Selenalysine and protein synthesis.

Selenalysine is a lysine analog having the gamma-methylene group substituted by a selenium atom. It has been demonstrated that selenalysine is activated and transferred to tRNAlys by either Escherichia coli or rat liver aminoacyl-tRNA synthetases, and inhibits lysine incorporation into polypeptides in protein-synthesizing systems from E. coli, rat liver or rabbit reticulocytes. All tests were performed in comparison with thialysine, a lysine analog having the gamma-methylene group substituted by a sulfur atom. In all the reactions studied, both thialysine and selenalysine act as competitive inhibitors of lysine. With respect to thialysine, selenalysine act as competitive inhibitors of lysine. With respect to thialysine, selenalysine shows a slightly lower activity as lysine inhibitor.     

Action of thiazolidine-2-carboxylic acid, a proline analog, on protein synthesizing systems.

Thiazolidine-2-carboxylic acid, or beta-thiaproline, is a proline analog in which the beta methylene group of proline is substituted by a sulfur atom. It has been deomonstrated that beta-thiaproline is activated and transferred to tRNAPro by Escherichia coli and rat liver aminoacyl-tRNA synthetases, and inhibits proline incorporation into polypeptides in protein synthesizing systems from E. coli, rat liver or rabbit reticulocytes. In mammalian systems beta-thiaproline inhibits also leucine incorporation; in rabbit reticulocyte lysate it inhibits ribosome run-off. Both these effects may be explained by the fact that beta-thiaproline once incorporated into the growing polypeptide chain impairs its further elongation, as shown by experiments made with puromycin. All tests were performed in comparison with thiazolidine-4-carboxylic acid, or gamma-thiaproline, another proline analog having the gamma methylene group substituted by a sulfur atom; it was shown that in all the reactions studied both compounds act as competitive inhibitors of proline. Some differences in the effects of the two analogs have been evidenced: in almost all the reactions and mainly in the whole protein synthesizing systems, beta-thiaproline shows an higher inhibitory activity.     

Role for Free Isoleucine or Glycyl-Leucine in the Repression of Threonine Deaminase in Escherichia coli

In Escherichia coli, the three branched-chain amino acid activating enzymes appear to be essential for multivalent repression of the isoleucine- and valine-forming enzymes. The results of experiments with a mutant, strain CU18, having an altered threonine deaminase, indicate that free isoleucine and some form of threonine deaminase (the product of the ilvA gene) are also involved in multivalent repression. This strain exhibits abnormally high derepressibility but normal repressibility of its ilv gene products, and its threonine deaminase is inhibited only by high concentrations of isoleucine. In strain CU18, the isoleucine analogue, thiaisoleucine, is incapable of replacing isoleucine in the multivalent repression of the ilv genes, whereas the analogue can fully replace the natural amino acid in repression in other strains examined. The dipeptide, glycyl-leucine, which, like isoleucine, is a heterotropic negative effector of threonine deaminase but is not a substrate for isoleucyl-transfer ribonucleic acid synthetase, can completely prevent the accumulation of threonine deaminase-forming potential during isoleucine starvation in strains with normal threonine deaminases. It can not, however, prevent such accumulation in strain CU18 or in other strains in which threonine deaminase is insensitive to any concentration of isoleucine.     

The metabolic effects of thia fatty acids in rat liver depend on the position of the sulfur atom

