Biomedical subjects
Publications and source records attributed to N K Gupta.
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The characteristics of Met-tRNAfMet binding to ribosomes (40 S and 80 S) were studied using a two-stage assay method (Gupta, N.K., Chatterjee, B. and Majumdar, A. (1975) Biochem. Biophys. Res. Commun. 65, 797) and the complexes formed were analyzed either by Millipore filtration or by sucrose density gradient centrifugation. The results are summarized as follows: (a) with both assay methods, Met-tRNAfMet binding to 40 S ribosomes was entirely dependent upon addition of a partially purified mixture of initiation factors and AUG codon; (b) this binding occurred over a wide Mg2+ concentration range; significant binding was observed even at 20 mM Mg2+; (c) upon addition of 60 S ribosomes, a significant part of Met-tRNAfMet bound to 40 S ribosomes was transferred to 80 S complex. This transfer reaction had a sharp Mg2+ optimum around 2 mM. Met-tRNAfMet-80 S-AUG complex thus formed was active in Met-puromycin synthesis; (d) Met-tRNAfMet deacylase present in crude 0.5 M KCl ribosomal wash is a potent inhibitor of the binding reaction as it deacylates Met-tRNAfMet in the Met-tRNAfMet-40 S-AUG complex; (e) glutaraldehyde (0.5%) degrades Met-tRNAfMet-40 S-AUG complex but increases the background binding of Met-tRNAfMet to 40 S ribosomes in the absence of AUG codon; (f) polynucleotides containing uracil and adenosine are strong inhibitors of Met-tRNAfMet binding to 40 S ribosomes. The order of inhibitory activities of the polynucleotides tested was as follows: poly(rU)-poly(rA) (2:1) greater than poly(rU)-poly(rA) (1:1) greater than poly(rU) greater than poly(rA). Other RNAs tested such as poly(rC), poly(rI)-poly(rC) and phi6 bacteriophage RNA (double-stranded) were without significant effects on the Met-tRNAfMet-binding reaction.
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A stable Escherichia coli IF-2-fMet-tRNAfMet complex is formed upon incubation of IF-2 (prokaryotic initiation factor) with fMet-tRNAfMet is the presence of 50 mM Tris-HCl, pH 7.0, 100 mM NH4Cl, and 7 mM 2-mercaptoethanol. The complex thus formed is retained on a Millipore filter and is assayed accordingly. Complex formation does dot require GTP, is unstable in the presence of 5 mM Mg2+, and is specific for fMet-tRNAfMet. Other amino acyl-tRNAs or deacylated tRNAs do not form such a complex with IF-2. A crude ribosomal high salt wash preparation contains other protein factors which bind unspecifically to RNAs under the above binding conditions. One of these factors elute similarly to IF-1 on DEAE-cellulose chromatography. Extensively purified IF-1 and IF-3 show weak and unspecific RNA-binding activities. The RNA-protein complex formed in each of the above cases, like the IF-2-fMet-tRNAfMet complex, is retained on Millipore filter and is sensitive to Mg2+.
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A preliminary study of iron absorption by whole body counting was carried on a group of 16 women. The cases included 8 patients suffering from iron deficiency anaemia and various infections as well as 8 healthy controls. High iron absorption is associated with iron deficiency, these changes being more marked in iron deficient controls than in those with infection or malignancy. In iron deficient controls results of whole body counting correlate very well with other haematological investigations.
The effects of additions of Mg-2+, ribosomes, and AUG codon on the Met-tRNAf Met-initiation factor-GTP complex were studied using a Millipore filtration method (J. Biol. Chem. 248, 4500 (1973)). Upon addition of increasing concentration of Mg-2+, the Met-tRNAf Met-initiation factor-GTP complex dissociates into free Met-tRNAf Met and initiation factor (GTP), with an infection around 1.5 to 2 mM Mg-2+. The Mg-2+-induced dissociation of Met-tRNAf Met-initiation factor-GTP complex was enhanced at ice bath temperature. At 37 degrees and in the presence of 1.5 to 2mM Mg-2+, the Met-tRNAf Met-initiation factor-GTP complex catalyzes the transfer of Met-tRNAf Met to ribosomes and AUG codon. Ribosome bound Met-tRNAf Met is stable to Mg-2+ and low temperature. A Millipore filtration assay for studies of (35S)Met-tRNAf Met binding to ribosomes and Aug codon has been developed. The assay procedure is carried out in three stages. In Stage I, the Met-tRNAf Met is bound to initiation factor in the presence of GTP, AUG codon (required for Stage II reaction), and 3.7 times 10-5 M aurintricarboxylic acid. The incubation is carried out at 37 degrees for 5 min. In Stage II, ribosomes and Mg-2+ (1.5 to 2mM final concentration) are added and the incubation is continued at 37 degrees for 10 min. In Stage III, more Mg-2+ is added to make the final Mg-2+ concentration of the incubation mixture 5 mM, and the reactions are further incubated at ice bath temperature for 10 min. The reactions are then terminated by addition of excess cold wash buffer and filtered through Millipore filters. Under the standard assay conditions, the radioactivity bound to Millipore filters in the absence of ribosomes and AUG codon is markedly reduced. Addition of ribosomes alone gave a significant increase in the radioactivity bound to Millipore filters. A further 2- to 3-fold stimulation of binding of (35S)Met-tRNAf Met to Millipore filters was observed when both ribosomes and AUG codon were added. The Met-tRNAf Met bound to ribosomes under the assay condition was reactive with puromycin. Upon DEAE-cellulose chromatography of a partially purified mixture of initiation factors (IF), Met-tRNAf Met binding activities separate into two forms, and are designated as IF-1A and IF-1B. These two forms can be distinguished by the stabilities of their respective Met-tRNAf Met-IF-1-GTP complexes to Mg-2+. The Met-tRNAf Met-IF-1A-GTP complex is distinctly more stable in the presence of Mg-2+ than Met-tRNAf Met-IF-1B-GTP complex. Continue.
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