E.s.r. and spin-trapping studies of the reactions of hydrated electrons with dipeptides.

The reactions of hydrated electrons (eaq-) with 55 dipeptides and 25 acetyl and formyl amino acids have been studied by e.s.r. and spin-trapping techniques. Gamma-radiolysis of deaerated aqueous solutions was used to generate eaq-, and sodium formate or t-BuOH was added to scavenge the OH radicals. t-Nitrosobutane was employed as the spin-trapping reagent. The radical,--CO---NH--, which is the initial product of the reactions of eaq- with dipeptides, was observed only for val-gly, val-ala, val-leu and ile-ala. For most of the dipeptides this radical converts to the primary deamination radical, CHR'-CONH-CHR-COO-, where R and R' are the side-chains of the common amino acids. In many cases a radical of the type CHR-COO-, formed by secondary deamination, was also observed. Only secondary deamination reactions were observed for dipeptides containing beta-alanine as the amino terminal residue and for acetyl and formyl amino acids. The secondary deamination reactions of eaq- with dipeptides, acetyl and formyl amino acids in aqueous solutions have not been observed previously. This type of reaction is of interest since it brings about main-chain scission in polypeptides and proteins.     

E.S.R. of spin-trapped radicals in gamma-irradiated polycrystalline amino acids. Chromatographic separation of radicals

The free radicals produced by gamma-radiolysis of polycrystalline amino acids (L-valine, L-leucine, L-isoleucine and L-proline) at room temperature in the absence of air were investigated by spin trapping with 2-methyl-2-nitrosopropane (MNP). The spin adducts produced by dissolving the irradiated solids in aqueous MNP solutions were separated by high-performance liquid chromatography and then identified by e.s.r. Deamination (ring-opening reaction for L-proline) was observed for all amino acid. For L-valine and L-leucine, H-abstraction from the tertiary carbon in the side chains occurred. For isoleucine, H-abstractions from the alpha-carbon of the amino acid and from a non-terminal carbon in the side chain were found.     

E.s.r. of spin-trapped radicals in gamma-irradiated polycrystalline amino acids, N-acetyl amino acids and dipeptides

The radicals produced in several polycrystalline amino acids, N-acetyl amino acids and dipeptides by gamma-radiolysis at room temperature were investigated by spin-trapping. After irradiation in the solid state, the samples were dissolved in aqueous solutions f t-nitrosobutane and the trapped radicals identified by e.s.r. For alpha-amino acids, deamination radicals were found, and in some cases H-abstraction radicals were also observed. No decarboxylation radicals could be detected. For N-acetyl amino acids, except for N-acetylglycine, the major radical was the decarboxylation radical. For N-acetyglycine the H-abstraction radical from the glycine residue was observed. For dipeptides of the x-glycine, the radical formed by removal of H from the alpha-carbon of the carboxyl-terminal residue was always spin-trapped. Some primary deamination radicals and minor amounts of decarboxylation radicals could also be observed. For dipeptides of the type x-alanine, glycine-x and alanine-x, the decarboxylation radical was always the major spin-trapped radical. Some primary and secondary deamination radicals were also detected.     

Inhibition of urease activity by hydroxamic acid derivatives of amino acids.

Hydroxamic acids have been reported to be potent and specific inhibitors of urease (EC 3.5.1.5) activity of plant and bacterial origin. The present investigation was performed on the inhibitory effect of hydroxamic acid derivatives of naturally occurring amino acids on the urease activity of the Jack Bean and the alimentary tracts of rats. Methionine-hydroxamic acid was the most powerful inhibitor (I50=3.9 X 10(-6) M) among nineteen alpha-aminoacyl hydroxamic acids. Phenylalanine-, serine-, alanine-, glycine-, histidine-, threonine-, leucine-, and arginine-hydroxamic acids followed, in order of decreasing inhibitory power. The inhibition proceeded with time at a comparable rate to fatty acyl hydroxamic acid inhibition. The I50 values of alpha-aminoacyl hydroxamic acids were found to be almost equal to those of the corresponding fatty acyl hydroxamic acids. This fact shows that the alpha-amino group did not affect inhibitory power. However, aspartic-beta-, lysine-, and glutamic-gamma-hydroxamic acids, in descending order, were much less inhibitory, probably due to the presence of a carboxyl or omega-amino group. Furthermore, the pH optimum of the inhibition shifted to lower pH in the presence of a carboxyl group, and to a higher pH in e presence of an amino group. The results suggest that the dissociation of an acidic or a basic group reduces the inhibitory power of hydroxamic acid. Hydroxamic acid inhibits urease activity with strict specificity, excpet for aspartic-beta-hydroxamic acid, which inhibited asparaginase competitively. Hydroxamic acid derivatives of amino acids inhibited not only the urease activity of the Jack Bean, but also that of the caecum and ileum parts of the rat intestine.     

