NISCAIR Online Periodicals Repository Collection: IJBB Vol.36(6) [December 1999]

Inhibition of thymidylate synthase by pergularinine, tylophorinidine and deoxytubulosine

Title: Inhibition of thymidylate synthase by pergularinine, tylophorinidine and deoxytubulosine

Authors: Rao, K Narasimha; Bhattacharya, R K; Veankatachalam, S R

Abstract: The activity of thymidylate synthase (TS) purified in our laboratory from Lactobacillus leichmannii was inhibited by pergularinine (PGL) and tylophorinidine (TPD) and deoxytubulosine (DTB) isolated froml the Indian medicine plants Pergularia pallida and Alanguml lamarckii respectively. Cytotoxicity studies showed that cell growth of L. leichmannii was inhibited (IC₅₀) = 40-45 μM) by all the three alkaloids, the concentrations > 80-90 μM resulting in complete loss of the enzyme activit y. K_i values of the enzyme calculated from Lineweaver-Burk and Dixon plots for PGL, TPD and DTB were l0× 10^-6M, 9× 10^-6M and 7× 10^-6M respectively. These are typed as ‘non-competitive’ inhibitors of TS. All the three alkaloids, inhibited (IC₅₀ = 50 μM) the elevated TS activity of leukocytes in cancer patients with clinically diagnosed chronic myelocytic leukemia (n= 10), acute lymphocytic leukemia (n=8) and metastatic solid tumours (n=3).

Page(s): 442-448

Metal ion specificity in anaesthetic induced increase in the rate of monensin and nigericin mediated H⁺/ M⁺ exchange across phospholipid vesicular membranes

Title: Metal ion specificity in anaesthetic induced increase in the rate of monensin and nigericin mediated H⁺/ M⁺ exchange across phospholipid vesicular membranes

Authors: Prabhananda, B S; Kombrabail, Mamata H

Abstract: From a study or the decay or the pH difference across vesicular membranes (Δ pH) it has been possible to show that H⁺and alkali metal ion (M⁺) concentration gradients across bilayer membranes (which are responsible for driving important biochemical processes) can be selectively perturbed by anaesthetics such as chloroform and benzyl alcohol by combining them with a suitable exchange ionophore. On adding the anaesthetic to the membrane in an environment containing metal ions M⁺=K⁺. the rate or Δ pH decay by H⁺/M⁺ exchange increases by a larger factor or by a smaller factor (when compared to that in a membrane environment with M⁺=Na⁺) depending on whether the exchange ionophore chosen is monensin or nigeriein. A rational explanation of this "metal ion specificity" can be given using the exchange ionophore mediated ion transport scheme in which the equilibrations at the "interfaces" are fast compared to the "translocation equilibration" between the species in the two layers of the membrane. The following three factors are responsible for the observed "specificity": On adding the anesthetic (i) translocation rate constants increase. (ii) the concentrations of the M⁺ bound ionophores increase at the expense of H⁺ bound ionophores. (iii) Under our experimental conditions the rate determining species are the complexes monensin-K (Mon-K) and nigeriein-H (Nig-H) for M⁺=K⁺ whereas they are monensin -H (MonH) and nigeriein-Na (Nig-Na) for M⁺=Na⁺ Possible anesthetic induced membrane perturbations contributing to the above mentioned changes in the membrane are (A), the loosening of the membrane structure and (B ), an associated increase in the membrane hydration (and membrane dielectric constant ). An analysis of the consequent changes in the various transport steps shows the following: (a), The anaesthetic induced changes in the translocation rates of electrically charged species are not relevant in the explanation or the observed changes in the Δ pH decay rates. (b), Changes in the rates of fast equilibria at the interface contribute to changes in K_H and K_M (c), A suggestion made in the literature, that a significant interaction between the dipole moment of the monensin-K complex and the membrane slows down its translocation, is not valid. (d), The ability to explain rationally all the Δ pH decay data confirms the validity or the transport scheme used. In our experiments Δ pH across the vesicular membrane was created by pH jump coming from a temperature jump.

