Structure-activity relationships of human urotensin II and related analogues on rat aortic ring contraction

The sequence of human urotensin II (UII) has been recently established as H-Glu-Thr-Pro-Asp-Cys-Phe-Trp-Lys-Tyr-Cys-Val-OH, and it has been reported that UII is the most potent mammalian vasoconstrictor peptide identified so far. A series of UII analogues was synthesized, and the contractile activity of each compound was studied in vitro using de-endothelialised rat aortic rings. Replacement of each amino acid by an L-alanine or by a D-isomer showed that the N- and C-terminal residues flanking the cyclic region of the amidated peptide were relatively tolerant to substitution. Conversely, replacement of any residue of the cyclic region significantly reduced the contractile activity of the molecule. The octapeptide UII(4-11) was 4 times more potent than UII, indicating that the C-terminal region of the molecule possesses full biological activity. Alanine or D-isomer substitutions in UII(4-11) or in UII(4-11)-NH2, respectively, showed a good correlation with the results obtained for UII-NH2. Disulfide bridge disruption or replacement of the cysteine residues by their D-enantiomers markedly reduced the vasoconstrictor effect of UII and its analogues. In contrast, acetylation of the N-terminal residue of UII and UII-NH2 enhanced the potency of the peptide. Finally, monoiodination of the Tyr6 residue in UII(4-11) increased by 5 fold the potency of the peptide in the aortic ring bioassay. This structure-activity relationship study should provide useful information for the rational design of selective and potent UII receptor agonists and antagonists.     

Studies on the synthesis and properties of new PNA analogs consisting of L- and D-lysine backbones

Homopentamers of PNA consisting of L- and D-lysine were synthesized. It was found that the intramolecular hydrogen bonding has no significant influence on intermolecular interaction. These pentamers show a tendency to form complex with polyA in tris(pH 8.0) solution. The L-conformer has more potential than D-isomer.     

D-amino acid and alanine scans of the bioactive portion of porcine motilin.

A recent systematic study of porcine motilin fragments has clearly shown that biological activity resides in the amino-terminal end. The amino-terminal tetradecapeptide retains more than 90% of the potency of the full molecule. We now examined the effect of replacement of residues 1 through 11 by either their D-isomer or by alanine in [Leu13]pMOT(1-14). Peptides were synthesized using Fmoc solid phase methodology, purified by HPLC, and assayed for their ability to displace bound motilin (rabbit antral smooth muscle homogenate) and to induce contractions (isolated rabbit duodenal segments). The negative logarithm of the concentration displacing 50% of the tracer (pIC50), or producing 50% of the maximal contractile response (pEC50), was determined. All compounds were still full agonists. A reduction in potency of more than two log units was seen for the compounds in which residues 1 (Phe), 4 (Ile), and 7 (Tyr) were replaced by Ala and residues 3 (Pro), 4 (Ile), and 6 (Thr) by their D-isomer. The largest drop was noted for the analogs substituted at position 4. For all compounds there was an almost perfect correlation between the pIC50 and the pEC50 values (r = 0.96), although the pEC50 was consistently smaller. These results show that the biological activity of motilin is mainly determined by the first seven residues. The pharmacophore consists of the aromatic rings from Phe1 and Tyr7 and the aliphatic side chains from Val2 and Ile4. Pro3, Phe5, and Thr6 may stabilize the bioactive conformation.     

D-amino acid formation induced by a chiral field within a human lens protein during aging.

We have previously shown that Asp-151 in alphaA-crystallin from aged human lens are converted to the biologically uncommon D-isomer to a high degree, showing that the formation of D-isomer was not simple racemization, but stereoinvertion. This suggests that alphaA-crystallin has a chiral reaction field which promotes the inversion of L-Asp to D-Asp residues in the native higher order structure of alphaA-crystallin itself. Here, we show that when the aged human alphaA-crystallin, enriched at Asp-151 with the D-isomer (D/L ratio of 5.7), was unfolded by heating at 70 degrees C or 6 M urea, the D-Asp-151 in the unfolded alphaA-crystallin was rapidly racemized (D/L ratio of 2.17 to 1.21). This presumably reflects a relaxation of the chiral field that was initially inducing the stereoinversion from the natural L-isomer to the D-isomer.     

