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Biomedical subjects

S M Hecht

Publications and source records attributed to S M Hecht.

At least 127 records · Page 7Linked to original sources
PubMed1977

Preparation of Escherichia coli tRNAs terminating of modified nucleosides by the use of CTP(ATP):tRNA nucleotidyltransferase and polynucleotide phosphorylase.

Two procedures were investigated for the modification of tRNAs at the 3'-terminal nucleoside. The first involved the incubation of an enzymatically abreviated tRNA (tRNA-C-COH) with appropriate nucleoside triphosphates in the presence of CTP(ATP):tRNA nucleotidyltransferase from Escherichia coli and yeast. The E. coli enzyme did not utilize 2'- or 3'-deoxyadenosine 5'-triphosphate as substrates, but affected incorporation of the 2'- and 3'-O-methyladenosine triphosphates onto tRNA-C-Cou to the extent of 30 and 37%, respectively. Although incorporation of the deoxynucleotides could not be effected using the E. coli enzyme, yeast CTP(ATP:tRNA nucleotidyltransferase produced the desired tRNAs in yields of 45-65%. The second modification procedure involved incubation of tRNA-C-COH with (appropriately blocked) nucleoside diphosphates in the presence of polynucleotide phosphorylase. This procedure afforded the tRNAs terminating in 2'- and 3'-deoxyadenosine in yields of 4% (and the yield of the former was increased to 36% when the incubation was carried out in the presence of 20% methanol). The yields of tRNAs terminating in 2'- and 3'-O-methyladenosing produced by this procedure were 55 and 17%, respectively. Because only single isomers of most of the tRNAs terminating in 2'- and 3'-deoxy- and O-methyladenosine are aminoacylated, attempts were made to obtain the other isomericaminoacyl-tRNA by enzymatic introduction of chemically preaminoacylated nucleotides onto tRNA-C-COH. Although incubation of tRNA-C-COH with three aminoacylated nucleoside 5'-triphosphates and E. coli CTP(ATP):tRNA nucleotidyltransferase did not result in production of the desired tRNAs to a detectable extent, incubation with 2'-deoxy-3'-O-L-phenylalanyladenosine 5'-diphosphate and polynucleotide phosphorylase afforded E. coli tRNA terminating with the corresponding aminoacylated deoxynucleoside.

Adenosine Diphosphate↗
PubMed1977

Initial position of aminoacylation of individual Escherichia coli, yeast, and calf liver transfer RNAs.

Transfer RNAs from Escherichia coli, yeast (Sacharomyces cerevisiae), and calf liver were subjected to controlled hydrolysis with venom exonuclease to remove 3'-terminal nucleotides, and then reconstructed successively with cytosine triphosphate (CTP) and 2'- or 3'-deoxyadenosine 5'-triphosphate in the presence of yeast CTP(ATP):tRNA nucleotidyltransferase. The modified tRNAs were purified by chromatography on DBAE-cellulose or acetylated DBAE-cellulose and then utilized in tRNA aminoacylation experiments in the presence of the homologous aminoacyl-tRNA synthetase activities. The E. coli, yeast, and calf liver aminoacyl-tRNA synthetases specific for alanine, glycine, histidine, lysine, serine, and threonine, as well as the E. coli and yeast prolyl-tRNA synthetases and the yeast glutaminyl-tRNA synthetase utilized only those homologous modified tRNAs terminating in 2'-deoxyadenosine (i.e., having an available 3'-OH group). This is interpreted as evidence that these aminoacyl-tRNA synthetases normally aminoacylate their unmodified cognate tRNAs on the 3'-OH group. The aminoacyl-tRNA synthetases from all three sources specific argining, isoleucine, leucine, phenylalanine, and valine, as well as the E. coli and yeast enzymes specific for methionine and the E. coli glutamyl-tRNA synthetase, used as substrates exclusively those tRNAs terminating in 3'-deoxyadenosine. Certain aminoacyl-tRNA synthetases, including the E. coli, yeast, and calf liver asparagine and tyrosine activating enzymes, the E. coli and yeast cysteinyl-tRNA synthetases, and the aspartyl-tRNA synthetase from yeast, utilized both isomeric tRNAs as substrates, although generally not at the same rate. While the calf liver aspartyl- and cysteinyl-tRNA synthetases utilized only the corresponding modified tRNA species terminating in 2'-deoxyadenosine, the use of a more concentrated enzyme preparation might well result in aminoacylation of the isomeric species. The one tRNA for which positional specificity does seem to have changed during evolution is tryptophan, whose E. coli aminoacyl-tRNA synthetase utilized predominantly the cognate tRNA terminating in 3'-deoxyadenosine, while the corresponding yeast and calf liver enzymes were found to utilize predominantly the isomeric tRNAs terminating in 2'-deoxyadenosine. The data presented indicate that while there is considerable diversity in the initial position of aminoacylation of individual tRNA isoacceptors derived from a single source, positional specificity has generally been conserved during the evolution from a prokaryotic to mammalian organism.

