• Volume 7, Issue 3-4

      June 1985,   pages  257-431

    • Purification and properties of an α-amylase inhibitor specific for human pancreatic amylase from proso (Panicium miliaceum) seeds

      R H Nagaraj T N Pattabiraman

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      An α-amylase inhibitor was purified to homogeneity by acid extraction, ammonium sulphate fractionation, chromatography on carboxymethyl-cellulose, diethylaminoethyl-cellulose and Sephadex G-100 from proso grains (Panicium miliaceum). The calculated molecular weight was 14000. The inhibitor was fairly heat stable and stable under acidic and neutral conditions. The factor was more effective by two orders of magnitude in its action on human pancreatic amylase than on human salivary amylase. It did not inhibit onA. oryzae,B. subtilis and porcine pancreatic amylases. Pepsin rapidly inactivated the inhibitor. Chemical modification studies revealed that amino and guanido groups are essential for the action of the inhibitor. The inhibitor was found to protect both human salivary and pancreatic amylases against inactivation by acid. The mode of inhibition was found to be uncompetitive

    • Role of glutamine synthetase in citric acid fermentation byAspergillus niger

      N S Punekar C S Vaidyanathan N Appaji Rao

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      The activity of glutamine synthetase fromAspergillus niger was significantly lowered under conditions of citric acid fermentation. The intracellular pH of the organism as determined by bromophenol blue dye distribution and fluorescein diacetate uptake methods was relatively constant between 6·0–6·5, when the pH of the external medium was varied between 2·3–7·0.Aspergillus niger glutamine synthetase was rapidly inactivated under acidic pH conditions and Mn2+ ions partially protected the enzyme against this inactivation. Mn2+-dependent glutamine synthetase activity was higher at acidic pH (6·0) compared to Mg2+-supported activity. While the concentration of Mg2+ required to optimally activate glutamine synthetase at pH 6·0 was very high (≥ 50 mM), Mn2+ was effective at 4 mM. Higher concentrations of Mn2+ were inhibitory. The inhibition of both Mn2+ and Mg2+-dependent reactions by citrate, 2-oxoglutarate and ATP were probably due to their ability to chelate divalent ions rather than as regulatory molecules. This suggestion was supported by the observation that a metal ion chelator, EDTA also produced similar effects. Of the end-products of the pathway, only histidine, carbamyl phosphate, AMP and ADP inhibitedAspergillus niger glutamine synthetase. The inhibitions were more pronounced when Mn2+ was the metal ion activator and greater inhibition was observed at lower pH values. These results permit us to postulate that glutamine synthesis may be markedly inhibited when the fungus is grown under conditions suitable for citric acid production and this block may result in delinking carbon and nitrogen metabolism leading to acidogenesis

    • Purification and properties of a carboxylesterase from germinated finger millet (Eleusine coracana Gaertn.)

      G Aravinda Upadhya L Govardhan P S Veerabhadrappa

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      A carboxylesterase (EC 3.1.1.1) was purified from germinated finger millet by ammonium sulphate fractionation, diethylaminoethyl-cellulose chromatography and Sephadex G-200 filtration. The homogeneity of the enzyme was established by Polyacrylamide gel electrophoresis, isoelectric focussing and sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The enzyme has a single polypeptide chain with a molecular weight of 70,000. The amino acid analysis of the purified enzyme revealed that it contained a greater number of neutral and acidic, compared to, basic amino acid residues. The isoelectric pH of the enzyme was found to be 5·1. Studies with different organophosphate and carbamate inhibitors showed that this enzyme was more sensitive to organophosphate inhibitors than carbamates. The rate constantski andl50 for different inhibitors were calculated. The product inhibition studies with this enzyme showed linear competitive inhibition with acetate and linear noncompetitive inhibition with 1-naphthol

    • Activity of radiation degradation products of vitamins A and E to haemolyse erythrocyte

