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R Nagaraj

Publications and source records attributed to R Nagaraj.

88 records · Page 5Linked to original sources
PubMed1982

Ionophore-mediated transmembrane movement of divalent cations in small unilamellar liposomes: an evaluation of the chlortetracycline fluorescence technique and correlations with black lipid membrane studies.

Conceptual advances in the field of membrane transport have, in the main, utilized artificial membranes, both planar and vesicular. Systems of biological interest, viz., cells and organelles, resemble vesicles in size and geometry. Methods are, therefore, required to extend the results obtained with planar membranes to liposome systems. In this report we present an analysis of a fluorescence technique, using the divalent cation probe chlortetracycline, in small, unilamellar vesicles, for the study of divalent cation fluxes. An ion carrier (X537 A) and a pore former (alamethicin) have been studied. The rate of rise of fluorescence signal and the transmembrane ion gradient have been related to transmembrane current and potential, respectively. A second power dependence of ion conduction - including the electrically silent portion thereof - on X537 A concentration, has been observed. An exponential dependence of "current" on "transmembrane potential" in the case of alamethicin is also confirmed. Possible errors in the technique are discussed.

Alamethicin↗
PubMed1981

Fluorescent alamethicin fragments. A study of membrane activity and aqueous phase aggregation.

The linear polypeptide antibiotic alamethicin is known to form channels in artificial lipid membranes. Synthetic 13- and 17-residue alamethicin fragments, labelled with a fluorescent dansyl group at the N-terminus, have been shown to translocate divalent cations across phospholipid membranes and to uncouple oxidative phosphorylation in rat liver mitochondria, in a manner analogous to the parent peptides. From studies of the aqueous phase aggregation behavior of the peptides, as well as their interaction with rat liver mitochondria, it is concluded that the interaction of the peptides with membranes is a complex process, probably involving both aqueous and membrane phase aggregation.

Alamethicin↗
PubMed1981

Conformations of synthetic alamethicin fragments. Evidence for 310 helical folding from 270-MHz hydrogen-1 nuclear magnetic resonance and circular dichroism studies.

1H NMR studies at 270 MHz on the synthetic alamethicin fragments Z-Aib-Pro-Aib-Ala-Aib-Ala-OMe (1-6), Boc-Gln-Aib-Val-Aib-Gly-Leu-Aib-OMe (7-13), Boc-Leu-Aib-Pro-Val-Aib-OMe (12-16), and Boc-Gly-Leu-Aib-Pro-Val-Aib-OMe (11-16) have been carried out in CDCl3 and (CD3)2SO. The intramolecularly hydrogen bonded amide hydrogens in these peptides have been delineated by using solvent titration experiments and temperature coefficients of NH chemical shifts in (CD3)2SO. All the peptides adopt highly folded structures, characterized by intramolecular 4 leads to 1 hydrogen bonds. The 1-6 fragment adopts a 310 helical conformation with four hydrogen bonds, in agreement with earlier studies (Rao, Ch. P., Nagaraj, R., Rao, C. N. R., & Balaram, P. (1980) Biochemistry 19, 425-431]. The 7-13 fragment also appears to be folded in 310 helical fashion, although some intramolecular hydrogen bonds are loosened in (CD3)2-SO). The 11-16 fragment favors a structure with three intramolecular hydrogen bonds of the 4 leads to 1 and 5 leads to 1 types. CD studies in trifluoroethanol suggest a helical structure for the 1-13 and 1-17 fragments and alamethicin, while IR studies support a helical structure for the 1-13 peptide, stabilized by intramolecular hydrogen bonding. On the basis of fragment conformations and earlier studies of the stereochemistry of alpha-aminoisobutyric acid (Aib) containing peptides, a structure is suggested for the alamethicin backbone. A largely 310 helical folding pattern is postulated for the hydrophobic 1-17 segments, with a polar flexible C-terminal tripeptide.

Alamethicin↗
PubMed1981

Determination of beta-turn conformation by laser Raman spectroscopy.

To correlate the Raman frequencies of the amide I and III bands to beta-turn structures, three peptides shown to contain beta-turn structure by x-ray diffraction and NMR were examined. The compounds examined were tertiary (formula: see text). The amide I band of these compounds is seen at 1,668, 1,665, and 1,677 cm-1, and the amide III band appears at 1,267, 1,265, and 1,286 cm-1, respectively. Thus, it is concluded that the amide I band for type III beta-turn structure appears in the range between 1,665 and 1,677 cm-1 and the amide III band between 1,265 and 1,286 cm-1.

Amides↗
PubMed1981

1H N.M.R. studies of protected alpha-aminoisobutyric acid containing peptides. Chemical shift nonequivalence of benzyloxycarbonyl methylene protons.

The benzylic methylene protons in a large number of benzyloxycarbonyl alpha-aminoisobutyric acid (Z-Aib) containing peptides, show chemical shift nonequivalence. The magnitude of the geminal nonequivalence is correlated with the involvement of the urethane carbonyl group, in an intramolecular hydrogen bond. Studies of the model compounds Z-Aib-Aib-Ala-NHMe, and Z-Aib-Aib-Aib-Pro-OMe clearly establish the presence of intramolecular hydrogen bonds, involving the urethane CO group. In both compounds marked anisochrony of the benzylic methylene protons is demonstrated. In Z-Aib-Aib-Pro-OMe, where a 4 leads to 1 hydrogen bonded beta-turn is not possible, the benzylic-CH2-protons appear as a singlet in CDCl3 and have a very small chemical shift difference in (CD3)2SO. The observation of such nonequivalence is of value in establishing whether the amino terminal Aib-Pro beta-turn is retained in large peptide-fragments of alamethicin.

