Enantiomer selection in the reaction of N-methyl-α-amino acid N-carboxyanhydride and 3-methyl-5-substituted hydantoin: A model reaction for the stereoselective polymerization of α-amino acid N-carboxyanhydride

The addition reaction to N-methyl-(S)-alanine or N-methyl-(S)-phenylalanine N-car-boxyanhydride (NCA) of 3-methyl-5-substituted hydantoin (HDT) catalyzed by a tertiary amine was investigated as a model reaction for the propagation reaction of NCA according to the activated-NCA mechanism. Several activated HDTs having the (S)-configuration of the asymmetric carbon atom were found to react more rapidly than their activated enantiomers. This experimental result indicates that the enantiomer selection by terminal-unit control takes place in the propagation reaction according to the activated-NCA mechanism in which an activated NCA is added to a terminal acylated NCA ring of the growing chain. The enantiomer excess of the HDT recovered from the reaction mixture of N-methyl-(S)-phenylalanine NCA and racemic HDTs activated by a tertiary amine was determined. The extent of the enantiomer selection in the polymerization was found to be 3–10 times as large as that in the model reaction. From these results, it was concluded that the chirality of the penultimate unit, as well as that of the terminal NCA ring, plays an important role in determining the enantiomer selection in the NCA polymerization.     

Enantiomer selection in the addition reaction of optically active 3-methyl-5-substituted hydantoins to N-[(S)-α-methylbenzyl]glycine N-Carboxyanhydride: Model Reaction for the Stereoselective Polymerization of α-amino acid N-carboxyanhydride

In order to investigate the contribution from the chiral penultimate unit to the enantiomer selection in the activated N-carboxyanhydride (NCA) polymerizations, the addition reaction to N-[(S)-methylbenzyl]glycine NCA of various α-amino acid hydantoins activated by the tertiary amines was investigated in different solvents. The reactions of activated Ala, Val, and Phe hydantoins were stereoselective and suggested the participation of the penultimate unit in the enantiomer selection of the activated NCA type of polymerization. The degree of enantiomer selection was not well correlated with the structure of hydantoins. Taking into account the dipole repulsion and the orbital overlapping between the reaction species, the transition-state model was proposed, which gave a good explanation of the selectivity for (R)-hydantoin in PhNO₂ and CH₃CN and the selectivity for (S)-hydantoin in AcNMe₂ and HCONMe₂. In these two types of solvents the orientation of the methylbenzyl group with respect to the NCA ring is so different that the direction of the approach of the activated hydantoin to the NCA is different. This difference leads to the inversion of enantiomer selection in amide solvents and in others. Cationic species derived from tertiary amines and the chiral amide compound were found to affect the enantiomer selection in the model reaction. The implications of these findings with regard to enantiomer selection in the activated NCA type of polymerization are discussed.     

Block copolymerization of α-amino acid N-carboxyanhydride initiated by vinyl polymers having a terminal amino group and the characterization of the block copolymers

Poly(methyl methacrylate) and polystyrene having terminal amino groups were synthesized by the radical polymerization of those monomers in the presence of 2-mercaptoethylammonium chloride as a chain-transfer agent. By the terminal group analysis and the molecular weight determination of the polymers, 0.5–1.3 amino groups were found in a chain of poly(methyl methacrylate) and 0.5–2.5 amino groups in a chain of polystyrene. Using these polymers having a terminal amino group as an initiator, the block polymerization of α-amino acid N-carboxyanhydride (NCA) was carried out. In the polymerizations of Glu(OBzl) NCA and Lys(Z) NCA by the poly(methyl methacrylate) initiator, the terminal amino group underwent a nucleophilic addition reaction to NCA and initiated the polymerization, yielding A-B-type block copolymers in a high yield. The same was observed in the polymerizations of Gly(OBzl) NCA and Lys(Z) NCA by the polystyrene initiator. By eliminating the protecting groups of the side chains of the polypeptide segment, the block copolymers poly(methyl methacrylate)-poly(Glu), poly(methyl methacrylate)-poly(Lys), polystyrene-poly(Glu) and polystyrene-poly(Lys) were synthesized with little side reactions. The side chain amino groups of poly(Lys) segment in the poly(methyl methacrylate)-poly(Lys) block copolymers were sulphonated or stearoylated successfully.     