Amino Acids↗
PubMed1977

Isomeric aminoacyl-tRNAs are both bound by elongation factor Tu.

Recent suggestions that elongation factor Tu (EF-Tu) is specific for 2'-O-aminoacyl-tRNA, as compared with the 3'-isomer, prompted us to assay [3H]aminoacyl-tRNAs from Escherichia coli terminating in 2'- or 3'-deoxyadenosine for binding to EF-Tu to determine the possible positional specificity of the factor. Binding of modified aminaocyl-tRNAs to EF-Tu-GTP was measured both as a function of the ability of EF-Tu-GTP to diminish the rate of chemical deacylation of [3H]aminoacyl-tRNAs and by gel filtration of the individual ternary complexes. Fifteen different tRNA isoacceptors were tested by the deacylation procedure, including three (tRNAAsp, tRNACys, and tRNATyr) for which isomeric modified aminoacyl-tRNAs were available. All of the modified aminoacyl-tRNAs were protected fromdeacylation, although generally to a lesser extent than the corresponding unmodified species. Six modified tRNA isoacceptors (including tRNATrp and tRNATyr, for which both modified aminoacyl-tRNAs were accessible by enzymatic aminoacylation) were used in gel filtration experiments to permit direct measurement of the individual aminoacyl-tRNA-EF-Tu-GTP complexes. These experiments were also done in the presence of equimolar amounts of the corresponding unmodified [14C]aminoacyl-tRNAs, and the relative affinities for a limiting amount of EF-Tu-GTP were measured. The results were completely consistent with those obtained by the deacylation procedure and indicated that EF-Tu can bind to both positional isomers of aminoacyl-tRNA with no obvious preference for either.

Amino Acids↗
PubMed1976

Synthesis and biological activity of pyrazolo[3,4,-d]pyrimidine nucleosides and nucleotides related to tubercidin, toyocamycin, and sangivamycin.

The 6-aza analogues of toyocamycin and sangivamycin were prepared as potential cytotoxic agents. The toyocamycin analogue (4-amino-1-(beta-D-ribofuranosyl)pyrazolo[3,4-d]pyrimidine-3-carbonitrile) could not be obtained directly from its O-acetylated precursor but was accessible via 4-amino-1-(beta-D-ribofuranosyl)pyrazolo[3,4-d]pyrimidine-3-thiocarboxamide. The identity of the nitrile was verified by its ultraviolet, infrared, and mass spectra, and by its conversion to the corresponding 3-carboxamide and thiocarboxamide when treated with water or hydrogen sulfide, respectively. Bioassay of the synthetic compounds in comparison with 4-amino-1-(beta-D-ribofuranosyl)pyrazolo[3,4-d]pyrimidine (6-azatubercidin) and 4-amino-2-(beta-D-ribofuranosyl)pyrazolo[3,4-d]pyrimidine revealed that the 3-thiocarboxamido derivative was more cytotoxic to the growth of mouse fibroblasts than 6-azatubercidin, effecting killing of 3T6 cells at less than or equal to 1 mug/ml. 4-Amino-1-(beta-D-ribofuranosyl)pyrazolo[3,4-d]pyrimidine (but not its 2-ribofuranosyl isomer) was shown to act as a substrate for adenosine deaminase from calf intestinal mucosa with an apparent Km of 125 (vs. 20 for adenosine) and the corresponding 5'-diphosphate of 6-azatubercidin was polymerized by polynucleotide phosphorylase (Micrococcus luteus) in the presence of Mn2+ to afford a homopolymer and copolymers with adenosine. The copolymers directed the binding of [3H]lysyl-tRNA to the A-site of ribosomes from Escherichia coli, but could not be used for the synthesis of polylsine in a cellfree system. The copolymer consiting of adenosine and 6-azatubercidin in a 2:1 ratio was found to form a 1:1 complex with poly(uridylic acid) at 4degreesC.

Antibiotics, Antineoplastic↗
PubMed1976

Position of aminoacylation of individual Escherichia coli and yeast tRNAs.