      Brij Bhushan V Ninjoor G B Nadkarni

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      Exposure of vitamin A acetate in freely dissolved state to γ-radiationin vitro caused a dose dependent degradation accompanied by the formation of new products. The radiation degradation products were separated by chromatography using step gradient elution. The parent molecule, vitamin A acetate, induced negligible haemolysis of erythrocytes. In contrast, the polar products formed by irradiation were found to be potent haemolysing agents. A highly polar product, eluted with methanol revealed maximum haemolytic activity. Acetylation of these products resulted in loss of their haemolytic properties. Similarly, vitamin E acetate, a known stabilizer of the biomembranes, after irradiation yielded products which caused haemolysis of erythrocytes. It was demonstrated that irradiation introduces hydroxyl groups which impart haemolytic properties to the radiation degradation products of vitamin A

    • Theoretical prediction of antigenic sites in the Β-subunits of human choriogonadotropin and luteinizing hormone

      Aftab A Ansari

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      Using hydrophilicity and recognition values of amino acids, the antigenic sites of theΒ-subunits of human choriogonadotropin and luteinizing hormone were computed from their amino acid sequences. Six antigenic sites were calculated for human choriogonadotropinΒ-subunits: residues 3–8, 17–22, 59–65,100–106,110–116 and 134–139. For luteinizing hormoneΒ-chain three antigenic sites were calculated: residues 17–22,59–65, and 100–106; all these three sites of luteinizing hormoneΒ being identical to the corresponding sites in human choriogonadotropinΒ. There was no antigenic site in luteinizing hormone that was also not found in human choriogonadotropin. On the other hand, there were unique determinants in human choriogonadotropin that were not found in luteinizing hormone; these determinants were residues 3–8, 110–116 and 134–139

    • Gramicidin-S: Structure-activity relationship

      G Nagamurthi S Rambhav

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      The two side chain amino groups of the two L-ornithine residues in gramicidin-S seem to be important for the antibacterial activity of the molecule, since complete acetylation, formylation, carbamylation, deamination, trinitrophenylation, succinylation, maleylation of the antibiotic caused 90–95 % loss of the antibacterial activity of the antibiotic. However this modification leads to only 12–30% loss of the hemolytic activity. Monoacetyl- and monoformyl gramicidin-S with a free amino group retains nearly 50% of the antibacterial activity of the molecule. It seems, therefore, that the two amino groups contribute equally to the antibacterial activity of gramicidin-S

    • Purification and characterization of arginine decarboxylase from cucumber (Cucumis sativus) seedlings

      G L Prasad P R Adiga

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      A simple, reproducible and rapid protocol for the purification of arginine decarboxylase fromCucumis sativus seedlings has been standardised. The purification steps involved ion-exchange chromatography on diethylaminoethyl-cellulose followed by gel filtration on Sephadex G-l 50. The purified enzyme preparation migrated as a single stainable band on Polyacrylamide gels at both basic and acidic pH, but under denaturing and reducing conditions on sodium dodecyl sulphate-polyacrylamide gels resolved into polypeptides of molecular weight 48,000,44,000 and 15,000. However, in the absence of 2-mercaptoethanol on electrophoresis on sodium dodecyl sulphate-polyacrylamide gels, the enzyme moved as single band with a molecular weight of 150,000. Evidence was obtained to indicate that these three polypeptides were probably derived from a single larger molecular weight enzyme. On storage of the purified protein, the 48,000 species was preferentially degraded to smaller polypeptides. The preliminary data suggested that the 48,000 and 44,000 species shared many common tryptic peptides as revealed by finger printing of the [125I ]-labelled protein. The purified enzyme was a glycoprotein and had aKm of 0.5 mM for arginine. Its activity was stimulated by dithiothrietol and pyridoxal phosphate. EDTA did not inhibit the enzyme activity. Mn2+ at 1 mM stimulated arginine decarboxylase activity but was inhibitory at higher concentration

    • A quantitative analysis of germ cells and the histone variants in the testes of vitamin A-deficient rats and during subsequent repletion with vitamin A

      Emmanual Unni M R S Rao

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      A quantitative analysis of the different types of germ cells present in the seminiferous tubules of vitamin A-deficient-retinoate maintained rats revealed that the number of pachytene spermatocytes and spermatogonia was greatly reduced in the deficient rats. Spermatids were virtually absent in the deficient tubules which contained mostly spermatogonia and preleptotene spermatocytes along with the Sertoli cells. There was no change in the number of Sertoli cells present in the tubules of deficient rats as compared to that of normal rats. Following supplementation of retinyl acetate to vitamin A-deficient-retinoate maintained rats, there was an immediate thinning of the germinal epithelium resulting from the sloughing off of the damaged spermatocytes which were beyond repair. However, after 12 days of vitamin A supplementation fresh batch of pachytene spermatocytes started appearing while by day 16 round spermatids could be seen.