Asymmetric selection in the copolymerization of N-carboxyl-L- and D-alanine anhydride

The copolymerization of N-carboxy-L - and D -alanine anhydride with methanol as initiator was carried out. The enantiomer excess in the starting monomer mixture is preferentially incorporated into polymer chains, demonstrating asymmetric selection during the D - and L -copolymerization. The mechanism of asymmetric-selective polymerization of α-amino acid NCA is discussed in terms of the stereoregulation by molecular asymmetry of the growing polymer chain.     

Solvent effect in the stereoselective polymerization of α-amino acid N-carboxyanhydride and some considerations on the mechanism of stereoregulation

In the Polymerization of phenylalanine N-carboxyanhydride (NCA) in No₂Oh initiated by MeNHBzl, L -,D -, and DL -NCA As were polymerized at the same rate, and no stereoselectivity was observed. When the same experiment was carried out in HCONEt₂, however, L - and D -NCA were both polymerized at a rate which was about twice as large as that of DL -NCA. In this case, the polymerization is stereoselective, ascribable to a preferable reaction between the optical enantiomorphs of the terminal residue of the growing chain and the NCA of the same chirality. On the other hand, the polymerization initiated by SarNMe₂ and MeNH(CH₂)₂CONMe₂ were stereoselective in NO₂Ph and HCONEt₂, but they were not stereoselective in m-(MeO)₂Ph. These findings indicate that the polymerizations initiated by a strong base in highly dipolar solvents are stereoselective. Apparently, the reaction between a chiral, cyclic terminal of growing chain and a chiral, cyclic activated NCA in the activated-NCA mechanism is highly stereoselective. In addition, from a kinetic investigation on on the copolymerization between L - and D -NCAs, the penultimate chiral centers were also suggested to contribute to the stereoselection. Stereoselection by the α-helical conformation of the growing chain and by a chiral, linear terminal amine have been considered so far, and the contribution from the present type of stereoselection must have been overlooked.     

Stereochemistry of ATP and GTP bound to fish haemoglobins. A transferred nuclear overhauser enhancement, 31P-nuclear magnetic resonance, oxygen equilibrium and molecular modelling study

This study was undertaken in order to test the models of ATP and GTP binding to carp deoxyhaemoglobin proposed by Perutz & Brunori (1982) and to find out why GTP is a more potent allosteric effector than ATP. We have determined the conformations of both nucleoside triphosphates by nuclear magnetic resonance studies and found them to be the same. The purines are in anti conformation about the glycosidic bond that links them to the ribose; the pentose ring is 3'-endo; the P-O5'-C5'-C4' torsion angle lies in the trans domain (180 degrees +/- 20 degrees); the P alpha-O-P beta and P beta-O-P gamma angles are as in the free nucleotides, i.e. the trinucleotide chain is fully extended. Models having this conformation were fitted, first manually and then by energy refinement, to the effector site of an atomic model of human deoxyhaemoglobin in which the side-chains in the NA, EF and H segments had been replaced by those of carp. The results showed the location of the polar groups in carp haemoglobin to be such that (PO4) gamma can accept hydrogen bonds from Val NA1 beta 2 and from Arg H21 beta 1, while (PO4) beta and (PO4) alpha can accept hydrogen bonds from Lys EF6 beta 1 and beta 2. In ATP, the 6-amino group of the purine can donate a hydrogen bond to Glu NA2 beta 1. In GTP, the 2-amino group can donate a hydrogen bond to Glu NA2 beta 1; in addition, Val Na1 beta 1 can donate a hydrogen bond to O2' of the ribose. This additional hydrogen bond may explain why in carp haemoglobin GTP is a stronger allosteric effector than ATP. We have found the influence of the two allosteric effectors on the oxygen affinity of trout IV haemoglobin to be the same, even though the only difference in the lining of the allosteric effector sites lies in the replacement of Glu Na2 beta in carp by Asp in trout IV haemoglobin. Model building then showed that formation of a hydrogen bond between Asp Na2 beta and the 2-amino group of guanine precludes formation of a hydrogen bond between Val NA1 beta and O2' of the ribose or vice versa, which makes the number of hydrogen bonds formed between trout IV haemoglobin and GTP the same as those formed with ATP.     

