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D Kern

Publications and source records attributed to D Kern.

At least 91 records · Page 5Linked to original sources
PubMed1983

Large scale purification and structural properties of yeast aspartyl-tRNA synthetase.

A large scale purification procedure of baker's yeast aspartyl-tRNA synthetase is described which yields more than 200 mg pure protein starting from 30 Kg of wet commercial cells. The synthetase is an alpha 2 dimer of Mr = 125,000 +/- 5,000 which can be crystallized (J. Mol. Biol. 138, 1980, 129-135). The enzyme has an elongated shape with a Stokes radius of 50 A and a frictional ratio of 1.5. The synthetase has a tendency to aggregate but methods are described where this effect is overcome.

Amino Acyl-tRNA Synthetases↗
PubMed1983

Interaction of tRNAPhe and tRNAVal with aminoacyl-tRNA synthetases. A chemical modification study.

The alkylation by ethylnitrosourea of phosphodiester bonds in tRNAPhe from yeast and in tRNAVal from yeast and from rabbit liver and that by 4-(N-2-chloroethyl-N-methylamino)-benzylamine of N-7 atoms of guanosine residues in yeast tRNAVal have been used to study the interaction of these tRNAs with aminoacyl-tRNA synthetases. The modifications occurring at low yield were carried out on 3' and/or 5' end-labelled tRNAs either free or in the presence of cognate or non-cognate synthetases. After splitting of the tRNAs at the alkylated positions, the position of the modification sites in the tRNA sequences were detected by acrylamide gel electrophoresis. It was found that the synthetases protect against alkylation certain phosphate or guanosine residues in their cognate tRNAs. Non-cognate synthetases failed to protect efficiently specific positions in tRNA against modification. In yeast tRNAPhe the cognate phenylalanyl-tRNA synthetase protects certain phosphates located in all four stems and in the anticodon and extra-loop of the tRNA. Particularly strong protections occur on phosphate 34 in the anticodon loop and on phosphates 23, 27, 28, 41 and 46 in the D and anticodon stems. In yeast tRNAVal complexed with yeast valyl-tRNA synthetase the protected phosphates are essentially located in the corner between the amino-acid-accepting and D stems, in the D loop, anticodon stem and in the variable region of the tRNA. Three guanosine residues, located in the D stem, and another one in the 3' part of the anticodon stem were also found protected by the synthetase. In mammalian tRNAVal, complexed with the cognate but heterologous yeast valyl-tRNA synthetase, the protected phosphates lie in the anticodon stem, in the extra-loop and in the T psi arm. The location of the protected residues in the structure of three tRNAs suggests some common features in the binding of tRNAs to aminoacyl-tRNA synthetases. These results will be discussed in the light of informations on interaction sites obtained by nuclease digestion and ultraviolet cross-linking methods.

Alkylation↗
PubMed1983

Lack of prognostic value of T-, B- and null-lymphocytes in adult acute leukemia.

The distribution of T-, B- and null-lymphocytes was studied in the peripheral blood of 38 adult patients with acute nonlymphocytic leukemia (ANLL) and 15 with acute lymphocytic leukemia (ALL) at first diagnosis, during induction treatment, and in remission. Thirteen ANLL and 9 ALL patients were followed until relapse and during reinduction therapy. T- and B-cells were detected by specific membrane marker. The pre- and posttreatment pattern of lymphocyte subpopulations was analyzed to determine their prognostic significance for remission incidence, remission duration, and survival. It was observed that in both types of leukemia, T-cells are more affected by the leukemic process and cytostatic drugs than B-cells. Nonresponding patients possibly have a reduced potential for recruiting precursor T- and B-cells. At first diagnosis, no significant correlation was found between pre- or posttreatment variables and prognosis. At relapse, ANLL patients had a longer second remission when a high proportion of B-cells was found; ALL patients with a high lymphocyte count before and after treatment, experienced longer survival.

Acute Disease↗
PubMed1983

Effects of ochratoxin A metabolites on yeast phenylalanyl-tRNA synthetase and on the growth and in vivo protein synthesis of hepatoma cells.

The ochratoxin A (OTA) metabolite (4R)-4-hydroxyochratoxin A [4R)-OTA) inhibits the aminoacylation of phenylalanine tRNA catalyzed by phenylalanyl-tRNA synthetase (PheRS) with a Ki-value of 0.9 mM as compared to 1.3 mM for OTA. It also inhibits protein synthesis and cell growth in the same manner as OTA. Ochratoxin alpha (OT alpha) does not affect either protein synthesis or cell growth.

Amino Acyl-tRNA Synthetases↗
PubMed1983

Comparative study of the effect of ochratoxin A analogues on yeast aminoacyl-tRNA synthetases and on the growth and protein synthesis of hepatoma cells.

