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D Chatterji

Publications and source records attributed to D Chatterji.

54 records · Page 3Linked to original sources
PubMed1988

An improved method for the purification of DNA-dependent RNA polymerase from Escherichia coli.

DNA-dependent RNA polymerase from Escherichia coli was purified further by elution through heparin-Sepharose CL-6B column after the enzyme was obtained, partially purified, using Burgess and Jendrisak's method [(1975)Biochemistry 14, 4634] The total yield of the pure protein was 10 mg from 50 g of E.coli cells. The method was found to be very reproducible and convenient. The enzyme preparation had 60% active molecules and the elongation rate of RNA synthesis by this enzyme was measured to be 11 bases/s over delta D111 T7 DNA.

Chromatography, Affinity↗
PubMed1987

DNA bound RNA polymerase activity of the yeast nuclei.

A preparation of nuclei from yeast cells is shown to be capable of autodigesting its chromatin in the presence of a series of magnesium containing buffers having different concentrations of potassium. The 'digests' or 'extracts' prepared in this manner contain an active transcription complex which can transcribe endogenous as well as exogenous DNA template. The effects of heparin and actinomycin D on the in vitro transcription by these complexes confirm the presence of RNA polymerase in DNA bound form. Further purification of this transcription complex is attempted by sucrose density gradient centrifugation. Circular dichroic measurements show that the nuclear DNA exists in compact state in the extracts.

Cell Nucleus↗
PubMed1986

Terbium(III)-induced conformational transition in poly(dG-dC) fluorescence and circular dichroic studies.

Poly(dG-dC) in 60% aqueous alcohol exhibits the characteristic inversion of the circular dichroism spectrum associated with the formation of left-handed helix. Upon complexation with Tb3+, poly(dG-dC) in this medium induces marked enhancement of the Tb(III) fluorescence emission at 488 and 545 nm, when excited at 290 nm. The degree of fluorescence enhancement is dependent on the concentration of Tb(III) at a fixed poly(dG-dC) concentration. Neither poly(dG-dC) in water nor poly(dA-dT) in water or 60% alcohol, causes any significant fluorescence spectral changes of Tb3+. Tb(III)-poly(dG-dC) in 60% alcohol shows circular dichroic spectra associated with a broad positive molar ellipticity ranging from 6000 to 10 000 degree X cm2 X dmol-1 between 270 and 280 nm, and a small negative band around 240 nm.

Circular Dichroism↗
PubMed1986

Spatial relationship between the intrinsic metal in the beta subunit and cysteine-132 in the sigma subunit of Escherichia coli RNA polymerase: a resonance energy transfer study.

Fluorescence excited-state energy transfer measurements were carried out between the N-(1-pyrene)maleimide (PM)-labeled sigma subunit and Co in the beta subunit of Co-Zn RNA polymerase (RPase). sigma subunit with or without PM labeling was cleaved with 2-nitro-5-thiocyanobenzoic acid, and the reaction products were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. One molecule of the fluorescent probe (PM) was found to be attached to the cysteine-132 residue of the sigma subunit. When excited at 340 nm, the fluorescence emission bands from 380 to 420 nm of PM-labeled sigma overlap with the charge transfer absorption band of Co-Zn RPase around 400 nm. Based on Förster's equation, the R0 values for the donor-acceptor pair were calculated to be 21.5 and 22 A in the absence and presence of template analog (dA-dT)60, respectively. Using these R0 values and the observed energy transfer efficiencies, the distance between the cysteine-132 of the sigma subunit and Co located at the initiation site of the beta subunit was calculated to be 22 A with or without the template present, indicating that no major conformational change of the enzyme was induced upon template binding. However, a small but significant change in the above distance was observed upon the addition of ATP to RPase in the presence (dA-dT)60 but not in the absence of (dA-dT)60 template. The biological implications of these observations are discussed.

Binding Sites↗
PubMed1985

Interaction of bovine seminalplasmin with Escherichia coli RNA polymerase in the presence of rifampicin.

The interaction of bovine seminalplasmin and rifampicin with E. coli RNA polymerase was studied using fluorescence spectroscopy. Both seminalplasmin and rifampicin are known to be the inhibitors for the initiation of RNA synthesis in E. coli. Rifampicin quenced the intrinsic fluorescence of RNA polymerase and seminalplasmin when excited at 280 nm. However, excess of seminalplasmin reversed the quenching of RNA polymerase fluorescence by rifampicin. Upon addition of rifampicin to the seminalplasmin-RNA polymerase complex, no change in fluorescence spectrum was observed. It appeared that although rifampicin could form complexes with RNA polymerase and seminalplasmin alone, no binding domain was available for rifampicin in the RNA polymerase-seminalplasmin complex. These observations are discussed in the light of the 'initiation site' of E. coli RNA polymerase.

