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H Sternbach

Publications and source records attributed to H Sternbach.

At least 19 recordsLinked to original sources
PubMed1999

Heterologous overexpression of human NEFA and studies on the two EF-hand calcium-binding sites.

Human NEFA is an EF-hand, leucine zipper protein containing a signal sequence. To confirm the calcium binding capacity of NEFA, recombinant NEFA analogous to the mature protein and mutants with deletions in the EF-hand domain were expressed in Pichia pastoris and secreted into the culture medium at high yield. The calcium binding activity of each purified protein was measured by a modified equilibrium dialysis using the fluorescent Ca2+ indicator FURA-2 and atomic absorption spectroscopy. A stoichiometry of 2 mol Ca2+/mol NEFA was determined. The Ca2+ binding constants were resolved by intrinsic fluorescence spectroscopy. Fluorescence titration exhibited two classes of Ca2+ binding sites with Kd values of 0.08 microM and 0.2 microM. Circular dichroism (CD) spectroscopy showed an increase from 30 to 43% in the amount of alpha-helix in NEFA after addition of calcium ions. Limited proteolytic digestion indicated a Ca2+ dependent conformational change accompanied by an altered accessibility to the enzyme.

Binding Sites↗
PubMed1998

Accuracy of protein biosynthesis: quasi-species nature of proteins and possibility of error catastrophes.

Yeast aminoacyl-tRNA synthetases act in a multi-step process when recognizing their cognate amino acids; this identification event includes "physical" binding and "chemical" proof-reading steps. However, the various enzymes use these single steps at different degrees, and their specificities with regard to the 20 naturally occurring amino acids deviate considerably from each other. The characteristic discrimination factors D were determined for seven synthetases in vitro: the highest specificity with D values between 28,000 and > 500,000 were observed with tyrosyl-tRNA synthetase, the lowest values between 130 and 1700 for lysyl-tRNA synthetase. The tested class I enzymes are more specific than the investigated class II enzymes, and it may be put into discussion whether this observation can be generalized. Error rates in amino acid recognition differ not only between the individual aminoacyl-tRNA synthetases but also considerably for different amino acids sorted by the same enzyme. Strikingly, all investigated enzymes exhibit a poor specificity in discrimination of cysteine and tryptophan from their cognate substrates, and these cases may be regarded as "specificity holes". In view of the observed specificities a protein consisting of 700 amino acids would contain maximally up to five "incorrect" residues, if the in vitro error rates are also valid under in vivo conditions. Therefore the terminus "quasi-species", an expression which was originally created for nucleic acids, is justified. The "quasi-species" nature of proteins may become important when genes are translated in different organisms with different accuracies of the translation apparatus. In such cases different "quasi-species" will be obtained. Using our data in mathematical models which predict the stability of protein synthesizing systems, we find that they are consistent with a stable yeast organism which is not prone to die by an "error catastrophe". However, this appears only if average values from our experiments are used for calculations. If a single compound, e.g. the arginine analog canavanine, is discriminated very poorly from the cognate substrate, or if the "specificity holes" get larger, an "error catastrophe" must be envisaged.

Amino Acids↗
PubMed1998

Age-associated testosterone decline in men: clinical issues for psychiatry.

OBJECTIVE: The author summarizes current knowledge about the diagnosis and treatment of testosterone decline in healthy aging men and the associated clinical issues for psychiatry. METHOD: A MEDLINE search was conducted in which the search terms "male climacteric," "male menopause," "andropause," "viropause," "low-testosterone syndrome," and "testosterone replacement therapy" were used. Literature published before 1966 was identified by reviewing the reference lists of later publications. RESULTS: Manifestations of testosterone deficiency have included depression, anxiety, irritability, insomnia, weakness, diminished libido, impotence, poor memory, reduced muscle and bone mass, and diminished sexual body hair. Although testosterone levels decline with age, there is great interindividual variability, and the connection between serum testosterone levels and clinical psychiatric signs and symptoms is not clear-cut, since other hormonal changes are implicated as well. Testosterone replacement therapy may offer hypogonadal men benefit, but long-term studies on its efficacy and safety are lacking. Comprehensive biopsychosocial assessment should be a routine part of the evaluation of complaints of low-testosterone syndrome in men. CONCLUSIONS: Testosterone decline/deficiency is not a state strictly analogous to female menopause and may exhibit considerable overlap with primary and other secondary psychiatric disorders.

