Search PubMed⌕ Search

Biomedical subjects

R Nagaraj

Publications and source records attributed to R Nagaraj.

At least 73 records · Page 4Linked to original sources
PubMed1993

Delta-toxin, unlike melittin, has only hemolytic activity and no antimicrobial activity: rationalization of this specific biological activity.

The antimicrobial activity of a synthetic peptide corresponding to delta-hemolysin had been examined. The peptide did not exhibit antimicrobial activity against gram negative and gram positive micro-organisms unlike other hemolytic peptides like melittin. This lack of antibacterial activity arises due to the inability of delta-hemolysin to perturb the negatively charged bacterial cell surface and permeabilize the bacterial plasma membrane. However, the red blood cell surface has a structure considerably different from bacteria and does not act as a barrier to molecules reaching the lipid membrane. Hence delta-toxin can lyse erythrocytes. Thus, the specificity in biological activity has been rationalized in terms of differences in the interaction of the toxin with the bacterial and red blood cell surfaces.

Amino Acid Sequence↗
PubMed1992

The antibacterial peptide seminal plasmin alters permeability of the inner membrane of E. coli.

Seminal plasmin (SPLN) a 47-residue peptide, isolated from bovine seminal plasma, exhibits antibacterial activity against Gram-positive and Gram-negative bacteria. Although SPLN strongly inhibits the transcription of various natural and synthetic templates by E. coli RNA polymerase in vitro, it also associates with model membranes of phosphatidylcholine and phosphatidic acid. We have undertaken experiments to ascertain whether SPLN permeabilizes the bacterial inner membrane and thereby exerts its antibacterial activity, as in the case of recently isolated antibacterial peptides from mammalian sources. Our results show that SPLN affects the permeability properties of the bacterial inner membrane which is reflected by increased uptake of ortho-nitrophenylgalactoside (ONPG), which can normally be translocated only by protein transporters. SPLN has also been shown to act on the outer membrane, since divalent cations inhibit antibacterial activity.

Animals↗
PubMed1992

Change of glutamic acid to lysine in a 13-residue antibacterial and hemolytic peptide results in enhanced antibacterial activity without increase in hemolytic activity.

A 13-residue peptide corresponding to a hydrophobic segment of the antimicrobial 47-residue peptide seminalplasmin, PKLLETFLSKWIG (SPF), has been shown to have antibacterial and hemolytic activities (N. Sitaram and R. Nagaraj, J. Biol. Chem. 265:10438-10442, 1990). In an effort to get an insight into the structural and charge requirements for these biological activities, an analog of SPF in which Glu has been replaced with Lys has been synthesized and its antibacterial and hemolytic properties have been examined. It has been demonstrated that the analog, SPFK, exhibits potent antibacterial activity at concentrations at which hemolysis does not occur.

Amino Acid Sequence↗
PubMed1992

Unfolding of lysozyme by breaking its disulphide bridges results in exposure of hydrophobic sites.

The interaction of 1-anilino-naphthalene-8-sulphonate (ANS), a probe whose fluorescence is strongly dependent on hydrophobicity of the environment, with native lysozyme and lysozyme partially unfolded by breaking the disulphide bridges and reacting the free -SH groups with iodoacetamide, has been investigated. Monitoring the intensity of ANS fluorescence and the position of the emission maximum in the presence of native and partially unfolded lysozyme indicated that unfolding resulted in the exposure of hydrophobic sites. Hydrophobic sites could not be detected when native and partially unfolded lysozyme were denatured with urea or guanidinium chloride. Protein components of the cells export machinery like 'chaperones' associate only with partially unfolded proteins and not native, folded proteins. Hence, hydrophobic regions of proteins, exposed on partial unfolding, could be the sites of recognition by 'chaperone' proteins.

Anilino Naphthalenesulfonates↗
PubMed1991

Anomalous stimulation of Escherichia coli alkaline phosphatase activity in guanidinium chloride. Modulation of the rate-limiting step and negative cooperativity.

