X-Ray Studies on Crystalline Complexes Involving Amino Acids and Peptides XXXI. Effect of Chirality on Ionization State,Stoichiometry and Aggregation in the Complexes of Oxalic Acid with L-and DL-histidine

Abstract

Crystals of the oxalic acid complex of L-histidine (orthorhombic P2₁2₁2₁; a=5.535(4), b=6.809(4), c=26.878(3) Å) R= 3.6% for 1188 observed reflections) contain histidine molecules and semi-oxalate ions in the 1:1 ratio, while the ratio is 1:2 in the crystals of the DL-histidine complex (monoclinic P2₁ lc; a=6.750(7), b=10.139(2), c=19.352(2) Å, β= 90.8°; R= 3.7% for 3176 observed reflections). The histidine molecule in the latter has an unusual ionization state with positively charged amino and imidazole groups and a neutral carboxyl group. The molecule has the sterically least favourable allowed conformation with the side chain imidazole ring staggered between the α-amino and the α- carboxyl (carboxylate) groups, in both the structures. The unlike molecules aggregate into separate alternating layers in both of them. There are elements of similarity in the aggregation patterns in the semi-oxalate layers in the two complexes, but the patterns in the amino acid layers are entirely different. Interestingly, the crystal structure of L-histidine semi-oxalate has broad similarities with that of DL-histidine glycolate, demonstrating how broad features of aggregation could be retained inspite of changes in chirality and composition. The unusual ionization state of the amino acid molecule in the DL-histidine complex is reflected in a hitherto unobserved aggregation pattern in its crystal structure.     

The Bacteriophage-Inactivating Effect of Basic Amino Acids; Arginine,Histidine, and Lysine

Some physicochemical properties and substrate specificity of acid protease B isolated from Scytalidium lignicolum were investigated.

The molecular weight determined by the sedimentation equilibrium and sedimentation velocity method was 21,000 and 19,000~20,000, respectively. The isoelectric point was determined as 3.0 using the Tiselius electrophoresis apparatus, 3.2 by isoelectric focusing, respectively.

The enzyme did not contain histidine and was composed of 188 amino acid residues. Substrate specificity against various synthetic peptides was different from those of the acid proteases which were inactivated by S–PI and DAN.     

Complex Patterns of Histidine, Hydroxylated Amino Acids and the GxxxG Motif Mediate High-affinity Transmembrane Domain Interactions

Specific interactions of transmembrane helices play a pivotal role in the folding and oligomerization of integral membrane proteins. The helix-helix interfaces frequently depend on specific amino acid patterns. In this study, a heptad repeat pattern was randomized with all naturally occurring amino acids to uncover novel sequence motifs promoting transmembrane domain interactions. Self-interacting transmembrane domains were selected from the resulting combinatorial library by means of the ToxR/POSSYCCAT system. A comparison of the amino acid composition of high-and low-affinity sequences revealed that high-affinity transmembrane domains exhibit position-specific enrichment of histidine. Further, sequences containing His preferentially display Gly, Ser, and/or Thr residues at flanking positions and frequently contain a C-terminal GxxxG motif. Mutational analysis of selected sequences confirmed the importance of these residues in homotypic interaction. Probing heterotypic interaction indicated that His interacts in trans with hydroxylated residues. Reconstruction of minimal interaction motifs within the context of an oligo-Leu sequence confirmed that His is part of a hydrogen bonded cluster that is brought into register by the GxxxG motif. Notably, a similar motif contributes to self-interaction of the BNIP3 transmembrane domain.     

Visualization of Drug-Nucleic Acid Interactions at Atomic Resolution. IX. Structures of Two N,N-Dimethylproflavine: 5-Iodocytidylyl (3′-5′) Guanosine Crystalline Complexes

Abstract

This paper describes two complexes containing N,N-dimethylproflavine and the dinucleoside monophosphate, 5-iodocytidylyl(3′-5′)guanosine (iodoCpG). The first complex is triclinic, space group PI, with unit cell dimensions a = 11.78 Å, b = 14.55 Å, c = 15.50 Å, a = 89.2°, β = 86.2°, γ = 96.4°. The second complex is monoclinic, space group P2₁, with a = 14.20 Å, b = 19.00 Å, c = 20.73 Å, β = 103.6°. Both structures have been solved to atomic resolution and refined by Fourier and least squares methods. The first structure has been refined anisotropically to a residual of 0.09 on 5,025 observed reflections using block diagonal least squares, while the second structure has been refined isotropically to a residual of 0.13 on 2,888 reflections with full matrix least squares. The asymmetric unit in both structures contains two dimethylproflavine molecules and two iodoCpG molecules; the first structure has 16 water molecules (a total of 134 non-hydrogen atoms), while the second structure has 18 water molecules (a total of 136 non-hydrogen atoms). Both structures demonstrate intercalation of dimethylproflavine between base-paired iodoCpG dimers. In addition, dimethylproflavine molecules stack on either side of the intercalated duplex, being related by a unit cell translation along b and a axes, respectively.

