Department of Biochemistry

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    Modifying effects of divalent ions on the sulfhydryl content of normal and tumorous beet root tissue under thermal and γ-irradiation stress
    ( 1981-02-01) Ramaiah, K. V.Atchuta ; Mookerjee, Anjali
    Temperature and γ-irradiation stresses result in the loss of DTNB (5-5′ Dithionitrobenzoic acid) - reactive sulfhydryl groups in the TCA (trichloroacetic acid)-insoluble protein of normal and tumorous beet root tissue. Metal ions like Ca2+, Zn2+, and Pb2+ have been found to partially prevent the change of-SH quantity under stress conditions. An attempt has been made to correlate the observed loss of DTNB - reactive sulfhydryl content with the loss of membrane permeability under thermal and irradiation conditions. © 1981 Birkhäuser Verlag.
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    A comparative study of membrane related phenomena in normal and crown gall tissues of red beet (Beta vulgaris L.)
    ( 1982-11-01) Atchuta Ramaiah, K. V. ; Mookerjee, A.
    Divalent metal ions have been found to protect membranes of red beet crown gall tissue more than their adjacent normal regions from thermal or gamma radiation stress, suggesting the possibility of an alteration on the surface charge accompanying tumor formation in plants. Further, tumor tissue has been observed to possess enhanced membrane ATPase activity, a higher tissue sulfhydryl content and increased protein levels, thus suggesting a model of 'source' (normal tissue) and 'sink' (tumor tissue) relationship. © 1982 Birkhäuser Verlag.
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    Wounding of aged pea epicotyls enhances the reinitiating ability of isolated ribosomes
    ( 1985-10-01) Ramaiah, K. V.A. ; Davies, Eric
    Total ribosomes (monosomes plus polysomes) isolated from wounded pea epicotyls are more efficient at supporting protein synthesis in a wheat germ S30 system (containing wheat ribosomes) than are total ribosomes from aged (control) pea tissue. This increased efficiency is seen when enriched large polysomes, almost devoid of monosomes, are used to program a wheat germ S300 system, from which the wheat germ ribosomes have been removed. Reactions primed by enriched polysomes from wounded tissue, but not aged tissue, continue for at least 30 min, suggesting that reinitiation is occurring during the reaction, albeit in the initial absence of monosomes from wheat or pea. Wheat germ ribosomes, but not monosomes from either aged or wounded pea tissue, are able to translate pea poly(A) RNA and globin mRNA. Aurintricarboxylic acid reduces protein synthesis in a rather indiscriminate manner, whereas, pactamycin seems to have an inhibitory effect specific for initiation, and it is much more effective on wounded than on control tissue polysomes. We interpret these results to imply that polysomal ribosomes from wounded tissue are more efficient at initiation than are polysomal ribosomes from control tissue or than non-polysomal ribosomes (monosomes) from either tissue. © 1985 The Japanese Society of Plant Physiologists.
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    Wounding inhibits protein synthesis yet stimulates polysome formation in aged, excised pea epicotyls
    ( 1986-01-01) Davies, Eric ; Ramaiah, K. V.A. ; Abe, S.
    Wounding of aged, previously-excised pea epicotyl segments by removal of the basal 1-2 mm resulted in a rapid (beginning within 15 min) recruitment of monosomes on to polysomes and an even more rapid (maximal between 6-12 min) inhibition of protein synthesis in the remaining tissue. This inhibition of protein synthesis in vivo did not appear to be an artefact caused by the removal of highly active tissue (e.g., callus, contaminating bacteria), since wounds inflicted at a site distant from the region analyzed still elicited the response, and protein synthesis in the 1-2 mm slices (normally discarded) was inhibited even more strongly than it was in the remaining tissue. The proportion of radioactive methionine in nascent chains (bound to polysomes) increased, while the production of completed polypeptides decreased, after wounding. Cycloheximide, a known inhibitor of the ribosome translocation/release process mimicked some of the effects of wounding. We interpret the results to indicate that the initial effect of wounding is to inhibit translation by inhibiting the ribosome translocation/release process, whereas the subsequent recovery in protein synthesis is brought about partly by a recovery in ribosome translocation/release and partly by enhanced initiation. © 1986 The Japanese Society of Plant Physiologists.
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    The nature of lectins from Dolichos lablab
    ( 1986-03-01) Siva Kumar, N. ; Rajagopal Rao, D.
