• Volume 3, Issue 2

      June 1981,   pages  89-214

    • Purification and properties of diaminopimelate decarboxylase ofMicrococcus glutamicus

      Meena Lakshman B C Shenoy M R Raghavendra Rao

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      Diaminopimelate decarboxylase (EC 4.1.1.20) ofMicrococcus glutamicus ATCC 13059 was purified to homogeneity. The enzyme had an apparent molecular weight of 191,000 as determined by gel filtration on Sephadex G-200. At protein concentrations of 20 and 10 μg per ml and in the absence of pyridoxal-5′-phosphate, it dissociated into a species of molecular weight 94,000. The polypeptide chain molecular weight as determined by sodium dodecyl sulphate Polyacrylamide gel electrophoresis was 100,000. TheKm formeso diaminopimelate was 0.5 mM and that for pyridoxal-5′-phosphate was 0.6 μI. Sulphydryl groups and pyridoxal-5′-phosphate were essential for activity and stability. The enzyme was inhibited significantly by L-lysine and DL-aspartic β-semialdehyde.

    • Purification and physicochemical properties of α -amylase from irradiated wheat

      J P Machaiah U K Vakil

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      α-Amylases from control and gamma-irradiated (at 0.2 and 2.0 kGy dose levels) wheat seedlings were purified to homogeneity and characterized. The molecular weight of the enzyme from a 2 kGy irradiated sample was slightly lower than that of the control; other general and catalytic properties also showed some differences. α-Amylase from the irradiated (2 kGy) sample had a narrow range of pH optimum and was inactivated faster at alkaline pH and by heat treatment than the enzyme from unirradiated wheat. A high apparent Michaelis constant (Km) and a low maximal velocity (Vmax) for the hydrolysis of soluble starch catalyzed by the enzyme from irradiated (2 kGy) wheat, suggested some modifications in the formation of the substrate α-amylase complex. Further, of the total number of amino acid residues lost on irradiation, dicarboxylic amino acids constituted the largest percentage; these structural alterations in the enzyme may be responsible for its partial inactivation. The total sugars liberated upon amylolysis of starch with the 2 kGy irradiated enzyme were lower than control, and there was accumulation of higher maltodextrins in the place of maltose.

    • In vitro andin vivo nitrogenase activity ofRhizobium mutants and their symbiotic effectivity

      K R Dadarwal B S Kundu P Tauro

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      A slow growing nitrogen-fixing strain ofVigna radiata var.aureus (mung bean)Rhizobium which expressed nitrogenase activity in a synthetic medium was isolated from its native population. Mutants with decreased and increased nitrogenase activity were derived from this strain by treatment with acridine orange and ethidium bromide. These mutants were tested for symbiotic effectivity invivo. The effectivity of mutants with decreased nitrogenase activity in the culture medium was lower than the parent strain; however, the effectivity of mutants with higher nitrogenase activity did not increase above that of the parent. This suggests that the plant is perhaps a limiting factor in the full expression of rhizobial nitrogenase in the nodules.

    • Localization of glucose-6-phosphatase and fructose-1-6-diphosphaiase in subcellular fractions from different regions of the rat brain

      Gurucharan Kaur Rameshwar Singh N Z Baquer

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      Two key enzymes of gluconeogenesis, glucose-6-phosphatase and fructosp-1-6-diphosphatase, were present in the cerebral hemispheres, the cerebellum and the brain stem of the rat brain. Significant activities of these-enzymes were associated with the particulate fraction.

    • Toluene toxicity: Effects of sublethal levels on enzyme activities in seawater adapted tilapia(Sarotherodon mossambicus Peters)

      Ajit D Dange Vasant B Masurekar

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      The effects of exposing seawater adapted cichlid fish, tilapia(Sarotherodon mossambicus Peters) to sublethal concentrations of toluene on the activities of lactate dehydrogenase, succinate dehydrogenase and acetylcholinesterase were studied. The activity of lactate dehydrogenase increased while those of succinate dehydrogenase and acetylcholinesterase were inhibited in most tissues of the exposed fish. The alterations in the dehydrogenases suggested that some changes in carbohydrate metabolism may have occurred. Acetylcholinesterase inhibition in brain and other tissues indicated impairment of nervous function in toluene-intoxicated tilapia.