Adriamycin-liposome interactions. A magnetic resonance study of the differential effects of cardiolipin on drug-induced fusion and permeability

L-Glutamate and L-aspartate transport into osmotically active intestinal brush border membrane vesicles is specifically increased by Na+ gradient (extravesicular greater than intravesicular) which in addition energizes the transient accumulation (overshoot) of the two amino acids against their concentration gradients. The "overshoot" is observed at minimal external Na+ concentration of 100 mM for L-glutamate and 60 mM for L-aspartate; saturation with respect to [Na+] was observed at a concentration near 100 mM for both amino acids. Increasing amino acid concentration, saturation of the uptake rate was observed for L-glutamate and L-aspartate in the concentration range between 1 and 2 mM. Experiments showing mutual inhibition and transtimulation of the two amino acids indicate that the same Na+ -dependent transport system is shared by the two acidic amino acids. The imposition of diffusion potentials across the membrane vesicles artificially induced by addition of valinomycin in the presence of a K+ gradient supports the conclusion that the cotransport Na+/dicarboxylic amino acid in rat brush border membrane vesicles is electroneutral.     

Mutational analysis of the human immunodeficiency virus type 1 env gene product proteolytic cleavage site.

The structural requirements for proteolytic cleavage of the human immunodeficiency virus type 1 env gene product, gp160, to gp120 and gp41 have been assessed by specific mutagenesis of the sequence Lys Ala Lys Arg Arg Val Val Glu Arg Glu Lys Arg located between amino acids 500 and 511, i.e., at the putative C terminus of gp120. The basic amino acids underlined have been mutated, individually and in combination, to neutral amino acids, and the cleavability of the mutated env gene products was examined after expression in CV-1 cells. The results show that the replacement of Arg-511 (cleavage presumably occurs C terminal to this amino acid) with Ser completely abolishes recognition and cleavage by the cellular protease(s), i.e., the remaining basic amino acids in the vicinity do not serve as alternative substrates. However, Arg-508 and Lys-510 are important features of the recognition site since, when they are individually changed to neutral amino acids, cleavage is severely impaired. The basic amino acids 500, 502, and 504 are, individually, not important for cleavage, since their individual replacement by neutral amino acids does not impair cleavage. However, when all four basic amino acids 500, 502, 503, and 504 are changed to neutral amino acids, cleavage is almost completely abolished. This shows that the sequence Arg Glu Lys Arg at the cleavage site is alone not sufficient for cleavage but that a contribution of other amino acids is required, whether the other amino acids provide a basic character or a certain structure in the vicinity of the cleavage site. When noncleavable or poorly cleavable mutant env genes are expressed from the infectious plasmid pNL4-3 in CD4+ human lymphoblastoid cells, noninfectious virus, incapable of spread throughout the culture, is produced.     

Microcalorimetric determination of the kinetics of substrate utilisation by non-growing suspensions of Neisseria sicca

The metabolism of various substrates by non-growing suspensions of Neisseria sicca was investigated by a flow-microcalorimetric technique. Substrate utilisation showed Michaelis kinetics allowing determination of saturation constants (Km) and maximum specific rates of substrate utilisation (Vmax). Pyruvate, lactate, a number of tricarboxylic acid cycle intermediates, and amino acids (aspartate, glutamate and proline) were rapidly metabolised [Vmax 5-35 mumol (g dry wt cells)-1 min-1]; Km values were between 4 and 20 microM. Glucose, glycerol, acetate and the other amino acids investigated gave only a slight or no increase in power. The pattern of substrate utilisation is discussed in relation to the role of carbonic anhydrase in N. sicca.     

Transport of the aromatic amino acids into isolated rat liver cells. Properties of uptake by two distinct systems.

