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N K Gupta

Publications and source records attributed to N K Gupta.

At least 91 records · Page 5Linked to original sources
PubMed1985

Protein synthesis in rabbit reticulocytes. A study of the roles of Co-eIF-2, Co-eIF-2A80, and GDP in peptide chain initiation.

The roles of Co-eIF-2, Co-eIF-2A80, and GDP in ternary complex and Met-tRNAf X 40 S initiation complex formation were studied. 1) Partially purified eukaryotic initiation factor 2 (eIF-2) (50% pure) preparations contained 0.4-0.6 pmol of bound GDP/pmol of eIF-2. eIF-2 purity was calculated from ternary complex formation in the absence of Mg2+ and in the presence of excess Co-eIF-2. 2) In the absence of Mg2+, approximately 30% of the potentially active eIF-2 molecules formed ternary complexes, and both Co-eIF-2 and Co-eIF-2A80 were equally effective in full activation of the eIF-2 molecules for ternary complex formation. 3) In the presence of Mg2+, approximately 10% of the potentially active eIF-2 molecules formed ternary complexes in the absence of ancillary factors, and the ancillary factors Co-eIF-2A80 and Co-eIF-2 raised the incorporation to 20 and 50% of the eIF-2 molecules, respectively. 4) In the absence of Mg2+, [3H]GDP in preformed eIF-2 X [3H]GDP was readily displaced by GTP during ternary complex formation. 5) In the presence of Mg2+, [3H]GDP remained tightly bound to eIF-2 and ternary complex formation was inhibited. Co-eIF-2, but not Co-eIF-2A80, was effective in promoting [3H]GDP displacement and the former was more effective in promoting ternary complex formation than the latter. 6) eIF-2 X [3H]GDP was converted to eIF-2 X [3H] GTP by incubation in the presence of nucleoside-5'-diphosphate kinase and ATP, but the eIF-2 X [3H]GTP thus formed did not bind Met-tRNAf in the presence of Mg2+ and required exogeneous addition of Co-eIF-2 and GTP for ternary complex formation and GTP displacement. 7) In the absence of Mg2+, the increased ternary complex formed in the presence of eIF-2 X [3H] GDP and Co-eIF-2A80 (with accompanying loss of [3H] GDP) was inactive in a subsequent reaction, which involves Met-tRNAf transfer to 40 S ribosomes (in the presence of Mg2+), and required trace amounts of Co-eIF-2 for such activity. Based on the above observations, we have suggested a two-step activation of eIF-2 molecules by the Co-eIF-2 protein complex for functional ternary complex formation. One of these steps involves the Co-eIF-2A component of Co-eIF-2. This activation results in stimulated Met-tRNAf binding to eIF-2 and is most apparent in the absence of Mg2+ and with aged eIF-2 molecules.(ABSTRACT TRUNCATED AT 400 WORDS)

Eukaryotic Initiation Factor-2↗
PubMed1985

Isolation and characterization of human liver guanine deaminase.

Guanine deaminase (EC 3.5.4.3, guanine aminohydrolase [GAH]) was purified 3248-fold from human liver to homogeneity with a specific activity of 21.5. A combination of ammonium sulfate fractionation, and DEAE-cellulose, hydroxylapatite, and affinity chromatography with guanine triphosphate ligand were used to purify the enzyme. The enzyme was a dimer protein of a molecular weight of 120,000 with each subunit of 59,000 as determined by gel filtration and sodium dodecyl sulfate-gel electrophoresis. Isoelectric focusing gave a pI of 4.76. It was found to be an acidic protein, as evidenced by the amino acid analysis, enriched with glutamate, aspartate, alanine and glycine. It showed a sharp pH optimum of 8.0. The apparent Km for guanine was determined to be 1.53 X 10(-5) M at pH 6.0 and 2 X 10(-4) M for 8-azaguanine as a substrate at pH 6.0. The enzyme was found to be sensitive to p-hydroxymercuribenzoate inhibition with a Ki of 1.53 X 10(-5) M and a Ki of 5 X 10(-5) M with 5-aminoimidazole-4-carboxamide as an inhibitor. The inhibition with iodoacetic acid showed only a 7% loss in the activity at 1 X 10(-4) M and a 24% loss at 1 X 10(-3) M after 30 min of incubation, whereas p-hydroxymercuribenzoate incubation for 30 min resulted in a 91% loss of activity at a concentration of 1 X 10(-4) M. Guanine was the substrate for all of the inhibition studies. The enzyme was observed to be stable up to 40 degrees C, with a loss of almost all activity at 65 degrees C with 30 min incubation. Two pKa values were obtained at 5.85 and 8.0. Analysis of the N-terminal amino acid proved to be valine while the C-terminal residue was identified as alanine.

Aminohydrolases↗
PubMed1984

Protein synthesis in rabbit reticulocytes: requirements for Met-tRNAf . 40S preinitiation complex formation with AUG-codon and physiological mRNAs.