D-(-)-beta-hydroxybutyrate-induced effects on mouse embryos in vitro

The effects of the "physiologically occurring" D-isomer of beta-hydroxybutyrate (D-BOHB) were evaluated in neurulating mouse embryos by using the technique of whole embryo culture. Following a 24-hour culture period D-BOHB induced malformations and growth retardation in a concentration-dependent manner. At a 48 mM concentration essentially all embryos exhibited neural tube defects, and decreased rates of glucose metabolism by the pentose phosphate pathway (PPP) and Krebs cycle were observed when compared to controls. The relationship between an inhibition of the PPP and induction of malformations by DL-BOHB has been reported, and thereby suggests a similar mechanism may be operating for the D-isomer. In contrast, the effect of the D-isomer on the Krebs cycle may result from a replacement of glucose intermediates by those generated from metabolism of D-BOHB. Concentrations as high as 20 mM D-BOHB have been reported in the serum of uncontrolled diabetic patients, and since ketones rapidly equilibrate across extraembryonic membranes, embryos in vivo may be exposed to concentrations equivalent to those which induced malformations in vitro. However, the incidence of malformations induced by D-BOHB was less than that reported for the DL-racemic mixture at equivalent concentrations, thereby suggesting that the L-isomer is also teratogenic. Therefore, until the presence and concentration of L-BOHB in the serum of diabetics are documented, the assessment of the impact of maternal ketosis on the embryo remains incomplete.     

Purification and characterization of D-2-haloacid dehalogenase from Pseudomonas putida strain AJ1/23

A D-2-haloacid dehalogenase was isolated and purified to homogeneity from Pseudomonas putida strain AJ1/23. The enzyme catalysed the stereospecific dehalogenation of the D-isomer of 2-chloropropionate. Using a new ion-chromatograph assay, the enzyme was found to catalyse the dehalogenation of short-chain 2-halocarboxylic acids. Maximum enzyme activity occurred at pH 9.5 and 50 degrees C and the enzyme was insensitive to most -SH reagents. The enzyme has an Mr of about 135,000 and appears to be composed of four subunits of identical Mr.     

2'-Fluoro-4'-thio-2',3'-unsaturated nucleosides: anti-HIV activity, resistance profile, and molecular modeling studies

Both D- and L-2'-fluoro-4'-thio-2',3'-unsaturated nucleosides were synthesized and their anti-HIV activity against the drug sensitive virus and lamivudine-resistant mutant (M184V) were evaluated. In vitro antiviral evaluation indicated that the L-isomers are more potent than the D-isomers, but unfortunately all were cross-resistant with 3TC. Molecular modeling studies revealed that the unnatural sugar moiety of the L-nucleosides as well as 4'-sulfur atom of the D-isomer has a steric conflict with the bulky side chain of valine 184, resulting in cross-resistance.     

Utilization of D-asparagine by Saccharomyces cerevisiae.

Yeast strains sigma1278b and Harden and Young, which synthesize only an internal constitutive form of L-asparaginase, do not grow on D-asparagine, as a sole source of nitrogen, and whole cell suspensions of these strains do not hydrolyze D-asparagine. Strains X2180-A2 and D273-10B, which possess an externally active form of asparaginase, are able to grow slowly on D-asparagine, and nitrogen-starved suspensions of these strains exhibit high activity toward the D-isomer. Nitrogen starvation of strain X218O-A2 results in coordinate increase of D- and L-asparaginase activity; the specific activity observed for the D-isomer is approximately 20% greater than that observed for the L-isomer. It was observed, in studies with cell extracts, that hydrolysis of D-asparagine occurred only with extracts from nitrogen-starved cells of strains that synthesize the external form of asparaginase. Furthermore, the activity of the extracts toward the D-isomer was always higher than that observed with the L-isomer. A 400-fold purified preparation of external asparaginase from Saccharomyces cerevisiae X218U-A2 hydrolyzed D-asparagine with an apparent Km of 0.23 mM and a Vmax of 38.7 mumol/min per mg of protein. D-Asparagine was a competitive inhibitor of L-asparagine hydrolysis and the Ki determined for this inhibition was approximately equal to its Km. These data suggest that D-asparagine is a good substrate for the external yeast asparaginase but is a poor substrate for the internal enzyme.     