The effects on oxidation and composition of fatty acids in rat liver were compared after administration of fatty acids with sulfur substituted in different positions. It has been hypothesized that drugs with hydrophobic backbone have lipid-lowering effects because they are not easily catabolized by mitochondrial beta-oxidation. Thia fatty acids cannot be beta-oxidized when sulfur is in 3-position, but beta-oxidation is possible when sulfur is positioned further from the carboxyl group. To investigate whether catabolism of thia fatty acids would affect their ability to influence lipid metabolism, a series of thia fatty acids were synthesized and administered by oral gavage to male Wistar rats (300 mg/kg bodyweight/day for 7 days). Depending on the position of the sulfur atom and the chain length, the thia fatty acids were beta-oxidized, desaturated and/or elongated, and the accumulated amounts were lower as the sulfur atom were positioned further from the carboxyl group. All thia fatty acids led to high peroxisomal beta-oxidation of endogenous fatty acids, whereas the mitochondrial beta-oxidation was high when sulfur was in 3-position, low when sulfur was in 4-position and similar to controls when sulfur was in 5- or 7-position. The changes in hepatic fatty acid composition were more pronounced when sulfur was positioned close to the carboxyl group. In conclusion, both the position of the sulfur atom and the chain length appear to determine the catabolic fate of thia fatty acids, and the non-beta-oxidizable thia fatty acids were most potent in regulating oxidation and composition of endogenous fatty acids in rat liver.     

Subtle alteration of the active site of ribulose bisphosphate carboxylase/oxygenase by concerted site-directed mutagenesis and chemical modification

Both activities of ribulose bisphosphate carboxylase/oxygenase are dependent on carbamylation by CO2 of a specific lysyl epsilon-amino group (Lys-191 of the enzyme from Rhodospirillum rubrum). To examine the stringency of the requirement for this lysyl side chain, Lys-191 was converted to an aminoethylcysteinyl residue (net replacement of a gamma-methylene group by a sulfur atom) by a combination of site-directed mutagenesis and subsequent chemical modification. The purified Cys-191 mutant was totally devoid of both carboxylase and oxygenase activities. However, this mutant protein exhibited tight-binding of the transition-state analogue, 2-carboxyarabinitol bisphosphate, a property heretofore ascribed solely to the carbamylated form of the carboxylase. Treatment of the mutant protein with ethylene imine restored catalytic activity to 4-7% of the wild-type level. The carboxylase:oxygenase activity ratio of the aminoethylated protein was unperturbed relative to that of wild-type enzyme.     

Studies concerning the specificity of the effect of leucine on the turnover of proteins in muscles of control and diabetic rats.

The protein anabolic effect of branched chain amino acids was studied in isolated quarter diaphragms of rats. Protein synthesis was estimated by measuring tyrosine incorporation into muscle proteins in vitro. Tyrosine release during incubation with cycloheximide served as an index of protein degradation. In muscles from normal rats the addition of 0.5 mM leucine stimulated protein synthesis 36--38% (P less than 0.01), while equimolar isoleucine or valine, singly or in combination were ineffective. The three branched chain amino acids together stimulated no more than leucine alone. The product of leucine transamination, alpha-keto-isocaproate, did not stmino norborane-2-carboxylic acid (a leucine analogue) were ineffective. Leucine and isoleucine stimulated protein synthesis in muscles from diabetic rats.Leucine, isoleucine, valine and the norbornane amino acid but not alpha-ketoisocaproate or beta-hydroxybutyrate decreased the concentration of free tyrosine in tissues during incubation with cycloheximide; tyrosine release into the medium did not decrease significantly. Leucine caused a small decrease in total tyrosine release, (measured as the sum of free tyrosine in tissues and media), suggesting inhibition of protein degradation. The data suggest that leucine may be rate limiting for protein synthesis in muscles. The branched chain amino acids may exert a restraining effect on muscle protein catabolism during prolonged fasting and diabetes.     