Guanidinium chloride stimulates the activity of alkaline phosphatase from Escherichia coli, by 3-4-fold. Structural parameters of the enzyme, monitored by fluorescence and circular dichroism, indicate progressive denaturation. This unusual stimulation is shown to be independent of the nature of the substrate and source of the enzyme. Profiles of pH dependence and transphosphorylation reaction indicate that the dephosphorylation step of the catalysis is enhanced in the presence of guanidinium chloride. We demonstrate, by fast-flow kinetics and inhibitor titrations, that guanidinium chloride enhances activity by abolishing negative cooperativity and by accelerating the dissociation of rate-limiting enzyme and substrate (E.P) complex.

Alkaline Phosphatase↗
PubMed1990

A synthetic 13-residue peptide corresponding to the hydrophobic region of bovine seminalplasmin has antibacterial activity and also causes lysis of red blood cells.

Seminalplasmin (SPLN), a 47-residue peptide present in bovine seminal plasma, is one of the few proteins isolated from mammalian sources having potent antibacterial activity. SPLN also interacts with sperm acrosomal and plasma membranes. On the basis of analysis of the primary structure of SPLN with respect to its relative hydrophobicity and hydrophilicity, a region comprising of 13-amino acids, Pro-Lys-Leu-Leu-Glu-Thr-Phe-Leu-Ser-Lys-Trp-Ile-Gly, has been delineated. It is demonstrated that a synthetic peptide corresponding to this 13-residue region inhibits growth of Escherichia coli like SPLN and also has the ability to lyse red blood cells.

Alkaline Phosphatase↗
PubMed1989

A synthetic peptide corresponding to the hydrophobic amino terminal region of pardaxin can perturb model membranes of phosphatidyl choline and serine.

Peptides corresponding to the amino terminal region of pardaxin from Pardachirus pavoninus (Gly-Phe-Phe-Ala-Leu-Ile-Pro-Lys-Ile-Ile-Ser-Ser-Pro-Leu-Phe) have been synthesized and their interaction with model membranes of phosphatidyl choline and serine studied by 90 degrees C light scattering and fluorescence spectroscopy. The amino terminal 8-residue peptide and the protected 15-residue peptide cause only aggregation of lipid vesicles. The deprotected 15-residue peptide has the ability to cause aggregation and release of entrapped carboxyfluorescein with both phosphatidyl choline and serine lipid vesicles, like pardaxin. The membrane-perturbing ability of the amino terminal 15-residue peptide can be attributed to its ability to adopt an alpha-helical conformation which is amphiphilic in nature in a hydrophobic environment.

Amino Acid Sequence↗
PubMed1989

A recycling assay for alkaline phosphatase applied to studies on its transport in E. coli K12.

A recycling assay for alkaline phosphatase, based on its ability to hydrolyse NADP to NAD+, is presented. The product NAD+ is recycled in a coupled assay consisting of NADH regeneration and reduction of a nitroblue tetrazolium salt. This assay is 10-12 times more sensitive than the conventional assay. We demonstrate the role of energy poisons in transport of this protein into the periplasm by combining the improved detection with phase separation of the periplasmic and cytoplasmic alkaline phosphatase pools.

Alkaline Phosphatase↗
PubMed1989

Antigenic sites on ribonuclease A. Synthetic peptides corresponding to the sites 37-42 and 83-88 on ribonuclease A show immunogenicity.

Two hexapeptides NH2-Lys-Asp-Arg-Cys-Lys-Pro-COOH and NH2-Asp-Cys-Arg-Glu-Thr-Gly-COOH corresponding to the strong hydrophilic regions 37-42 and 83-88, respectively, on ribonuclease A were synthesized by solid-phase method. These synthetic peptides showed antigenic characteristics and provided an experimental validity to the prediction made earlier, supporting the view that highly hydrophilic regions on the protein have a good correlation with their being potentially antigenic.

Animals↗
PubMed1988

Interaction of a hydrophobic model peptide and its fatty acid derivative with lipid vesicles.

The interaction of a peptide [Lys-Phe-Phe-Phe-Ile-Ile-Trp-OCH3] and its fatty acid derivatives [Lys-(epsilon-palmitoyl)-Phe-Phe-Phe-Ile-Ile-Trp-OCH3 and Lys-(epsilon-12-(9-anthroyloxy)stearic acid)-Phe-Phe-Phe-Ile-Ile-OCH3] with model membranes was investigated by fluorescence spectroscopy. The emission characteristics of the Trp fluorophore indicated that only the peptide with the fatty acid chain is associated with lipid vesicles. Quenching experiments with spin probes suggest an orientation for the fatty acylated peptide wherein the fatty acid chain is perpendicular to the bilayer surface and the peptide chain parallel to the bilayer surface.

