Role of hydration of polyhydroxy compounds in biological systems.

The spin-lattice relaxation times, T1, of H2(17)O have been measured for aqueous solutions of 9 polyols as a function of the concentration at 25 degrees C. The dynamic hydration number, nDHN, for polyols were obtained. The values of nDHN increased with an increase in the number of OH groups and depended on the conformation of isomers. The value of nDHN for inositol was the largest. This means that the thermal motion of water molecules around inositol is most inhibited among the polyols studied. The increment of thermal stability of proteins by polyols and sugars, the equilibrium distribution coefficients of polyols and sugars in the denatured hemoglobin solution, and the effect of sugars on the osmotic flow of water were expressed by linear relation of nDHN. On the bases of these relations, a systematic treatment for the effect of hydration of polyols and sugars on these phenomena is discussed.     

Specifity of DNA-basic polypeptide interactions. Influence of neutral residues incorporated into polylysine and polyarginine.

An approach is described for evaluation of the specificity of basic polypeptides concerning the base pair composition of DNA. The polypeptides were covalently bound to CNBr activated agarose and two DNAs strongly different in base composition but of equal molecular weight were loaded and detached by a NaCl gradient. The difference in the NaCl concerntrations between the elution maxima of the two DNAs was taken as a measure for the recognition specificity. The results obtained confirmed the known AT- und GC-specificity of polylysine and polyarginine, respectively. Neutral residues incorporated into polylysine generally reduce the interaction affinity and also the AT-specificity of their host. This behavior is very pronounced with three homogeneous fractions of clupeine containing about one third of neutral aliphatic amino acids within clusters of arginine; the base pair specificity of these arginine copolymers was found to be practically nil.     

FREE AMINO ACID CHANGES DURING GERMINATION OF TELIOSPORES OF THE WHEAT BUNT FUNGUS,TILLETIA CARIES

Teliospores were aerated and agitated in a mineral salts medium and their free amino acid contents were analyzed at eight different times, from shortly after imbibition of water until just before germ tube emergence. In addition to the common amino acids, eight unidentified ninhydrin-positive components were detected. About 50 % or more of nearly each of the amino acids diffused out of the spores during the initial phase of germination. These released amino acids were actively taken up by the spores during the latter stages of germination. The free amino acids in largest amounts in the dormant spores of T. caries were arginine 15.0, glutamic acid 6.3, and alanine 3.7 μmoles per g dry spores. Together these three amino acids accounted for about 71 % of the total free amino acids in dormant spores of T. caries and T. controversa. The total amounts of free amino acids in spores of common bunt were much higher than in spores of dwarf bunt.     

Studies on the Nitrogen Metabolism in Ectomycorrhizae

Concentrations of free and bound amino acids were determined in 1) the mycorrhizal fungus Boletus variegatus Fr. 2) nonmycorrhizal root systems of aseptically grown Pinus sylvestris L. seedlings, and 3) mycorrhizal root systems of seedlings developed aseptically using the two symbionts. Arginine (total) was the major amino acid constituent in the mycelium of B. variegatus (18%–22%) during the exponential phase of growth. While 59%–86% of the available arginine was bound during the acceleration phase of growth, in the logarithmic phase 63%–75% was in the free pool. There were differences in the proportions between the individual amino acids in the bound fraction at different stages of growth suggesting production of diverse proteins. Twenty per cent of the amino acid content of uninfected P. sylvestris root systems was arginine. Infection of the root systems by the fungal symbiont did not result in an increase but a slight decrease in the free arginine content of the composite structure. Almost all other amino acids in the free pool were found in higher concentrations in the mycorrhizal root system. It is suggested that arginine synthesis in B. variegatus is repressed by the arginine available in the host. The mycorrhizal fungus possibly metabolizes the host arginine pool ultimately resulting in more efficient protein synthesis in both the partners.     