Energetics of side chain packing in staphylococcal nuclease assessed by exchange of valines, isoleucines, and leucines

To examine the importance of side chain packing to protein stability, each of the 11 leucines in staphylococcal nuclease was substituted with isoleucine and valine. The nine valines were substituted with leucine and isoleucine, while the five isoleucines, previously substituted with valine, were substituted with leucine and methionine. These substitutions conserve the hydrophobic character of these side chains but alter side chain geometry and, in some cases, size. In addition, eight threonine residues, previously substituted with valine, were substituted with isoleucine to test the importance of packing at sites normally not occupied by a hydrophobic residue. The stabilities of these 58 mutant proteins were measured by guanidine hydrochloride denaturation. To the best of our knowledge, this is the largest library of single packing mutants yet characterized. As expected, repacking stability effects are tied to the degree of side chain burial. The average energetic cost of moving a single buried methyl group was 0.9 kcal/mol, albeit with a standard deviation of 0.8 kcal/mol. This average is actually slightly greater than the value of 0.7-0.8 kcal/mol estimated for the hydrophobic transfer energy of a methylene from octanol to water. These results appear to indicate that van der Waals interactions gained from optimal packing are at least as important in stabilizing the native state of proteins as hydrophobic transfer effects.     

Escherichia coli K-12 Mutants Altered in the Transport of Branched-Chain Amino Acids

Two mutants of Escherichia coli K-12 are described which are resistant to the inhibition that valine exerts on the growth of E. coli. These mutants have lesions at two different loci on the chromosome. One of them, brnP, is linked to leu (87% cotransduction) and is located between leu and azi represented on the map at 1 min; the other, brnQ, is linked to phoA (96% cotransduction), probably between proC and phoA and represented at 10 min. These mutants are resistant to valine inhibition but are sensitive to dipeptides containing valine. Since it is known that dipeptides are taken up by E. coli through a transport system(s) different from those used by amino acids, this sensitivity to the peptides suggests an alteration in the active transport of valine. The mutants are resistant to valine only if leucine is present in the growth medium; the uptake of valine is less in both mutants than it is in wild-type E. coli, and it is reduced even further if leucine is present. Under these conditions the total uptake of valine is almost completely abolished in the brnQ mutant. The brnP mutant takes up about 60% as much valine as does the wild type, but no exogenous valine is incorporated into proteins. The apparent K(m) and V(max) of isoleucine, leucine, and valine for the transport system are reported; the brnP mutant, when compared to the wild type, has a sevenfold higher K(m) for isoleucine and a 17-fold lower K(m) for leucine; the V(max) for the three amino acids is reduced in the brnQ mutant, up to 20-fold for valine. The transport of arginine, aspartic acid, glycine, histidine, and threonine is not altered in the brnQ mutant under conditions in which that of the branched amino acids is. Evidence is reported that O-methyl-threonine enters E. coli through the transport system for branched amino acids, and that thiaisoleucine does not.     

Oxytocin analogues with amide groups substituted by tetrazole groups in position 4, 5 or 9

Eleven oxytocin analogues substituted in position 4, 5 or 9 by tetrazole analogues of amino acids were prepared using solid-phase peptide synthesis method and tested for rat uterotonic in vitro and pressor activities, as well as for their affinity to human oxytocin receptor. The tetrazolic group has been used as a bioisosteric substitution of carboxylic, ester or amide groups in structure-activity relationship studies of biologically active compounds. Replacement of the amide groups of Gln(4) and Asn(5) in oxytocin by tetrazole analogues of aspartic, glutamic and alpha-aminoadipic acids containing the tetrazole moiety in the side chains leads to analogues with decreased biological activities. Oxytocin analogues in which the glycine amide residue in position 9 was substituted by tetrazole analogues of glycine had diminished activities as well. The analysis of differences in rat uterotonic activity and in the affinity to human oxytocin receptors of analogues containing either an acidic 5-substituted tetrazolic group or a neutral 1,5- or 2,5-tetrazole nucleus makes it possible to draw some new conclusions concerning the role of the amide group of amino acids in positions 4, 5 and 9 of oxytocin for its activity. The data suggest that the interaction of the side chain of Gln(4) with the oxytocin receptor is influenced mainly by electronic effects and the hydrogen bonding capacity of the amide group. Steric effects of the side chain are minor. Substitution of Asn(5) by its tetrazole derivative gave an analogue of very low activity. The result suggests that in the interaction between the amide group of Asn(5) and the binding sites of oxytocic receptor hydrogen bonds are of less importance than the spatial requirements for this group.     