    • Spectrophotometric assay of immobilized tannase

      L C Katwa M Ramakrishna M R Raghavendra Rao

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      A procedure for the assay of immobilized tannase with Polyacrylamide gel, collagen and Duolite-S-762 as matrices is described. It is based on the spectrophotometric determination of gallic acid formed by the enzymatic hydrolysis of tannic acid. The kinetic parameters of the enzymatic reaction have been studied and an assay procedure has been formulated. This method appears to be much more accurate than those reported earlier.

    • Embryonic control of isocitrate lyase activity in cotyledons of germinating soybean

      Balwant Singh P S Sukhija I S Bhatia

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      The activity of isocitrate lyase (EC 4.1.3.1) in the cotyledons of germinating soybean is controlled by the embryonic axis. Plant growth regulators like gibberellic acid, indole acetic acid and 2,4-dichlorophenoxy acetic acid are able to increase the enzyme activity in cotyledons of whole seedlings but not in dissected cotyledons. The control of induction of the enzyme activity during germination by the embryo could be mediated by the elaboration of kinetin.

    • Cardiotoxin of the Indian cobra(Naja naja) is a pyrophosphatase

      K E Achyuthan L K Ramachandran

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      An inorganic pyrophosphatase has been purified to apparent homogeniety from Indian cobra(Naja naja) venom, with a ten-fold increase in specific activity. The enzyme activity is intrinsic to a protein fraction in the venom which is normally termed cardiotoxin, cobramine, cytotoxin and so on. The enzyme shows a lowKm (70 μI) and high heat stability. The enzyme was active against sodium pyrophosphate; it also hydrolyses a few mononucletides and sugar phosphates at much lower rates. The physiological significance of inorganic pyrophosphatase in venom is discussed.

    • Transport of retinol in the duck plasma

      B Sridhara Rao N Appaji Rao H R Cama

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      Retinol-binding protein and prealbumin were isolated from duck plasma by chromatography on DEAE-cellulose-and DEAE-Sephadex A-50, gel filtration on Sephadex G-100 and preparative Polyacrylamide gel electrophoresis. The molecular weights of the retinol-binding protein-prealbumin complex, prealbumin and retinol-binding protein were found to be 75,000, 55,0000 and 20,000, respectively. On sodium dodecyl sulphate Polyacrylamide gel electrophoresis, prealbumin dissociated into identical subunits exhibiting a molecular weight of 13,500. Retinol-binding protein exhibited microheterogeneity on electrophoresis, whereas prealbumin moved as a single band unlike the multiple bands observed in chicken and rat. The ultraviolet and fluorescence spectra of the two proteins were similar to those isolated from other species. No carbohydrate moiety was detected in either retinol-binding protein or prealbumin. Duck retinol-binding protein and prealbumin showed cross-reactivity with their counterparts in chicken but differed immunologically from those of goat and man. Retinol-binding protein and prealbumin could be dissociated at low ionic strength, in 2M urea, by CM-sephadex chromatography or on preparative electrophoresis. Although the transport of retinol in duck plasma is mediated by carrier proteins as in other species, it is distinguished by the absence of microheterogeneity in prealbumin and of an apo-retinol-binding protein form that could be transported in the plasma.