The basic structural feature of the sugar-phosphate chains accompanying dimethylproflavine intercalation in both structures is the mixed sugar puckering pattern: C3′ endo (3′-5′) C2′ endo. This same structural information is again demonstrated in the accompanying paper, which describes a complex containing dimethylproflavine with deoxyribo-CpG.

Similar information has already appeared for other “simple” intercalators such as ethidium, acridine orange, ellipticine, 9-aminoacridine, N-methyl-tetramethylphenanthrolinium and terpyridine platinum. “Complex” intercalators, however, such as proflavine and daunomycin, have given different structural information in model studies. We discuss the possible reasons for these differences in this paper and in the accompanying paper.     

氨基酸Schiff碱及其金属配合物的抑菌抗癌活性的研究进展

氨基酸Ｓｃｈｉｆｆ碱及其金属配合物具有良好的生理、生物活性,本文就近年来国内外在氨基酸Ｓｃｈｉｆ碱及其金属配合的抑菌抗癌活性的研究进展方面进行了综述     

Visualization of Drug-Nucleic Acid Interactions At Atomic Resolution. VIII. Structures of Two Ethidium/Dinucleoside Monophosphate Crystalline Complexes Containing Ethidium: Cytidylyl(3′-5′) Guanosine

Abstract

This paper describes two complexes containing ethidium and the dinucleoside monophosphate, cytidylyl(3′-5′)guanosine (CpG). Both crystals are monoclinic, space group P2₁, with unit cell dimensions as follows: modification 1: a = 13.64 Å, b = 32.16 Å, c - 14.93 Å, β = 114.8° and modification 2: a = 13.79 Å, b = 31.94 Å, c = 15.66 Å, β = 117.5°. Each structure has been solved to atomic resolution and refined by Fourier and least squares methods; the first has been refined to a residual of 0.187 on 1,903 reflections, while the second has been refined to a residual of 0.187 on 1,001 reflections. The asymmetric unit in both structures contains two ethidium molecules and two CpG molecules; the first structure has 30 water molecules (a total of 158 non-hydrogen atoms), while the second structure has 19 water molecules (a total of 147 non-hydrogen atoms). Both structures demonstrate intercalation of ethidium between base-paired CpG dimers. In addition, ethidium molecules stack on either side of the intercalated duplex, being related by a unit cell translation along the a axis.

The basic feature of the sugar-phosphate chains accompanying ethidium intercalation in both structures is: C3′ endo (3′-5′) C2′ endo. This mixed sugar-puckering pattern has been observed in all previous studies of ethidium intercalation and is a feature common to other drug-nucleic acid structural studies carried out in our laboratory. We discuss this further in this paper and in the accompanying papers.     

Studies on Tolfenamic Acid–Chitosan Intermolecular Interactions: Effect of pH, Polymer Concentration and Molecular Weight

Solid-state properties of tolfenamic acid (TA) and its complexes with chitosan (CT) have been studied. Effect of medium pH, molecular weight of polymer and its different concentrations on these TA–CT complexes were studied in detail. Low and medium molecular weight CT have been used in different ratios at pH ranging from 4 to 6 and freeze-drying technique has been employed to modify the appearance of crystalline TA. Physical properties of the formed complexes have been studied by employing X-ray diffraction, differential scanning calorimetry and scanning electron microscopy; chemical structure has been studied using Fourier transform infrared spectroscopy. The results showed that both forms of the polymer exhibited complete conversion in 1:8 ratio at pH 4, 1:4 at pH 5 and 1:1 at pH 6 indicating a marked effect of pH on drug–polymer complexation. The percent crystallinity calculations indicated low molecular weight CT slightly more effective than the other form. No changes in the complexes have been observed during the 12 week storage under controlled conditions. Both forms of CT at different pH values indicated retardation of recrystallization in TA during cooling of the melt from 1:1 ratios exhibiting formation of strong intermolecular hydrogen bonding between the drug and the polymer.     

