• Volume 5, Issue 4

      December 1983,   pages  279-375

    • Sugar-hydrolysing enzymes in gustatory mutants ofDrosophila

      P N Bhavsar V Rodrigues O Siddiqi

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      The distribution of the isozymes of α-glucosidase in different parts ofDrosophila melanogaster was examined. Two of the eight glucosidase bands are associated with tarsi. The gustatory mutations in the genesgustB andgustC greatly reduce tarsal glucosidase activity, but the enzyme deficiency is not correlated with inability to taste sugars.

    • Purification and kinetic mechanism of 5,10-metliyienetetrahydrofolate reductase from sheep liver

      K Varalakshmi H S Savithri N Appaji Rao

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      5,10-Methylenetetrahydrofolate reductase (EC 1.1.1.68) was purified from the cytosolic fraction of sheep liver by (NH4)2 SO4 fractionation, acid precipitation, DEAE-Sephacel chromatography and Blue Sepharose affinity chromatography. The homogeneity of the enzyme was established by sodium dodecyl sulphate-polyacrylamide gel electrophoresis, ultracentrifugation and Ouchterlony immunodiffusion test. The enzyme was a dimer of molecular weight 1,66,000 ± 5,000 with a subunit molecular weight of 87,000 ±5,000. The enzyme showed hyperbolic saturation pattern with 5-methyltetrahydrofolate.K0.5 values for 5-methyltetrahydrofolate menadione and NADPH were determined to be 132 ΜM, 2.45 ΜM and 16 ΜM. The parallel set of lines in the Lineweaver-Burk plot, when either NADPH or menadione was varied at different fixed concentrations of the other substrate; non-competitive inhibition, when NADPH was varied at different fixed concentrations of NADP; competitive inhibition, when menadione was varied at different fixed concentrations of NADP and the absence of inhibition by NADP at saturating concentration of menadione, clearly established that the kinetic mechanism of the reaction catalyzed by this enzyme was ping-pong.

    • Effect of sodium dodecyl sulphate on the high molecular weight protein fraction of mustard (Brassica juncea) and rapeseed (Brassica campestris)

      A Gururaj Rao M S Narasinga Rao

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      The effect of sodium dodecyl sulphate on mustard and rapeseed 12S protein has been monitored by the techniques of ultracentrifugation, viscosity, difference spectra and fluorescence spectrophotometry. At low concentration of sodium dodecyl sulphate (<3.47 mM) mustard protein undergoes aggregation and at higher concentrations it dissociates to 1.8 S protein, the dissociation being complete at 17.3 mM sodium dodecyl sulphate. The rapeseed protein, on the other hand, undergoes dissociation at all the concentrations of sodium dodecyl sulphate. The reduced viscosity values of mustard protein in the presence of the denaturant are higher than those of rapeseed protein. Similarly in difference spectra change in absorbance values of mustard protein are higher.’ The relative fluorescence intensity of the mustard protein increases with sodium dodecyl sulphate concentration, upto 0.87 mM and this is followed by fluorescence quneching at higher denaturant concentrations. However, with the rapeseed protein fluorescence quenching was observed at all concentrations of sodium dodecyl sulphate.

    • Denaturation of the high molecular weight protein fraction of mustard (Brassica juncea) and rapeseed (Brassica campestris) by urea or guanidinium hydrochloride

      A Gururaj Rao M S Narasinga Rao

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      Urea and guanidinium hydrochloride dissociate the 12S protein of mustard and rapeseed to 1.8 S protein and the extent of dissociation depends on the concentration of the denaturant. Mustard (Brassica juncea) protein is more readily dissociated than the rapeseed (Brassica campestris) protein. The reagents denature the protein as evidenced by increase in viscosity, appearance of difference spectra and quenching of fluorescence. Rapeseed protein is denatured more readily than the mustard protein. Analysis of visctosity, spectral and fluoresence data suggests that the first event in the denaturation reaction is the perturbation of the aromatic amino acid residues followed by their exposure to the solvent medium and unfolding of the protein molecule.

    • Effect of calcium on synthesis of dipicolinic acid inPenicillium citreoviride and its feedback resistant mutant

      G P Kalle Y M Deo

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      Dipicolinic acid synthesis inPenicillium citreoviride strain 3114 was inhibited by Ca2+ ions, but not by Ba2+, Cu2+or Fe2+. Among the metals tested, only Zn2+ inhibited the synthesis of dipicolinic acid and promoted sporulation. None of these metals reversed the inhibition by Ca2+ or Zn2+. A mutant 27133-dpa-ca selected for resistance to feedback inhibition by dipicolinic acid: Ca2+ complex showed cross-resistance to inhibition by dipicolinic acid: Zn2+. Both 3114 and271 33-dpa-ca excreted a number of aliphatic and amino acids during secondary metabolism of dipicolinic acid. In the presence of 1000 ppm of Ca2+, accumulation of citric acid and α-aminoadipic acid was completely inhibited under conditions of inhibition of dipicolinic acid in parent strain 3114 but not in the mutant. Citric acid with or without Ca2+ did not inhibit thede novo synthesis of dipicolinic acid in the strain 3114. In fact, citric acid in the presence of Ca2+ improved significantly rate of dipicolinic acid synthesis. Apart from resistance to feed back inhibition by dipicolinic acid: Ca2+ complex, mutant differed from the parent in three other aspectsviz. (i) dipicolinic acid synthesis was not subject to catabolite repression by glucose, (ii) sporulation as well as dipicolinic acid synthesis was dependent on the presence of Ca2+ ions in the medium and (iii) Mg2+ requirement for the mutant increased three fold. Higher requirement of the Mg2+ could be partially relieved by Ca2+ during secondary metabolism. The results support the inference thatde novo synthesis of dipicolinic acid is regulated through feedback inhibition by dipicolinic acid: Ca2+complex.