Ochratoxin A (OTA), a naturally occurring mycotoxin of Aspergillus and Penicillium species, consists of a 5' chlorinated dihydromethyl isocoumarin linked to L,beta-phenylalanine by an alpha-amide bond. 8 analogues of OTA were prepared in which the phenylalanine was always substituted by another amino acid. The effects of these analogues on yeast tRNA amino acylation reaction and on growth and protein synthesis of hepatoma culture cells were compared with those of OTA. In addition, Ochratoxin B (OTB) and ochratoxin alpha (OT alpha) were examined. All the analogues of OTA had inhibitory effects in the 3 test systems, although to a lesser degree than OTA. The degree of inhibition depended on the kind of substituted amino acid, the tyrosine, valine, serine and alanine analogues being most effective, in contrast to the proline analogue. OTB and OT alpha were ineffective.

Amino Acyl-tRNA Synthetases↗
PubMed1982

Covalent attachment of aspartic acid to yeast aspartyl-tRNA synthetase induced by the enzyme.

Aspartic acid can be covalently linked to yeast aspartyl-tRNA synthetase and to other proteins, in the absence of tRNA, under conditions where the synthetase activates the amino acid into aspartyl-adenylate, i.e., in the presence of ATP and MgCl2. The linkage between aspartic acid and the protein is acid and alkali resistant; thus it is likely a peptide-like amide bond formed between the activated carboxylate group of aspartic acid and the primary amine function of the side chain of lysine residues.

Amino Acids↗
PubMed1981

Catalytic mechanism of valyl-tRNA synthetase from baker's yeast. Reaction pathway and rate-determining step in the aminoacylation of tRNAVal.

The catalytic mechanism of valyl-tRNA synthetase from baker's yeast has been investigated by pre-steady-state and steady-state kinetic measurements and end product dissociation studies. The pre-steady-state kinetics show a lag period during the early time when the reaction is started with free enzyme. The preincubation of the synthetase with tRNAVal and/or valine or preformation of Val approximately AMP leads to a progressive suppression of the lag. This lag probably reflects conformational transitions of the enzyme-substrate complex necessary for the transfer. At low pH or at a low ionic strength, the tRNAVal charging occurs much faster at the pre steady state than at the steady state. We show that after the fast transfer of valine from adenylate to tRNAVal, followed by the fast dissociation of AMP and PPi, a new adenylate is synthesized which promotes the dissociation of the nascent Val-tRNAVal. This dissociation occurs in a multistep process. First ATP and magnesium promote the ejection of the valine moiety of Val-tRNAVal from the adenylate site. A new adenylate is then synthesized which promotes, in the presence of magnesium, several state changes of the end product complex. A complex is finally generated in which the enzyme-bound Val-tRNAVal is able to exchange rapidly with a tRNAVal molecule. The free tRNAVal plays an active role in this exchange. Depending upon the experimental conditions, one of these steps can determine the steady-state rate of tRNAVal charging. The dissociations of enzyme-bound uncharged tRNAVal or aa-tRNAs substituted on the amino acid or on the tRNA parts by noncognate parts as well as the effect of the replacement of the adenylates by wrong adenylates have been investigated. It is shown that the valine and the tRNA moieties of Val-tRNAVal and the valine moiety of the adenylate are involved in this mechanism of dissociation. Finally, the rate-determining step of the reversal of tRNAVal charging at the steady-state has been investigated. It is shown that this step is the dissociation of the deacylated tRNAVal from enzyme.

Acylation↗
PubMed1981

Purification and some properties of alanyl- and leucyl-tRNA synthetases from baker's yeast.

Alanyl- and leucyl-tRNA synthetases from baker's yeast were purified to homogeneity in the presence of the protease inhibitor phenylmethylsulfonyl fluoride. Both consist of single polypeptide chains of 118 000 and 125 000 daltons, respectively, as determined by polyacrylamide gel electrophoresis under denaturing conditions. The monomeric structure of leucyl-tRNA synthetase differs from the dimeric one obtained previously in the absence of protease inhibitors. This illustrates the sensitivity of the synthetases to proteolytic actions and indicates that native structures can only be obtained under optimal protecting conditions. Alanyl- and leucyl-tRNA synthetases differ with respect to pH optimum (6.5 and 8.5, respectively), Michaelis constant for amino acid (1 mM and 0.03, respectively) and in the rate-limiting step for the tRNA aminoacylation reaction. Whereas the catalytic step itself was rate-limiting for alanyl-tRNA synthetase, a step occurring after this was rate-limiting for leucyl-tRNA synthetase.

Alanine-tRNA Ligase↗
PubMed1981

The catalytic mechanism of glutamyl-tRNA synthetase of Escherichia coli. A steady-state kinetic investigation.

The sequence of substrate binding and of end-product dissociation at the steady state of the catalytic process of tRNAGlu aminoacylation by glutamyl-tRNA synthetase from Escherichia coli has been investigated using bisubstrate kinetics, dead-end and end-product inhibition studies. The nature of the kinetic patterns indicates that ATP and tRNAGlu bind randomly to the free enzyme, whereas glutamate binds only to the ternary enzyme . tRNAGlu . ATP complex. Binding of ATP to the enzyme hinders that of tRNAGlu and vice versa. After interconversion of the quaternary enzyme . substrates complex the end-products dissociate in the following order: PPi first, AMP second and Glu-tRNA last. In addition to its role as substrate and as effector with ATP for the binding of glutamate, tRNAGlu promotes the catalytically active enzyme state. Whereas at saturating tRNAGlu concentration the catalysis is rate-determining, this conformational change can be rate-determining at low tRNAGlu concentrations. The results are discussed in the light of the two-step aminoacylation pathway catalyzed by this synthetase.