Beta-selenaproline as competitive inhibitor of proline activation

Beta-Selenaproline, a proline analog having the beta-methylene group substituted by a selenium atom, has been tested in ATP-PPi exchange reaction catalyzed by either Escherichia coli or rat liver aminoacyl-tRNA synthetases. It has been shown that with both enzymatic systems beta-selenaproline does not give rise to ATP-PPi exchange, but specifically inhibits proline activation. The inhibition is of fully competitive type and the Ki values, lower than the Km values for proline, show that beta-selenaproline binds to the synthetases with high affinity. The inability to form the complex with AMP, taking into account also the behavior of gamma-selenaproline and other proline analogs, has been ascribed to the presence of the selenium atom in the beta-position.     

STRUCTURE ACTIVITY RELATIONS FOR THE INHIBITION OF 5-HT UPTAKE INTO RAT HYPOTHALAMIC HOMOGENATES BY SEROTONIN AND TRYPTAMINE ANALOGUES

Abstract— Various 5-HT and tryptamine analogues have been examined as inhibitors of [³H]5-HT uptake into rat hypothalamic homogenates. Acetylation of the terminal amino group or methylation of the hydroxyl group of 5-HT resulted in compounds having a reduced affinity for the serotonin uptake site. This also occurred when the hydroxyl group of 5-HT was substituted in other positions on the benzene ring. Substitution of the tryptamine side chain in the a-position by methyl or ethyl groups, but not by a carboxyl function, enhanced the affinity for the 5-HT uptake site. Increasing the tryptamine side chain by one carbon atom also resulted in a more potent compound. Several of the compounds tested are known to be either hallucinogens or antidepressants.     

Effect of tunicamycin on the secretion of serum proteins by primary cultures of rat and chick hepatocytes. Studies on transferrin, very low density lipoprotein, and serum albumin.

Using rat or chick hepatocyte monolayers, we have studied the effect of tunicamycin, a specific inhibitor of protein glycosylation, on the synthesis and secretion of serum proteins. Tunicamycin inhibited glucosamine incorporation into rat liver transferrin and the apoprotein B chain of chick liver very low density lipoprotein (VLDL) by 75 to 90%. In contrasts, amino acid incorporation into these two glycoproteins, as well as into the normally unglycosylated proteins, rat serum albumin and apoprotein A of chick liver VLDL, was decreased by only 10 to 25% in the presence of the antibiotic. Despite the inhibitory effect of tunicamycin on glycosylation, secretion of all four proteins was virtually unimpaired. Thus, the carbohydrate moieties of rat liver transferrin or apoprotein B of chick liver VLDL do not appear to play an essential role in the secretion process.     

Synthesis and evaluation of new omega-borono-alpha-amino acids as rat liver arginase inhibitors

Recent studies have demonstrated that arginase plays important roles in pathologies such as asthma or erectile dysfunctions. We have synthesized new omega-borono-alpha-amino acids that are analogues of the previously known arginase inhibitors S-(2-boronoethyl)-l-cysteine (BEC) and 2-amino-6-boronohexanoic acid (ABH) and evaluated them as inhibitors of purified rat liver arginase (RLA). In addition to the distance between the B(OH)(2) and the alpha-amino acid functions, the position of the sulfur atom in the side chain also appears as a key determinant for the interaction with the active site of RLA. Furthermore, substitution of the alkyl side chain of BEC by methyl groups and conformational restriction of ABH by incorporation of its side chain in a phenyl ring led to inactive compounds. These results suggest that subtle interactions govern the affinity of inhibitors for the active site of RLA.     