Degradation of Tau by Lysosomal Enzyme Cathepsin D: Implication for Alzheimer Neurofibrillary Degeneration

Abstract: The degradation of different isoforms of human recombinant tau (R-tau; T39, T40, and T44) and fetal tau (F-tau) by cathepsin D (CD) was investigated. Gel electrophoresis and Coomassie Blue staining of different R-tau species digested at pH 3.5 showed very little differences in CD susceptibility. Immunoblotting analyses revealed that amino and carboxy termini of tau were cleaved before other regions. F-tau was most vulnerable to proteolysis at both termini. Digestion of R-tau with 0.01 unit of CD/ml at pH 3.5 resulted in cleavage between Phe⁸-Glu⁹, Met⁴¹⁹-Val⁴²⁰, Thr⁴²⁷-Leu⁴²⁸-Ala⁴²⁹, and Leu⁴³⁶-Ala⁴³⁷ as determined by amino acid sequencing and mass spectroscopy (numbering of amino acids was based on T40). With higher concentrations of CD (1 unit/ml), additional sites of digestion were detected between amino acids 34–161, 200–257, and 267–358. The cleavage sites at amino acids 34–161 and 267–358 were observed at pH 3.5, whereas that at amino acids 200–257 was detected at pH 7.0. Our results suggest that CD cleavage of tau could generate tau fragments with intact microtubule binding domains, which could have a role in the pathogenesis of paired helical filaments (PHFs) in Alzheimer's disease. Such proteolysis might also contribute to the changes of PHF phenotype observed in intracellular and extracellular tangles.     

Nitrogen partitioning and blood meal utilization by Aedes aegypti (Diptera Culicidae)

The utilization of the blood meal by mosquitoes was investigated by first feeding females quantities of blood ranging from 1 to 5 mg, and then analyzing the faeces for the various by-products of protein catabolism that were subsequently eliminated. The nitrogeneous waste products in order of importance were uric acid, histidine, ammonia and arginine. Only traces of the other amino acids were excreted.The total amount of each faecal substance varied linearly with the quantity of blood ingested, however their relative proportions did not change. Regardless of blood meal size the quantily of uric acid and ammonia produced indicates that about 80% of the non-histidine and arginine amino acids are deaminated and utilized for metabolic purposes other than egg protein synthesis.Most of the histidine and about one half of the arginine content of the blood were excreted as free amino acids, but the other amino acids were lost in trace amounts.Nineteen per cent of the total ingested amino acids was incorporated into soluble yolk proteins and this proportion was constant even for small blood meals that result in a reduction in the numbers of eggs produced.The comparative aspects of nitrogen partitioning and blood meal utilization by haematophagous insects, as well as the factors that affect blood meal utilization and fecundity in A. aegypti are discussed.     

Biosynthesis of the cyanobacterial reserve polymer multi-L-arginyl-poly-L-aspartic acid (cyanophycin): mechanism of the cyanophycin synthetase reaction studied with synthetic primers.

Biosynthesis of the cyanobacterial nitrogen reserve cyanophycin (multi-L-arginyl-poly-L-aspartic acid) is catalysed by cyanophycin synthetase, an enzyme that consists of a single kind of polypeptide. Efficient synthesis of the polymer requires ATP, the constituent amino acids aspartic acid and arginine, and a primer like cyanophycin. Using synthetic peptide primers, the course of the biosynthetic reaction was studied. The following results were obtained: (a) sequence analysis suggests that cyanophycin synthetase has two ATP-binding sites and hence probably two active sites; (b) the enzyme catalyses the formation of cyanophycin-like polymers of 25-30 kDa apparent molecular mass in vitro; (c) primers are elongated at their C-terminus; (d) the constituent amino acids are incorporated stepwise, in the order aspartic acid followed by arginine, into the growing polymer. A mechanism for the cyanophycin synthetase reaction is proposed; (e) the specificity of the enzyme for its amino-acid substrates was also studied. Glutamic acid cannot replace aspartic acid as the acidic amino acid, whereas lysine can replace arginine but is incorporated into cyanophycin at a much lower rate.     