Polymerization of N-carboxyanhydrides of various α-amino acids using secondary amines as initiator

In the polymerizations of alanine, γ-ethyl glutamate, and leucine N-carboxyanhydrides (NCA's) initiated by tertiary amines and some secondary amines such as N-methyl-L -alanine dialkylamide, a stereoselectivity was observed: the polymerization rates of L - and D -NCA's were identical to each other and larger than that of DL -NCA. However, this selectivity was not observed in the polymerizations of valine and isoleucine NCA's initiated by N-methyl-L -alanine dialkylamide. The stereoselective polymerizations of valine and isoleucine NCA's were induced only with tetriary amines such as tri-n-butylamine. N-Methyl-L -alanine di-alkylamide has been shown to initiate the polymerization of usual α-amino acid NCA according to the activated-NCA mechanism, but it initiated the polymerizations of valine and isoleucine NCA's according to the primary amine-type mechanism. This is because in the latter NCA's the N–H group is masked by the adjacent C^β-branched alkyl substituent against the approach of the secondary amine. Poly(DL -alanine)s produced in the stereoselective polymerization had higher viscosities and were more stereoblock-like than those produced without the stereoselectivity. These experimental results indicate that the stereoselective polymerization is possible only when the polymerization proceeds through the activated-NCA mechanism.     

The amino acid sequence of human chorionic gonadotropin. The alpha subunit and beta subunit.

The amino acid sequences of both the alpha and beta subunits of human chorionic gonadotropin have been determined. The amino acid sequence of the alpha subunit is: Ala - Asp - Val - Gln - Asp - Cys - Pro - Glu - Cys-10 - Thr - Leu - Gln - Asp - Pro - Phe - Ser - Gln-20 - Pro - Gly - Ala - Pro - Ile - Leu - Gln - Cys - Met - Gly-30 - Cys - Cys - Phe - Ser - Arg - Ala - Tyr - Pro - Thr - Pro-40 - Leu - Arg - Ser - Lys - Lys - Thr - Met - Leu - Val - Gln-50 - Lys - Asn - Val - Thr - Ser - Glu - Ser - Thr - Cys - Cys-60 - Val - Ala - Lys - Ser - Thr - Asn - Arg - Val - Thr - Val-70 - Met - Gly - Gly - Phe - Lys - Val - Glu - Asn - His - Thr-80 - Ala - Cys - His - Cys - Ser - Thr - Cys - Tyr - Tyr - His-90 - Lys - Ser. Oligosaccharide side chains are attached at residues 52 and 78. In the preparations studied approximately 10 and 30% of the chains lack the initial 2 and 3 NH2-terminal residues, respectively. This sequence is almost identical with that of human luteinizing hormone (Sairam, M. R., Papkoff, H., and Li, C. H. (1972) Biochem. Biophys. Res. Commun. 48, 530-537). The amino acid sequence of the beta subunit is: Ser - Lys - Glu - Pro - Leu - Arg - Pro - Arg - Cys - Arg-10 - Pro - Ile - Asn - Ala - Thr - Leu - Ala - Val - Glu - Lys-20 - Glu - Gly - Cys - Pro - Val - Cys - Ile - Thr - Val - Asn-30 - Thr - Thr - Ile - Cys - Ala - Gly - Tyr - Cys - Pro - Thr-40 - Met - Thr - Arg - Val - Leu - Gln - Gly - Val - Leu - Pro-50 - Ala - Leu - Pro - Gin - Val - Val - Cys - Asn - Tyr - Arg-60 - Asp - Val - Arg - Phe - Glu - Ser - Ile - Arg - Leu - Pro-70 - Gly - Cys - Pro - Arg - Gly - Val - Asn - Pro - Val - Val-80 - Ser - Tyr - Ala - Val - Ala - Leu - Ser - Cys - Gln - Cys-90 - Ala - Leu - Cys - Arg - Arg - Ser - Thr - Thr - Asp - Cys-100 - Gly - Gly - Pro - Lys - Asp - His - Pro - Leu - Thr - Cys-110 - Asp - Asp - Pro - Arg - Phe - Gln - Asp - Ser - Ser - Ser - Ser - Lys - Ala - Pro - Pro - Pro - Ser - Leu - Pro - Ser-130 - Pro - Ser - Arg - Leu - Pro - Gly - Pro - Ser - Asp - Thr-140 - Pro - Ile - Leu - Pro - Gln. Oligosaccharide side chains are found at residues 13, 30, 121, 127, 132, and 138. The proteolytic enzyme, thrombin, which appears to cleave a limited number of arginyl bonds, proved helpful in the determination of the beta sequence.     