Animals↗
PubMed1984

Nuclear magnetic resonance studies on the role of intrinsic metals in Escherichia coli RNA polymerase. Effect of DNA template on the nucleotide-enzyme interaction.

Nuclear magnetic resonance studies were performed to investigate the effect of DNA template on the interaction of initiating nucleotide ATP with Escherichia coli RNA polymerase (RPase) in which one of the two intrinsic Zn ions was substituted with a Co(II) (Co-Zn RPase) or Mn(II) (Mn-Zn RPase) ion. This intrinsic metal ion is located at the initiation site in the beta subunit of RPase. The paramagnetic effects of Co-Zn and Mn-Zn RPases on the relaxation rates of 1H- and 31P-nuclei of ATP were used to determine the distances from the intrinsic metal to various atoms of ATP bound at the initiation sites in the presence of DNA. The distances from the metal to H2, H8, H1', alpha-P, beta-P, and gamma-P atoms were estimated to be 6.7 +/- 0.9, 4.1 +/- 0.6, 6.0 +/- 1.2, 7.5 +/- 0.8, 9.4 +/- 1.0, and 9.8 +/- 1.0 A, respectively. These distances were compared with those measured in the absence of DNA (Chatterji, D., and Wu, F. Y.-H. (1982) Biochemistry 21, 4657). In both the presence and absence of DNA, the close proximity between the intrinsic metal and the H8 atom strongly indicates that the metal is coordinated directly to the base moiety of ATP. Such a coordination may provide a structural basis for the selection of a purine nucleotide during the initiation process. The presence of DNA causes the H2 atom to move away (greater than 2 A) from the intrinsic metal, whereas all three phosphorus atoms shift closer (greater than 3 A) toward the metal. The possible mechanistic implications of the conformational alteration of ATP at the initiation site induced by the DNA template is discussed.

DNA↗
PubMed1984

Cytosine arabinoside cerebrospinal fluid kinetics.

To better characterize the disposition of cytosine arabinoside (Ara-C) in cerebrospinal fluid (CSF), its kinetics were studied in seven patients with meningeal leukemia in complete remission. After intraventricular injection of 30 mg Ara-C, CSF and plasma samples were obtained over a 24-hr period. Ara-C levels were measured by a reverse-phase HPLC assay (with a sensitivity of 0.5 microM in CSF and 1.0 microM in plasma) that readily separated Ara-C from its major metabolite uracil arabinoside (Ara-U). Elimination of Ara-C from CSF followed a biphasic pattern, with an initial t1/2 of 1 hr and a terminal t1/2 of 3.4 hr. Ara-C clearance from CSF was 0.42 ml/min, suggesting that drug elimination was primarily by CSF bulk flow. The ratio of the AUC of Ara-U to the AUC of Ara-C was 0.08, indicating only minor metabolism of Ara-C to Ara-U in CSF, in contrast to that after systemic Ara-C. Despite initial CSF Ara-C concentrations exceeding 2 mM, Ara-C was not detectable in plasma in any patient. Intraventricular Ara-C results in very high levels in CSF, but systemic tissues are relatively spared from exposure to Ara-C.

Adolescent↗
PubMed1983

Thermal stability of a long-acting analogue of luteinizing hormone releasing hormone (D-trp6-pro9-NEt-LHRH).

The stability of solutions of a long-acting analogue of luteinizing hormone releasing hormone (D-trp6-pro9-NEt-LHRH [LHRHa] after heating to 60C for 5 days, after repeated freezing and thawing, and after refrigeration at 4C for 8 days has been examined. None of the treatments caused a detectable alteration in the HPLC profile, and none caused a significant change in biological activity in vivo. It is concluded that D-trp6-pro9-NEt-LHRH can withstand mild heating, repeated freezing and thawing, and short-term refrigeration without apparent change in HPLC profile or biological activity. It is also concluded that the results obtained with the HPLC method correlate well with the results from the in vivo bioassay.

Animals↗
PubMed1982

Selective substitution in vitro of an intrinsic zinc of Escherichia coli RNA polymerase with various divalent metals.

A simple in vitro substitution method involving a sequential denaturation--reconstitution process was developed to substitute selectively one of the two intrinsic Zn ions in Escherichia coli RNA polymerase with Co, Mn, Ni, or Cu ion. The resultant metal hybrid Co-Zn, Mn-Zn, Ni-Zn, and Cu-Zn RNA polymerases possess 100, 100, 60, and 17% of the enzymatic activity of the reconstituted Zn-Zn enzyme, respectively. The substituted metal was found to be located in the beta subunit of the polymerase which contains the substrate binding site. The biochemical and physical properties of these metal-substituted polymerases were compared with those of the native Zn enzyme. Co-Zn and Ni-Zn core polymerases exhibit characteristic absorption spectra in the near-UV and visible region, while Mn-Zn and Cu-Zn enzymes do not. The Co-Zn enzyme shows two major peaks at 400 nm (epsilon = 3000) and 475 nm (epsilon = 2700), while the Ni-Zn enzyme exhibits a major peak at 462 nm (epsilon = 8000). The difference absorption spectrum of Ni-Zn core polymerase could be perturbed by the addition of substrate ATP but not by UTP in the absence of template and Mg(II) ion. These observations suggest that the substituted metal was located at the initiation site of the enzyme. The various metal hybrid enzymes do not differ appreciably in their abilities to incorporate noncomplementary nucleotide or deoxyribonucleotide into RNA product. It was found, however, that the difference in enzymatic activities of these metal hybrid enzymes resides at least partly in the initiation step of RNA synthesis.

