Morphology and structure of γ-benzyl-l-glutamate copolymerized with l-phenylalanine, l-valine and l-alanine

Observation of random copolypeptides of γ-benzyl-l-glutamate with l-phenylalanine, l-valine and l-alanine was carried out in an electron microscope with samples cast from dilute solution. The relationship between the morphology and the molecular conformation in solution was studied with mixed solvents composed of chloroform and trifluoroacetic acid; these show a preference for α-helix and random coil, respectively. From the solutions in which molecules take α-helical conformation, fibrous films of nematic structure were formed. From random coil solutions discrete precipitates with folded molecules such as lamellar single crystals, piles of lamellae and structureless particles were formed. A copolypeptide containing l-valine in sufficiently large quantity to form β-structure also showed a variation in morphology with solvent, from films to discrete precipitates. It is suggested that the change in stiffness of the molecules contributes to the morphological variation.     

ATP binding to brain l-glutamate decarboxylase: A study by affinity chromatography

The binding of ATP to brain l-glutamate decarboxylase (GAD) was studied by means of ATP-agarose chromatography, utilizing partially purified GAD from mouse brain after DEAE-cellulose chromatography and ammonium sulfate fractional precipitation. GAD was found to bind with a high affinity to the ATP-agarose with the ATP molecule linked to the beaded agarose through the N⁶-amino group. Agarose with ATP attached through the ribosyl hydroxyls was totally ineffective to bind the enzyme. GAD bound to the immobilized ATP could be dissociated by free ATP (10–50 mM), but not by ADP at a concentration as high as 100 mM. Mg²⁺ was not a required factor for the binding. The enzyme binding to the ATP-agarose occurred under a saturating concentration (50 μM) of pyridoxal 5′-phosphate (PLP). Moreover, GAD bound to the ATP-agarose was not dissociated by PLP even at 1.0 mM, indicating no competition of PLP with ATP for the same binding site on the enzyme. Kinetic characterization showed that binding of ATP raised the K_m of the enzyme for PLP. Our approach provides direct evidence that there is a specific binding site on GAD for ATP, which is distinct from the binding site for PLP.     

Single crystals of a random copolypeptide composed of γ-benzyl l-glutamate and l-phenylalanine

Lamellar single crystals were formed from a random copolypeptide composed of γ-benzyl l-glutamate and l-phenylalanine at the ratio of 4 to 1. The copolypeptide takes the αhelical structure. The crystals were formed by casting dilute solutions at room temperature from a solvent consisting of a 1 to 1 mixture of chloroform and trifluoroacetic acid and were observed by electron microscopy. The average crystal thickness was 670 a in the as-polymerized sample, and 580 a in a fractionated sample. The thickness was decreased by annealing at temperatures above 110 C. A hexagonal form, a group of three orthorhombic forms (group 1), and a group of an orthorhombic form and two monoclinic forms (group II) were observed by electron diffraction. The diversity of the crystal structures is suggested to be caused by a variation in crystallization conditions during evaporation of the solvent. The hexagonal form and the structures of group I are changed into the structures of group II by annealing. The crystal structures other than the hexagonal form indicate on ordered arrangements of side chains in the crystals.     

Silk fibroin model,poly(l-alanylglycyl-l-alanylglycyl-l-alanylglycyl-l-seryglycine)

l-Alanylglycyl-l-alanylglycyl-l-alanylglycyl-l-serylglycine and its pentachlorophenyl ester methanesulphonate have been synthesized as monomers for the preparation of silk fibroin model polypeptide. The former octapeptide was polymerized with diphenylphosphorylazide (DPPA) and triethylamine in DMSO or in HMPA—pyridine, and the latter octapeptide pentachlorophenylester was polymerized by adding triethylamine in DMSO to give poly(l-alanylglycyl-l-alanylglycyl-l-alanylglycyl-l-serylglycine). This sequential polypeptide gave a similar i.r. pattern to the crystalline part of Bombyx mori silk fibroin, which indicated antiparallel β-conformation. Dialysis of the solution of this polymer in 60%, aqueous LiBr against water gave mainly the polymer of α-form. O.r.d. measurements suggest that this polypeptide exists as a random structure in dichloroacetic acid on in 60% aqueous LiBr.     