    The lectins from the seeds of Dolichos lablab var. lignosus (field bean) and Dolichos lablab var. typicus (lablab bean) have been isolated in a homogeneous form by affinity chromatography on D-mannose linked Sepharose. Both the lectins are glycoproteins and have a molecular weight of 60,000 and S 20,w value of 5.2 and seem to be made up of 4 similar subunits (apparent molecular weight 15,000). The carbohydrate content ofthe lectins is mostly fucose (2-5 mol per mol of protein), mannose (5-8 mol per mol of protein) and N-acetyl glucosamine (1-2 mol per mol of protein). The amino acid composition of both the lectins was similar and methionine and half cystine could not be detected, Both the lectins have similar tryptic peptide map. Alanine and serine were the only N and C-terminal amino acids for both lectins. The lectins were found to contain low amounts of bound metals such as manganese, magnesium and calcium. The near ultra-violet circular dichroism spectra of the lectins are similar to that of Sainfoin. Circular dichroism data indicate that tyrosine and tryptophan residues are involved in sugar binding. The lectins are nonspecific for human blood groups and they agglutinate a variety of other erythrocytes. Among a number of sugars, D-glucose and D-mannose inhibited the haemag-glutinating activity ofthe lectins. The lectins were antigenically similar © 1986 Indian Academy of Sciences.
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    Mechanism of protein synthesis inhibition by vaccinia viral core and reversal of this inhibition by reticulocyte peptide chain initiation factors
    ( 1987-03-01) Chakrabarti, Debopam ; Ramaiah, Kolluru V.A. ; Roy, Ananda L. ; Bagchi, Milan ; Gupta, Naba K.
    Vaccinia viral core inhibits protein synthesis in heme-supplemented reticulocyte lysate. A reticulocyte cell suPernatant factor, which reversed Protein synthesis inhibition in heme-deficient reticulocyte lysate also reversed vaccinia viral core induced Protein synthesis inhibition in heme-suPPlemented reticulocyte lysate. Significant inhibition reversal activity was also observed with a partially purified eukaryotic initiation factor-2 PreParation and this activity was lost uPon further Purification of eukaryotic initiation factor-2.The ribosomal salt-wash factor Co-eukaryotic initiation factor-2 which like reticulocyte suPernatant factor contains guanine nucleotide exchange factor activity, was comPletely inactive. Vaccinia viral core induced detectable level of eukaryotic initiation factor-2 α-subunit phosphorylation when incubated in the heme-supplemented reticulocyte lysate. This lysate preparation contains guanine nucleotide exchange factor activity. However, when the same reticulocyte lysate was previously incubated with the vaccinia viral core, the guanine nucleotide exchange factor activity during subsequent incubation was almost comPletely inhibited. © 1987 Indian Academy of Sciences.
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    Antibodies against the cytoplasmic tail can differentiate between the quaternary forms of the M < inf > r < /inf > 46000 mannose 6-phosphate receptor
    ( 1991-03-11) Nadimpalli, Siva Kumar ; Schmidt, Bernhard ; von Figura, Kurt ; Hille, Annette
    An antiserum against a peptide of the cytoplasmic tail of the Mr 46000 mannose 6-phosphate receptor is described which recognizes preferentially the tetrameric versus the dimeric form of this receptor. This indicates that the conformation of the cytoplasmic tail, which harbours signals necessary for the trafficking of the receptor, depends on the quaternary structure of the receptor. © 1991.
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    Recycling and phosphorylation of eukaryotic initiation factor 2 on 60S subunits of 80S initiation complexes and polysomes
    ( 1992-01-01) Ramaiah, Kolluru V.A. ; Dhindsa, Rajinder S. ; Chen, Jane Jane ; London, Irving M. ; Levin, Daniel
    Phosphorylation of the α-subunit (38 kDa) of eukaryotic initiation factor 2 (eIF-2α) regulates initiation of protein synthesis in eukaryotic cells. This phosphorylation is enhanced in cycloheximide-treated heme-deficient reticulocyte lysates in which polysomes are maintained. In early heme deficiency prior to polysome disaggregation, eIF-2(αP) accumulates primarily on the 60S subunits of polysomes. Further, isolated polysomes contain eIF-2α that is efficiently phosphorylated in vitro by heme-regulated inhibitor (HRI). Immunoblot analysis of eIF-2 distribution in sucrose gradients of actively protein-synthesizing lysates indicates that eIF-2 is distributed at low levels throughout the polysome profiles. These findings suggest that polysome-bound eEF-2α is a target of HRI under physiological conditions. The presence of eIF-2 on the 60S subunits of polysomes is incompatible with the conventional model in which eIF-2 is recycled during the joining of the 48S preinitiation complex and the 60S subunit to form the 80S initiation complex. A modified model is presented with emphasis on the translocation of eIF-2 from the 40S ribosomal subunit of the 48S preinitiation complex (eIF-2·GTP·Met-tRNA·40S·mRNA) to the 60S subunit of the 80S initiation complex. (.