The Effect of Nitrogen Nutrition on Cytokinin Activity and Free Amino Acids inBetula pendulaRoth, andAcer pseudoplatanusL.

A comparison was made of the levels of extractable cytokinin-likecompounds and free nitrate in the tissues of Betula pendulaand Acer pseudoplatanus at two levels of nitrogen nutrition.In further studies comparisons were made of the levels of cytokinin-likecompounds and free amino acids extracted from the tissues ofB. pendula supplied with nitrogen as ammonium sulphate, ammoniumnitrate, or calcium nitrate. These results were then relatedto growth differences between the treatments. B. pendula supplied with a low level of nitrogen as ammoniumnitrate continued to make slow growth whereas A. pseudoplatanusformed terminal resting buds after 2–3 weeks, furthergrowth being arrested. The low levels of extractable cytokinin-likecompounds found in the leaves of B. pendula receiving a lownitrogen supply were in contrast to the results from A. pseudoplatanus,where only a small reduction was found in response to low nitrogensupply. Analysis of the plant tissue showed that free nitrateand amino acids were depleted to a greater extent from B. pendulathan from A. pseudoplatanus under conditions of low nitrogensupply. It is suggested that the ability of B. pendula to continueactive growth with low nitrogen supply may be related to thereadiness with which nitrogen can be mobilized and re-circulatedin the plant, a process including sequential leaf senescence. Contrasting growth habits were seen in B. pendula in ammoniumnitrate- and ammonium sulphate-fed plants. Lateral shoot growthin ammonium sulphate-fed plants was completely inhibited andno detectable cytokinin activity was found in tissue extracts,whereas ammonium nitrate-fed plants grew rapidly, and showedconsiderable development of laterals. Changes in the levelsof individual amino acids show that the form in which nitrogenis taken up by plants affects the subsequent pathways of metabolism.     

In Vivo Studies on the Extracellular, and Veratrine-Releasable, Pools of Endogenous Amino Acids in the Rat Striatum: Effects of Corticostriatal Deafferentation and Kainic Acid Lesion

The effects of corticostriatal deafferentation (decortication) and destruction of intrinsic neurons (intrastriatal kainate injection) on the extracellular concentration, and veratrine-releasable pools, of endogenous amino acids in the rat striatum were examined using the in vivo brain dialysis technique. Intracellular amino acid content was also determined. Decortication reduced selectively intra- and extracellular levels of glutamate (Glu) and aspartate (Asp). Extracellular changes were more pronounced than those in tissue content. gamma-Aminobutyric acid (GABA), taurine (Tau), and phosphoethanolamine (PEA) levels were not affected, whereas nonneuroactive amino acids were increased at 1 week but not at 1 month post-lesion. The intracellular pool of Glu and Asp was also reduced in kainate-lesioned striata. However, extracellular levels of these compounds were not affected significantly by this treatment. The tissue content of all other amino acids was decreased, the most prominent change being in the concentration of GABA. Extracellular GABA concentration was also reduced dramatically, whereas the concentrations of noneuroactive amino acids were increased to varying degrees. These data suggest that transmitter pools of neuroactive amino acids are an important supply for their extracellular pools. Lesion-induced alterations in nonneuroactive amino acids are discussed with regard to the loss of metabolic pools, glial reactivity, and changes in blood-brain barrier transport. Veratrine induced a massive release of neuroactive amino acids such as Glu, Asp, GABA, and Tau into the extracellular fluid, and a delayed increase in PEA. Extracellular levels of neuroactive amino acids were raised slightly. Decortication reduced, selectively, the amounts of Glu and Asp released by veratrine.(ABSTRACT TRUNCATED AT 250 WORDS)     

Studies on a Model of Bitter Peptides Including Arginine,Proline and Phenylalanine Residues. II. Bitterness Behavior of a Tetrapeptide (Arg-Pro-Phe-Phe) and Its Derivatives

In order to investigate the production of a strong bitter taste of the tetrapeptide, Arg-Pro-Phe-Phe (1), we synthesized 16 kinds of analogs and tasted them. From the results, it was clarified that all the constituent amino acid residues in Arg-Pro-Phe-Phe (1) were necessary for its strong bitter taste. For a further increase in bitterness potency, it was found that the bitterness production units necessary should be concentrated together. In addition, Arg-Pro-Gly-Gly (6) and Gly-Gly-Arg-Pro (7) were found to have no bitterness. This will be very useful not only for studies on debittering of food but also for basic studies on the taste production mechanism.     