D-aspartic acid in purified myelin and myelin basic protein

The presence of the biologically uncommon D-isomer of aspartic acid in the white matter of human brains has been reported previously from this laboratory (1). We now report that the level of D-aspartate in human brains is higher in purified myelin than in white matter and is even higher in the myelin basic protein fraction. There also appears to be a difference in the level of D-aspartate found in human brain as compared to bovine brain, possibly a species or age-related difference.     

D-1-amino-2-propanol:NAD+ oxidoreductase. Purification and general properties of the large molecular form of the enzyme from Escherichia coli K12

Growth of Escherichia coli K12 under relatively anaerobic conditions in a medium containing casein hydrolysate, 0.8% glycerol, and 0.8% hydroxyacetone has been found to induce the level of D-1-amino-2-propanol oxidoreductase activity 50- to 100-fold over that in cells grown in casein hydrolysate alone or with 0.8% glycerol added. A large molecular weight form of this oxidoreductase (designated Form L) has been purified to apparent homogeneity in good yield by three simple steps designed to obviate its conversion to a smaller species. The molecular weight of native Form L and its basic subunit are 417,000 +/- 20,700 and 50,500 +/- 2,770, respectively; hence Form L would appear to consist of eight identical subunits. The pH activity profile for Form L shows one optimum in the range of 8.3 to 8.6 and another at pH 10.0 to 10.2. This form of the oxidoreductase has no apparent requirement for added metal ions (rather, numerous divalent transition metal ions are strongly inhibitory) or thiol compounds; it catalyzes the oxidation of several vic-glycols but is completely stereospecific for the D-isomer of 1-amino-2-propanol, utilizes only NAD+ as cosubstrate in the oxidation reaction (Km for NAD+ with DL-1-amino-2-propanol = 1.23 mM), but both NADH and NADPH serve as cosubstrate in the reduction of hydroxyacetone. Oxidoreductase activity of Form L is highly sensitive to inhibition by Hg2+, p-mercuribenzoate, or dithiodipyridine; inhibition by the latter two compounds is completely reversed by adding a thiol in excess.     

Structural elucidation of a novel exopolysaccharide produced by a mucoid clinical isolate of Burkholderia cepacia. Characterization of a trisubstituted glucuronic acid residue in a heptasaccharide repeating unit.

The structure of the exopolysaccharide (EPS) produced by a clinical isolate of Burkholderia cepacia isolated from a patient with fibrocystic lung disease has been investigated. By means of methylation analyses, carboxyl reduction, partial depolymerization by fuming HCl and chemical degradations such as Smith degradation, lithiumethylenediamine degradation and beta-elimination, supported by GC/MS and NMR spectroscopic analyses, the repeat unit of the EPS has been identified and was shown to correspond to the acidic branched heptasaccharide with the following structure: [formula: see text]. This partially acetylated acidic polymer, distinguished by the presence of the less usual D-isomer of rhamnose and of a trisubstituted glucuronic acid residue, could represent the main EPS produced by this bacterial species.     

Purification, separation, and characterization of two molecular forms of D-1-amino-2-propanol:NAD+ oxidoreductase activity from extracts of Escherichia coli K-12.

D-1-Amino-2-propanol:NAD+ oxidoreductase activity, which catalyzes the second step in a pathway wherein L-threonine is converted to D-1-amino-2-propanol via the intermediate formation of aminoacetone, has been purified 500-fold from Escherichia coli K-12. Although the enzyme catalyzes the oxidation of certain diols as well as 1-amino-2-propanol, it is completely specific for the D-isomer of the amino alcohol and for NAD+. Two molecular forms (designated Form L and Form S) of the oxidoreductase, both of which are catalytically active, have been separated by gel filtration on Sephadex G-200; apparently, Form L is converted to Form S by dissociation (Form L leads to Form S). Molecular weight determinations indicate that the two forms of the enzyme are different not only in size but also in shape; Form L apparently is an asymmetric tetramer of Form S. The two molecular species have similar catalytic properties. Both exhibit the same pH optimum of 8.6, have nearly identical apparent Km values for substrate and cosubstrate, are equally sensitive to inhibition by p-mercuribenzoate and N-ethylmaleimide, and show the same specificity for cosubstrate. Neither form of the enzyme has an absolute requirement for added thiol compounds or divalent metal ions.     

