional structures of different proteins can be different.

Animals↗
PubMed1993

Studies on the interaction of the dye, stains-all, with individual calcium-binding domains of calmodulin.

We show that the calcium-mimic dye, Stains-all, is a convenient probe to study the structural features of the individual calcium-binding sites of calmodulin (CaM) and related calcium-binding proteins (CaBP). These peptides bind the dye in their calcium-binding sites, and induce a circular dichroism (CD) band in the bound dye in the 620 nm (J band) region, which is abolished upon the addition of calcium. Replacement of Asp by Asn in the + x position of the weaker calcium-binding site (site I of CaM) abolishes the dye binding, while the same change in the higher affinity site IV attenuates the binding of the dye and does not abolish it. Replacement of Tyr in site IV with Trp does not distort the geometry, although it increases the dye binding affinity.

Amino Acid Sequence↗