• Volume 6, Issue 2

      June 1984,   pages  155-248

    • Purification and characterization of chymotrypsin inhibitors from marine turtle egg white

      M K Guha N K Sinha

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      The egg white of marine turtle(Caretta caretta Linn.) contains two chymotrypsin inhibitors and one trypsin inhibitor. The two chymotrypsin inhibitors were purified to homogeneity, as judged by ion-exchange chromatography, Polyacrylamide gel and sodium dodecyl sulphate-gel electrophoresis, isoelectric focusing, immunochemical tests and sedimenttation in the ultracentrifuge. Their sedimentation coefficient values were independent of protein concentration. Their amino acid composition was similar, and contained seven disulphide bonds, and methionine and carbohydrate moiety were absent. Each inhibitor consisted of a single polypeptide chain of 117 amino acids. The average molecular weight of each inhibitor, calculated from sedimentation and diffusion coefficient values, amino acid composition and sodium dodecyl sulphate-gel electrophoresis was 13000. Both the inhibitors were stable over the pH range of 2–11. They inhibited α-chymotrypsin by forming enzymeinhibitor complexes at a molar ratio of unity. The dissociation constant of each complex was 1.06 × 10−10 M. Both the inhibitors were indistinguishable in their physical, chemical and inhibitory properties except for their isoelectric points which were pH 5.23 for inhibitorA and pH 6.0 for inhibitorB. Chemical modification of all amino groups with trinitrobenzene sulphonate had no effect on their inhibitory activity

    • Evaluation of fractionatedWuchereria bancrofti microfilarial excretory-secretory antigens for diagnosis of bancroftian filariasis by enzyme linked immunosorbent assay

      M V R Reddy Ashok Malhotra G B K S Prasad B C Harinath

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      TheWuchereria bancrofti microfilarial excretory-secretory antigens were fractionated into ES1, ES2, ES3 and ES4 by ultra-membrane filtration and evaluated for their diagnostic utility by enzyme linked immunosorbent assay. Three of the four fractions showed antigenic activity (ES2, ES3 and ES4). The antigen fractions ES2 and ES4 were highly active in the detection of filarial IgM antibody in clinical filariasis and microfilaraemia respectively. The chemical characterization of the ES2 and ES4 antigen fractions showed that they were glycoproteins

    • Cyclic AMP transport across membrane vesicles of ultra-violet light irradiatedEscherichia coli

      D Bhatnagar A K Bhattacharya

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      Transport of cyclic AMP acrossEscherichia coli membrane was studied using membrane vesicles. Uptake of cyclic AMP was measured using normally oriented vesicles, whereas uptake in everted vesicles was taken as a measure of the efflux of cyclic AMP. Ultra-violet irradiation of the cells led to an inhibition of both uptake and efflux of cyclic AMP across the membrane. The presence of cyclic AMP in the growth medium prior to ultra-violet irradiation caused an enhancement of the uptake and efflux. The uptake and efflux of cyclic AMP were less in vesicles from glucose grown cells as compared to the uptake and efflux by the vesicles prepared from glycerol grown cells. Similarly both uptake and efflux of cyclic AMP were more in vesicles prepared from cells grown on glycerol or glucose in the presence of cyclic AMP than in vesicles from cells grown in absence of cyclic AMP. It is suggested that the number of cyclic AMP carrier molecules were reduced in cells under catabolite repression by glucose as well as by ultra-violet irradiation

    • Diverse effects of formate on the assimilatory metabolism of nitrate and nitrite inRhizobium

      R K Singh R M Singh

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      Two strains ofRhizobium, cowpeaRhizobium 32H1 andRhizobium japonicum CB 1809, showed a marked stimulation in growth on addition of formate to the minimal medium containing nitrate as the sole source of nitrogen. The amount of accumulated nitrite and specific nitrate reductase activity was much higher in cultures supplemented with formate than in the control medium. In contrast, growth, consumption of nitrite and specific nitrite reductase activity in minimal medium + nitrite was greatly reduced by the addition of formate. A chlorate resistant mutant (Chl-16) was isolated spontaneously which contained a nitrite reductase which was not inhibited by formate. The results suggest that formate serves as an electron donor for nitrate reductase and inhibits nitrite assimilation inRhizobium

    • Ureide biogenesis and the enzymes of ammonia assimilation and ureide biosynthesis in nitrogen fixing pigeonpea (Cajanus cajan) nodules

      Amarjit Randhir Singh

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      Allantoic acid production from IMP, XMP, inosine, xanthosine, hypoxanthine, xanthine, uric acid and allantoin was investigated by incubating each of these substrates withCajanus cajan cytosol and bacteroid fractions separately in the presence and absence of NAD+ and allopurinol. Allantoic acid synthesis by bacteroid fraction could only be observed with uric acid and allantoin as substrates. Addition of NAD+ or allopurinol to the reaction mixtures had no effect. However, with cytosol fraction, allantoic acid was produced by each of these substrates, with maximum rate with allantoin. With NAD+ or with allopurinol, allantoic acid was produced only with uric acid and allantoin as substrates. NADH production with cytosol fraction could again be observed with all the substrates. Except with uric acid and allantoin, allopurinol completely inhibited NADH formation. Regardless of the presence or absence of allopurinol, none of the substrates exhibited significant activity with bacteroid fraction. Based on the activities of glutamine synthetase, glutamate synthase, glutamate dehydrogenase, aspartate aminotransferase, asparagine synthetase, nucleotidase, nucleosidase, xanthine de-hydrogenase, uricase and allantoinase and their intracellular localisation in various nodule fractions, a probable pathway for the biogenesis of ureides in pigeonpea nodules has been proposed

