Biomedical subjects
Publications and source records attributed to H Sternbach.
The overnight dexamethasone suppression test (DST) has emerged as a useful clinical test in identifying a proportion of patients with primary affective disorder. This test may also be helpful in predicting response to tricyclic antidepressants, MAO inhibitors, and/or electroconvulsive therapy. This review summarized the recent clinical studies on the DST.
The binding of N-acetyl-Phe-tRNAPhe (an analogue of peptidyl-tRNA), Phe-tRNAPhe, and deacylated tRNAPhe to poly(U)-programmed tightly coupled 70S ribosomes was studied. The N-acetyl-Phe-tRNAPhe binding is governed by an exclusion principle: not more than one N-acetyl-Phe-tRNAPhe can be bound per ribosome, although this peptidyl-tRNA analogue can be present either at the aminoacyl-tRNA (A) site or the peptidyl-tRNA (P) site. Two Phe-tRNAPhe molecules are accepted by one ribosome in the presence of poly(U). This aminoacyl-tRNA binds enzymatically (in the presence of elongation factor Tu and GTP) and nonenzymatically to the A site and is then transferred to the P site, if that site is free. If this elongation factor G-independent movement is hampered, either by using an incubation temperature of 0 degrees C or by the addition of the translocation inhibitor viomycin, only one Phe-tRNAPhe per ribosome can be bound. The effect of the peptidyltransferase inhibitor chloramphenicol on the binding is similar to that of viomycin. In the absence of poly(U), Phe-tRNAPhe cannot bind to the ribosome. Deacylated [14C]tRNAPhe can bind in three copies to one ribosome. The new third tRNA binding site is called the "E" site. The sequence of filling the sites is P, E, and A. The apparent binding constants for the P and the E sites are both approximately 9 X 10(6) M-1 and that for the A site is 1.3 X 10(6) M-1. In the absence of poly(U), only one deacylated tRNAPhe can be bound per ribosome. This tRNAPhe most likely occupies the P site.
The order of substrate addition to arginyl-tRNA synthetase from baker's yeast has been investigated by bisubstrate kinetics, product inhibition and inhibition by three different inhibiting ATP analogs, the 6-N-benzyl, 8-bromo and 3'-deoxy derivatives of ATP, each acting competitively with respect to one of the substrates. The kinetic patterns are consistent with a random ter-ter mechanism, an addition of the three substrates and release of the products in random order. The different inhibitors are bound to different enzyme . substrate complexes of the reaction sequence. Addition of inorganic pyrophosphatase changes the inhibition patterns and addition of methylenediphosphonate as pyrophosphate analog abolishes the effect of pyrophosphatase, showing that the concentration of pyrophosphate is determinant for the mechanism of catalysis.
The authors studied 19 inpatients with major depressive episode or schizoaffective disorder, depressed type, and compared their response to the dexamethasone suppression test (DST) with their response to the methylphenidate stimulation test. They found a significant negative correlation between responses to DST and to methylphenidate.
The substrate specificity of twenty aminoacyl-tRNA synthetases from E. coli and thirteen enzymes from baker's yeast with regard to eight ATP analogs is investigated for a comparison of the active-site topography. The enzymes are arranged in a scheme of possible "enzyme families" and compared to earlier schemes.
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2-Thiocytidine 5'-triphosphate, s2CTP, is able to replace CTP as a substrate for tRNA nucleotidyltransferase. s2CMP can be incorporated into both cytidine sites of the C-C-A terminus common to all tRNAs, and in the absence of ATP into at least two additional positions. This was shown by alkylation of the 2-thiocytidine residues with iodo[14C]acetamide, total nucleoside analysis, microgel electrophoresis and analysis of RNase T1 fragments of these tRNAs. The incorporation of the 3'-terminal AMP is not influenced by the additional s2CMP residues at pH 9.0. However, at pH 7.6 the additional s2CMP residues are hydrolysed and AMP can be incorporated into the normal position. Two different tRNAs with terminal 2-thiocytidine alkylated by iodoacetamide inhibit tRNA nucleotidyltransferase. This inhibition is significantly slower if an elongated species is used compared to a tRNA with alkylated 2-thiocytidine in the normal position 75. The addition of 2-mercaptoethanol reactivates the enzyme and leads to a cytidine containing tRNA. This reaction identifies the attacking nucleophile of the enzyme as cysteine residue, which is probably identical to a cysteine residue found in a similar experiment reported previously. The mechanism of the enzymatic and chemical reactions is discussed.
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Yeast tRNAPhe containing a phosphorothioate modified -CS-CS-A terminus binds two moles of chloroterpyridineplatinum(II). This result was determined by titrating the tRNA with [3H](terpy)PtCl] Cl, removing excess platinum by cation exchange chromatography, and determining the amount of bound platinum by radiocounting techniques. It has thus been established that adjacent phosphorothioate modified nucleotides can be labeled with an electron dense stain, a necessary requirement for electronmicroscopic sequencing of polynucleotides to become practical.