Page(s): 415-421

A perspective of biological supramolecular electron transfer

Title: A perspective of biological supramolecular electron transfer

Authors: Ramasarma, T

Abstract: Electron transfer is an essential activity in biological systems. The migrating electron originates from water-oxygen in photosynthesis and reverts to dioxygen in respiration. In this cycle two metal porphyrin complexes possessing circular conjugated system and macrocyclic π-clouds, chlorophyll and heme, play a decisive role in mobilising electrons for travel over biological structures as extraneous electrons. Transport of electrons within proteins (as in cytochromes) and within DNA (during oxidative damage and repair) is known to occur. Initial evaluations did not favour formation of semiconducting pathways of delocalized electrons of the peptide bonds in proteins and of the bases in nucleic acids.Direct measurement of conductivity of bulk material and quantum chemical calculations of their polymeric structures also did not support electron transfer in both proteins and nucleic acids.

New experimental approaches have revived interest in the process of charge transfer through DNA duplex. The fluorescence on photoexcitation of Ru-complex was found to be quenched by Rh-complex, when both were tethered to DNA and intercalated in the base stack. Similar experiments showed that damage to G-bases and repair of T-T dimers in DNA can occur by possible long range electron transfer through the base stack. The novelty of this phenomenon prompted the apt name, "chemistry at a distance".

Based on experiments with ruthenium modified proteins, intramolecular electron transfer in proteins is now proposed to use pathways that include C-C σ-bonds and surprisingly hydrogen bonds which remained out of favour for a long time. In support of this, some experimental evidence is now available showing that hydrogen bond-bridges facilitate transfer of electrons between metal-porphyrin complexes. By molecular orbital calculations over 20 years ago we found that "delocalization of an extraneous electron is pronounced when it enters low-lying virtual orbitals of the electronic structures of peptide units linked by hydrogen bonds". This review focuses on supramolecular electron transfer pathways that can emerge on interlinking by hydrogen bonds and metal coordination of some unnoticed structures with π-clouds in proteins and nucleic acids, potentially useful in catalysis and energy missions.

Page(s): 379-397

UDP-galactose 4-epimerase from Kluyveromyces fragilis: Equilibrium unfolding studies

Title: UDP-galactose 4-epimerase from Kluyveromyces fragilis: Equilibrium unfolding studies

Authors: Maity, Nilesh Ranjan; Barat, Bhaswati; Bhattacharyya, Debasish

Abstract: UDP-galactose 4-epimerase from yeast (Kluyveromyces fragilis) is a homodimer of total molecular mass 150 kDa having possibly one mole of NAD/dimer acting as a cofactor. The molecule could be dissociated and denatured by 8 M urea at pH 7.0 and could be functionally reconstituted after dilution with buffer having extraneous NAD. The unfolded and refolded equilibrium intermediates of the enzyme between 0-8 M urea have been characterized in terms of catalytic activity. NADH like characteristic coenzyme fluorescence, interaction with extrinsic fluorescence probe I- anilino 8-naphthelene sulphonic acid (ANS), far UV circular dichroism spectra, fluorescence emission spectra of aromatic residues and subunit dissociation. While denaturation monitored by parameters associated with active site region e.g. inactivation and coenzyme fluorescence, were found to be cooperative having ΔG between -8.8 to -4.4 kcals/mole, the overall denaturation process in terms of secondary and tertiary structure was however continuous without having a transition point. At 3 M urea a stable dimeric apoenzyme was formed having 65% of native secondary structure which was dissociated to monomer at 6 M urea with 12% of the said structure. The unfolding and refolding pathways involved identical structures except near the final stage of refolding where catalytic activity reappeared.

Page(s): 433-441

NISCAIR Online Periodicals Repository Collection: IJBB Vol.36(6) [December 1999]

Inhibition of thymidylate synthase by pergularinine, tylophorinidine and deoxytubulosine

Metal ion specificity in anaesthetic induced increase in the rate of monensin and nigericin mediated H+/ M+ exchange across phospholipid vesicular membranes

A perspective of biological supramolecular electron transfer

UDP-galactose 4-epimerase from Kluyveromyces fragilis: Equilibrium unfolding studies

Metal ion specificity in anaesthetic induced increase in the rate of monensin and nigericin mediated H⁺/ M⁺ exchange across phospholipid vesicular membranes