Synthesis and interaction with metal ions of cyclic oligopeptides bearing carboxyl groups

Cyclic di- and tetrapeptides bearing carboxyl or carboxylate groups, cyclo[Glu(OBzl)-Glu(OMe)], cyclo[Glu-Glu(OMe)], cyclo(Glu-Glu), cyclo[Glu(OMe)-Pro)2, and cyclo(Glu-Pro)2, were synthesized and investigated on the intramolecular interaction of carboxyl side chains in the complexation with metal ions in relation with the conformation. The three kinds of cyclic dipeptides were found to take a flagpole boat conformation. Folded conformation of side chains was predominant for cyclo[Glu(OBzl)-Glu(OMe)] and cyclo[Glu-Glu(OMe)]. However, cyclo(Glu-Glu) took an unfolded conformation. Intramolecular interaction of carboxyl groups was observed neither in free state nor in complexation with metal ions. The intramolecular interaction of carboxyl groups was observed in the case of cyclo(Glu-Pro)2 in the absence of metal ions added. Cyclo[Glu(OMe)-Pro]2 and cyclo(Glu-Pro)2 formed a complex with Ca2+ and Ba2+ without participation of side chains.     

Synthesis of side chain-protected amino acid phenylthiohydantoins and their use in quantitative solid-phase Edman degradation

Solid-phase Edman degradation of synthetic peptidyl-resins has been used advantageously to detect errors of deletion which might occur during Merrifield peptide synthesis. To facilitate complete quantitation of the resulting phenylthiohydantoin(PTH)-amino acids, the PTH derivatives of the following side chain-protected amino acid residues have been synthesized: Arg(Tos), Asp(OBzl), Cys(3,4-(CH3)2-Bzl), Glu(OBzl), Lys(2-ClZ), Ser(Bzl), Thr(Bzl), Tyr(2-BrZ), and Tyr(2,6-Cl2Bzl). For each derivative, a melting point, elemental analysis, and extinction coefficient were obtained. With these new compounds as HPLC standards, an unequivocal assignment and quantification of each side chain protected amino acid was possible. A quantitative analysis was performed for six model peptides with the general formula Ala-X-Leu-Y-Ala-Gly-NHCH2-resin (where X and Y represented different side chain-protected amino acyl residues). We have found solid-phase Edman degradation to be a useful aid for the characterization of peptides when they are used unpurified as synthetic antigens.     