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    Tail-specific antibodies that block return of 46,000 M(r) mannose 6- phosphate receptor to the trans-Golgi network
    ( 1993-01-01) Schulze-Garg, C. ; Boker, C. ; Nadimpalli, S. K. ; Von Figura, K. ; Hille-Rehfeld, A.
    Recycling of 46,000 M(r) mannose 6-phosphate receptor (MPR 46) was investigated by microinjection of F(ab) fragments against small epitopes within the cytoplasmic domain of the receptor. F(ab) fragments against the peptide 43-47 (Ala-Tyr-Arg-Gly-Val) efficiently blocked return of MPR 46 to the TGN. Antibody-induced redistribution resulted in accumulation of MPR 46 within an endosomal compartment, from which it recycled to the plasma membrane. Rab5 and rab7, markers for early and late endosomes, respectively, were not detectable in the compartment of redistributed MPR 46, suggesting that it represents a specialized endosomal subcompartment. The bulk of redistributed MPR 46 did not colocalize with endocytosed fluid-phase marker, suggesting that it accumulates at a site where MPR 46 has been segregated from endocytosed material, which is destined for transport to lysosomes. Peptide 43-47 contains a tyrosine (residue 44) which has been shown earlier to be part of an internalization signal for MPR 46 (Johnson, K. F., W. Chan, and S. Kornfeld. 1990. Proc. Natl. Acad. Sci. USA. 87:10010-10014). The role of tyrosine residue 44 as part of a putative multifunctional sorting signal is discussed.
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    Studies on enzyme electrodes
    (University of Hyderabad, 1993-10-23) Narasaiah, Dontha ; Chanchal K Mitra
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    Structure-function relationship in ribosomal protein s1 from E. coli and evolution of its domain structure
    (University of Hyderabad, 1993-11-23) Ravi, Kambampati ; Suryanarayana, T
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    Expression of mutant eukaryotic initiation factor 2 α subunit (eIF-2α) reduces inhibition of guanine nucleotide exchange activity of eIF-2B mediated by eIF-2α phosphorylation
    ( 1994-01-01) Ramaiah, Kolluru V.A. ; Davies, Monique V. ; Chen, Jane Jane ; Kaufman, Randal J.
    The inhibition of protein synthesis that occurs upon phosphorylation of the alpha subunit of eukaryotic initiation factor 2 (eIF-2α) at serine 51 correlates with reduced guanine nucleotide exchange activity of eIF-2B in vivo and inhibition of eIF-2B activity in vitro, although it is not known if phosphorylation is the cause of the reduced eIF-2B activity in vivo. To characterize the importance of eIF-2α phosphorylation in the regulation of eIF-2B activity, we studied the overexpression of mutant eIF-2α subunits in which serine 48 or 51 was replaced by an alanine (48A or 51A mutant). Previous studies demonstrated that the 51A mutant was resistant to phosphorylation, whereas the 48A mutant was a substrate for phosphorylation. Additionally, expression of either mutant partially protected Chinese hamster ovary (CHO) cells from the inhibition of protein synthesis in response to heat shock treatment (P. Murtha-Riel, M. V. Davies, J. B. Scherer, S. Y. Choi, J. W. B. Hershey, and R. J. Kaufman, J. Biol. Chem. 268:12946-12951, 1993). In this study, we show that eIF-2B activity was inhibited in parental CHO cell extracts upon addition of purified reticulocyte heme-regulated inhibitor (HRI), and eIF-2α kinase that phosphorylates Ser-51. Preincubation with purified HRI also reduced the eIF-2B activity in extracts from cells overexpressing wild-type eIF-2α. In contrast, the eIF-2B activity was not readily inhibited in extracts from cells overexpressing either the eIF-2α 48A or 51A mutant. In addition, eIF-2B activity was decreased in extracts prepared from heat-shocked cells overexpressing wild-type eIF-2α, whereas the decrease in eIF-2B activity was less in heat-shocked cells overexpressing either mutant 48A or mutant 51A. While the phosphorylation at serine 51 in eIF-2α impairs the eIF-2B activity, we propose that serine 48 acts to maintain a high affinity between phosphorylated eIF-2α and eIF-2B, thereby inactivating eIF-2B activity. These findings support the hypothesis that phosphorylation of eIF-2α inhibits protein synthesis directly through reducing eIF-2B activity and emphasize the importance of both serine 48 and serine 51 in the interaction with eIF-2B and regulation of eIF-2B activity.