The transport of the aromatic amino acids into isolated rat liver cells was studied. There was a rapid and substantial binding of the aromatic amino acids, L-alanine and L-leucine to the plasma membrane. This has important consequences for the determination of rates of transport and intracellular concentrations of the amino acids. Inhibition studies with a variety of substrates of various transport systems gave results consistent with aromatic amino acid transport being catalysed by two systems: a 2-aminobicyclo(2,2,1)heptane-2-carboxylic acid (BCH)-insensitive aromatic D- and L-amino acid-specific system, and the L-type system (BCH-sensitive). The BCH-insensitive component of transport was Na+-independent and facilitated non-concentrative transport of the aromatic amino acids; it was unaffected by culture of liver cells for 24 h, by 48 h starvation, dexamethasone phosphate or glucagon. Kinetic properties of the BCH-inhibitable component were similar to those previously reported for the L2-system in liver cells. The BCH-insensitive component was a comparatively low-Km low-Vmax. transport system that we suggest is similar to the T-transport system previously seen only in human red blood cells. The results are discussed with reference to the importance of the T- and L-systems in the control of aromatic L-amino acid degradation in the liver.     

An e.s.r. and spin-trapping study of the reactions of the SO4- radical with protein and nucleic acid constituents.

Reactions of the SO4- radical, generated by U.V. photolysis of Na2S2O8, were studied in aqueous solutions of amino acids, dipeptides, nucleic acid bases, nucleosides and nucleotides. The transient free radicals so formed were spin-trapped by t-nitrosobutane and identified by e.s.r. spectroscopy. The amino acids primarily undergo oxidative decarboxylation. The pKs of the ammonium groups of the spin-trapped decarboxylated radicals of glycine and alanine in D2O were determined to be 8.3 +/- 0.2. An oxidation product, which is the precursor of the decarboxylated radical, is tentatively identified for alanine, valine and isoleucine. Radicals formed by hydrogen abstraction by SO-4 are identified for leucine, serine, phenylalanine and 4-hydroxyproline. In dipeptides, SO-4 produces decarboxylation of the amino acid located at the carboxylate terminal residue. For gly-ala and ala-ala, radicals generated by hydrogen abstraction from the carboxylate terminal residue alanine were also characterized. Radicals centered on the C(5) carbon were observed for uracil, cytosine and thymine. For nucleosides and nucleotides, radicals situated on the base and/or the sugar moiety were assigned.     

Genomic DNA sequence for human C-reactive protein

The gene for the prototype acute phase reactant, C-reactive protein, has been isolated from two lambda phage libraries containing inserted human DNA fragments using synthetic oligonucleotide probes. Nucleotide sequence analysis indicates that after coding for a signal peptide of 18 amino acids and the first two amino acids of the mature protein, there is an intron of 278 base pairs followed by the nucleotide sequence for the remaining 204 amino acids. The intron is unusual in that it contains on the positive strand a poly(A) stretch 16 nucleotides long and a poly(GT) region 30 nucleotides long which could adopt the Z-form of DNA. The nucleotide sequence reported here confirms the amino acid sequence of mature C-reactive protein as originally reported except that it codes for an additional 19 amino acids beginning at position 62. Thus DNA sequence analysis predicts that the mature protein consists of 206 amino acids rather than 187 as originally reported. The mRNA cap site is located 104 nucleotides from the start of the signal peptide and there is a 3' noncoding region 1.2 kilobase pairs in length. The gene has a typical promoter containing the sequences TATAAAT and CAAT 29 and 81 base pairs upstream, respectively, of the cap site.     

Unusual regulation of the uptake system for branched-chain amino acids in Corynebacterium glutamicum

The uptake of branched-chain amino acids in threonine-dehydratase deficient mutants of Corynebacterium glutamicum is dependent on the presence of relatively high (>1 mM) intracellular concentrations of isoleucine, valine or leucine. This indicates that the respective uptake-system is induced by its substrate, i.e. branched-chain amino acids, at the internal side. This unusual regulation presumably is the reason for the failure to obtain mutants deficient in isoleucine uptake by use of a selection scheme which starts from isoleucine auxotroph mutants. The physiological meaning of this regulation is discussed with respect to isoleucine efflux and the cyclic retention hypothesis.Abbreviations amp ampicillin - dw dry weight - Km kanamycin - kb kilobase(s) - NMG N-methyl-N-nitro-N-nitrosoguanidine - ®, resistant resistance     

44-amino-acid E5 transforming protein of bovine papillomavirus requires a hydrophobic core and specific carboxyl-terminal amino acids.

The 44-amino-acid E5 protein of bovine papillomavirus type 1 is the shortest known protein with transforming activity. To identify the specific amino acids required for in vitro focus formation in mouse C127 cells, we used oligonucleotide-directed saturation mutagenesis to construct an extensive collection of mutants with missense mutations in the E5 gene. Characterization of mutants with amino acid substitutions in the hydrophobic middle third of the E5 protein indicated that efficient transformation requires a stretch of hydrophobic amino acids but not a specific amino acid sequence in this portion of the protein. Many amino acids in the carboxyl-terminal third of the protein can also undergo substitution without impairment of focus-forming activity, but the amino acids at seven positions, including two cysteine residues that mediate dimer formation, appear essential for efficient transforming activity. These essential amino acids are the most well conserved among related fibropapillomaviruses. The small size of the E5 protein, its lack of similarity to other transforming proteins, and its ability to tolerate many amino acid substitutions implies that it transforms cells via a novel mechanism.     