The metabolism of deoxyguanosine in mitochondria: relationship of the uptake of deoxyguanosine to the electron transport chain and adenosine triphosphate

That deoxyguanosine is taken up by isolated rat liver mitochondria has been shown. This report describes the relationship between that uptake and oxidative phosphorylation. By measuring this process in the presence of standard inhibitors of oxidative phosphorylation it was determined that a functional electron transport chain, but not the phosphorylation of ADP, was essential for uptake. ATP analogs adenyl(beta,gamma-methylene)diphosphate and adenylimidodiphosphate blocked uptake, indicating that ATP hydrolysis was required. ADP also proved to be an inhibitor. Exogenous UTP slightly stimulated deoxyguanosine uptake, as did added ATP, but several other nucleotides (dGTP, dATP, UDP, dGDP) were inhibitory. A sulfhydryl group was important for deoxyguanosine uptake and inhibition of uptake by N-ethylmaleimide was protected by both deoxyguanosine and ATP. These data show that deoxyguanosine uptake by mitochondria is a process which is coordinated and, perhaps, regulated by other events which take place in the organelle.     

O-Methylthreonine Inhibition of Growth and of Threonine Deaminase in Escherichia coli

O-methylthreonine (OMT), an isosteric analogue of isoleucine, markedly inhibited growth of Escherichia coli 15. This inhibition was overcome most effectively by addition of isoleucine, valine, or leucine to the medium and less effectively by addition of threonine. The dipeptide, valylleucine, also relieved the OMT-induced inhibition but only after a lag period, suggesting that valine and leucine, liberated by dipeptidase action, compete with OMT for entry into the cell. OMT was activated and transferred to transfer ribonucleic acid (RNA) by isoleucyl-RNA synthetase in vitro. The rate of OMT incorporation into protein of intact cells was comparable to that of isoleucine. In contrast to isoleucine, very high concentrations of OMT were required to inhibit threonine deaminase, and the inhibition was strictly competitive with threonine. In addition, OMT inhibited a threonine deaminase preparation desensitized to isoleucine inhibition.     

ilvU, a locus in Escherichia coli affecting the derepression of isoleucyl-tRNA synthetase and the RPC-5 chromatographic profiles of tRNAIle and tRNAVal.

A mutation in the ilvU locus of Escherichia coli has led to a complex phenotype that included resistance to thiaisoleucine, a loss of derepressibility of isoleucyl tRNA synthetase, and an alteration of the RPC-5 chromatographic profile of the branched-chain aminoacyl-tRNA's. The alterations were manifest in an increase in the amount of Species 2 of both tRNAIle and tRNAVal at the expense of Species 1. A similar alteration, but independent of (and additive to) that caused by the ilvU mutation, was observed upon limitation of either isoleucine or valine. The shift in profile caused by limitation was also independent of the reduced growth rate or the derepression of the isoleucine and valine biosynthetic enzymes that also result from limitation. During chloramphenicol treatment nearly all tRNAIle and tRNAVal formed appears as species 2. Upon recovery from chloramphenicol, Species 2 of both acceptors are converted to Species 1. It is proposed that the ilvU product not only allows derepression of isoleucyl-tRNA synthetase but also retards the conversion of tRNA2Ile to tRNA1Ile and that of tRNA2Val to tRNA1Val. The mutated ilvU loci abolish the derepression and are more efficient in retarding the conversion.     

Study on the Correlation of Chemial Structure and Ovicidal Activities of 2-Bromoethylthiobenzenes

Ovicidal and chemical properties of many analogs of 2-bromoethylthiobenzenes were investigated. Replacement of the sulfur atom of 2-bromoethylthiobenzenes by an oxygen atom or a sulfone group, and that of the ethylene group by a trimethylene or a tetramethylene group lowered both the ovicidal activity and the chemical reactivities. Branching of methyl group(s) in the ethylene chain of a certain substituted 2-bromoethylthiobenzenes extremely enhanced the chemical reactivities, but lowered the ovicidal activity of the parent compound.     