Nuclear magnetic resonance of protamines. A 1H-NMR study of the interaction of clupeine fractions with mononucleotides

The interaction of the three clupeine fractions, YI, YII, Z, and salmine fraction AI with mononucleotides has been examined by means of 1H nuclear magnetic resonance. The results obtained are interpreted in terms of electrostatic interactions between positive arginine guanidinyl groups and negative nucleotide phosphates. In addition, clupeine fraction YI and salmine fraction AI exhibit with guanine and adenine nucleotides a more specific interaction that leads to the formation of large aggregates in solution. The experimental data presented in this work demonstrate that the strength of interaction between clupeine YI and salmine AI with mononucleotides follows the order: 5'-dTMP approximately equal to 5'-dCMP much less than 5'-dAMP less than 5'-dGMP approximately equal to 5'-GMP.     

Solution structure of biopolymers: a new method of constructing a bead model

We propose a new, automated method of converting crystallographic data into a bead model used for the calculations of hydrodynamic properties of rigid macromolecules. Two types of molecules are considered: nucleic acids and small proteins. A bead model of short DNA fragments has been constructed in which each nucleotide is represented by two identical, partially overlapping spheres: one for the base and one for the sugar and phosphate group. The optimum radius sigma = 5.0 A was chosen on the basis of a comparison of the calculated translational diffusion coefficients (D(T)) and the rotational relaxation times (tau(R)) with the corresponding experimental data for B-DNA fragments of 8, 12, and 20 basepairs. This value was assumed for the calculation D(T) and tau(R) of tRNA(Phe). Better agreement with the experimental data was achieved for slightly larger sigma = 5.7 A. A similar procedure was applied to small proteins. Bead models were constructed such that each amino acid was represented by a single sphere or a pair of identical, partially overlapping spheres, depending on the amino acid's size. Experimental data of D(T) of small proteins were used to establish the optimum value of sigma = 4.5 A for amino acids. The lack of experimental data on tau(R) for proteins restricted the tests to the translational diffusion properties.     

Arginyl-tRNA synthetase from yeast. Discrimination between 20 amino acids in aminoacylation of tRNA(Arg)-C-C-A and tRNA(Arg)-C-C-A(3'NH2)

For discrimination between arginine and 19 other amino acids in aminoacylation of tRNA(Arg)-C-C-A by arginyl-tRNA synthetase from baker's yeast, discrimination factors (D) have been determined from kcat and Km values. The lowest values were found for Trp, Cys, Lys (D = 800-8500), showing that arginine is 800-8500 times more often incorporated into tRNA(Arg)-C-C-A than noncognate acids at the same amino acid concentrations. The other noncognate amino acids exhibit D values between 10,000 and 60,000. In aminoacylation of tRNA(Arg)-C-C-A(3'NH2) discrimination factors D1 are in the range 10-600. From these values and AMP formation stoichiometry, pretransfer proof-reading factors II1 were determined; from D values and AMP stoichiometry in aminoacylation of tRNA(Arg)-C-C-A, posttransfer proof-reading factors II2 could be calculated, II1 values between 2 and 120 show that pretransfer proof-reading is the main correction step, posttransfer proof-reading (II2 approximately 1-10) plays a marginal role. Initial discrimination factors due to different Gibbs free energies of binding between arginine and the noncognate amino acids were calculated from discrimination and proof-reading factors. According to a two-step binding process, two factors (I1 and I2) were determined. They can be related to hydrophobic interaction forces and hydrogen bonds that are especially formed by the arginine side chain. A hypothetical 'stopper' model of the amino acid recognition site is discussed.     

Selection of Lysine Plus Threonine-Resistant Mutant of Maize

Resistance to certain amino acids or amino acid analogs can lead to overproduce specific 'free amino acids. By selection-Mutagenic treatment-Selection, lysine plus threonine-resistant mutant (RLT) was obtained from tissue culture of maize, W77-R3019V The resistance of RLT was 20 times higher than that of wild type. The levels of all free aspartate family amino acids in RLT were higher than those in wild type. Especially, threonine was 20 times higher. The resistance was inheritable and segregation in progenies, RLT1 and F1, was approximate to 3:1 and 1:1 resistant/sensitive ratio, respectively. The resistance was inherited as a single dominant or semidominant nuclear gene. In RLT2 embryo cultures, the resistance and free threonine levels in resistant callus were 20 and 23 times higher than those in sensitive one, respectively. In the homozygous seeds of RLT2, the levels of free threonine, arginine, lysine, methionine and isoleucine were 11, 8, 5, 5 and 3 times higher than those of wild type.     