Acylation↗
PubMed1987

Perturbation of the lipid bilayer of model membranes by synthetic signal peptides.

The interaction of synthetic peptides corresponding to the signal sequences of Escherichia coli alkaline phosphatase: Lys-Gln-Ser-Thr-Ile-Ala-Leu-Ala-Leu-Leu-Pro-Leu-Leu-Phe-Thr-Pro-Val-Thr- Lys-Ala - OCH3, chicken lysozyme: Met-Lys-Ser-Leu-Leu-Ile-Leu-Val-Leu-Cys(Bzl)-Phe-Leu-Pro-Leu- Ala-Ala-Leu-Gly-OCH2-C6H5 and variant of the chicken lysozyme signal sequence with a charged residue in the hydrophobic region: Lys-Leu-Leu-Ile-Ala-Leu-Val-Leu-Lys-Phe-Leu-Pro-Leu-Ala-Ala- Leu-Gly-OCH3 with model membranes of brain phosphatidylserine (PS) and egg phosphatidylcholine (PC) have been investigated by 90 degrees light scattering and fluorescence spectroscopy. Our results indicate that the association of signal peptides with model membranes results in extensive perturbation of the lipid bilayer so as to cause fusion of PS vesicles and aggregation of PC vesicles. The vesicles are also rendered permeable to hydrophilic molecules like carboxyfluorescein. The variant peptide with the lysine residue in the hydrophobic region also has the ability to perturb lipid bilayers of model membranes.

Alkaline Phosphatase↗
PubMed1987

Circular dichroism studies on a synthetic peptide corresponding to the membrane-spanning region of vesicular stomatitis virus G protein and its fatty acyl derivative.

The conformations of synthetic peptides Lys-Phe-Phe-Phe-Ile-Ile-Gly-Leu-Ile-Ile-Gly-Leu-Phe-OCH3 and Lys(epsilon-palmitoyl)-Phe-Phe-Phe-Ile-Ile-Gly-Leu-Ile-Ile-Gly-Leu-Phe-O CH3, which constitute a part of the membrane-spanning region of the vesicular stomatitis virus G protein, have been studied by circular dichroism (CD) spectroscopy. Secondary structural features are observed for both peptides in trifluoroethanol, methanol, aqueous mixtures of trifluoroethanol and methanol and in a micellar environment. In trifluoroethanol, the CD spectra indicate the presence of a helical conformation, whereas in aqueous mixtures of organic solvents, both helical and beta-conformations are observed. While fatty acid acylation does not directly modulate peptide conformation, it promotes self-association of the acylated peptide and association with micelles. In a micellar environment, the acylated peptide adopts an alpha-helical conformation.

Circular Dichroism↗
PubMed1987

Circular dichroism studies on synthetic peptides corresponding to the cleavage site region of precursor proteins.

The conformations of synthetic peptides which span the region in which the precursor part of proteins (signal sequences) destined for export are cleaved by signal peptidases, were investigated by circular dichroism spectroscopy. Pentapeptides comprising amino acids only from the carboxy-terminus of signal sequences or the amino terminus of the mature protein do not have any preferred conformation in a variety of solvents. Octa- and nonapeptides containing amino acids from the carboxy-terminal protion of signal sequences and the amino-terminus of the mature portions of precursor proteins tend to adopt beta-turn conformations in trifluoroethanol and micelles of sodium dodecylsulphate. Hence, in addition to the distribution of amino acids with small side chains at the carboxy terminus of signal sequences, it is conceivable that signal peptidases also recognize a beta-turn conformation in the cleavage site region of precursor proteins.

Amino Acid Sequence↗
PubMed1986

Circular dichroism studies on the signal sequence of E. coli alkaline phosphatase indicate the presence of both alpha-helix and beta-structure in hydrophobic environments.