Aged↗
PubMed1996

Phenylalanyl-tRNA synthetase from yeast and its discrimination of 19 amino acids in aminoacylation of tRNA(Phe)-C-C-A and tRNA(Phe)-C-C-A(3'NH2).

For discrimination between phenylalanine and 18 other naturally occurring non-cognate amino acids by the class II aminoacyl-tRNA synthetase specific for phenylalanine, discrimination factors, D, of 190-6300 have been determined from kcal and K(m) values. Generally, phenylalanyl-tRNA synthetase is more specific than the class II enzymes specific for Lys and Thr, but works with lower accuracy than the class I enzymes specific for IIe, Tyr, and Arg. In aminoacylation of tRNA(Phe)-C-C-A(3'NH2) discrimination factors D1 vary between 80-1610. Pre-transfer proof-reading factors II1 are in the range 2.3-74, post-transfer proof-reading factors II2 in the range 1.0-4.6, showing that pre-transfer proof-reading is the main correction step, post-transfer proofreading is less effective or negligible. Initial discrimination factors (I1 and I2) caused by differences in Gibbs free energies of binding between phenylalanine and non-cognate amino acids have been calculated assuming a two-step binding process. Factors I1 can be related to hydrophobic-interaction forces depending on accessible surface areas of the amino acids, factors I2 scatter about a low mean value and do not show any relation to amino acid structures or surfaces, indicating less checking of amino acid side chains in the putative second binding step.

Acylation↗
PubMed1994

Threonyl-tRNA synthetase from yeast. Discrimination of 19 amino acids in aminoacylation of tRNA(Thr)-C-C-A and tRNA(Thr)-C-C-A(2'NH2).

For discrimination between threonine and 18 other naturally occurring non-cognate amino acids by the class II aminoacyl-tRNA synthetase specific for threonine, discrimination factors (D) have been determined from Kca and Km values. The lowest values were found for Cys, Met, Val (D = 70-280), indicating that threonine is only 70-280-times more often esterified to tRNA(Thr)-C-C-A than are these non-cognate compounds at the same amino acid concentrations. The highest D values have been observed for Gly, Pro, Gln, Leu, Phe, and Lys (D = 1000-2000), for the other non-cognate amino acids D values are in the medium range 300-1000. Generally, threonyl-tRNA synthetase is less specific than the class I enzymes specific for Ile, Val, Tyr, Arg, but more specific than the only investigated class II enzyme specific for Lys. In aminoacylation of tRNA(Thr)-C-C-A(2'NH2) discrimination factors D1 are in the range 2-170. From D1 values and AMP-formation stoichiometry, pre-transfer proof-reading factors II1, were determined; post-transfer proof-reading factors II2 were determined from D values and AMP-formation stoichiometry in acylation of tRNA(Thr)-C-C-A. II1 values are in the range 1.8-33, II2 values in the range 1.4-22, thus threonyl-tRNA synthetase shows the highest post-transfer proof-reading activity of six investigated synthetases (specific for Ile, Val, Tyr, Arg, Lys). Initial discrimination factors caused by differences in Gibbs free energies of binding between threonine and non-cognate amino acids have been calculated from discrimination and proof-reading factors. Assuming a two-step binding process, two factors (I1 and I2) have been determined which can be related to hydrophobic interaction forces depending on accessible surface areas of the amino acids. The threonine side chain must be bound by hydrophobic forces and two hydrogen bonds. In contrast to proof-reading factors obtained with the synthetases specific for Ile, Val, Tyr, Arg, and Lys, proof-reading factors II1 and II2 obtained with threonyl-tRNA synthetase are also related to hydrophobic interaction of the amino acid side chains and the enzyme. Threonyl-tRNA synthetase examines side chain structures of amino acids in the four postulated recognition steps, for each step the enzyme uses special distinct structures or conformations of the binding cleft.