Cations, Divalent↗
PubMed1982

Direct coordination of nucleotide with the intrinsic metal in Escherichia coli RNA polymerase. A nuclear magnetic resonance study with cobalt-substituted enzyme.

Nuclear magnetic resonance studies were performed with Escherichia coli RNA polymerase (RPase) in which one of the two intrinsic Zn ions was substituted with Co(II) ion (Co-Zn RPase). The Co ion was located in the beta subunit which contains the initiation site of the enzyme. The paramagnetic effect of Co-Zn RPase on the relaxation rates of rapidly exchanging water protons indicated that the Co ion was accessible to solvent. There were approximately two water molecules in the inner coordination sphere of the Co ion, one of which could be replaced by the substrate adenosine 5'-triphosphate (ATP) or the initiator adenylyl-(3' leads to 5')-adenine (ApA) but to a much less extent by uridine 5'-triphosphate. The effects of ATP and ApA did not require the presence of DNA or Mg (II) ions, and their Kd values were estimated to be 0.15 and 0.075 mM, respectively. These results showed that the Co ion was at the initiation site. From the measurements of the paramagnetic effects of Co-Zn RPase on the relaxation rates of 1H and 31P nuclei of ATP, the distances from the intrinsic Co ion to H2, H8, and H1' were determined to be 4.1 +/- 0.6, 3.6 +/- 0.5, and 6.8 +/- 0.8 A, respectively, and those to the alpha-, beta-, and gamma-phosphorus atoms were 10.5 +/- 0.7, 15.1 +/- 1.1, and 14.1 +/- 0.8 A, respectively. These spatial relationships clearly indicate that the Co ion is directly coordinated to the base moiety of ATP bound at the initiation site. Thus, the intrinsic metal in the beta subunit of RNA polymerase may play a regulatory role in the recognition of the initiating nucleotide and may orient the nucleotide in a stereospecific position for the initiation.

Adenosine Monophosphate↗
PubMed1979

Site of action of RNase I on the 50 S ribosome of Escherichia coli and the association of the enzyme with the partially degraded subunit.

The 50 S ribosome of Escherichia coli is partially degraded by RNase I in presence of a high concentration of Mg2+ (10 to 20 mM); the partially degraded subunit becomes resistant to the further action of RNase I. The latter remains latent in association with the subparticle as in case of 30 S ribosome (Neu, H.C., and Heppel, L.A. (1954) Proc. Natl. Acad. Sci. U.S.A. 51, 1267-1274). As a result of nucleolytic action, 23 S RNA is degraded to a smaller size and four proteins (L4, L10, L7/L12) are released from the subunit. From the location of these proteins, it appears that the primary site of action of RNase I is the central protuberance of the armchair model proposed for the subunit (Stoffler, G., and Whitman, H.G. (1977) in Molecular Mechanisms of Protein Biosynthesis (Weissbach, H., and Pestka, S., eds) pp. 117-144, Academic Press, New York).

Escherichia coli↗
PubMed1975

Acylation of ascorbic acid in water.

The acylation of ascorbic acid by acetic anhydride was studied in water at 25 degrees. The results indicate that the initial products of reactions between these two compounds were acetic acid and 3-o-acetylascorbic acid. The latter product then underwent two parallel reactions: hydrolysis to ascorbic and acetic acids and an intramolecular O leads to O acyl migration to yield-O-acetylascorbic acid. The former reaction was predominant at pH values below 4, whereas the latter reaction predominated at pH values between 4 and 7. These results are used as a basis for questioning the structural assignments previously made to some ascorbic acid esters.

Acetylation↗
PubMed1975

Stability of sodium oxacillin in intravenous solutions.

The influence of concentration, vehicle, dextrose and temperature on the stability of sodium oxacillin solutions was determined using a chemical kinetic approach. Sodium oxacillin degraded faster in dextrose solution than in sodium chloride injection. This finding was attributed to the catalytic effect of dextrose on sodium oxacillin hydrolysis. Solutions of 1-50 mg/ml oxacillin in each vehicle were found to be stable for 24 hours at 23 C. The degradation of sodium oxacillin in various dextrose solutions was independent of oxacillin concentration, and followed first order kinetics.

Drug Stability↗