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Biomedical subjects

R K Pandey

Publications and source records attributed to R K Pandey.

At least 73 records · Page 4Linked to original sources
PubMed1986

NMR study of the molecular and electronic structure of the heme cavity of Aplysia metmyoglobin. Resonance assignments based on isotope labeling and proton nuclear Overhauser effect measurements.

The 1H NMR characteristics of the high-spin metmyoglobin from the mollusc Aplysia limacina have been investigated and compared with those of the myoglobin (Mb) from sperm whale. Aplysia metMb exhibits a normal acid----alkaline transition with pK approximately 7.8. In the acidic form, the heme methyl and meso proton resonances have been assigned by 1H NMR using samples reconstituted with selectively deuterated hemins and in the latter case by 2H NMR as well. On the basis of the methyl peak intensities and shift pattern, heme rotational disorder could be established in Aplysia Mb; approximately 20% of the protein exhibits a reversed heme orientation compared to that found in single crystals. Three meso proton resonances have been detected in the upfield region between -16 and -35 ppm, showing that the chemical shift of such protons can serve as a diagnostic probe for a pentacoordinated active site in hemoproteins, as previously shown to be the case in model compounds. The temperature dependence of the chemical shift of the meso proton signals deviates strongly from the T-1 Curie behavior, reflecting the presence of a thermally accessible Kramers doublet with significant S = 3/2 character. Nuclear Overhauser effect, NOE, measurements on Aplysia metMb have provided the assignment of individual heme alpha-propionate resonances and were used to infer spatial proximity among heme side chains. The hyperfine shift values for assigned resonances, the NOE connectivities, and the NOE magnitudes were combined to reach a qualitative picture of the rotational mobility and the orientation of the vinyl and propionate side chains of Aplysia metMb relative to sperm whale MbH2O.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1985

Proton NMR study of yellowfin tuna myoglobin in whole muscle and solution. Evidence for functional metastable protein forms involving heme orientational disorder.

Proton NMR spectra of met-aquo-myoglobin have been recorded in whole dark muscle from yellowfin tuna for the first time; in addition, spectra of the met-aquo, met-cyano, and deoxy forms were recorded in solution. The number of resolved methyl resonances in the met-aquo and met-cyano derivatives of the purified protein indicates a molecular heterogeneity, with the two species present in a ratio of approximately 3:2. These same two species, in very similar environments, are found in whole muscle. Upon reconstitution of the protein, the ratio of the two species is approximately 1:1. Specific isotope labeling in the met-cyano form reveals an interchange of 5-methyl and 8-methyl environments that is characteristic of heme orientational disorder about the alpha, gamma-meso axis. Incubation of the met-cyano protein at 37 degrees C shows that the two species are interconvertible, with the minor component declining to about one-eighth of that of the major component. This indicates that one of the two orientations is favored thermodynamically. The rate of equilibration for the met-aquo protein is exceedingly slow (half-life greater than 30 days), some 10(2) slower than for the analogous sperm whale derivative. The "reversed" heme orientation is therefore present in muscle as a kinetically trapped or metastable species, suggesting that the last step in the biosynthesis of myoglobin is similar to that of in vitro reconstitution. The presence of both heme orientations in myoglobin in whole muscle proves that heme orientational disorder is a physiological phenomenon.

Amino Acid Sequence↗
PubMed1985

1H NMR characterization of metastable and equilibrium heme orientational heterogeneity in reconstituted and native human hemoglobin.

A proton nuclear magnetic resonance study of the reaction of apohemoglobin A with both oxidized and reduced hemes reveals that at least two slowly interconverting species are initially formed, only one of which corresponds to the native proteins. Reconstitutions with isotope-labeled hemes reveal that the hyperfine-shift patterns for heme resonances in the metazido derivatives differ for the two species by interchange of heme environment characteristic of heme orientational disorder about the alpha, gamma-meso axis, as previously demonstrated for myoglobin [La Mar, G. N., Davis, N. L., Parish, D. W., & Smith, K. M. (1983) J. Mol. Biol. 168, 887-896]. Careful scrutiny of the 1H NMR spectrum of freshly prepared hemoglobin A (Hb A) reveals that characteristic resonances for the alternate heme orientation are present in both subunits, clearly demonstrating that "native" Hb A possesses an important structure heterogeneity. It is observed that this heterogeneity disappears with time for one subunit but remains unchanged in the other. This implies that a metastable disordered state in vivo involves the alpha subunit and an equilibrium disordered state both in vivo and in vitro is involved within the beta subunit. The presence of metastable disorder in fresh blood suggests an in vivo hemoglobin assembly from apoprotein and heme that is similar to the in vitro reconstitution process. The slow equilibration and known lifetimes for erythrocytes provide a rationalization for the presence of detectable metastable states. The implications of such heme disorder for Hb function are discussed.