Cysteine desulfhydrase activity in cucurbit plants: Stimulation by preincubation with l- or d-cysteine

Cysteine desulfhydrase activity in leaf discs of cucurbit plants is enhanced 2–4-fold by preincubation with l or d-cysteine. Preincubation with structural analogs of cysteine also stimulated the activity of the enzyme, but to a smaller extent. Maximal increase in cysteine desulfhydrase activity was observed by preincubation with 5 mM or higher concentrations of cysteine. Although not caused by activation, stimulation of the enzyme activity was half-maximal within less than 15 min. Whereas the increase in cysteine desulfhydrase activity by preincubation of leaf discs with cysteine was light independent, pretreatment of the entire plant with light or dark determined the leaf discs' potential for stimulation of the enzyme. Exposure to darkness for 4 hr reduced this potential by 60%. It is concluded that the potential for stimulation of cysteine desulfhydrase activity by preincubation with cysteine is regulated by a compound not synthesized, but metabolized, in the leaf tissue. This regulatory compound may be supplied to the leaves by long-distance transport.     

Phenylalanine ammonia-lyase: Mirror-image packing of d- and l-phenylalanine and d- and l-transition state analogs into the active site

The binding of substrate and product analogs to phenylalanine ammonia-lyase (EC 4.3.1.5) from maize has been studied by a protection method. The ligand dissociation constants, K_L, were estimated from the variation with [L] of the pseudo-first-order rate constants for enzyme inactivation by nitromethane. The phenylalanine analogs d- and l-2-aminooxy-3-phenylpropionic acid showed K_L, values over 20,000-fold lower than the K_m for l-phenylalanine. From these and other K_L values it is deduced that when the enzyme binds l-phenylalanine the structural free energy stored in the protein is higher than when it binds the superinhibitors. Models for binding d- and l-phenylalanine and the superinhibitors are described. The enantiomeric pairs are considered to have similar K_L values because they pack into the active site in a mirror-image relationship. If the elimination reaction approximates to the least-motion course deduced on stereoelectronic grounds, the mirror-image packing of the superinhibitors into the active site mimics the conformation inferred for a transition state in the elimination. It appears, therefore, that structural changes take place in the enzyme as the transition state conformation is approached causing stored free energy to be released. This lowers the activation free energy for the elimination reaction and accounts for the strong binding by the above analogs.     

A new fluorescence assay for dipeptidyleptidase IV using tripeptide l-prolyl-l-prolyl-l-alanine as substrate

We have developed a new fluorescence assay for dipeptidylpeptidase IV using a tripeptide, l-prolyl-l-prolyl-l-alanine, which might be one of the potential natural substrates. The principle of the assay is based on the measurement of fluorescent adduct between alanine liberated from the tripeptide by enzymatic hydrolosis and o-phthaldialdehyde in the presence of 2-mercaptoethanol in aqueous alkaline medium. This new assay is sensitive enough to measure the enzyme activity in as little as 0.01 μl of human serum and in crevicular fluid obtained from human gingival sulcus. The K_m value for the tripeptide was 1.7 · 10^?5 M which is less than one-tenth of that obtained with other chromogenic or fluorogenic substrates. The interference by serum was overcome by simply incorporating the same amount of serum in the standards.     

Pyrrolizidine alkaloid biosynthesis: Derivation of retronecine from l-arginine and l-ornithine

The relative retention of ³H and ¹⁴C on incorporation of d-, l- and dl-isomers of [¹⁴C]arginine and [¹⁴C]ornithine into retrorsine using L-[5-³H]arginine as an internal standard has been measured. The retronecine portion of the pyrrolizidine alkaloid retrorsine, present in Senecio isatideus plants, is shown to be derived from l-arginine and l-ornithine.     