Synthesis and solubility properties of peptide fragments of human hemoglobin alpha-chain (123-136)

The solubility prediction method for protected peptides was successfully applied to relatively small peptide fragments of human hemoglobin alpha-chain (123-136) which contained various polar amino acid residues such as Asp(OBzl), Glu(OBzl), Lys(Z), Ser(Bzl), and Thr(Bzl). As reported previously for hydrophobic peptides and human proinsulin C-peptide fragments, solubility data indicated that the insolubility of protected peptides having a mean value of Pc value below 0.90 appeared to begin at the octa- or nonapeptide sequence level and that beta-sheet structure played an important role in the insolubility of peptides. When a peptide has a beta-sheet structure in the solid state, we can clearly determine the critical chain length for peptide insolubility, the solubility dependence on solvent properties, and the solubility independence of amino acid compositions of peptides.     

A convenient,rapid method for the resolution of enantiomeric amino acids using chiral phases on stainless-steel capillary gas chromatographic columns

An improved method is described for the resolution of enantiomeric isopropyl esters of N-trifluoroacetyl-α-amino acids of nonbasic amino acids using N-docosanoyl-l-valyl-t-butylamide and N-octadecanoyl-l-valyl-l-valine cyclohexyl ester as mixed chiral phases on 150-ft stainless-steel capillary columns. Enantiomers of Ala, Val, Ile, Leu, Ser, Thr, Asp, Met, Glu, and Phe are resolved in 105 min. This method avoids the fractionation problems and high costs encountered with the diastereometric method and difficulties and costs encountered in loading and maintaining glass capillary columns. It is particularly useful for studies involving a large number of resolutions as in a study of the kinetics of racemization of amino acids.     

Detailed characterization of the cooperative mechanism of Ca(2+) binding and catalytic activation in the Ca(2+) transport (SERCA) ATPase

Expression of heterologous SERCA1a ATPase in Cos-1 cells was optimized to yield levels that account for 10-15% of the microsomal protein, as revealed by protein staining on electrophoretic gels. This high level of expression significantly improved our characterization of mutants, including direct measurements of Ca(2+) binding by the ATPase in the absence of ATP, and measurements of various enzyme functions in the presence of ATP or P(i). Mutational analysis distinguished two groups of amino acids within the transmembrane domain: The first group includes Glu771 (M5), Thr799 (M6), Asp800 (M6), and Glu908 (M8), whose individual mutations totally inhibit binding of the two Ca(2+) required for activation of one ATPase molecule. The second group includes Glu309 (M4) and Asn796 (M6), whose individual or combined mutations inhibit binding of only one and the same Ca(2+). The effects of mutations of these amino acids were interpreted in the light of recent information on the ATPase high-resolution structure, explaining the mechanism of Ca(2+) binding and catalytic activation in terms of two cooperative sites. The Glu771, Thr799, and Asp800 side chains contribute prominently to site 1, together with less prominent contributions by Asn768 and Glu908. The Glu309, Asn796, and Asp800 side chains, as well as the Ala305 (and possibly Val304 and Ile307) carbonyl oxygen, contribute to site 2. Sequential binding begins with Ca(2+) occupancy of site 1, followed by transition to a conformation (E') sensitive to Ca(2+) inhibition of enzyme phosphorylation by P(i), but still unable to utilize ATP. The E' conformation accepts the second Ca(2+) on site 2, producing then a conformation (E' ') which is able to utilize ATP. Mutations of residues (Asp813 and Asp818) in the M6/M7 loop reduce Ca(2+) affinity and catalytic turnover, suggesting a strong influence of this loop on the correct positioning of the M6 helix. Mutation of Asp351 (at the catalytic site within the cytosolic domain) produces total inhibition of ATP utilization and enzyme phosphorylation by P(i), without a significant effect on Ca(2+) binding.     