    • Allosteric serine hydroxymethyltransferase from monkey liver: Correlation of conformational changes caused by denaturants with the alterations in catalytic activity

      Kashi S Ramesh V S Anantanarayanan N Appaji Rao

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      The far-ultraviolet region circular dichroic spectrumof serine hydroxymethyltransferase from monkey liver showed that the protein is in an α-helical conformation. The near ultraviolet circular dichoric spectrum revealed two negative bands originating from the tertiary conformational environment of the aromatic amino acid residues. Addition of urea or guanidinium chloride perturbed the characteristic fluorescence and far ultraviolet circular dichroic spectrum of the enzyme. The decrease in (θ)222 and enzyme activity followed identical patterns with increasing concentrations of urea, whereas with guanidinium chloride, the loss of enzyme activity preceded the loss of secondary structure. 2-Chloroethanol, trifluoroethanol and sodium dodecyl sulphate enhanced the mean residue ellipticity values. In addition, sodium dodecyl sulphate also caused a perturbation of the fluorescence emission spectrum of the enzyme. Extremes of pH decreased the — (θ)222 value. Plots of — (θ)222and enzyme activity as a function of pH showed maximal values at pH 7.4–7.5. These results suggested the prevalence of “conformational flexibility” in the structure of serine hydroxymethyltransferase.

    • Allosteric serine hydroxymethyltransferase from monkey liver: Temperature induced conformational transitions

      Kashi S Ramesh V S Ananthanarayanan N Appaji Rao

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      The homogeneous serine hydroxymethyltransferase from monkey liver was optimally activate at 60°C and the Arrhenius plot for the enzyme was nonlinear with a break at 15°C. The monkey liver enzyme showed high thermal stability of 62°C, as monitored by circular dichroism at 222 nm, absorbance at 280 nm and enzyme activity. The enzyme exhibited a sharp co-operative thermal transition in the range of 50°–70°(Tm= 65°C), as monitored by circular dichroism. L-Serine protected the enzyme against both thermal inactivation and thermal disruption of the secondary structure. The homotropic interactions of tetrahydrofolate with the enzyme was abolished at high temperatures (at 70°C, the Hill coefficient value was 1.0). A plot ofh values vs. assay temperature of tetrahydrofolate saturation experiments, showed the presence of an intermediate conformer with anh value of 1.7 in the temperature range of 45°–60°C. Inclusion of a heat denaturation step in the scheme employed for the purification of serine hydroxymethyltransferase resulted in the loss of cooperative interactions with tetrahydrofolate. The temperature effects on the serine hydroxylmethyltransferase, reported for the first time, lead to a better understanding of the heat induced alterations in conformation and activity for this oligomeric protein.

    • Expression of Hb β-T and Hb β-E genes in Eastern India—Family studies

      Manju Ajmani Geeta Talukder Archana Sharma D K Bhattacharya

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      The distribution patterns of different haemoglobins were observed amongst the family members of β-thalassaemia homozygous and HbE-β-thalassaemia patients with the aid of gel electrophoretic and alkali denaturation techniques. Of the 18 families studied, four belonged to β-thalassaemia homozygous and 14 to HbE-β-thalassaemia patients. Interaction of HbE and β-thalassaemia genes resulted in major clinical abnormalities with increase in the percentages of haemoglobins F and E. The percentages of HbA2 in homozygous β-thalassaemia were within the normal range. Although in Southeast Asia the β° type of HbE-thalassaemia is more prevalent, only one individual with this type of thalassaemia was observed during this survey. In the rest of the patients examined the percentages of adult haemoglobin ranged from 5.2 to 42.5 indicating the presence of a β+ type gene.

    • A probability model for the number of births in an equilibrium birth process

      S N Singh V K Singh

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      A probability model for the number of complete conceptions (that is, live births) taking into account foetal wastages, occurring in a couple during a specified period of time (T0, T0+T) is developed assuming that the data was collected starting a long time after marriage. A method of estimating some of the underlying parameters is given. The model is applied to data obtained in a Varanasi Survey in 1969–70.

    • A conception dependent probability distribution of couple fertility

      I J Singh

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      A probability distribution for the number of conceptions during a specified period of time is derived assuming that the fecundability as well as the proportion of incomplete conceptions vary between conceptions. A procedure for finding the estimate of some of the parameters has been outlined. The probability model was tested with the date from the Varnasi survey.

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