Homochirality and long-range transfer in biological systems.

It has been shown that the chiral purity of biomacromolecules has important biological significance not only from the standpoint of lock-and-key stereocomplementarity, but also as a basis for long-range communication in biosystems. An explicit demonstration is given for the case of proton transfer along the hydrogen-bonded chain that is formed by amino acids containing OH groups. It is found that the replacement of the L-amino acid residue by the D-isomer in a peptide chain suppresses proton transport through the hydrogen bond network.     

3-Deoxy-D-pentulosonic acid aldolase and its role in a new pathway of D-xylose degradation

Evidence is presented for a new pathway of D-xylose metabolism. Cell-free extracts prepared from a pseudomonad grown on D-xylose as a sole carbon source contain prominent D-xylose dehydrogenase, D-xylo-aldonate dehydrase and a new aldolase which cleaves 2-keto-3-deoxy-D-xylonate (3-deoxy-D-pentulosonic acid) to pyruvate and glycolaldehyde. The aldolase is specific for the D-isomer and is distinguishable from a previously described enzyme present in the same organism which is correspondingly specific for the L-isomer. The subsequent conversion of 3-deoxy-D-pentulosonic acid to α-keto-glutarate which has been reported in other organisms could not be demonstrated. Thus, the pathway of D-xylose degradation in this pseudomonad is believed to be: D-xylose → D-xylonate → 3-deoxy-D-pentulosonic acid → pyruvate + glycolaldehyde.     

Cysteine residues are involved in structure and function of melanocortin 1 receptor: Substitution of a cysteine residue in transmembrane segment two converts an agonist to antagonist

Reduction of disulfide bonds in human melanocortin 1 receptor (hMC1R) with increasing concentrations of DTT (dithiothreitol) resulted in a decrease in the binding of [125I]-ACTH (adrenocorticotropic hormone, L-isomer) in an uniphasic manner and a decrease in [125I]-NDP-MSH ([Nle(4),D-Phe(7)]-alpha-melanocyte stimulating hormone; D-isomer) binding in a biphasic manner. Pretreatment of hMC1R with 10 mM DTT resulted in a 36-fold loss of affinity for alpha-MSH (L-isomer) without affecting the affinity of NDP-MSH (D-isomer). To characterize the role of individual cysteine residues, we employed site-directed mutagenesis to substitute cysteine by glycine at all fourteen positions in hMC1R and analysed wild-type and mutant receptors for ligand binding and cAMP signalling. Single point mutation of four cysteine residues in extracellular loops to glycine (C35G, C267G, C273G, and C275G) resulted in a complete loss of binding for [125I]-NDP-MSH. Moreover, mutants with normal ligand binding, at positions C191G (transmembrane segment 5), C215G (third intracellular loop), and C315G (C-terminal loop) failed to generate cAMP signal in response to both agonists alpha-MSH and NDP-MSH. Mutant at position C78G (with wild-type binding to alpha-MSH as well as NDP-MSH) generated a cAMP signal in response to alpha-MSH (identical to wild-type hMC1R) but interestingly could not be stimulated by NDP-MSH. Moreover, this single amino acid substitution converted NDP-MSH from being an agonist to antagonist at the C78G mutant receptor. These findings demonstrate that (i) alpha-MSH and ACTH (L-isomers) are different from D-isomer NDP-MSH in their sensitivity to DTT for receptor binding, (ii) cysteine residues in N-terminus and extracellular loop three make disulfide bridges and are needed for structural integrity of hMC1R, (iii) cysteine residues in transmembrane segments and intracellular loops are required for receptor-G-protein coupling, (iv) C78 in transmembrane segment two is required for generating a functional response by D-isomer agonist (NDP-MSH) but not by L-isomer agonist (alpha-MSH), and (v) wild-type receptor agonist NDP-MSH is an antagonist at the mutant C78G receptor.     

Glycerate kinase from spinach leaves: Partial purification, characterization and subcellular localization

Spinach ( Spinacia oleracea L. cv. Melody hybrid) leaf glycerate kinase (EC 2.7.1.31) was partially purified and characterized. The enzyme did not exhibit any absolute stereospecificity towards the two enantiomers of glycerate, but the affinity for the D-isomer was 15-fold greater. The enzyme exhibited a broad pH optimum of 7.5–9.0 and a requirement for divalent cation, satisfied by Mg₂₊. The reaction product was identified as 3-phosphoglyceric acid. The observed high glycerate kinase activity together with its strategic localization exclusively in the chloroplast stroma are considered adequate for an efficient coupling of photosynthetic and photorespiratory carbon pathways.     