Control by amino acids of the activity of system A-mediated amino acid transport in isolated rat hepatocytes.

The effect of amino acids, in concentrations corresponding to those found in the portal vein of rats given a high-protein diet, was investigated on the activity of system A amino acid transport in hepatocytes from fed rats. Amino acids counteracted the induction of system A by insulin or glucagon. This effect was observed at all concentrations of hormones tested, up to 1 microM. Amino acids did not affect the basal cyclic AMP concentration in hepatocytes, or the large rise in cyclic AMP elicited by glucagon. The reversal of system-A induction was observed at relatively low concentration of amino acids, corresponding to plasma values reported in rats given a basal diet. Amino acids were separately tested: substrates of system A were particularly efficient, but so were glutamine and histidine. Non-metabolizable substrates of system A, such as 2-aminoisobutyrate, were also inhibitory, suggesting that a part of the effect of amino acids is independent of their cellular metabolism. Provision of additional energy substrates such as lactate and oleate did not affect induction of system A or the inhibitory effects of amino acids. Thus amino acids do not act by serving as an energy source and by maintaining the integrity of hepatocytes. Inhibition of mRNA synthesis by actinomycin practically abolished the effect of amino acids on the induction of system A by glucagon. The results suggest that amino acids may promote the synthesis of protein(s) affecting the activity of system A either directly at the carrier unit or at an intermediate stage of its emergence.     

The mechanism of action of dipeptidyl aminopeptidase. Inhibition by amino acid derivatives and amines; activation by aromatic compounds.

A variety of amino acid and peptide amides have been shown to be inhibitors of dipeptidyl aminopeptidase. Among these compounds derivatives of strongly hydrophobic amino acids are the strongest inhibitors (Phe-NH2, Ki = 1.0 +/- 0.2 mM), while amides of basic amino acids were somewhat less effective (Lys-NH2, Ki = 36 +/- 3 mM). Short chain amino acid amides are notably weaker inhibitors (Gly-NH2, Ki = 293 +/- 50 mM). The interaction of the side chains of compounds with the enzyme appears to be at a site other than that at which the side chain of the amino-penultimate residue of the substrate interacts since the specificity of binding is different. Primary amines have been shown to inhibit, e.g., butylamine, Ki = 340 +/- 40 mM, and aromatic compounds have been shown to stimulate activity toward Gly-Gly-NH2 and Gly-Gly-OEt (phenol, 35% stimulation of activity at a 1:1 molar ratio with the substrate). The data suggest that inhibition involves binding at the site occupied by the free alpha-amino group and the N-terminal amino acid.     

Postnatal amino acid uptake by the rat small intestine. Energetics of membrane transport systems for amino acids in the developing jejunum

The energetics of amino acid uptake by the developing small intestine was investigated in vitro. L-valine, L-leucine, L-phenylalanine, L-methionine, L-lysine and L-arginine were all actively transported by the newborn rat jejunum. Metabolic inhibitors (e.g. 2,4-dinitrophenol) significantly reduced uptake of all amino acids but uptake against a concentration gradient was not totally abolished. Uptake of all amino acids was reduced at low[Na+]. Inhibition of transport of neutral amino acids by reduced luminal [Na+] was greater than that of basic amino acids, and the tissue was barely able to concentrate the neutral amino acids. [Na+] affected the Michaelis constant (Km) of neutral transport systems for their substrates; for the basic amino acids Km values were unaffected by the presence or absence of Na+. Ouabain significantly inhibited neutral amino acid uptake but had no effect on L-lysine or L-arginine uptake. These results are discussed in terms of the Na+ gradient hypothesis for amino acid transport, and the site of energy input to active transport. The role of glycolysis in providing energy for intestinal transport in the neonatal rat and the efficiency of Na+ dependent and independent transport mechanisms are considered. It is concluded that the energetics of amino acid transport systems in neonatal and adult rats are essentially similar.     

Primary structure of the alanine carrier protein of thermophilic bacterium PS3.