Under standard conditions, in the presence of GTP, highly purified eIF-2 and Co-eIF-2 factor preparations efficiently stimulated AUG-codon dependent but not physiological mRNA-dependent Met-tRNAf binding to 40S ribosomes. Replacement of GTP by a nonhydrolyzable GTP analog, GMP-PNP, in the above system, gave significant stimulation of Met-tRNAf binding to 40S ribosomes dependent on physiological mRNAs. Lower but significant stimulation of Met-tRNAf binding to 40S ribosomes was also observed when GTP was used in the presence of nucleoside 5'-diphosphate kinase (NDK) and ATP. ATP alone in the absence of NDK had no significant effect. This is the first report on the formation of a stable Met-tRNAf . 40S initiation complex dependent on physiological mRNAs and the factor requirements for such complex formation.

Animals↗
PubMed1984

Protein synthesis in rabbit reticulocytes: a study of the mechanism of action of the protein factor RF that reverses protein synthesis inhibition in heme-deficient reticulocyte lysates.

A eukaryotic initiation factor 2 (eIF-2)-ancillary protein factor Co-eIF-2 promotes displacement of GDP from eIF-2 X GDP and facilitates ternary complex (Met-tRNAf X eIF-2 X GTP) formation in the presence of Mg2+. Heme-regulated protein synthesis inhibitor, HRI, phosphorylates the alpha-subunit of eIF-2 and thus inhibits ternary complex formation as Co-eIF-2 does not displace GDP from eIF-2 alpha (P) X GDP. RF, a high molecular weight cell supernatant factor, reverses protein synthesis inhibition in heme-deficient reticulocyte lysates and also reverses HRI inhibition of ternary complex formation. RF contains Co-eIF-2 activity. In addition, an active RF preparation contains excess alpha-subunit of eIF-2 in the free and unphosphorylated form and this alpha-subunit of eIF-2 is not phosphorylated by HRI and ATP. In this paper we report (i) an active RF preparation contains excess alpha-subunit of eIF-2 and this alpha-subunit can be phosphorylated by HRI and ATP in the presence of GDP; (ii) RF promotes ternary complex formation by eIF-2 X [3H]GDP with accompanying GDP displacement; (iii) in the presence of HRI and ATP, RF promotes ternary complex formation by eIF-2 X [3H]GDP without accompanying GDP displacement; (iv) in the presence of HRI and ATP, the ternary complex formed using RF is active in Met-tRNAf X 40S initiation complex formation; (v) both the ternary complex and the Met-tRNAf X 40S complex formation in the presence of HRI and ATP are completely inhibited by prior incubation of RF with GDP; (vi) upon further fractionation of an active RF fraction, a preparation can be obtained that contains HRI-sensitive Co-eIF-2 activity. However, this preparation does not efficiently reverse protein synthesis inhibition in heme-deficient reticulocyte lysates and does not contain excess alpha-subunit of eIF-2. Based on these observations, we have suggested (a) RF provides the unphosphorylated alpha-subunit to eIF-2 alpha (P) X GDP and restores eIF-2 activity. This RF activity is inhibited as the alpha-subunit in the RF preparation becomes phosphorylated by HRI and ATP in the presence of GDP; (b) RF contains Co-eIF-2 activity, which has dual functions: (i) stimulation of ternary complex formation by eIF-2 and (ii) GDP displacement from eIF-2 X GDP during ternary complex formation. In the presence of HRI and ATP, Co-eIF-2 but does not displace GDP from eIF-2 alpha(P) X GDP.

Adenosine Triphosphate↗
PubMed1982

Protein synthesis in rabbit reticulocytes: characteristics of CO-eIF-2 protein complex.

A high molecular weight reticulocyte protein factor, named Co-eIF-2, contains Co-eIF-2A, Co-eIF-2B, and Co-eIF-2C activities and stimulates Met-tRNAf binding to eIF-2 both in the presence and absence of Mg2+. Some characteristics of this stimulation in the absence of Mg2+ are: (1) Stimulation is most pronounced at low eIF-2 levels. (2) Stimulation is partially resistant to heat and NEM treatment, and thus appears to be due to the combined action of both heat and NEM-insensitive Co-eIF-2A, and heat and NEM-sensitive Co-eIF-2C activities. (3) [3H]GDP bound in eIF-2 . [3H]GDP complex is rapidly displaced by unlabelled GTP during ternary complex formation Co-eIF-2 stimulates Met-tRNAf binding to eIF-2 even when added after the [3H]GDP from eIF-2 . [3H]GDP has been completely displaced. This indicates that Co-eIF-2-stimulation is not due to GDP displacement from eIF-2 . GDP. We propose that eIF-2 molecules become inactive in the presence of Mg2+ and at high dilution, and Co-eIF-2 restores the inactive eIF-2 molecules into an active form.