Adenosine Triphosphate↗
PubMed1981

Glycyl-tRNA synthetase from baker's yeast. Interconversion between active and inactive forms of the enzyme.

Glycyl-tRNA synthetase from baker's yeast has been purified to homogeneity. This synthetase was found to be very sensitive to proteases present in the yeast extracts and to oxidizing agents of thiol groups. In the absence of protease inhibitors and/or dithioerythritol, the enzyme rapidly lost its activity and could not be isolated. The use of these protectors allowed us to obtain different oligomeric structures of the synthetase. In the presence of a minimal concentration of dithioerythritol but in the absence of protease inhibitors, a tetrameric glycyl-tRNA synthetase of the alpha 2 beta 2 type (alpha = 67 600, beta = 57 500) with a very low specific activity was recovered. With high concentrations of both protectors, a dimeric enzyme was isolated with a specific activity comparable to that for other yeast synthetases. The enzyme was of the alpha 2 type where alpha = 70 000--80 000 daltons, depending on whether phenylmethanesulfonyl fluoride or diisopropyl fluorophosphate was used as the protecting agent. The native form of the enzyme (alpha 2 = 160 000) associated easily with other proteins in various complexes of molecular weights from 250 000 to 300 000, some of them containing valyl-tRNA synthetase. The dimeric glycyl-tRNA synthetase was found in equilibrium with its subunits. Diluting the enzyme solution or increasing the salt concentration displaced the equilibrium toward the monomers, which are catalytically inactive for both the tRNA aminoacylation and the PPi-ATP exchange reactions. Addition of both tRNAGly and ATP.MgCl2 plus glycine displaced the equilibrium toward the dimeric form of the enzyme. Thiol groups were found to be involved in the association between the two subunits and in both activities of the synthetase. The results are interpreted in the light of possible regulatory mechanisms of the activity of this synthetase.

Amino Acyl-tRNA Synthetases↗
PubMed1980

The glutaminyl-transfer RNA synthetase of Escherichia coli. Purification, structure and function relationship.

Glutaminyl-tRNA synthetase from Escherichia coli has been purified to homogeneity with a yield of about 50%. It is a monomer of about 69 000 daltons. Arginyl and glutamyl-tRNA synthetases are also monomeric synthetases of molecular weight significantly lower than 100 000. In addition it is well known that these three synthetases require their cognate tRNA to catalyze the [32P]PPi-ATP exchange. Like arginyl-tRNA synthetase, but unlike glutamyl-tRNA synthetase, glutaminyl-tRNA synthetase seems to contain some repeated sequences. Therefore no correlation can be established between the tRNA requirement of these synthetases for the catalysis of the isotope-exchange and the presence or the absence of sequence duplication. In the native enzyme four sulfhydryl groups react with dithiobisnitrobenzoic acid causing a loss of both the aminoacylation and the [32P]PPi-ATP exchange activities. The rate-limiting steps of the overall aminoacylation and its reverse reaction correspond, respectively, to the catalysis of the aminoacylation of tRNA Gln and of the the deacylation of glutaminyl-tRNA Gln. At acidic pH, glutaminyl-tRNA synthetase catalyzes the synthesis of the glutaminyl-tRNA Gln and its deacylation at significantly lower rates than the [32P]PPi-ATP exchange, indicating than glutaminyl-tRNA Gln cannot be an obligatory intermediate in this isotope exchange. These results suggest the existence of a two-step aminoacylation mechanism catalyzed by this enzyme.

Amino Acids↗
PubMed1980

The catalytic mechanism of the glutamyl-tRNA synthetase from Escherichia coli. Detection of an intermediate complex in which glutamate is activated.

Up to now it was not possible to isolate an enzyme . adenylate complex after mixing the glutamyl-tRNA synthetase from Escherichia coli with ATP, MgCl2, and glutamate. This enzyme catalyzes an AMP-dependent and PPi-independent deacylation of Glu-tRNAGlu. The labeled glutamate which disappears from Glu-tRNAGlu in the presence of AMP remains linked to the enzyme in a complex isolated by filtration on nitrocellulose discs. The addition of tRNAGlu to this reaction mixture at the deacylation plateau gives rise to a synthesis of Glu-tRNAGlu, via an ATP-independent reaction. These results indicate the existence of the following equilibrated reaction catalyzed by the glutamyl-tRNA synthetase E + Glu-tRNAGlu + AMP in equilibrium E . AMP approximately Glu + tRNAGlu. This transfer of glutamate from an activated complex to tRNAGlu indicates that the formation of glutamyl-tRNA is catalyzed via a two-step reaction mechanism. The AMP-dependent and PPi-independent deacylation of Glu-tRNAGlu is the rate-limiting step of the reverse of the AMP- and PPi-dependent deacylation.

Adenosine Monophosphate↗