The relaxin receptor-binding site geometry suggests a novel gripping mode of interaction

Relaxin has a unique, clearly identifiable, mixed function receptor-binding region comprising amino acid residues that evolve sequentially from the central portion of the B chain alpha-helix. Two arginine residues in positions B13 and B17 that project like forefinger and middle finger from the helix provide the electrostatic element opposed by the hydrophobic (thumb) element isoleucine (B20), offset from the arginines by about 40 degrees. The binding intensity of relaxin to its receptor decreases by 3 orders of magnitude if alanine is substituted for the newly discovered binding component isoleucine in position B20. The arginine residues cannot be replaced by other positive charges, nor can the guanidinium group be presented on a longer or shorter hydrocarbon chain. In contrast, the hydrophobic interaction is incremental in nature, and the contribution to the total binding energy is roughly proportional to the number of hydrocarbon units in the side chain. It appears that a hydrophobic surface exists on the receptor that offers optimal van der Waals' interaction with beta-branched hydrophobic amino acids. The binding energy increases roughly 10-fold with each methylene group whereby beta-branching is more effective per surface unit than chain elongation. Aromatic side chains appear to demarcate the extent of the binding region in so far as residues larger than phenylalanine decrease receptor binding. The exceptional clarity of binding site geometry in relaxin makes for an excellent opportunity to design peptido-mimetics.     

Synthesis and biological property of carba and 20-deoxo analogues of arenastatin A

The carba analogue, in which a methylene group is substituted for the oxygen atom linked to C-15, and 20-deoxo analogue of arenastatin A, a potent cytotoxic spongean depsipeptide, were synthesized. Both analogues lacking the 15,20-ester function, which was easily metabolized in serum, showed good stability in serum as well as moderate cytotoxic activity against KB cells and better solubility.     

Stereoselectivity of ectonucleotidases on vascular endothelial cells.

We have investigated the stereoselectivity of ectonucleotidases (nucleoside triphosphatase, EC 3.6.1.15; nucleoside diphosphatase, EC 3.6.1.6; 5'-nucleotidase, EC 3.1.3.5) on pig aortic endothelial cells using two classes of nucleotide analogue. In experiments with nucleotide enantiomers in which the natural D-ribofuranosyl moiety is replaced by an L-ribofuranosyl moiety, the rate of catabolism of 100 microM-L-ATP was one-fifth that of D-ATP, the rate of catabolism of 100 microM-L-ADP was one-fifteenth that of D-ADP and there was no detectable catabolism of 100 microM-L-AMP. Each of the L-enantiomers inhibited, apparently competitively, the catabolism of the corresponding D-enantiomer; Ki values were approx. 0.6 mM, 1.0 mM and 3.9 mM for L-ATP, L-ADP and L-AMP respectively. Experiments with adenosine 5'-[beta, gamma-imido]triphosphate and with D- and L-enantiomers of adenosine 5'-[beta, gamma-methylene]triphosphate revealed modest ectopyrophosphatase activity, undetectable in experiments with natural nucleotides, which was also stereoselective. Use of phosphorothioate nucleotide analogues demonstrated that ATP catabolism was virtually stereospecific with respect to the geometry of the thiol group substituted on the beta-phosphate: the Rp isomer was degraded, whereas there was little or no breakdown of the Sp isomer. ADP catabolism was also stereospecific with respect to the geometry of the thiol group substituted on the alpha-phosphate: the Sp isomer but not the Rp isomer was degraded. The geometry of thiol-group substitution on the alpha-phosphate had no effect on ATP catabolism to ADP. There was no detectable catabolism of analogues with thiol-group substitution on the terminal phosphate. Each of the phosphorothioate analogues that was catabolized broke down at a rate similar to that of the natural nucleotide from which it was derived. These results demonstrate that the ectonucleotidases on pig aortic endothelial cells exhibit a high degree of stereoselectivity, characteristic for each enzyme, both with respect to the ribofuranosyl moiety and to the phosphate side chain.