The conformations of a synthetic peptide corresponding to the signal sequence of E. coli alkaline phosphatase, Lys-Gln-Ser-Thr-Ile-Ala-Leu-Ala-Leu-Leu-Pro-Leu-Leu-Phe-Thr-Pro-Val-Thr- Lys- Ala-OCH3, have been examined in different environments by circular dichroism spectroscopy. In trifluoroethanol, methanol and aqueous mixtures of these solvents, the signal peptide has largely random conformation (approximately 80%) with small amounts of alpha-helix and beta-structure. However, in micellar environment, there is a significant increase in ordered conformation with both alpha-helix and beta-structure being present, unlike in other signal sequences reported in the literature, where only the alpha-helical conformation has been observed. Hence, an alpha-helical conformation may not be as stringent a requirement as overall hydrophobicity for recognition of signal sequences by the cell's export machinery.

Alkaline Phosphatase↗
PubMed1985

Circular dichroism studies on synthetic signal peptides.

Circular dichroism studies on synthetic peptides corresponding to the signal sequences of chicken lysozyme and Escherichia coli proteins, lambda-receptor and lipoprotein, have been carried out in trifluoroethanol. The peptides, (CH3)3-C-O-CO-Thr-Leu-Lys-Lys-Leu-Pro-Leu-Ala-Val-Ala-Val-Ala-Ala-Gly- Val-Met-Thr-Ala- Ala-Met-Ala-OCH3, (CH3)3-C-O-CO-Met-Lys-Ser-Leu-Leu-Ile-Leu-Val-Leu-Cys(benzyl)- Phe-Leu-Pro- Leu-Ala-Ala-Leu-Gly-OH and (CH3)3-C-O-CO-Leu-Val-Leu-Gly-Ala-Val-Ile-Leu-Gly- Thr-Thr-Leu-Leu- Ala-Gly-OCH3, corresponding to the signal sequences of lambda-receptor, lysozyme and the hydrophobic region of lipoprotein, respectively, show two negative bands at approx. 205 and 220 nm, characteristic of an alpha-helical conformation. Secondary structural features are discernible even in the shorter, 12-residue carboxy-terminal fragments of these signal peptides. A comparison of the conformation of the amino-terminal, central and carboxy-terminal fragments of lipoprotein signal sequence indicates that the central octapeptide fragment is more structurally ordered compared to the amino- and carboxy-terminal fragments.

Amino Acid Sequence↗
PubMed1984

Interaction of synthetic signal sequence fragments with model membranes.

Peptide fragments corresponding to the signal sequence of chicken lysozyme, labelled with the fluorescent 5-dimethylaminonaphthalene-1-sulfonyl (dansyl) group have been synthesized. The emission characteristics and fluorescence polarization of the dansyl group have been used to study the interaction of signal sequence fragments with liposomes. The peptide fragments bind to liposomes and are associated with the hydrophobic core of the bilayer.

Dansyl Compounds↗
PubMed1982

Membrane channel-forming polypeptides. Aqueous phase aggregation and membrane-modifying activity of synthetic fluorescent alamethicin fragments.

Synthetic fragments of the membrane channel-forming polypeptide, alamethicin, have been labeled with a fluorescent 5-dimethylaminonaphthalene-1-sulfonyl (dansyl) group at the NH2-terminal. Seventeen and 13-residue fluorescent peptide esters have been shown to translocate divalent cations in unilamellar liposomes and uncouple oxidative phosphorylation in rat liver mitochondria. The corresponding peptide acids also exhibit membrane-modifying activity, whereas the shorter fragments are inactive. Aggregation of fluorescent peptides in aqueous solutions leads to a marked blue shift and enhancement of the dansyl group emission spectrum. "Critical micelle" concentrations may be determined for the association of peptides. The longer peptides aggregate at lower concentrations than the short peptides, with the ease of aggregation following a trend similar to that for functional activity. The peptide acids aggregate only in media of high ionic strength. The peptide ester aggregates are stabilized by increasing salt concentration and dissociated by urea, suggestive of hydrophobic stabilization of the aggregates. The enthalpy of association for the 10- and 17-residue peptide esters is estimated to be between -1 and -3 kcal mol-1 of monomer. The aqueous phase aggregation of channel-forming peptides at low concentrations suggests that preformed aggregates may be inserted into the membrane to constitute functional channels.

Alamethicin↗