Effect of Motor and Premotor Cortex Ablation on Concentrations of Amino Acids, Monoamines, and Acetylcholine and on the Ultrastructure in Rat Striatum. A Confirmation of Glutamate as the Specific Cortico-Striatal Transmitter

At 1, 2, and 4 weeks after unilateral premotor and motor cortex ablation in rats, a significant and lasting decrease in glutamate levels in the ipsilateral versus contralateral striatum was observed. A significant corresponding fall in aspartate was seen only after 1 week. In contrast, there was a large increase in the striatal concentrations of lysine, threonine, alanine, and glutamine 1 week after the cortical ablation. This correlates with the extensive glial proliferation in the deafferented ipsilateral striatum. Four weeks after cortical ablation the GABA concentration was significantly increased. There was no decrease in other putative transmitters (dopamine, serotonin, acetylcholine, glycine and taurine), nor was a glutamate decrease observed in the hippocampus or in the hypothalamus, which do not receive direct premotor and motor cortical inputs. Both biochemical and morphological evidence for a minor contralateral cortico-striatal projection was obtained. Correlating with the fall in glutamate, ultrastructural observations indicated the degeneration of two types of striatal synapses, i.e., those of the axo-spinous type III and of the axo-dendritic type VII. Frontal cortex ablation clearly affects, in opposite directions, the metabolism of various striatal amino acids but not that of acetylcholine and the monoamine transmitters. The results strongly support the view that glutamate is the transmitter of the cortico-striatal fibers.     

Interactions of Zn(II) Ions with Humic Acids Isolated from Various Type of Soils. Effect of pH,Zn Concentrations and Humic Acids Chemical Properties

The main aim of this study was the analysis of the interaction between humic acids (HAs) from different soils and Zn(II) ions at wide concentration ranges and at two different pHs, 5 and 7, by using fluorescence and FTIR spectroscopy, as well as potentiometric measurements. The presence of a few areas of HAs structures responsible for Zn(II) complexing was revealed. Complexation at α-sites (low humified structures of low-molecular weight and aromatic polycondensation) and β-sites (weakly humified structures) was stronger at pH 7 than 5. This trend was not observed for γ-sites (structures with linearly-condensed aromatic rings, unsaturated bonds and large molecular weight). The amount of metal complexed at pH5 and 7 by α and γ-structures increased with a decrease in humification and aromaticity of HAs, contrary to β-areas where complexation increased with increasing content of carboxylic groups. The stability of complexes was higher at pH 7 and was the highest for γ-structures. At pH 5, stability decreased with C/N increase for α-areas and -COOH content increase for β-sites; stability increased with humification decrease for γ-structures. The stability of complexes at α and β-areas at pH 7 decreased with a drop in HAs humification. FTIR spectra at pH 5 revealed that the most-humified HAs tended to cause bidentate bridging coordination, while in the case of the least-humified HAs, Zn caused bidentate bridging coordination at low Zn additions and bidentate chelation at the highest Zn concentrations. Low Zn doses at pH 7 caused formation of unidentate complexes while higher Zn doses caused bidentate bridging. Such processes were noticed for HAs characterized by high oxidation degree and high oxygen functional group content; where these were low, HAs displayed bidentate bridging or even bidentate chelation. To summarize, the above studies have showed significant impact of Zn concentration, pH and some properties of HAs on complexation reactions of humic acids with zinc.     

Poster Sessions AP02: Proteins,Amino Acids,Peptides and Metabolism. The effect of proline rich polypeptide on the interaction between activated factor X and anti‐thrombin III

The effect of the hypothalamic neurosecretory Proline Rich Polypeptide (PRP) on the interaction of human Factor Xa (Xa) with human antithrombin III (ATIII) was investigated by SDS–PAGE and Western Blot analysis. Xa runs as two bands with masses of ～51 and ～47 kDa. A fraction of the ATIII (～55 kDa) undergoes a conformational change upon interaction with Xa to run slower at ～58 kDa. Xa and ATIII form a 1° duplex complex of ～109 and ～104 kDa, a 2° duplex complex of ～99 and ～94 kDa, and a 3° duplex complex of ～66 and ～62 kDa. Additionally, a Xa degradation product (～35 kDa) is noted. Pre‐incubation of 1.5 μg of either Xa or ATIII with 8 μg PRP for 15′ at R.T., or essentially simultaneous addition of all three, increases the 2° complex upper band, 3° complex lower band, ATIII conformational change, and the Xa degradation product. It also decreases the 3° complex upper band and the native ATIII. Xa/PRP premixtures cause a decrease in unreacted Xa. ATIII/PRP premixtures produce an increase in the 1° complex lower band and a decrease in the unreacted Xa upper band. Xa/ATIII/PRP mixtures increase both bands of the 1° complex. Thus, 8 μg PRP promotes 1° complex formation as well as formation of the 2° and 3° degradation complexes. It also promotes modification of ATIII and degradation of Xa, possibly by autolysis and/or promotion of proteolysis of the 1° complex by free Xa. Data obtained indicate the role of PRP in the molecular mechanisms of blood clotting. (Supported, in part, from a grant by the Office of Sponsored Programs and Research, BGSU.)     