Binding Sites↗
PubMed1984

The oxidation of hemins by microsomal heme oxygenase. Structural requirements for the retention of substrate activity.

The substrate specificity of microsomal heme oxygenase from rat liver was studied by introducing systematic structural changes in the array of substituents of the protohemin IX rings. Replacement of the vinyls by methyl groups resulted in hemins which were excellent substrates of the heme oxygenase. Replacement of the 4-vinyl group by a propionic acid chain (harderohemin), decreased substrate activity to 40%. The replacement of the vinyls by formyl residues strongly decreased substrate activity but the hemins were still substrates of heme oxygenase. The oxidation rates of Spirographis hemin and of 2,4-diformyldeuterohemin IX showed a time lag which was absent when isoSpirographis hemin was used as a substrate. This lag could be attributed to the formation of a transient hemiacetal between the 2-formyl group and the alpha-mesohydroxy residue. The isomeric protohemins I, XI, and XIV (Fischer's notation) were examined as possible substrates of microsomal heme oxygenase. In these protohemins the array of substituents of rings A and B was the same as in protohemin IX, but the methyl and propionic acid residues of rings C and D were at different positions from those of protohemin IX. None of them had substrate activity, indicating that the presence of two vicinal propionic acid side-chains at C6 and C7 was necessary for substrate activity. A hemin with only one propionic acid residue at C5 was not a substrate of the enzyme, either. When the propionic acid residues of protohemin IX were replaced by butyric acid residues, substrate activity decreased to 50% (as compared to protohemin IX), while when they were replaced by acetic acid residues, the substrate activity was entirely suppressed. The addition of dimethyl sulfoxide (25 mM) to the incubation mixture enhanced the oxidation of hemins with non-polar substituents in rings A and B by about 35%, while it was without effect on hemins with polar substituents in the same rings.

Animals↗
PubMed1984

The specificity of biliverdin reductase. A study with different biliverdin types.

The specificity of rat liver biliverdin reductase was examined with the help of a series of synthetic biliverdins. The mixture of the four biliverdin isomers obtained by the chemical oxidation of protohemin I, protohemin XI, protohemin XIV and harderohemin were used as substrates of biliverdin reductase and were compared with the mixture of biliverdins IX alpha-delta. Biliverdin reductase (molecular form 1) from rat liver efficiently reduced the isomer mixtures of biliverdins I, XI, XIV and harderobiliverdins to the bilirubins in the presence of NADPH. The enzymatic reduction of the different biliverdin types was studied in the presence of different NADPH analogues. NADPH could be replaced by NADH, 3-acetyl NADPH and deamino-NADPH with retention of a good substrate activity only in the case of biliverdins of types I and IX and harderobiliverdins. Biliverdins XI and XIV were efficiently reduced only in the presence of NADPH and an excess of NADH. Bactobilin III-alpha was also very efficiently reduced by biliverdin reductase in the presence of both NADPH and NADH but not in the presence of the other analogues. These results indicate that biliverdin reductase reduced bilitriene acids substituted with non-polar and polar residues.

Animals↗
PubMed1982

Spore differentiation in relation to certain antibiotics in the blue-green alga Nodularia spumigena Mertens.

Induction of spore differentiation is achieved within three days in Nodularia spumigena by incubating the cultures at 35 degrees C in the light. Morphologically detectable sporulation and spore germination could not occur in the presence of chloramphenicol, streptomycin and penicillin. But chloramphenicol-supplemented cultures developed prominent cyanophycin granules. Synthesis of these granules seems to be a non-ribosomal phenomenon.

Anti-Bacterial Agents↗
PubMed1981

Hepatic copper in Indian childhood cirrhosis.

Forty-three liver biopsies histologically diagnosed as Indian childhood cirrhosis were studied with the object of investigating accumulation of copper and copper-associated protein in the hepatocytes as demonstrated by the rhodanine and orcein stains respectively. The findings reveal that, in almost all cases, there was an increased accumulation of copper and copper-associated protein, accompanied by the presence of numerous Mallory bodies. It is postulated that the accumulation of copper in the hepatocytes might be the result of a metabolic defect and might be aetiologically related to the liver cell injury and the subsequent development of Indian childhood cirrhosis.

Biopsy↗
PubMed1981

Poly (delta-L-ornithine).

Poly (delta-L-Orn) is an example of an iso-polypeptide (i.e. variant of the usual poly a-peptide chain), where the a-carboxyl and delta-amino groups of ornithine are used in polymerization while the a-amino groups form the side chain. A procedure for the synthesis of this iso-polypeptide is described. Circular dichroism studies of poly (delta-L-Orn) and its Na-Boc derivative suggest that these polymers might adopt a conformation in solution similar to the beta-pleated sheet.

Circular Dichroism↗