Development of an interference-free biosensor for l-glutamate using a bienzyme salicylate hydroxylase/l-glutamate dehydrogenase system

An amperometric biosensor was developed for the interference-free determination of l-glutamate with a bienzyme-based Clark electrode. This sensor is based on the specific dehydrogenation by l-glutamate dehydrogenase (GLDH, EC 1.4.1.3) in combination with salicylate hydroxylase (SHL, EC 1.14.13.1). The enzymes were entrapped by a poly(carbamoyl) sulfonate (PCS) hydrogel on a Teflon membrane. The principle of the determination scheme is as follows: the specific detecting enzyme, GLDH, catalyses the specific dehydrogenation of l-glutamate consuming NAD⁺. The product, NADH, initiates the irreversible decarboxylation and the hydroxylation of salicylate by SHL in the presence of oxygen. This results in a detectable signal due to the SHL-enzymatic consumptions of dissolved oxygen in the measurement of l-glutamate. The sensor has a fast steady-state measuring time of 20 s with a quick response (1 s) and a short recovery (1 min). It shows a linear detection range between 10 μM and 1.5 mM l-glutamate with a detection limit of 3.0 μM. A Teflon membrane, which is used to fabricate the sensor, makes the determination to avoid interferences from other amino acids and electroactive substances.     

Demonstration of aromatic l-amino acid decarboxylase activity in human brain with l-dopa and l-5-hydroxytryptophan as substrates by high-performance liquid chromatography with electrochemical detection

The enzymatic decarboxylations of l-DOPA and l-5-hydroxytryptophan (l-5-HTP) by aromatic l-amino acid decarboxylase (AADC) were measured with homogenates from human brain regions, caduate nucleus and hypothalamus, using our new and highly sensitive methods for l-DOPA decarboxylase and l-5-HTP decarboxylase by high-performance liquid chromatography with electrochemical detection (HPLC-ED). Dopamine formed from l-DOPA as substrate was measured for DOPA decarboxylase activity using d-DOPA for the blank. For 5-HTP decarboxylase activity, serotonin (5-HT) formed from l-5-HTP was measured, and the blank value in presence of NSD-1055 was subtracted. NSD-1055 inhibited 5-HTP decarboxylase activity completely at a concentration of 0.2 mM. In this study, the properties of l-5-HTP decarboxylase activity in human caudate nucleus were first examined. AADC activities in human brains were found to be widely variable for both l-DOPA and l-5-HTP as substrates. The ratio of the activities for l-DOPA and l-5-HTP were found to be significantly higher in hypothalamus than in caudate nucleus. AADC activity for l-DOPA in the brain was found to be linear up to 40 min of incubation, while that for l-5-HTP was found to be linear up to 240 min of incubation. The optimum pyridoxal phosphate concentration was found to be similar for both substrates and was between 0.01 and 0.1 mM. The optimum pH values were found to be 7.2 and 8.2 for l-DOPA decarboxylase and l-5-HTP decarboxylase, respectively. K_m and V_max values for a human caudate nucleus l-DOPA decarboxylase were found to be 414 μM and 482 pmol/min/g wet weight, respectively, while those for l-5-HTP decarboxylase were found to be 90 μM and 71 pmol/min/g wet weight, respectively.     

Enzymatic synthesis of l-cysteine by O-acetylserine sulfhydrylase of 3-chloro-l-alanine resistant Bacillus sphaericus L-118

The regulatory properties of serine-O-transacetylase and O-acetylserine sulfhydrylase have been investigated with 3-chloro-l-alanine resistant Bacillus sphaericus L-118. The enhancement of O-acetylserine sulfhydrylase formation by 3-chloro-l-alanine was observed and this effect was counteracted by corepressor l-cysteine. O-Acetylserine sulfhydrylase occurring in B. sphaericus L-118 can catalyse β-replacement reaction of 3-chloro-l-alanine in the presence of a high concentration of sodium hydrosulfide to form l-cysteine. The optimal reaction conditions for l-cysteine production were studied using resting cells. Under optimal conditions, about 80% of the added 3-chloro-l-alanine could be converted to l-cysteine. The highest yield achieved was 70 mg of l-cysteine per 1.0 ml of the reaction mixture.     