Transfer RNAs terminating 2'-or 3'-deoxyadenosine were prepared from unfractionated E. coli and yeast (Saccharomyces cerevisiae) tRNAs and purified to remove unmodified tRNAs. The modified tRNA species were assayed for aminoacylation with each of the 20 amino acids to determine the initial position of tRNA aminoacylation. The E. coli and yeast aminoacyl-tRNA synthetases specific for arginine, isoleucine, leucine, methionine, phenylalanine, and valine, as well as the E. coli glutamyl-tRNA synthetase, aminoacylated only those cognate tRNAs terminating in 3'-deoxyadenosine (i.e., those having a 2'-OH group). On the other hand, those E. coli and yeast synthetases specific for alanine, glycine, histidine, lysine, proline, serine, and threonine, as well as the yeast synthetase specific for glutamine, utilized exclusively those tRNAs having an available 3'-OH group on the 3'-terminal nucleoside, while the E. coli and yeast synthetases specific for asparagine, cysteine, and tyrosine, and the yeast aspartyl-tRNA synthetase, utilized both of the modified cognate tRNAs. The only observed difference in specificity between the E. coli and yeast systems was for tRNATrp, which was aminoacylated on the 2'-position in E. coli and the 3'-position in yeast. The results indicate that the initial position of aminoacylation is not uniform for all tRNAs, although for individual tRNAs the specificity has been conserved during the evolution from a prokaryotic to eukaryotic organism.

Amino Acyl-tRNA Synthetases↗
PubMed1975

On the "activation" of cytokinins.

A number of cytokinin analogs containing modifications in the heterocyclic moiety were prepared. These compounds were tested for activity as cytokinins and anticytokinins in the tabacco bioassay and the results were used to determine whether any position(s) of the heterocyclic nucleus of cytokinins may require derivatization as part of an over-all "activation" process. 3-substituted 4-alkylaminopyrazolo [3,4-d]pyrimidines and 4-alkylaminopyrrolo[2,3-d]pyrimidines, for example, have (substituted) carbon rather than nitrogen atoms at positions 3 and 5, respectively (analogous to position 7 in purines) and would be predicted to be metabolically stable at these positions. The finding that these compounds had cytokinin activity suggested that modification at the metabolically stable positions. The finding that these compounds had cytokinin activity suggested that modification at the metabolically stable position, and by extension at position 7 in cytokinin analogues which are purines, is not a prerequisite for the expression of cytokinin activity. Similar consideration of other heterocyclic analogs which have cytokinin activity suggests that the active form of a cytokinin can be the exogenous compound itself. Certain structural analogs of cytokinins were found to inhibit the growth of tobacco callus promoted by 6-(3-methyl-2-butenylamino)purine. These compounds were studied as potential cytokinin antagonists, i.e. having activity analogous to the 7-alkylamino-3-methylpyrazolo[4,3-d]pyrimidines (Hecht, S. M., 2068-2610; Skoog, F., Schmitz, R.Y., Hecht, S.M., and Bock, R. M. (1973) Phytochemistry 12, 25-37). The activity of these compounds is discussed and criteria are proposed to distinguish between those species which are specific anticytokinins and those which otherwise inhibit growth.

Biological Assay↗
PubMed1975

Hydrolysis of ribonucleoside 3'-diphosphates by rye grass 3'-nucleotidase.

Rye grass 3'-nucleotidase has been purified to apparent homogeneity on Sephadex A-25 and CM-cellulose columns and shown to hydrolyze 2'-O-methyladenosine 3'-monophohate and 2'-deoxyadenosine 3'-monophosphate 35.8 and 542 times more slowly than the normal substrate (3'-AMP), verifying the importance of the 2'-beta-OH group of the substrate in the overall hydrolysis process. Although neither was hydrolyzed as rapidly as 3'-AMP, both the 2'-O-methyl and 2'-deoxy analogs acted as competitive inhibitors of the hydrolysis of 3'-AMP (K-m equals 0.12 mM), with apparent K-i's of 0.39 and 0.51 nM, respectively. In order to determine the possible susceptibility of naturally occurring ribonucleoside 3'-diphosphates, such as guanosine tetraphosphate (ppGpp), to 3'-phosphohydrolase activities, the 3'-nucleotidase was also employed in the attempted pyrophosphorolysis of adenosine 3'-diphosphate and guanosine tetraphosphate. Neither adenosine 3'-diphosphate nor guanosine tetraphosphate was degraded at a significant rate by the nucleotidase, relative to the normal substrate.

Adenine Nucleotides↗
PubMed1975

Ribonucleoside 3'-di- and -triphosphates. Synthesis of guanosine tetraphosphate (ppGpp).

