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1.
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. 相似文献
2.
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. Km and Vmax 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. 相似文献
3.
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. 相似文献
4.
Kenneth R. Hanson 《Archives of biochemistry and biophysics》1981,211(2):575-588
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, KL, 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 KL, values over 20,000-fold lower than the Km for l-phenylalanine. From these and other KL 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 KL 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. 相似文献
5.
6.
M. Recasens F. Saadoun V. Varga F.V. DeFeudis P. Mandel G. Lynch G. Vincendon 《Neurochemistry international》1983,5(1):89-94
Binding of l-[3H]cysteine sulfinic acid (CSA) and l-[3H]glutamate were compared in various subcellular fractions and in the presence of a variety of pharmacological and ionic manipulations in order to test the possibility that the two amino acids possessed separate binding sites.The specific l-[3H]cysteine sulfinate binding was found to be enriched maximally in medium and high density synaptic membranes, while the crude mitochondrial synaptosomal fraction displayed the highest l-[3H]glutamate binding. The ratio of l-[3H]cysteine sulfinate binding/l-[3H]glutamate binding was variable across brain regions. Several compounds differentially affected l-[3H]cysteine sulfinate and l-[3H]glutamate binding. l-cysteine sulfinate was the most potent displacer regardless of the binding considered. Finally, while cations produced qualitatively similar effects on the binding of the two amino acids, quantitative differences were evident.In sum, these data revealed the complexity of l-[3H]cysteine sulfinate and l-[3H]glutamate binding. They suggest the existence of several binding sites and that some of these are shared by both substances. However, the results also indicate that separate binding sites for the two amino acids exist in synaptic membrane, giving further support to the hypothesis that cysteine sulfinate serves a neurotransmitter role in the central nervous system. 相似文献
7.
The first chemical synthesis of l-2-oxalylamino-3-aminopropionic acid, an isomer of the Lathyrus sativus neurotoxin, is described. Studies on its biological properties are reported. Experiments with l-3-[14C-oxalyl]amino-2- aminopropionic acid show that the amount of 2-oxalylamino isomer detectable in seed extracts can be accounted for by rearrangement which occurs during isolation. 相似文献
8.
l-(+)-Glutamate oxidation that is non-pyridine nucleotide dependent is readily carried out by a membrane-bound enzyme in Azotobacter vinelandii strain O. Enzyme activity concentrates in a membranous fraction that is associated with the Azotobacter electron transport system. This l-glutamate oxidation is not dependent on externally added NAD+, NADP+, FAD, or FMN for activity. O2, phenazine methosulfate and ferricyanide all served as relatively good electron acceptors for this reaction; while cytochrome c and nitrotetrazolium blue function poorly in this capacity. Paper chromatographic analyses revealed that the 2,4-dinitrophenylhydrazine derivative formed from the enzymatic oxidation of l-glutamate was α-ketoglutarate, while microdiffusion studies indicated that ammonia was also a key end product. These findings suggest that the overall reaction is an oxidative deamination. Ammonia formation was found to be stoichiometric with the amount of oxygen consumed (2 : 1 respectively, on a molar basis). The oxidation of glutamate was limited to the l-(+)-enantiomer indicating that this reaction is not the generalized type carried out by the l-amino acid oxidase. This oxidoreductase is functionally related to the Azotobacter electron transport system: (a) the activity concentrates almost exclusively in the electron transport fraction; (b) the l-glutamate oxidase activity is markedly sensitive to electron transport inhibitors, i.e. 2-n-heptyl-4-hydroxyquinoline-N-oxide, cyanide, and 4,4,4-trifluoro-1-(2-thienyl)-1,3-butanedione; and (c) spectral studies on the Azotobacter R3 fraction revealed that a substantial amount of the flavoprotein (non-heme iron) and cytochrome (a2, a1, b1, c4 and c5) are reduced by the addition of l-glutamate. 相似文献
9.
Robin E. Mitchell 《Phytochemistry》1985,24(2):247-249
Addition of either l-[U-14C]threonine or l-[U-14C]isoleucine to 2.7-day-old shaking liquid cultures of Pseudomonas syringae pv. atropurpurea resulted in incorporation of radioactivity into coronatine, but not into N- coronafacoylvaline, another phytotoxin excreted by P.s. atropurpurea. In contrast, addition ofl-[U-14C]valine did not lead to incorporation of radioactivity into coronatine, but instead into coronafacoylvaline. Acid hydrolysis of the purified [14C] coronatine obtained after incorporation of either [14C]isoleucine or [14C]threonine demonstrated that > 94% of the radioactivity was present in the 1-amido-1-carboxy-2-ethylcyclopropyl moiety of coronatine, and < 6 % was in the coronafacoyl moiety. These findings are used to propose a biosynthetic pathway for coronatine. 相似文献
10.
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. 相似文献
11.
S.S. Kerwar Richard J.M. Marcel Richard A. Salvador 《Archives of biochemistry and biophysics》1976,172(2):685-688
Various proline analogs have been tested in vitro for their ability to inhibit the enzymatic aminoacylation of tRNA by proline. Of these, l-3,4-dehydroproline is the most potent inhibitor. This inhibition is competitive; the Ki is 100 μm. It was shown that l-3,4-dehydroproline can serve as substrate in the aminoacylation reaction. However, the incorporation of radioactivity from l-3,4-[14C]dehydroprolyl-tRNA into protein occurs at one-fifth the rate observed for l-prolyl-tRNA. The addition of l-3,4-dehydroproline in vitro inhibits the synthesis of collagen to a greater extent than non-collagen protein. 相似文献
12.