Increasing dispensable amino acids in diets of kittens fed essential amino acids at or below their requirement increases the requirement for arginine

Summary Kittens fed diets containing 0.75 × the NRC (1986) essential amino acid requirement (EAArq) and 210 to 560g crude protein(CP)/kg diet exhibited, with increasing CP: 1) decreasing weight gain, 2) decreasing plasma arginine concentrations, 3) increasing urinary orotic acid excretion, 4) increasing plasma glutamic acid concentrations, and 5) plasma isoleucine concentrations at levels that suggest a marginal isoleucine deficiency. Kittens fed a control diet (CD) containing 1.5 × EAArq and 350 g CP/kg diet had maximal weight gains and no orotic aciduria. It was concluded that the decreased weight gain and adverse metabolic effects were caused by arginine deficiency and possibly glutamic acid toxicity induced by high dietary dispensable amino acids. Kittens fed the diets containing 1.0 × EAArq and 350 and 560 g CP/kg diet had depressed plasma arginine and elevated glutamic acid concentrations and orotic aciduria. These results indicate that 10 g arg/kg diet is not adequate at CP concentrations above 280 g/kg and the calculated requirement of arginine is (0.02 g arginine/g CP) × (Y g CP/kg diet) + (4.0 g arginine/kg diet) where Y is the dietary CP level.Abbreviations CD control diet - CP crude protein (g CP/kg diet = g nitrogen/kg diet × 6.25) - DAA dispensable amino acids - EAA essential amino acids - EAArq essential amino acid requirement     

Free amino acids in the late embryogenesis and pre-hatching stage of two coregonid fishes

Developing eggs of vendace (Coregonus albula L.) and whitefish (C. lavaretus L.) were experimentally delayed in hatching by incubation at low water temperature (1–2°). Some eggs were taken during this period to a water temperature which was gradually raised up to 8° to provoke mass hatching of embryos. The pattern of free amino acids was followed in eggs incubated at both temperatures. During a 56 days period, the content of several essential amino acids significantly decreased in eggs of both species. For instance, the lysine content dropped from 703 to 270 mg/g dry matter and the arginine content from 257 to 13.3 mg/100 g dry matter in whitefish eggs. A similar pattern of decreasing level of free amino acids in embryonated ova up to hatching was characteristic for essential amino acids and serine. Methionine was exceptional; its level remained approximately the same. On the other hand, non-essential amino acids showed a significant increase in concentration during the experimental period. For instance, the glycine level increased 4.9 and 2.1 times in whitefish and vendace eggs, respectively. Transfer of eggs to 8° accelerated the decrease of nearly all free amino acids before hatching. The consequence of such amino acid metabolism for newly hatched larvae is discussed.     

Studies on Amino Acid Contents of Processed Soybean

The influence of heat treatment on defatted soybean flour was studied. The eighteen kinds of amino acids were determined by microbiological assay method.

The heat destruction of the amino acids was found to occur when defatted soybean flour was autoclaved. At 0 Kg/cm² (100°C) for one, two, and four hours, no remarkable destruction was observed on the amino acids. On the other hand, cystine, lysine and arginine were destroyed under following heat conditions at 0.35 Kg/cm² (108°C), 0.7 Kg/cm² (115°C)and 1.4 Kg/cm² (126°C). Especially, heating at 1.4Kg/cm² (126°C) for four hours, a large amount of cystine, lysine and arginine and a small amount of tryptophan and serine were destroyed, but all other amino acids were not destroyed by any heat treatment.

The different types of heat destruction of cystine, lysine and arginine were observed when defatted soybean flour was autoclaved under the systematically specified heating conditions.