Purified alanine carrier proteins were cleaved into peptides either chemically after solubilization in 1,1,1,3,3,3-hexafluoro-2-propanol or proteolytically with lysylendopeptidase. From the amino acid sequence analyses of these peptides, we synthesized a DNA probe and utilized it for successful cloning of a gene encoding the alanine carrier protein (acp gene). The 5'-flanking region was determined by an inverse polymerase chain reaction, and an open reading frame consisting of 1,335 nucleotides was found. The amino acid sequence deduced from the open reading frame consists of 445 amino acids, and all the partial amino acid sequences determined are included in the sequence. Although the calculated M(r) of 47,803 is significantly larger than the apparent M(r) of 42,500 as reported previously (Hirata, H., Kambe, T., and Kagawa, Y. (1984) J. Biol. Chem. 259, 10653-10656), an in vitro translation experiment revealed that the product of the acp gene migrates at a position coinciding with that of the purified alanine carrier. Hydropathy analysis suggests that the protein contains at least 8 hydrophobic segments presumably spanning membrane. A homology search on a database reveals relatively high scores of homology with either the Escherichia coli melibiose carrier or the human Na+/glucose symporter, particularly in the region from Leu246 to Glu286. Furthermore, the region also reveals low but significant similarities to other Na(+)-coupled symporters.     

Importance of the prime subsites of the C1s protease of the classical complement pathway for recognition of substrates

The classical complement pathway, which plays a vital role in preventing infection, is initiated by the action of the serine proteases C1r and C1s. We have examined the hydrolysis of substrates representing cleavage sequences in the physiological substrates for C1s, C2 and C4. These studies showed that the P(1)'-P(4)' substrate residues of C2 and C4 conferred greater affinity of substrate for enzyme and also induced a sigmoidal dependence of enzyme velocity on substrate concentration. This indicates that the substrate gave rise to homotropic positive cooperative behavior in the enzyme. When C1s was in complex with C1q and C1r, as would occur under physiological conditions, the same behavior was observed, indicating that this mechanism is relevant in the complement pathway in vivo. We further investigated the requirements of C1s for prime side amino acids by examining a substrate library in which each of the P(1)'-P(4)' positions had been substituted by different classes of amino acids. This revealed that the P(1)' position was a major determinant of the selectivity of the enzyme, while certain substitutions at the P(1)'-P(4)' positions abolished the allosteric behavior, indicating that contact residues at these positions in the C1s enzyme must mediate the cooperativity. The studies reported here highlight the importance of prime subsites in C1s for interaction with its cognate substrates in the complement pathway and therefore yield greater understanding of the mechanism of interaction between this vital protease and its physiological substrates.     

Distribution of mapping points of 20 amino acids in the tetrahedral space

Summary Based on the genetic codes and a simple theorem for the geometrical property of the regular tetrahedron, each amino acid is mapped onto a unique point in a 3-dimensional tetrahedral space. The distribution of the 20 mapping points for 20 amino acids is studied in detail. It is found that the mapping points for the hydrophobic and hydrophilic amino acids are distributed at distinct regions in the 3-dimensional space. A plane separating the two kinds of points satisfactorily based on the Fisher's algorithm has been calculated. It is shown that the codons coding for the hydrophobic amino acids are constituted dominantly by the bases of keto group, i.e., G and T. While the codons coding for the hydrophilic amino acids are constituted dominantly by the bases of amino group, i.e., A and C. The biological implication of the mapping points and the separating plane has been discussed in some details.     

TRANSPORT OF AMINO ACIDS BY THE SOIL ALGA STICHOCOCCUS BACILLARIS1

The green alga Stichococcus bacillaris Naeg. is able to take up at least eleven amino acids. All of these except glutamic and aspartic acids are transported by carrier systems that obey saturation kinetics. The acidic amino acids enter the cell by passive diffusion. Michaelis-Menten parameters (K_s and V_max) were calculated for several amino acids. All obey simple Michaelis-Menten behavior except for 2-methylalanine and leucine which may have double carrier systems of different affinities. Interactions between pairs of amino acids suggest that there is at least one carrier system specific for basic amino acids and probably several systems specific for neutral amino acids. Further analysis of neutral amino acid interactions reveal that the uptake of several amino acids is incompletely inhibited by competitor uptake at infinite concentration. The simplest interpretation of the data is the operation of three carrier systems for neutral amino acids, one of which has higher affinity and broader specificity than the other two. The amino acid carrier systems appear to operate by an active mechanism. The metabolic poison DCCD inhibits uptake up to 99%. The capacities of the neutral amino acid carrier systems are increased when cells are grown in medium containing suboptimal concentrations of nitrogen.