      Analysis of the acid soluble proteins from nuclei on different types of Polyacrylamide gel electrophoretic systems has revealed that the levels of the testis specific histone variants Hlt, TH2A and TH2B, synthesized predominantly in the pachytene spermatocytes were greatly reduced in the testes of retinoate maintained rats. Following supplementation of retinyl acetate for either 4 days or 8 days the levels of these histone variants further decreased which correlated with the decrease in the number of pachytene spermatocytes. However, by day 12 of supplementation onwards, their levels started increasing and reached near normal levels by day 24 of vitamin A-supplementation

    • Interaction of pyridoxal phosphate with the amino groups at the active site of 5- aminolevulinic acid dehydratase in maize

      G B Maralihalli A S Bhagwat

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      Pyridoxal 5′-phosphate strongly and reversibly inhibited maize leaf 5-amino levulinic acid dehydratase. The inhibition was linearly competitive with respect to the substrate 5-aminolevulinic acid at pH values between 7 to 9.0. Pyridoxal was also effective as an inhibitor of the enzyme but pyridoxamine phosphate was not inhibitory. The results suggest that pyridoxal 5′-phosphate may be interacting with the enzyme either close to or at the 5-aminolevulinic acid binding site. This conclusion was further corroborated by the detection of a Schiff base between the enzyme and the substrate, 5-aminolevulinic acid and by reduction of pyridoxal phosphate and substrate complexes with sodium borohydride

    • Isolation and characterization of plasma membrane fromAcanthamoeba culbertsoni

      Sanjay Kumar Mishra N K Garg A M Kidwai

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      A rapid method for preparation of plasma membrane fromAcanthamoeba culbertsoni involving toluene treatment followed by lithium bromide extraction is described. In the plasma membrane preparation, 5′-nucleotidase, Na+ + K+ -ATPase, Mg2+ -ATPase and glucose-6-phosphatase activities were enriched. The membrane preparation was free from nucleic acid, cytochrome P-450 and cytochrome b5. Amino acid (14C-Ieucine) was not incorporated in the plasma membrane in 2 min. Succinic dehydrogenase was not detectable in the plasma membrane preparation. The molar ratio of cholesterol and phospholipids was 0.95 which is characteristics for plasma membranes. Under electronmicroscopy the preparation was homogenous without any other component of the cell. Plasma membrane proteins and glycoproteins were separated on acrylamide gel electrophoresis

    • Enzymes of ammonia assimilation and ureide biogenesis in developing pigeonpea (Cajanus cajan L.) nodules

      Amarjit Randhir Singh

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      Ammonia assimilatory and ureide biogenic enzymes were measured in the cytosol fraction of pigeonpea nodules during the period 15–120 days after sowing. The activity of enzymes involved in the initial assimilation of ammonia, i.e. glutamine synthetase, glutamate synthase, asparagine synthetase and aspartate aminotransferase, substantially increased activities during the period of plant growth and reached a maximum value around 105 days after sowing. These increases paralleled the increase in nodule mass, nitrogenase activity and ureide content in nodules. Though no regular pattern was obtained for their specific activities, yet these activities when expressed relative to the specific activity of nitrogenase were many fold higher at each stage of development. Similar increases were observed in the activities of enzymes associated with the formation of ureides from purines. In almost all cases, the activities were again maximum around 90–105 days after sowing. The specific activities of nucleotidase, nucleosidase, xanthine dehydrogenase, uricase and allantoinase, when expressed relative to the specific activity of nitrogenase at vegetative, flowering and podsetting stages were again many fold higher indicating the sufficiency of the levels of these enzymes for the biosynthesis of ureides. The data presented are consistent with the proposal that in ureide producing legumes, ammonia is initially assimilated into glutamine, aspartate, etc., which are metabolised for the denovo synthesis of purines. The purines are then utilised for the production of ureides by a group of enzymes investigated here