Animals↗
PubMed1982

Protein synthesis in rabbit reticulocytes: characteristics of the protein factor RF that reverses inhibition of protein synthesis in heme-deficient reticulocyte lysates.

During heme deficiency in reticulocyte lysates, the heme-regulated translational inhibitor of protein synthesis (HRI) is activated and shuts off protein synthesis. In partial reactions, HRI phosphorylates the Mr 38,000 subunit (alpha subunit) of eukaryotic initiation factor 2 (eIF-2), which forms a ternary complex, Met-tRNAf X eIF-2 X GTP. The eIF-2 alpha (P) thus formed is not recognized by two eIF-2 ancillary factors, Co-eIF-2B (which promotes the dissociation of the ternary complex at high Mg2+) and Co-eIF-2C (which reverses the inhibition of ternary complex formation), and thus, is presumably inactive in peptide chain initiation. A protein factor, designated RF, which reverses inhibition of protein synthesis in heme-deficient reticulocyte lysates, has been purified from reticulocyte cell supernatant. RF is a high molecular weight (Mr approximately equal to 450,000) protein complex composed of multiple polypeptides. An active RF preparation contains Co-eIF-2B and Co-eIF-2C activities, and these two activities in RF preparation are not inhibited by HRI and ATP--i.e., eIF-2 alpha (P) is recognized. During purification, RF remains associated with eIF-2 activity (eIF-2 X RF) and can be freed of this eIF-2 activity by CM-Sephadex chromatography. Both eIF-2 X RF and RF contain a Mr 38,000 polypeptide component that is indistinguishable from the Mr 38,000 subunit of eIF-2 by two-dimensional gel electrophoresis. It has been observed that a significant part of this Mr 38,000 polypeptide component in eIF-2 X RF and almost the entire Mr 38,000 polypeptide component in RF remain unphosphorylated after prolonged incubation with HRI and ATP. A possible role of this free Mr 38,000 polypeptide in RF action is discussed.

Animals↗
PubMed1981

Protein synthesis in rabbit reticulocytes. Purification and characterization of a double-stranded RNA-dependent protein synthesis inhibitor from reticulocyte lysates.

Reticulocyte lysates contain a latent form of eukaryotic peptide chain initiation factor 2 (eIF-2) kinase (dsI) which becomes activated in the presence of double-stranded RNA and ATP and inhibits protein synthesis. The latent form of dsI has been partially purified from reticulocyte ribosomal salt wash. The purified dsI has been activated by incubation in the presence of poly(rI).poly(rC) and [gamma 32P]ATP and the activated dsI has been further purified to near homogeneity. Upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis, purified [32P]dsI shows an intensely staining 67,000-dalton polypeptide band which corresponds to a single 67,000-dalton radioactive band. During Sephadex (G-200) gel filtration, both the latent form of dsI and the activated dsI elute similarly with a peak corresponding to a molecular weight of 67,000. Purified dsI phosphorylates the 38,000-dalton subunit of eIF-2 and, under conditions of eIF-2 phosphorylation, dsI strongly inhibits AUG-dependent Met-tRNAf binding to 40 S ribosomes. Also, in partial reactions, eIF-2 alpha (P) formed by phosphorylation of eIF-2 using dsI and ATP, is not recognized by two eIF-2 ancillary factors, Co-eIF-2B and Co-eIF-2C. These results are similar to those reported previously for the heme-regulated eIF-2 kinase (Das, A., Ralston, R. O., Grace, M., Roy, R., Ghosh-Dastidar, P., Das H. K., Yaghmai, B., Palmieri, S., and Gupta, N. K. (1979) Proc. Natl. Acad. Sci. U. S. A. 76,5076-5079) and suggest that dsI, like the heme-regulated eIF-2 kinase phosphorylates eIF-2 and eIF-2 alpha (P) thus formed, in both cases, is not recognized by Co-eIF-2B and Co-eIF-2C, and is inactive in some step(s) of Met-tRNAf.40 S initiation complex formation.

Adenosine Triphosphate↗
PubMed1981

Protein synthesis in rabbit reticulocytes. Co-eIF-2A reverses mRNA inhibition of ternary complex (Met-tRNAf.eIF-2.GTP) formation by eIF-2.

mRNAs, at low concentrations, drastically inhibit ternary complex formation by eIF-2 (Met-tRNAf.eIF-2.GTP) and, when added to the preformed ternary complex, cause extensive dissociation of the complex. Co-eIF-2A stimulates (2- to 4-fold) Met-tRNAf binding to eIF-2 and, in the presence of excess Co-eIF-2A, the stimulated Met-tRNAf binding to eIF-2 is fully resistant to mRNAs. Other cofactors tested such as Co-eIF-2B and Co-eIF-2C do not reverse mRNA inhibition of ternary complex formation.

Animals↗