Adenosine Triphosphate↗
PubMed1994

Channel active mammalian porin, purified from crude membrane fractions of human B lymphocytes and bovine skeletal muscle, reversibly binds adenosine triphosphate (ATP).

A new aspect of mammalian porin (mammalian VDAC = mammalian voltage-dependent anion channel) is presented: channel active VDAC binds adenosine triphosphate (ATP) in the absence of Ca2+. Channel active "Porin 31HL" or "Porin 31BM", enriched from crude membranes of human B lymphocytes or whole cell lysates of bovine skeletal muscle, respectively, was bound to a nine atoms spacer ATP-agarose at pH 7.4 or 5.0 and reeluted from the resin by 10 mM ATP disodium salt. Furthermore, channel active "Porin 31BM" was labelled by [32P]ATP in a 1:1 stoichiometric relation. Binding of ATP to human porin was confirmed by studying the interaction of the synthetic porin fragment Type-1/Ac-35, comprising the putative nucleotide binding site G Y G F G, with trinitrophenyl-ATP (TNT-ATP) by scanning fluorometry. Peptide/TNP-ATP complexes clearly show enhancement of fluorescence intensity and a spectral shift of the fluorescence maximum. In a control experiment, using a porin fragment lacking the putative nucleotide binding site, no change of fluorescence emission was observed. Further confirmation for ATP binding by human VDAC arose from an autoradiographic experimental approach: the porin fragment Type-1/Ac-35 could be labelled by [32P]ATP, while a second porin fragment ending immediately before the putative nucleotide binding site could not; nor could a synthetic non porin peptide.

Adenosine Triphosphate↗
PubMed1992

Lysyl-tRNA synthetase from yeast. Discrimination of amino acids by native and phosphorylated species.

Discrimination factors (D) which are characteristic for discrimination between lysine and 19 naturally occurring non-cognate amino acids have been determined from kcat and Km values for native and phosphorylated lysyl-tRNA synthetases from yeast. Generally, both species of this class II aminoacyl-tRNA synthetase are considerably less specific than the class I synthetases specific for isoleucine, valine, tyrosine, and arginine. D values of the native enzyme are in the range 90-1700, D values of the phosphorylated species in the range 40-770. The phosphorylated enzyme acts faster and less accurately. In aminoacylation of tRNALys-C-C-A(2'NH2) discrimination factors D1 vary over 30-980 for the native and over 8-300 for the phosphorylated enzyme. From AMP formation stoichiometry and D1 values pretransfer proof-reading factors (II1) of 1.1-56 were calculated for for the native enzyme, factors of 1.0-44 for the phosphorylated species. Post-transfer proof-reading factors (II2) were calculated from D values and AMP formation stoichiometry in acylation of tRNALys-C-C-A. Pretransfer proof-reading is the main correction step, posttransfer proof-reading is less effective or negligible (II2 approximately 1-8). Initial discrimination factors (I), which are due to differences in Gibbs free energies of binding between lysine and noncognate substrates (delta delta GI), were calculated from discrimination and proof-reading factors. In contrast to class I synthetases, for lysyl-tRNA synthetase only one initial discrimination step can be assumed and amino acid recognition is reduced to a three-step process instead of the four-step recognition observed for the class I synthetases. Plots of delta delta GI values against accessible surface areas of amino acids show clearly that phosphorylation of the enzyme changes the structures of the amino acid binding sites. This is illustrated by a hypothetical 'stopper model' of these sites.

Acylation↗
PubMed1992

Apparent association constants of tRNAs for the ribosomal A, P, and E sites.