Energization of mitochondrial inner membranes caused by l-malate

It was found that 0.06 μg antimycin A/mg mitochondrial protein, an amount sufficient to inhibit electron transfer between cytochromes b and c₁ completely, fully reversed the oxidation of cytochrome a caused by L-malate in anaerobic mitochondria. The effect of L-malate on cytochrome a was insensitive to oligomycin, but all the uncouplers and detergents tested reversed the oxidation of cytochrome a caused by L-malate in anaerobic mitochondria. It was also found that addition of L-malate to anaerobic mitochondria, like addition of ATP, decreased the fluorescence of 1-anilinonaphthalene-8-sulphonate, and that subsequent addition of uncouplers reversed this effect. The effect of L-malate on the fluorescence of the dye was insensitive to oligomycin. The present findings suggest that addition of L-malate may cause energization of the mitochondrial inner membranes and that the oxidation of cytochrome a caused by L-malate in anaerobic mitochondria may result from an L-malate-induced, energy-linked reversal of electron transfer in site II.     

l-DOPA (l-3,4-dihydroxyphenylalanine) uptake by human red blood cells

The uptake of l-DOPA (l-3,4-dihydroxyphenylalanine) was studied in normal human red blood cells in vitro using l-[3-¹⁴C]DOPA. Uptake was slow, tending towards a distribution ratio close to unity with a half-time to equilibrium of one hour. Uptake was not Na⁺-dependent. Concentration dependence studies showed both saturable and non-saturable components of uptake, and inhibition studies using l-leucine and l-tryptophan suggest that the L and T systems of red cell amino acid uptake are involved. A powerful inhibitor of both systems, 3,4-dihydroxy-2-methylpropriophenone (U-0521), is described. It is concluded that uptake is by carrier-mediated facilitated diffusion via the L and T systems for which l-DOPA has low affinity.     

Design,synthesis, and evaluation of l-cystine diamides as l-cystine crystallization inhibitors for cystinuria

To overcome the chemical and metabolic stability issues of l-cystine dimethyl ester (CDME) and l-cystine methyl ester (CME), a series of l-cystine diamides with or without N^α-methylation was designed, synthesized, and evaluated for their inhibitory activity of l-cystine crystallization. l-Cystine diamides 2a–i without N^α-methylation were found to be potent inhibitors of l-cystine crystallization while N^α-methylation of l-cystine diamides resulted in derivatives 3b–i devoid of any inhibitory activity of l-cystine crystallization. Computational modeling indicates that N^α-methylation leads to significant decrease in binding of the l-cystine diamides to l-cystine crystal surface. Among the l-cystine diamides 2a–i, l-cystine bismorpholide (CDMOR, LH707, 2g) and l-cystine bis(N′-methylpiperazide) (CDNMP, LH708, 2h) are the most potent inhibitors of l-cystine crystallization.     

Solvolysis of charged active esters of carboxylic acids with cyclo (l- or d-Glu-l-His)

Cyclic dipeptide cyclo(l- or d-Glu-l-His) carrying an anionic site and a nucleophilic site has been synthesized and used as a catalyst for the solvolysis of cationic esters in aqueous alcohols. In the solvolysis of 3-acyloxy-N-trimethylanilinium iodide (S⁺_n, n = 2 and 10) and Cl^?H₃N⁺(CH₂)₁₁COOPh(NO₂), no efficient nucleophilic catalysis was observed. On the other hand, in the solvolysis of Gly-OPh(NO₂)·HCl, Val-OPh(NO₂)·HCl and Leu-OPh(NO₂)·HCl a very efficient general base-type catalysis by cyclo(l-Glu-l-His) was observed. In particular, with the latter two substrates the catalysis by cyclo(l-Glul-His) was more efficient than that by imidazole, although the catalysis was not enantiomer-selective. The diastereomeric cyclic dipeptide cyclo(d-Glu-l-His) was almost inactive under the same conditions. Confomation of cyclo(l- or d-Glu-l-His) in aqueous solution was investigated and the structure/catalysis relationship is discussed.     

Sister-chromatid exchange induced by X-ray of human lymphocytes and the effect of l-cysteine

A staining technique that detects sister-chromatid exchanges (SCEs) has been used to examine the response of human lymphocyte chromosomes to various dosages of X-irradiation. The SCE frequency was markedly increased following irradiation. However, the increase was of a significantly smaller magnitude when irradiation occurred in the presence of an antimutagenic agent. Scoring SCEs may provide a useful technique for assaying the mutagenic effects of environmental carcinogens as well as the protective effects of antimutagenic agents.     