The influences of the added water, the period of heating and the temperature on destruction of amino acids of defatted soybean flour were studied. The eighteen amino acids available to the microbiological assay using lactic acid bacteria were assayed with respect to those products treated under different heating conditions in the presence of water 3 and 6 times the weight of the soybean flour, and in the absence of water under the pressure of 0.7 Kg/cm² (115°C) and 1.4 Kg/cm² (126°C) for various heating periods.

Amino acids except for lysine, arginine, cystine, tryptophan and serine were not destroyed in any heat treatment examined. The destruction of lysine and arginine was mainly due to the conditions of the amount of the added water, and those of cystine, tryptophan and serine were chiefly due to the period and the temperature of heat treatment.     

Arginine kinase: a common feature for management of energy reserves in African and American flagellated trypanosomatids

This work reports the characterization of an arginine kinase in the unicellular parasitic flagellate Trypanosoma brucei, the etiological agent of human sleeping sickness and Nagana in livestock. The arginine kinase activity, detected in the soluble fraction obtained from procyclic forms, had a specific activity similar to that observed in Trypanosoma cruzi, about 0.2 micromol min(-1) mg(-1). Western blot analysis of T. brucei extracts revealed two bands of 40 and 45 kDa. The putative gene sequence of this enzyme had an open reading frame for a 356-amino acid polypeptide, one less than the equivalent enzyme of T. cruzi. The deduced amino acid sequence has an 82% identity with the arginine kinase of T. cruzi, and highest amino acid identities of both trypanosomatids sequences, about 70%, were with arginine kinases from the phylum Arthropoda. In addition, the amino acid sequence possesses the five arginine residues critical for interaction with ATP as well as two glutamic acids and one cysteine required for arginine binding. The finding in trypanosomatids of a new phosphagen biosynthetic pathway, which is not present in mammalian host tissues, suggests this enzyme as a possible target for chemotherapy.     

Arginine increases the solubility of alkyl gallates through interaction with the aromatic ring

We have recently proposed the application of solubilizing effects of arginine to poorly soluble aromatic compounds for drug discovery research. In this study, we compared the solubilizing effects of arginine with those of other amino acids, salts and a surfactant using alkyl gallates as model drug substances of low aqueous solubility. The solubilizing effects of arginine on the alkyl gallates were distinct compared with those of other amino acids and salts; the effects were even greater than those achieved using a strongly chaotropic guanidinium ion. Transfer free energy of the alkyl gallates from water to arginine solution depended weakly on their dissolution free energy in water, which is in contrast to sodium dodecyl sulphate that showed strong dependence. The present results suggest that arginine solubilizes alkyl gallates through interaction with the aromatic moiety and sodium dodecyl sulphate does so by interacting with alkyl groups.     

Cu(II)–protamine interaction. II. The formation and structure of Cu(II) complexes of clupeine YII and of peptides mimicking clupeine N-terminals

The interaction of Cu(II) with the protamine clupeine YII (containing proline at the N-terminal) and with four peptides (H-Ala-Arg-OMe, H-Ala-Arg₂-OMe, H-Pro-Arg-OMe, and H-Arg₄-Tyr) has been studied by means of absorption, CD, and pH neasurements. The first two peptides mimic clupeine YI and Z N-terminals; the third, the clupeine YII N-terminal. At 1:1 molar ratio, clupeine YII yields two complexes: the first (I), at pH 6.6, through coordination via the N-terminal and the contiguous peptide nitrogen forming a five-membered chelate; the second (II), at pH 8.5, through the occupancy of the other two corners of the coordination square by amino nitrogens of the lateral chains. These complexes are strictly analogous and occur at the same pH as those formed with clupeine Z. Under the same conditions, all the peptides yield complex I in the first step, although the pH at which this complex is fully defined depends on the number of residues in the chain. It is 8.5 for dipeptides, decreases to 6.5 by the addition of a third residue to the chain, and remains constant when the number of residues is three or more. The amino nitrogens of lateral chains are unable to coordinate to the metal in a second step unless one additional peptide bond lies between the N-terminal residue and that containing the lateral chain bound to the metal. Thus, H-Ala-Arg-OMe and H-Pro-Arg-OMe form hydroxyl complexes in a second step (pH 11), by deprotonation of one of the water molecules coordinated to the metal; one of the lateral chains of H-Ala-Arg₂-OMe is able to coordinate in a second step (pH 8.5), but it is only with H-Arg₄-Tyr that a second complex (II) is obtained in which two amino nitrogens of lateral chains supersede the oxygens of water molecules in I, at pH 8.5.     