    • Interaction of lysozyme with antibiotics-binding of penicillins to lysozyme

      M Subramanian B S Sheshadri M P Venkatappa

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      Binding of lysozyme with the antibiotics such as penicillin-G, penicillin-V and methicillin at different concentrations and pH was studied by equilibrium dialysis. Co-operative binding isotherms were observed at pH 5.0,7.0 and 9.0 with all the penicillins and the binding ratios decreased slightly with the increase of pH. The Gibbs free energy change calculated on the basis of Wyman’s binding potential concept decreased slightly with the increase of pH indicating slight decrease in the binding strength at higher pH in the case of all penicillins.

      The ultra-violet difference spectra of lysozyme-penicillin complexes showed a less intense peak in the region of 284–300 nm at pH 5.0. Only penicillin-G complex had a peak at pH 7.0 at these wavelengths with less intensity compared to that at pH 5.0. However, none of the penicillins showed discrete peaks in this region at pH 9.0. The appearance of peaks in the difference spectra of all these complexes at pH 5.0 and with only penicllin-G complex at pH 7.0 in the aromatic region indicated hydrophobic interactions with tryptophan residues as the binding sites. In addition, the ionic interactions with lysine residues in lysozyme were also occurring.

      The conformational changes induced by the binding of penicillins to lysozyme monitored by circular dichroism showed a slight decrease in the aromatic bands in the 320–250 nm region. However, in the 250–200 nm region, [θ]222nm values obtained at various concentrations of penicillins in the complex indicated an increased α-helical content generating a more ordered structure. These results led to the conclusion that both the hydrophobic and electrostatic interactions prevail in the binding of penicillins to lysozyme.

    • A new DNA-cloning vector forHaemophilus influenzae Rd.

      V P Joshi N K Notani

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      A new, high-efficiency, DNA-cloning vector pJ1-8 was derived in two steps from the chimeric plasmid pD7 consisting of RSF 0885(ampr) andHaemophilus influenzae chromosomal DNA. pJl-8 has only oneEcoRI site and a molecular weight of only 2.5 × 106. No detectableampr transformation was obtained with pJl-8 DNA. However,ampr transformation increases markedly ifHaemophilus influenzae chromosomal DNA segments are spliced into it, providing a very facile assay for detecting inserts.

    • RNA polymerase activity in isolated nuclei ofNicotiana sanderae callus: Characteristics and modulation during differentiation

      P K Asha M S Shaila C S Vaidyanathan T Ramakrishnan

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      Isolated nuclei from differentiating cultures ofNicotiana sanderae showed increased levels of RNA polymerase activity as compared to the nuclei from callus cultures. The RNA synthetic activity was dependent on nucleotide triphosphates and Mg2+ and was destroyed by RNase. Maximum activity was obtained in the presence of 50 mM (NH4)2 SO4 and α-amanitin inhibited 40% and 55% of the activity in the nuclei from callus and differentiating tissue respectively. The nuclei from differentiating tissue elicited a 3-fold increase in RNA polymerase I and a 4-fold augmentation in RNA polymerase II activities.

    • On the presence of sulphate in pituitary lutropin

      A S Arunasmithasri T Rajendrakumar K Muralidhar

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      The presence of sulphate in the carbohydrate of pituitary lutropin from different species has been investigated using a biosynthetic approach. Pituitaries from rats, rabbits, goats, and buffaloes were incubated in the presence of35SO4- and the35SO4- -labelled proteins in the tissue immunoprecipitated with a well characterized anti-sheep lutropin serum. The incorporation into immunoreactive lutropin was low in the case of rat, rabbit and goat pituitaries while, it was considerable in the case of buffalo pituitaries. Hence further characterization studies were carried out on35SO4- -labelled proteins of buffaloes. The physico-chemical, immunological and biological properties of radio-labelled buffalo pituitary material were shown to be similar to those of standard lutropin. Inin vitro conditions of incubations, most of the incorporation of35SO4- was observed into tissue lutropin while under similar conditions of incubation, [14C]-amino acids were found to get incorporated mostly into medium lutropin. The physiologically specific releasing hormone, lutropin-releasing hormone was found to stimulate the release of35SO4--labelled lutropin from the rabbit pituitaries into the medium. These results give indirect evidence that sulphate could be present in pituitary lutropin.

    • Transcription in ultra violet-irradiated plasmodia ofPhysarum polycephalum

      P A Valsala Kumari Vimala R Nair (nee Devi)

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      InPhysarum polycephalum, transcription is significantly inhibited for the whole of the ultra violet-induced, extended G2 phase. Although, there is some increase in the rate of transcription closer to the first post-irradiation mitosis, thereafter, ovrall synthesis continues to be low. Also, the plasmodia developed significant resistance to actinomycin D during this period. The resistance is both in terms of mitosis and RNA synthesis. The overall depression in transcription and the drug resistance are apparently related phenomena.

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