    • Adrenocorticotrophin secreting cells in the hypophysis of the brown spiny mouseMus platythrix (Bennett)

      N H Gopal Dutt N A Madhyastha

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      Adrenocorticotrophin secreting cells are identified in the hypophysis of the brown spiny mouseMus platythrix by conventional methods of light microscopy. Quantitative data showed that certain smaller acidophilic cells in thepars distalis, under conditions provoking their hypersecretion such as unilateral adrenalectomy and metopirone treatment, increase in number and size from the pre-existing corticotrophs. There is no evidence for the transmigration of these cells from the chromophobes, basophils or any other cell type. Thepars intermedia revealed two types of cells of which the type II cells are histochemically identical to adrenocorticotrophin secreting cells of thepars distalis

    • Localization of 3-phosphoglyceric acid synthesis in the mother cell compartments and forespores ofBacillus megaterium and the effects of manganous ions on its metabolism

      Ravendra Pal Singh

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      Rapidly metabolizable compounds such as glucose or glycerol were not utilized byBacillus megaterium in the absence of manganese when grown in the supplemented nutrient broth medium. Under these conditions, growth ceased at low cell titre, 3-phosphoglyceric acid accumulated inside the cells and normal sporulation process was arrested. Addition of manganese to the medium caused disappearance of 3-phosphoglyceric acid, growth resumed and normal sporulation was observed. Synthesis of 3-phosphoglyceric acid occurred only in the mother cell compartments and it was transported for accumulation inside the forespores ofBacillus megaterium when grown in supplemented nutrient broth medium. Incubation of forespores in the presence of glucose or glycerol had no effect on 3-phosphoglyceric acid synthesis/accumulation, but it was completely utilized when forespores were incubated with manganese plus ionophore (X 537A). No other metal(s) could substitute for manganese suggesting that manganese plays crucial role in 3-phosphoglyceric acid metabolism

    • Studies on the effects ofin vitro methyiation on aminoacylation of transfer RNA

      B R Vani T Ramakrishnan

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      In vitro methyiation ofEscherichia coli transfer ribonucleic acid by cell free extracts ofMycobacterium smegmatis leads exclusively to the formation of 1-methyl adenine [Vani, B. R., Ramakrishnan, T., Taya, Y., Noguchi, S., Yamaiuzumi, Z. and Nishimura, S. (1978)J. Bact., 137, 1085]. We have studied the effect of this modification on aminoacylationof Escherichia coli tRNA by mycobacterial enzymes. Aminoacylation with total algal protein hydrolysate as well as several individual aminoacids like methionine, valine, tyrosine, aspartic acid and lysine were monitored. In all the cases methyiation had a positive effect on the extent of aminoacylation by mycobacterial enzymes. Decreased aminoacylationin vitro was observed when hypomethylated transfer RNA from ethionine treated cells was used as the substrate for aminoacylation

    • Anin vitro test using cholesterol metabolism of macrophages to determine drug sensitivity and resistance ofMycobacterium leprae

      Ishwari Nair P R Mahadevan

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      Macrophages that have ingested liveMycobacterium leprae show a preferential accumulation of cholesterol ester. Such an accumulation is not seen, on the ingestion of dead bacteria. Among the macrophages that ingest liveMycobacterium leprae, the presence of dapsone or rifampicin prevents largely the alteration in the anticipated increase in the cholesterol ester indicating the sensitivity of the bacteria to the drug. In the small number of relapsed patients, the presence of dapsone did not reduce the cholesterol ester increase, suggesting that theMycobacterium leprae present are either resistant or escaped detection. The method provides a rapid drug screening system foranti-Mycobacterium leprae activity of known and unknown compounds

    • Purification and regulation of aspartate transcarbamylase from germinated mung bean (Vigna radiata) seedlings

      P V Prasad N Appaji Rao

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      Aspartate transcarbamylase (EC 2.1.3.2) was purified to homogeniety from germinated mung bean seedlings by treatment with carbamyl phosphate. The purified enzyme was a hexamer with a subunit molecular weight of 20,600. The enzyme exhibited multiple activity bands on Polyacrylamide gel electrophoresis, which could be altered by treatment with carbamyl phosphate or UMP indicating that the enzyme was probably undergoing reversible association or dissociation in the presence of these effectors. The carbamyl phosphate stabilized enzyme did not exhibit positive homotropic interactions with carbamyl phosphate and hysteresis. The enzyme which had not been exposed to carbamyl phosphate showed a decrease in specific activity with a change in the concentration of both carbamyl phosphate and protein. The carbamyl phosphate saturation and UMP inhibition patterns were complex with a maximum and a plateau region. The partially purified enzyme also exhibited hysteresis and the hysteretic response, a function of protein concentration, was abolished by preincubation with carbamyl phosphate and enhanced by preincubation with UMP. All these observations are compatible with a postulation that the enzyme activity may be regulated by slow reversible association-dissociation dependent on the interaction with allosteric ligands

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