Association constants for tRNA binding to poly(U) programmed ribosomes were assessed under standardized conditions with a single preparation of ribosomes, tRNAs, and elongation factors, respectively, at 15 and 10 mM Mg2+. Association constants were determined by Scatchard plot analysis (the constants are given in units of [10(7)/M] measured at 15 mM Mg2+): the ternary complex Phe-tRNA.elongation factor EF-Tu.GTP (12 +/- 3), Phe-tRNA (1 +/- 0.4), AcPhe-tRNA (0.7 +/- 0.3), and deacylated tRNA(Phe) (0.4 +/- 0.15) bind with decreasing affinity to the A site of poly(U)-programmed ribosomes. tRNA(Phe) (7.2 +/- 0.8) binds to the P site with higher affinity than AcPhe-tRNA (3.7 +/- 1.3). The affinity of the E site for deacylated tRNA(Phe) (1 +/- 0.2) is about the same as that of the A site for AcPhe-tRNA (0.7 +/- 0.3). At lower Mg2+ concentrations the affinity of the E site ligand becomes stronger relative to the affinities of the A site ligands. Phe-tRNA and ternary complexes can occupy the A site at 0 degrees C in the presence of poly(U) even if the P site is free, whereas, as already known, deacylated tRNA or AcPhe-tRNA bind first to the P site of programmed ribosomes. Hill plot analyses of the binding data confirm an allosteric linkage between A and E sites in the sense of a negative cooperativity.

Acylation↗
PubMed1991

The serotonin syndrome.

OBJECTIVE AND METHOD: A review of the literature on the serotonin syndrome in animals and human beings was conducted, and 12 reports of 38 cases in human patients were then analyzed to determine the most frequently reported clinical features and drug interactions, as well as the incidence, treatment, and outcome of this syndrome. FINDINGS: The serotonin syndrome is most commonly the result of the interaction between serotonergic agents and monoamine oxidase inhibitors. The most frequent clinical features are changes in mental status, restlessness, myoclonus, hyperreflexia, diaphoresis, shivering, and tremor. The presumed pathophysiological mechanism involves brainstem and spinal cord activation of the 1A form of serotonin (5-hydroxytryptamine, or 5-HT) receptor. The incidence of the syndrome is not known. Both sexes have been affected, and patients' ages have ranged from 20 to 68 years. Discontinuation of the suspected serotonergic agent and institution of supportive measures are the primary treatment, although 5-HT receptor antagonists may also play a role. Once treatment is instituted, the syndrome typically resolves within 24 hours, but confusion can last for days, and death has been reported. CONCLUSIONS: The serotonin syndrome is a toxic condition requiring heightened clinical awareness for prevention, recognition, and prompt treatment. Further work is needed to establish the diagnostic criteria, incidence, and predisposing factors, to identify the role of 5-HT antagonists in treatment, and to differentiate the syndrome from neuroleptic malignant syndrome.

Adult↗
PubMed1990

Casein kinase II phosphorylates DNA-polymerase-alpha--DNA-primase without affecting its basic enzymic properties.

Immunoaffinity-purified DNA-polymerase-alpha--DNA-primase complex from calf thymus was phosphorylated in vitro by highly purified casein kinase II from the same tissue. Specific phosphorylation of the DNA-polymerizing alpha subunit and the primase-associated gamma subunit was observed. About 1 mol phosphate/mol polymerase--primase was incorporated. Despite this effect, neither the DNA polymerase nor the DNA primase activity were changed after phosphorylation by casein kinase II. Furthermore, dephosphorylation of polymerase--primase with alkaline phosphatase did not change the polymerase or the primase activity to a significant extent. Moreover, both alkaline phosphatase and casein kinase II had no effect on the processivity of DNA synthesis and on the lengths and amounts of primers formed by the DNA primase. Because DNA polymerase alpha maintained all its basic properties even after extensive treatment with alkaline phosphatase, it is unlikely that phosphorylation has a direct influence on the activities of the DNA-polymerase-alpha--DNA-primase complex. The possible influence of post-translational phosphorylation on the formation of a complex of polymerase alpha and its accessory proteins is discussed.

Animals↗