Growth of Rhodopseudomonas capsulata on l- and d-malic acid

d-malate replaced l-malate in supporting both photosynthetic (anaerobic, light) and heterotrophic (aerobic, dark) growth of Rhodopseudomonas capsulata. Growth rates and cell yields were nearly equivalent with both enantiomorphs. Addition of glucose to malate culture media increased the growth rate and doubled the cell yield of heterotrophic cultures, but had little effect on photosynthetic cultures. Aerobically-grown cells showed a higher level of substrate-dependent oxygen uptake with l-malate than with d-malate. This preference for l-malate occured even in cells grown on d-malate. No malic racemase activity was detected in extracts of heterotrophically- or photosynthetically-grown cells.     

Free-calcium and tension responses in single barnacle muscle fibres following the application of l-glutamate

The effect of the putative transmitter, l-glutamate, on free intracellular Ca²⁺, tension and membrane potential in single muscle fibres from the barnacle Balanus nubilus has been investigated. External application of l-glutamate (0.1–10 mM) resulted in a transient increase in free intracellular Ca²⁺, monitored by the Ca²⁺-activated protein aequorin. This increase in free intracellular Ca²⁺ was associated with membrane depolarization and force development, and was followed by a period of ‘desensitization’ in which the preparation was unresponsive to l-glutamate. This could be reversed by removing l-glutamate from the external saline. External application of a number of closely related compounds, including d-glutamate and l-aspartate, were ineffective for initiating the transient light response. The l-glutamate response was virtually abolished in Na-free (Li) medium and completely abolished in Ca-free (Na) medium. The responses to l-glutamate were not reduced in Mg-free medium. The fibre's response to 1 mM l-glutamate was also inhibited by D-600 (10 μM) or by La³⁺ (1 mM), suggesting that Ca was directly involved in the underlying ionic conductance changes brought about by this putative excitatory transmitter.     

A new assay for l-asparagine synthetase

A fast, relatively inexpensive method of measuring the enzymatic formation of l-asparagine from l-aspartate is presented. This radiochemical assay requires simple manipulations making it ideal for working with large numbers of samples, while maintaining high sensitivity and reproducibility. A mechanism similar to the enzymatic β-decarboxylation of aspartate is utilized but in a nonenzymatic reaction. In the presence of pyridoxal and Al³⁺ ions, the ¹⁴C of l-[4-¹⁴C]aspartate is decarboxylatd while l-[4-¹⁴C]asparagine remains intact. This assay is shown to be suitable for measuring mammalian l-asparagine synthetase activity, while not requiring the isolation of assay enzymes.     

Structure of l-arabino-d-galactan-containing glycoproteins from radish leaves

Two l-arabino-d-galactan-containing glycoproteins having a potent inhibitory activity against eel anti-H agglutinin were isolated from the hot saline extracts of mature radish leaves and characterized to have a similar monosaccharide composition that consists of l-arabinose, d-galactose, l-fucose, 4-O-methyl-d-glucuronic acid, and d-glucuronic acid residues. The chemical structure features of the carbohydrate components were investigated by carboxyl group reduction, methylation, periodate oxidation, partial acid hydrolysis, and digestion with exo- and endo-glycosidases, which indicated a backbone chain of (1→3)-linked β-d-galactosyl residues, to which side chains consisting of α-(1→6)-linked d-galactosyl residues were attached. The α-l-arabinofuranosyl residues were attached as single nonreducing groups and as O-2- or O-3-linked residues to O-3 of the β-d-galactosyl residues of the side chains. Single α-l-fucopyranosyl end groups were linked to O-2 of the l-arabinofuranosyl residues, and the 4-O-methyl-β-d-glucopyranosyluronic acid end groups were linked to d-galactosyl residues. The O-α-l-fucopyranosyl-(1→2)-α-l-arabinofuranosyl end-groups were shown to be responsible for the serological, H-like activity of the l-arabino-d-galactan glycoproteins. Reductive alkaline degradation of the glycoconjugates showed that a large proportion of the polysaccharide chains is conjugated with the polypeptide backbone through a 3-O-d-galactosylserine linkage.