Unique Induction of CA1 LTP Components After Intake of Theanine, an Amino Acid in Tea Leaves and its Effect on Stress Response

Theanine, γ-glutamylethylamide, is one of the major amino acid components in green tea. This study was undertaken to evaluate the effect of theanine intake on long-term potentiation (LTP) induction at hippocampal CA1 synapses and exposure to acute stress. Young rats were fed water containing 0.3% theanine after birth. Key findings: Serum corticosterone level was markedly decreased by theanine intake. Because this decrease can modify synaptic plasticity, the effect of theanine intake was examined focused on CA1 LTP induction. CA1 LTP induced by a 100-Hz tetanus for 1 s was almost the same extent in hippocampal slices from theanine-administered rats, whereas that induced by a 200-Hz tetanus for 1 s was significantly attenuated. 2-Amino-5-phosphonovalerate (APV), an N-methyl-d-aspartate (NMDA) receptor antagonist, significantly attenuated CA1 LTP induced by a 200-Hz tetanus in the control rats, but not in theanine-administered rats. Interestingly, APV completely blocked CA1 LTP induced by a 100-Hz tetanus in the control rats, while scarcely blocking it in theanine-administered rats. These results indicate that theanine intake reduces NMDA receptor-dependent CA1 LTP, while increasing NMDA receptor-independent CA1 LTP. Furthermore, neither 100-Hz tetanus-induced LTP nor 200-Hz tetanus-induced LTP was attenuated in theanine-administered rats after exposure to tail suspension stress, suggesting that the lack of NMDA receptor-dependent CA1 LTP by theanine intake is involved in ameliorating the attenuation of CA1 LTP after tail suspension. This study is the first to indicate that theanine intake modifies the mechanism of CA1 LTP induction.     

Studies on Feeding and Digestion in Protozoa. VII. Ingestion of Polystyrene Latex Particles and its Early Effect on Acid Phosphatase in Paramecium multimicronucleatum and Tetrahymena pyriformis

SYNOPSIS. Food vacuole-free P. multimicronucleatum and T. pyriformis readily ingest non-nutritive Dow polystyrene latex particles (PLP) and form vacuoles containing PLP at a rate comparable to the formation of vacuoles containing bacteria. The particles aggregate within the vacuoles and are egested as balls of the size of the vacuoles. PLP containing vacuoles rapidly acquire acid phosphatase activity, which is demonstrated by histochemical (alpha-naphthyl phosphatehexazonium salt or lead phosphate) methods as a peripheric staining. The total activity of the cell does not significantly change as a consequence of PLP uptake as suggested by the histochemical preparations and confirmed in T. pyriformis by measuring the splitting of p-nitrophenyl phosphate at pH 5. Accordingly, no selection between nutritive and non-nutritive particles could be revealed. The vacuole formation is induced by the mechanical action of the particles. The appearance of acid phosphatase activity in the vacuole seems to be dependent on the vacuole formation and not on its content. This early appearance of activity is due to a redistribution of the preexistent activity.     

Regulation of Bud Rest in Tubers of Potato, Solanum tuberosum L: VII. Effect of Abscisic and Gibberellic Acids on Nucleic Acid Synthesis in Excised Buds

The effect of gibberellin A₃ (10⁻⁴m) and abscisic acid (10⁻⁴m), applied separately and together, on incorporation of ³H-thymidine and ³H-uridine into DNA and RNA of buds from freshly harvested potatoes was investigated. In some treatments apical buds in intact tubers were treated three times daily for 3 days with test solution before the buds were excised and treated an additional 12 hours in Petri dishes. In other treatments, untreated buds were excised and treated 12 hours. Irrespective of length of treatment, gibberellin A₃ slightly promoted synthesis of DNA and RNA, and abscisic acid essentially blocked such synthesis, in both the presence and absence of gibberellin A₃.