A procedure has been outlined for the synthesis of ribonucleoside 3'-di- and -triphosphates. The synthetic scheme involves the conversion of a ribonucleoside 3'-monophosphate to its 2'-(5'-di)-O-(1-methoxyethyl) derivative, followed by successive treatments of the blocked ribonucleotide with 1,1'-carbonyldiimidazole and mono(tri-n-butylammonium) phosphate or pyrophosphate. The resulting ribonucleoside 3'-di- and -triphosphate derivatives are then deblocked by treatment with dilute aqueous acetic acid, pH 3.0. The use of this procedure is illustrated for adenosine 3'-monophosphate, which has been converted to its corresponding 3'-di- and -triphosphates in 61% overall yield. The decomposition of adenosine 3'-di- and -triphosphates to adenosine 2'-monophosphate, adenosine 3'-monophosphate, and adenosine cyclic 2',3'-monophosphate as a function of pH at 100 degrees has been studied as has the attempted polymerization of adenosine 3'-diphosphate with polynucleotide phosphorylase. Also prepared was guanosine 5'-diphosphate 3'-diphosphate (guanosine tetraphosphate; ppGpp), which was accessible via treatment of 2'-O-(1-methoxyethyl)guanosine 5'-monophosphate 3'-monophosphate with the phosphorimidazolidate of mono(tri-n-butyl ammonium) phosphate. The resulting blocked tetraphosphate was deblocked in dilute aqueous acetic acid to afford ppGpp in an overall yield of 18%.

Adenine Nucleotides↗
PubMed1975

Anticytokinin activity of substituted pyrrolo[2,3-d]pyrimidines.

Ten substituted pyrrolo[2,3-d]pyrimidines were tested as cytokinins and anticytokinins in the tobacco bioassay. Eight new anticytokinins were identified and two were found to be highly active. The most potent species were 4-cyclohexylamino- and 4-cyclopentylamino-2-methylthiopyrrolo[2,3-d]pyrimidine, of which 0.05 and 0.009 muM concentrations, respectively, were required to produce detectable inhibition of the growth of tobacco callus cultured on a medium containing 0.003 muM 6-(3-methyl-2-butenylamino)purine. The inhibition of growth by moderate (</=6.6 muM) concentrations of these compounds was reversible by equal or higher concentrations of 6-(3-methyl-2-butenylamino)purine, but not by indole-3-acetic acid or gibberellic acid. These substituted pyrrolo[2,3-d]pyrimidines were also found to enhance bud formation at high cytokinin concentrations, suggesting that a cytokinin may act at more than one cellular site in exerting its growth-promoting and morphogenetic effects.

Journal Article↗
PubMed1974

Isomeric phenylalanyl-tRNAs. Position of the aminoacyl moiety during protein biosynthesis.

The preparation of phenylalanyl-tRNA terminating in 3'-deoxyadenosine has been achieved by incubation of abbreviated tRNA (tRNA-CpC(OH)) with 3'-deoxyadenosine 5'-diphosphate and polynucleotide phosphorylase (EC 2.7.7.8), followed by aminoacylation. The isomeric phenylalanyl-tRNA terminating in 2'-deoxyadenosine was constructed by incubation of tRNA-CpC(OH) with 2'-deoxy-3'-O-phenylalanyladenosine 5'-diphosphate and polynucleotide phosphorylase. While tRNA is aminoacylated at the 2'-position, only the 3'-aminoacyl-tRNA is active as a peptide acceptor in the peptidyltransferase reaction. Both modified tRNAs were bound to the A-site as efficiently as unmodified tRNA, but neither was so efficient at P-site binding or as an acceptor in the peptidyltransferase reaction. Neither of the modified tRNAs acted as a donor in the peptidyltransferase reaction.

Amino Acyl-tRNA Synthetases↗
PubMed1974

Competitive inhibition of beef heart cyclic AMP phosphodiesterase by cytokinins and related compounds.

Two cytokinins and four related analogs, none of which is a cyclic ribonucleotide, have been shown to act as competitive inhibitors of the high K(m) cyclic-AMP phosphodiesterase (3':5'-cyclic-AMP 5'-nucleotidohydrolase, EC 3.1.4.17) activity from beef heart. Weak inhibition of the low K(m) cyclic AMP phosphodiesterase activity was also observed, suggesting a possible mechanism for regulation of intracellular cyclic AMP levels by the exogenously added compounds. In addition to the kinetic data, obtained on the six inhibitors in four different heterocyclic series, 15 other cytokinins and related compounds have been shown to inhibit the high K(m) cyclic AMP phosphodiesterase activity at single concentrations of substrate and inhibitor. Heterocycles such as adenosine and 7-amino-3-methylpyrazolo[4,3-d]pyrimidine, which lack the N-substituent, were inactive as cyclic AMP phosphodiesterase inhibitors. The observed inhibition of cyclic AMP phophodiesterase supports prior observations which implicate exogenously added cytokinins in cyclic AMP metabolism.

Animals↗