Stereoselectivity in the nucleophilic addition-type polymerization of α-amino acid N-caboxyanhydride initiated by diastereomeric dipeptide amines

In order to investigate the effect of the chiral penultimate unit on the stereoselection of α-amino acid N-carboxyanhydride (NCA) by the terminal unit of a growing chain in the nucleophilic addition-type polymerization, the diastereomers of dipeptide amines, H-(R)-Phe-(S)-Phe-Mo and H-(S)-Phe-(S)-Phe-Mo, in which Mo represents a morpholine residue, were synthesized, and the stereoselectivity in their nucleophilic addition reactions to NCA was investigated and compared with that of a monopeptide amine H-(S)-Phe-OEt. In the reaction with Phe NCA in nitrobenzene, either of the dipeptide amines reacted preferentially with an enantiomer of NCAs having a configuration opposite to the N-terminal unit of the dipeptide amine. The preference of enantiomeric NCA and the extent of stereoselectivity were nearly the same as those found with H-(S)-PheOEt. The opposite-enantiomer selectivity of the dipeptide amines was also observed in the reaction with N-MePhe NCA, and the extent of stereoselectivity was found to increase very much in the reaction of H-(R)-PHe-(S)-Phe-Mo compared with that of H-(S)-Phe-OEt. Therefore, the enhancement of the stereoselectivity of the N-terminal unit by the penultimate unit was shown experimentally. On the other hand, the stereoselectivity of H-(S)-Phe-(S)-Phe-Mo was not very different from that of H-(S)-Phe-OEt. These results were obtained either in nitrobenze or in m-dimethoxybenzene. H-(S)-Phe-(S)-Phe-OEt tends to aggregate by an intermolecular hydrogen bond in aqueous and tetrahydrofuran solutions. Its pK_a value and nucleophilicity towards NCA were much lower than H-(R)-Phe-(S)-Phe-Mo, which was free from the aggregation under similar conditions. These experimental results suggest that the major product in the polymerization of (RS)-Phe NCA by amine should be an alternating copolymer. However, this prediction was not verified experimentally, and the important contributions from the aggregation and the molecular weight distribution of growing chains were suggested.     

Block copolymerization of vinyl compounds by the terminal-group activation of poly(α-amino acids) and the characterization of block copolymers

The terminal amino groups of polysarcosine, poly(γ-benzyl l-glutamate), and poly(ε-benzyloxycarbonyl-l-lysine) were haloacetylated. The mixture of the terminally haloacetylated poly(α-amino acid) and styrene or methyl methacrylate was photoirradiated in the presence of Mn₂(CO)₁₀, or heated with Mo(CO)₆, yielding A-B-A-type block copolymers consisting of poly(α-amino acid) (the A component) and vinyl polymer (the B component). The block copolymers were characterized, and the present investigation revealed that the thermally initiated polymerization of vinyl compounds by the trichloroacetyl poly(α-amino acid)/Mo(CO)₆ system was the most suitable for the synthesis of the α-amino acid/vinyl compound block copolymers. The A-B-A type block copolymers showed higher antithrombogenicity than the corresponding homopolymers. In particular, a film of the A-B-A-type block copolymer of poly[Glu(OBzl)] and polystyrene possessed a microphase-separated structure and did not induce a conformational change of fibrinogen adsorbed, leading to a high antithrombogenicity.     

Intra- and intermolecular charge-transfer interactions between terminal electron donor and acceptor attached to poly(γ-benzyl-L-glutamate) in solution

Poly(γ-benzyl-L -glutamate) having a terminal dimethylaminoanilide group as an electron donor (D) and a terminal 3,5-dinitrobenzoyl group as an electron acceptor (A) (A-[Glu(OBzl)]_n-D) was synthesized by the N-carboxyanhydride method. Polymer samples were fractionated by gel chromatography and their number-average degrees of polymerization n were determined by the absorbances of the terminal chromophores. These polymers in chloroform and dimethylformamide solutions showed a charge-transfer (CT) absorption band around 455 nm, and the fraction of the polymer forming the CT complex was evaluated as a function of the chain length. CT absorption for short chains (n = 5 ～ 20) was attributed to intramolecular CT complex in which the A-[Glu(OBzl)]_n-D chain takes cyclic conformations. An optimum chain length for the intramolecular CT was found to be n ? 10, where the [Glu(OBzl)]_n chain may most easily bend back to form cyclic conformations. Stronger CT absorption observed for longer chains than n = 20 was shown to be intermolecular, and an intermolecular head-to-tail aggregation was found to be a cause of the strong CT interaction. All helical A-[Glu(OBzl)]_n-D chains were found to form the head-to-tail dimers in chloroform solution.     