Aminoisobutyric Acids↗
PubMed1980

Infrared studies on the conformation of synthetic alamethicin fragments and model peptides containing alpha-aminoisobutyric acid.

Infrared studies of synthetic alamethicin fragments and model peptides containing alpha-aminoisobutyric acid (Aib) have been carried out in solution. Tripeptides and larger fragments exhibit a strong tendency to form beta turns, stabilized by 4 leads to 1 10-atom hydrogen bonds. Dipeptides show less well-defined structures, though C5 and C7 conformations are detectable. Conformational restrictions imposed by Aib residues result in these peptides populating a limited range of states. Integrated intensities of the hydrogen-bonded N-H stretching band can be used to quantitate the number of intramolecular hydrogen bonds. Predictions made from infrared data are in excellent agreement with nuclear magnetic resonance and X-ray diffraction studies. Assignments of the urethane and tertiary amide carbonyl groups in the free state have been made in model peptides. Shifts to lower frequency on hydrogen bonding are observed for the carbonyl groups. The 1--6 segment of alamethicin is shown to adopt a 3(10) helical structure stabilized by four intramolecular hydrogen bonds. The fragments Boc-Leu-Aib-Pro-Val-Aib-OMe (12--16) and Boc-Gly-Leu-Aig-Pro-Val-Aib-OMe (11--16) possess structures involving 4 leads to 1 and 5 leads to 1 hydrogen bonds. Supporting evidence for these structures is obtained from proton nuclear magnetic resonance studies.

Alamethicin↗
PubMed1980

Rotational isomerism about the C alpha-CO bond in proline derivatives. 1H and 13C N.m.r. studies of benzyloxycarbonyl-Pro-N-methylamide and pivaloyl-pro-N-methylamide.

The 270 MHz 1H n.m.r. spectrum of benzyloxycarbonyl-Pro-N-methylamide in CDCl3 is exchange broadened at 293 degrees K. Spectral lines due to two species are frozen out at 253 degrees K and a dynamically averaged spectrum is obtained at 323 degrees K. A selective broadening of the C beta and C gamma resonances in the 13C n.m.r. spectrum is observed at 253 degrees K, with a splitting of the C beta and C gamma resonances into a pair of lines of unequal intensity. A similar broadening of C beta and C gamma peaks is also detected in pivaloyl-Pro-N-methylamide where cis-trans interconversion about the imide bond is precluded by the bulky t-butyl group. The rate process is thus attributed to rotation about the C alpha-CO bond (psi) and a barrier (delta G#) of 14 kcal mol-1 is estimated. 13C n.m.r. data for pivaloyl-Pro-N-methylamide in a number of solvents is presented and the differences in the C beta and C gamma chemical shifts are interpreted in terms of rotational isomerism about the C alpha-CO bond.

Calorimetry↗
PubMedDate not supplied

Immunobiology of a synthetic luteinizing hormone receptor peptide 21-41.

Immunization of adult male rabbits with a synthetic luteinizing hormone-receptor peptide (LH-RP; representing amino-acids 21-41 of the extracellular domain of the rat LH receptor) resulted in production of high-titer antibodies capable of interacting with particulate and cell-based LH receptors. The antibody produced was able to inhibit binding of 125I-labeled human chorionic gonadotropin (hCG) to a particulate sheep luteal LH receptor preparation by 40%-50%. Maximal inhibitory activity was correlated with high antibody titer. Immunocytometry revealed that the antibody could directly bind to cells having LH receptors, such as rat granulosa and Leydig cells. The antibodies recognized a 77-kilodalton membrane protein in Western blots of mouse testicular extracts. Interaction of endogenous Leydig cell LH receptor with the LH-RP antibody resulted in both hormone agonist and antagonistic activities. The hormone-mimicking activity (increase in serum testosterone over control) was confined only to the early phase of immunization when the antibody titer was low. Blockade of LH receptor during the later part of immunization resulted in a significant reduction in serum testosterone over controls and inhibition of spermatogenesis. DNA flow cytometry showed that a specific and significant inhibition of meiosis (transformation of primary spermatocytes to round and elongated spermatids P < .01) and spermiogenesis (transformation of round spermatids to elongated spermatids P < .0001) occurred following blockade of LH function.

Amino Acid Sequence↗
PubMedDate not supplied

Is the role of fatty acids only to provide membrane-anchor in fatty acylated proteins?

A large number of proteins on the eukaryotic cell surface that play an important role in cellular metabolism are covalently modified with fatty acids like palmitic and myristic acids. While some of these proteins have transmembrane spanning segments, many others do not. Early hypothesis was that this co or posttranslational modification helped in membrane-association and the fatty acyl chain provided a stable membrane anchor. We have investigated the structure of peptides with these modifications and also their interaction with membranes. Our results indicate that the fatty acylated peptides, especially when the peptide segment is not hydrophobic, do not have strong affinity for membranes. The recent observations about the dynamic nature of fatty acid acylation as well as the importance of protein-protein interactions for function in fatty-acylated proteins suggest that membrane-association may involve factors other than only the fatty acid, either myristic or palmitic. Revised models depicting the possible role of fatty acids in modulating protein-protein interaction and their dynamics is presented.

Acylation↗