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    Estrogen receptors of the goat uterus : emphasis on a receptor form that enters the nucles as a constituent of heterodimer
    (University of Hyderabad, 1994-10-11) Narayanan Karthikeyan ; Raghava Varman Thampan
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    Inhibition of protein synthesis in insect cells by baculovirus-expressed heme-regulated eIF-2α kinase
    ( 1994-10-14) Chefalo, Peter J. ; Yang, Jane M. ; Ramaiah, Kolluru V.A. ; Gehrke, Lee ; Chen, Jane Jane
    To study further the regulation of the heme-regulated eIF-2α kinase (HRI), we have produced functional wild type HRI using the baculovirus expression system. The amount of recombinant HRI protein expressed in insect cells is approximately 10 times higher than levels in reticulocytes. Baculovirus-expressed HRI (BV-HRI) is indistinguishable from HRI purified from rabbit reticulocytes. It is active both as an autokinase and an eIF-2α kinase. BV-HRI is regulated by heme in vitro as well as in intact insect cells. Coexpression of the wild type HRI with the inactive K199R HRI, S51A eIF-2α, or interleukin-1β (IL-1β) results in diminished expression of these proteins. Expression of wild type HRI also results in severe inhibition of general protein synthesis in Sf9 cells when compared with cells expressing K199R HRI or IL-β. In addition, the guanine nucleotide exchange activity of eIF-2B is suppressed in Sf9 cells expressing wild type HRI but not in cells expressing the K199R HRI or IL-1β. Furthermore, expression of wild type HRI is increased by coexpression with the nonphosphorylatable S51A eIF-2α or by the addition of hemin, which inhibits HRI activity. These results provide evidence that translational regulation by phosphorylation of eIF-2α and sequestration of eIF-2B can operate in insect cells.
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    Theoretical studies on protein sequences
    (University of Hyderabad, 1994-12-27) Meeta Rani ; Chanchal K Mitra
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    Phosphorylation of wheat germ initiation factor 2 (eIF-2) by N- ethylmaleimide-treated wheat germ lysates and by purified casein kinase II does not affect the guanine nucleotide exchange on eIF-2
    ( 1995-01-01) Janaki, Narahari ; Krishna, Vattem M. ; Ramaiah, Kolluru V.A.
    Phosphorylation of the small subunit of eukaryotic initiation factor-2 (eIF-2α) impairs protein synthesis in mammalian systems. It is not known, however, if a similar regulatory mechanism exists in plants. Previous reports indicate that one of the wheat germ eIF-2 subunits, the p40-41 doublet, is phosphorylated by heterologous eIF-2α kinases. Here we report that phosphorylation of the small subunit in wheat germ eIF-2, p36, occurs in translating wheat germ lysates which are pretreated with N-ethylmaleimide (NEM) and dithiothreitol. Also, a purified sea star casein kinase II (CKII) phosphorylates the p41-42 doublet and p36 subunits of wheat germ eIF-2. While heme-regulated eIF-2α kinase from reticulocyte lysates does not inhibit wheat germ protein synthesis, CKII and NEM are found to be inhibitory. To determine whether phosphorylation of the small subunit (p36) is the cause for protein synthesis inhibition, we have further studied the exchange of labeled GDP for unlabeled GDP in the preformed eIF-2 · [3H]GDP complex in vitro in the presence of CKII and ATP. The GDP exchange in eIF-2 GDP complex can occur without the addition of any protein factor and the exchange reaction is marginally inhibited by CKII. A 0-70% ammonium sulfate cut fraction, prepared from NEM-treated wheat germ lysate, also does not inhibit the guanine nucleotide exchange reaction. These findings suggest that the protein synthesis inhibition in these cases is not mediated by eIF-2 phosphorylation. © 1995 Academic Press, Inc.