Inversion and isomerization of Asp-58 residue in human alphaA-crystallin from normal aged lenses and cataractous lenses.

We have previously shown that L-Asp-151 in alphaA-crystallin from the human lens is converted to the biologically uncommon D-isomer. This process was not simple racemization, but stereoinversion, accompanied by isomerization to form the beta-Asp residue, such that L-beta-Asp, D-alpha-Asp and D-beta-Asp were formed. The present study shows that Asp-58 of human alphaA-crystallin is also converted to the D-isomer to a high degree to form the same isomers with age. The D/L ratio of beta-Asp-58 in aged normal lens increased to more than 3.0, showing stereoinversion by the 60 year range, then decreased to 1.0 in the 80 year range, while the isomerization of Asp-58 increased in the 80 year range. We also measured inversion and isomerization of the same residue from cataractous and normal human lenses of the 60 year range. The D/L ratio of Asp-58 from cataractous lenses was significantly lower than that from normal lenses, while the isomerization at Asp-58 in cataractous alphaA-crystallin was significantly higher than that of normal alphaA-crystallin. These results indicate that isomerization to the beta isomer of Asp-58 in cataractous alphaA-crystallin increased more than inversion to the D-isomer, suggesting that there are changes in the native structure of alphaA-crystallin in the human cataractous lens.     

Physiological basis for preferential uptake of D-alpha-aminoadipate over the L-isomer by Alcaligenes denitrificans.

Alcaligenes denitrificans, pre-incubated with D-alpha-aminoadipate and assayed for L-isomer uptake without removal of extracellular D-isomer, exhibits a reduced rate of uptake and a reduced level at which steady state is achieved. During D- or L-isomer uptake, intracellular alpha-aminoadipate is exclusively the L-configuration. These data are consistent with an intracellular, mediated reduction in L-isomer uptake as the physiological basis for preferential D-alpha-aminoadipate uptake by A. denitrificans growing on racemic alpha-aminoadipate. Translocated D-alpha-aminoadipate is rapidly metabolized to form an L-isomer pool which subsequently reduces the rate of L-isomer uptake and the level at which steady state occurs resulting in a preferred D-isomer uptake. Competitive inhibition of L-alpha-aminoadipate uptake by the D-isomer or a difference in the maximum rates of uptate uptake is an inducible process expressed only in the presence of that compound and while uptake of L-alpha-animoadipate is also inducible there is a low rate of constitutive uptake. While L-alpha-aminoadipate uptake occurs against a concentration gradient, uptake of the D-isomer is not against a gradient. D- and L-isomer uptake are active processes since both are inhibited by azide, cyanide and 2,4-dinitrophenol.     

Studies on a 2,3-diaminopropionate: ammonia-lyase from a Pseudomonad.

2,3-Diaminopropionate:ammonia-lyase, an induced enzyme in a Pseudomonas isolate, has been purified 40-fold and found to be homogeneous by disc gel electrophoresis and by ultracentrifugation. Some of its properties have been studied. The optimum pH and temperature for activity are 8 and 40 degrees C, respectively. The enzyme shows a high degree of substrate specificity, acting only on 2,3-diaminopropionate; the D-isomer is only one-eighth as effective as the L-form. L-Homoserine and DL-cystathionine are not substrates, and 3-cyanolalanine does not inhibit its activity. It is a pyridoxal phosphate enzyme which requires free enzyme sulphhydryls for activity. The Km values for L-2,3-diaminopropionate and pyridoxal phosphate are 1mM and 25 muM, respectively. The molecular weight of the enzyme is about 80 000 as determined by gel filtration. On treatment with 0.5M urea or guanidine by hydrochloride, the enzyme dissociates into inactive subunits with an approximate molecular weight of 45 000. One mole of the active enzyme binds one mole of pyridoxal phosphate. The bacterial enzyme seems to be quite different in many of its properties from the rat liver enzyme which also exhibits the substrate specificity of cystathionine gamma-lyase.