Sixteen analogues of ATP have been tested in the aminoacylation reaction of threonyl-tRNA, lysyl-tRNA, and arginyl-tRNA synthetases from baker's yeast. Two compounds are substrates for threonyl-tRNA and for lysyl-tRNA synthetases and five compounds for arginyl-tRNA synthetase. There are six inhibitors for threonyl-tRNA, nine for lysyl-tRNA, and six for arginyl-tRNA synthetase. Their Km and Ki values have been determined. Thus positions 2, 6, 7, 8 and 9 of the purine moiety and 2' and 3' of the sugar moiety of the ATP molecule are important for catalytic action of these aminoacyl-tRNA synthetases. Remarkably arginyl-tRNA synthetase is the first aminoacyl-tRNA synthetase which tolerates bulky substituents at the sugar moiety of ATP. These data fit with the idea that synthetases of subunit structure need magnesium-ion-ATP complexes with an anti conformation as substrates whereas single-chain enzymes accept this substrate in the syn conformation.
Isomer A of adenosine 5'-O-(1-thiotriphosphate) (ATP alpha S) is a substrate for tRNA nucleotidyltransferase from baker's yeast, whereas isomer B is a competitive inhibitor. The tRNA resulting from this reaction has a phosphorothioate instead of a phosphate diester linkage at the last internucleotidic linkage between cytidine and adenosine. On limited digestion of this tRNA with RNase A, one can isolate cytidine 2',3'-cyclic phosphorothioate which can be deaminated to uridine 2',3'-cyclic phosphorothioate. It can be shown that this compound is the endo isomer and that, therefore, the phosphorothioate diester bond in the tRNA must have had the R configuration. This result indicates that no racemization during the condensation of ATP alpha S, isomer A, onto the tRNA had occurred. Whether inversion or retention of configuration had taken place awaits elucidation of the absolute configuration of isomer A of ATP alpha S.
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Structural analogues of adenosine 5'-triphosphate and cytidine 5'-triphosphate were investigated as substrates for ATP(CTP):tRNA nucleotidyl transferase. Eight out of 26 ATP analogues and six out of nine CTP analogues were incorporated into the 3' terminus of tRNA. In general, for the recognition of the substrates the modification of the cytidine is less critical than is the modification of adenosine. An isosteric substitution on the ribose residue is possible in both CTP and ATP. The free hydroxyls of these triphosphates can be replaced by an amino group or hydrogen atom without loss of substrate properties. Modifications of positions 1, 2, 6, and 8 on the adenine ring of ATP are not allowed whereas modification on positions 2, 4 and 5 on the cytosine ring of CTP are tolerated by the enzyme. No differences can be observed in the substrate properties of ATP(CTP):tRNA nucleotidyl transferase isolated from different sources. Methods for preparation of tRNA species, which are shortened at their 3' end by one or more nucleotides, and analytical procedures for characterisation of these modified tRNAs are described.
2'-Deoxy-2'-amino-cytidylic acid can be incorporated into position 75 of tRNAPhe from yeast by tRNA nucleotidyltransferase yielding tRNAPhe-C-C(2'NH2). tRNAPhe-C-C(2'NH2) can be reacted with the N-hydroxysuccinimide esters of bromoacetic acid of mercuriacetic acid to yield the derivatives tRNAPhe-C-C(2'NHCOCH2Br) and tRNAPhe-C-C(2'NHCOCH2Hg+OH-). Each of these reactive tRNAs inactivates tRNA nucleotidyltransferase from yeast with similar kinetics. The enzyme can be protected against inhibition by its substrates tRNAPhe-C and tRNAPhe-C-C as well as ATP and CTP. A covalent, isolatable 1:1 complex between tRNAPhe-C-C(2'NHCOCH2Br) and the enzyme was formed, but could not be found when the enzyme had previously been inactivated with p-hydroxymercuribenzoate.
5-Formyl-1-(alpha-D-ribofuranosyl)uracil 5'=triphosphate has been used to affinity label E. coli DNA-dependent RNA polymerase. It is a noncompetitive inhibitor of the enzyme with Ki=0.54 mM. A short preincubation of the enzyme and alpha-fo5UTP is required to achieve maximum inhibition, and the entent of the inhibition is dependent upon the alpha-fo5UTP concentration. When a preincubation mixture of alpha-fo5UTP/enzyme is diluted, the enzyme regains activity with time showing that the inhibition is reversible, presumably occurring by Schiff base formation between an amino group on the enzyme and the formyl group. Upon sodium borohydride reduction of an enzyme/alpha-fo5UTP preincubation mixture the enzyme is irreversibly inhibited. alpha-fo5UTP is more effective in inhibiting the enzyme than alpha-fo5U, and the inhibition is decreased by the presence of ATP, UTP, or GTP in the preincubation mixture, suggesting that inhibition is occurring at a triphosphate binding site. The stoichiometry of binding of alpha-fo5UTP to the enzyme was determined using the gamma-32P-labeled derivative. After a 20-s preincubation of enzyme/alpha-fo5UTP followed by NaBH4 reduction the stoichiometry of binding was 1.1:1 (alpha-fo5UTP bound: inactivated enzyme), and this rose to 2.42:1 after a 10-min preincubation. After a 20-s preincubation the [gamma-32P]-alpha-fo5UTP was shown to be located on the beta subunit of RNA polymerase by cellulose acetate electrophoresis in 6 M urea.
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