Changes in the chemical composition of carbohydrates and proteins in surface water during a bloom ofMicrocystis in Lake Suwa

Carbohydrates and proteins in surface water during a bloom ofMictrocystis, which is the dominant summer phytoplankton in Lake Suwa, were analyzed in order to evaluate the function ofMicrocystis in organic matter metabolism. Glucose was the predominant sugar constituent of the cellular carbohydrate fraction and decreased in quantity from inside towards the outside of the cell through the slime layer. Other constituent sugars, on the other hand, were present in larger proportions in the lake water. Although the sugar composition of the cells did not change in July and August, during the first period of theMicrocystis bloom, it changed appreciably in September when the water temperature decreased below 20°C accompanied by the decrease in solar radiation and a marked change in nutrient concentration. It appears that the sugar composition of the cells may change in response to some environmental stresses. In addition, a temporal change in the sugar composition was found, particularly in the fraction containing the slime extracted by shaking. Among the constituent amino acids of the cells, the percentage of arginine, aspartic acid and leucine decreased from inside toward the outside of the cell, while glutamic acid, threonine, serine and glycine showed an opposite trend. In contrast to the carbohydrates, the percentage composition of each amino acid varied little throughout the period of the bloom.     

Important differences in cationic amino acid transport by lysosomal system c and system y+ of the human fibroblast

Superficial similarities led us to extend our designation for the transport of the plasma membrane for cationic amino acids, y+, to the lysosomal system also serving for such amino acids. Further study on the purified lysosomes of human skin fibroblasts leads us now to redesignate the lysosomal system as c (for cationic), rather than y+, to emphasize important contrasts. Lysosomal uptake of arginine at pH 7.0 was linear during the first 2 min, but attained a steady state in 6 min. This arginine uptake was Na+-independent and was tripled in rate when the lysosomes had first been loaded with the cationic amino acid analog, S-2-aminoethyl-L-cysteine. Uptake was slowed to one-third when 2 mM MgATP was added to the incubation mixture. The following differences in cationic amino acid influx between lysosomal System c and the plasma membrane System y+ became apparent: 1) arginine influx is increased 10-fold by raising the external pH from 5.0 to 7.0. This effect favors net entry of cationic amino acids under the H+ gradient prevailing in vivo. In contrast, arginine uptake across the plasma membrane is insensitive to pH changes in this range. 2) The Km of arginine uptake by lysosomal System c, 0.32 mM, is eight times that for System y+ arginine uptake by the fibroblast. 3) Certain neutral amino acids in the presence of Na+ are accepted as surrogate substrates by System y+, but not by lysosomal system c. 4) Cationic amino acids in which the alpha-amino group is monomethylated or the distal amino group is quaternary, also D-arginine, are recognized by lysosomal System c, whereas System y+ has little affinity for these analogs. This broader substrate specificity of lysosomal system c led us to discover that thiocholine serves to deplete accumulated cystine from cystinotic fibroblasts as effectively as does the therapeutic agent, cysteamine. The quaternary nitrogen of thiocholine renders the mixed disulfide formed when it reacts with cystine unsatisfactory as a substrate for System y+.     

A weighting method for predicting protein structural class from amino acid composition.

A protein is generally classified into one of the following four structural classes: all alpha, all beta, alpha+beta and alpha/beta. In this paper, based on the weighting to the 20 constituent amino acids, a new method is proposed for predicting the structural class of a protein according to its amino acid composition. The 20 weighting parameters, which reflect the different properties of the 20 constituent amino acids, have been obtained from a training set of proteins through the linear-programming approach. The rate of correct prediction for a training set of proteins by means of the new method was 100%, whereas the highest rate of previous methods was 82.8%. Furthermore, the results showed that the more numerous training proteins, the more effective the new method.