    • Carbohydrates, lipids and lipoproteins and islet changes in diabetes with superimposed myocardial infarction

      S D Bhatt P S Bora L M Srivastava

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      Short-term metabolic and concomitant morphologic effects of streptozotocin diabetes on isoproterenol-induced myocardial infarction was studied in Wistar rats, Of particular significance was the observation that myocardial infarction in concert with diabetes brought about a distinctive exacerbation of the severity and complexity of the histopathological lesions. Of all the biochemical parameters, serum glucose and free fatty acids registered maximum elevation and serum lactate and cardiac glycogen levels a maximum reduction. Among the lipoproteins, an inverse relationship was found between high density lipoproteins and low density and very low density lipoproteins; while high density lipoproteins, ratio of high density lipoprotein to low density lipoprotein and the percentage of high density lipoprotein were decreased, there was a significant increase in low density lipoprotein concentration and percentage values of low density and very low density lipoproteins. In diabetes, the B cell of the endocrine pancreas depicted selective necrosis. Loss of insulin granules and wide-spread necrobiosis of cellular elements of the pancreatic islets were observed, respectively, in myocardial infarction and in diabetes plus myocardial infarction combinations. Pathological evidence of chemical-induced mild toxicity was present in the exocrine parenchyma. Mitotic features and the presence of centroacinar cells in the damaged Langerhans’ islets supposedly formed the basis of regeneration of the tissue in diabetes, with or without vascular complications

    • Fungal glucoamylases

      B C Shenoy L C Katwa A G Appu Rao M R Raghavendra Rao

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      The purification and properties of glucoamylase (α-l,4-glucan glucohydrolase, EC 3.2.1.3) from different fungal sources have been compared. The studies on the conformation and activity of the native enzyme at a function of pH, temperature, substrate concentration and the effect of denaturants and on the role of carbohydrate moiety on structure and stability have been reviewed. The chemical modification of the active centre, binding kinetics of the substrate and active site and the mechanism of action have been summarized. They differ in their fine structure as revealed by their near ultra-violet circular dichroism spectra and contain 30–35 % α-helix, 24–36 %Β-structure and the rest aperiodic structure. The activity of the enzyme is very sensitive to the environment around aromatic aminoacid residues.

      The glucoamylases are glycoprotein in nature, differ in their content and nature of carbohydrate from different sources. The carbohydrate moiety plays an important role in stabilising the native conformation of the enzyme and is not involved in activity and antigenecity.

      At the active site of the enzyme, two tryptophan and two carboxyl (glutamate or aspartate) groups are present. It is likely that the histidine and tyrosine residues which are present away from the active site are involved in binding of the substrate. There seems to be seven subsites which are involved in binding of the substrate and the catalytic site is situated in between 1 and 2 subsites. In breaking of α-1,4-, α-1,3-, and α-l,6-bonds only one active centre is involved.

      Studies on the immobilization of either glucoamylase alone or as a part of a multienzyme system have been reviewed briefly

    • Metabolism of mycobacteria

      Rizwan Masood Yogesh Kumar Sharma T A Venkitasubramanian

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      The metabolism of mycobacteria have been studied with reference to carbohydrate, lipids, nitrogen metabolism and oxidative phosphorylation. Some of the enzymes of glycolytic pathway, tricarboxylic acid cycle and lypogenic enzymes were purified, characterized and their kinetic properties investigated. The effect of age of the culture and environmental factors on different aspects of metabolism of mycobacteria were also studied. A comparison of lipid profile in various species of mycobacteria grown in different culture conditions were made. The metabolism of spheroplasts isolated from mycobacteria has been established with respect to their energy charge and to synthesize peptidoglycan using D-alanine as the precursor

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