Structural and functional effects of disease-causing amino acid substitutions affecting residues Ala72 and Glu76 of the protein tyrosine phosphatase SHP-2

Mutations of the protein tyrosine phosphatase SHP-2 are implicated in human diseases, causing Noonan syndrome (NS) and related developmental disorders or contributing to leukemogenesis depending on the specific amino acid substitution involved. SHP-2 is composed by a catalytic (PTP) and two regulatory (N-SH2 and C-SH2) domains that bind to signaling partners and control the enzymatic activity by limiting the accessibility of the catalytic site. Wild type SHP-2 and four disease-associated mutants recurring in hematologic malignancies (Glu76Lys and Ala72Val) or causing NS (Glu76Asp and Ala72Ser), with affected residues located in the PTP-interacting region of the N-SH2 domain, were analyzed by molecular dynamics simulations and in vitro biochemical assays. Simulations demonstrate that mutations do not affect significantly the conformation of the N-SH2 domain. Rather they destabilize the interaction of this domain with the catalytic site, with more evident effects in the two leukemia associated mutants. Consistent with this structural evidence, mutants exhibit an increased level of basal phosphatase activity in the order Glu76Lys > Ala72Val > Glu76Asp > Ala72Ser > WT. The experimental data also show that the mutants with higher basal activity are more responsive to an activating phosphopeptide. A thermodynamic analysis demonstrates that an increase in the overall phosphopeptide affinity of mutants can be explained by a shift in the equilibrium between the inactive and active SHP-2 structure. These data support the view that an increase in the affinity of SHP-2 for its binding partners, caused by destabilization of the closed, inactive conformation, rather than protein basal activation per se, would represent the molecular mechanism, leading to pathogenesis in these mutants.     

Unique side-chain conformation encoding for chirality and azimuthal orientation in the molecular packing of skin collagen.

We used molecular mechanics to study the role of gly X-Y+ sequences, where X- was Asp or Glu and Y+ was Lys or Arg, in the molecular packing of type I collagen. In the minimal energy conformation of a triply stranded molecule having a coiled-coil configuration, the side-chains of these sequences segregated into two oppositely charged groupings of the forms X-Y+X- and Y+X-Y+. Groupings having the same net charge were clustered along two complementary azimuthal edges of the molecule. Intermolecular interactions, through these oppositely charged edges, align the molecules appropriately for the formation of the HHL crosslink of skin. This alignment also can account for the axial periodicity and chiral appearance of skin collagen fibrils.     

The Influence of Salinity Acclimation on Free Amino Acids and Enzyme Activities in the Intestinal Mucosa of Rainbow Trout,Oncorhynchus mykiss (Walbaum)

Postprandial changes of Arg, Leu, Val, Ala, Asp, Glu, Gly, Pro and Tau as well as activities of three enzymes of the transdeamination system in the midgut mucosa and, for comparison, in the liver of freshwater and seawater acclimated Oncorhynchus mykiss were studied. In the mucosa a postprandial increase of Arg, Leu, Val, Ala, Asp, Glu, Gly and Pro occurred. In contrast, only the postprandial Arg level increased strongly in the liver. Levels of Leu, Val, Ala, Asp, Glu, Gly, Pro and Tau remained stable. Concentrations of Ala, Asp, Glu and Pro are higher in the liver than the mucosa. Tau is the most important osmotic effector in both organs, but its concentration is much lower in the liver. Its postprandial concentrations remained stable in both tissues but were significantly higher in seawater trout. The trend of a stronger postprandial rise of Arg, Leu, Val, Ala, Asp, Glu, Gly and Pro levels in seawater trout than in freshwater trout was shown. In mucosa tissue aspartate aminotransferase activities were higher in seawater trout. Ratios of aspartate aminotransferase, alanine aminotransferase and glutamate dehydrogenase are similar to those of the gills.