Craig A. Luehr Sheldon M. Schuster 《Journal of biochemical and biophysical methods》1980,3(3):151-161
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. 相似文献
13.
Hiroyuki Yamamoto Tadao Hayakawa 《International journal of biological macromolecules》1982,4(5):258-262
The conformations of random and sequential copolypeptides containing l-β-3,4-dihydroxyphenyl-α-alanine (DOPA) and l-glutamic acid (Glu) as well as their protected precursors have been investigated mainly by means of circular dichroism (c.d.) spectroscopy in chloroform or trimethylphosphate as solvent. Protected and deprotected DOPA-containing polypeptides showed a positive ellipticity hand at 285 nm due to the stacked side-chains. The c.d. behaviour depended on the amino acid sequence as well as the amino acid composition. In random copolypeptides, ellipticities versus DOPA content showed a smooth variation, without any sharp changes. This supported the conclusion that poly(DOPA) is a right-handed helix. Although the ellipticities were low compared with the values of a typical α-helix, deprotected DOPA-containing sequential polypeptides are helical from the results of the induced dichroic band at 285 nm and the infrared amide I and II absorption bands. The results obtained were compared with those of sequential polypeptides containing l-tyrosine and l-Glu. 相似文献
14.
Aminotransferases are pyridoxal phosphate-dependent enzymes whose potential for the biocatalytic production of enantiopure amino acids is increasingly recognized. Because of this, there is a growing interest in engineering them to alter their substrate specificity and to increase their catalytic activity. Here, we report the development of a high-throughput assay for screening α-ketoglutarate-dependent aminotransferase mutant libraries. To achieve this, we exploited the l-glutamate dehydrogenase coupled assay that has previously been shown to allow for aminotransferase activity to be monitored in vitro. We adapted this assay to allow screening of mutant libraries of either l- or d-amino acid specific aminotransferases in a continuous fashion. This assay requiring clarified cell lysates is reproducible, rapid, and sensitive because it allowed for the identification of a catalytically active mutant of Bacillus sp. YM-1 d-amino acid aminotransferase displaying a decrease in kcat/KM of more than two orders of magnitude. In addition, this assay allowed us to discover a mutant of Escherichia coli branched-chain amino acid aminotransferase, F36W, which is approximately 60-fold more specific toward the natural substrate l-leucine than l-phenylalanine as compared with wild type. This result demonstrates the potential of our assay for the discovery of mutant aminotransferases displaying altered substrate specificity, an important goal of enzyme engineering. 相似文献
15.
Jens Kvist Nielsen Ole Olsen Lars Haastrup Pedersen Hilmer Sørensen 《Phytochemistry》1984,23(8):1741-1743
The cotyledons, leaves and inflorescences of the radish, Raphanus sativus, have been examined for their content of hydroxylated cinnamic acid malate esters. 2-O-(p-coumaroyl)-l-malate, 2-O-caffeoyl-l-malate and 2-O-feruloyl-l-malate were found to be quantitatively predominating compounds in the fraction of carboxylic acids isolated from radish leaves and inflorescences. Identifications were based on PC, TLC, HVE, GC, HPLC, UV and NMR spectroscopy. The l- or (2S)-configuration of malic acid released by hydrolysis from the esters was determined by using l-malate dehydrogenase. A combined column chromatography technique applied prior to HVE, HPLC and GC was useful for separating the malate esters from most other low MW plant constituents. The significance of the present investigation is briefly discussed in relation to the metabolism of phenolic constituents of glucosinolate-containing plants, and the effects of these compounds in relation to insect feeding behaviour. 相似文献
16.
Yoshiko Tajima James M. Anderson P.H. Geil 《International journal of biological macromolecules》1980,2(4):186-192
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. 相似文献
17.
B. Nakajima K. Hirata N. Nishi J. Noguchi 《International journal of biological macromolecules》1981,3(1):46-52
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. 相似文献
18.
Heinz Rennenberg 《Phytochemistry》1983,22(7):1557-1560
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. 相似文献
19.
Per Mølstad 《生物化学与生物物理学报:生物膜》1980,597(1):166-173
The efflux of l-[3H]carnitine was studied in cells from an established cell line from human heart (Girardi human heart cells, CCL 27). The cells were loaded with 4 μmol/l l-[3H]carnitine for 1 or 24 h, and the efflux of radioactivity into the medium was measured. The amount of intracellular l-[3H]carnitine retained was expressed as a function of time. The results were fitted to an exponential equation, from which efflux rate constants were computed.Increasing the extracellular concentration of butyrobetaine, l-carnitine, d-carnitine, betaine, dl-norcarnitine or 3-dimethylamino-2-hydroxypropionic acid each increased the observed efflux. This is most likely due to accelerated exchange diffusion. The substrate specificity of this accelerated exchange diffusion is different from what previously has been found in competitive uptake studies of l-carnitine. l-Carnitine was preferentially released to l-acetylcarnitine, and blocking the sulfhydryl groups with 5,5-dithiobis(2-nitrobenzoic acid) increased the efflux. 相似文献
20.
Membrane orientation induced by an alternating electric field has been examined for the l-enantiomer and racemic dipalmitoylphosphatidylcholine (DPPC) bilayers. The orientation effect was measured by bending curvature of hairpin-like deformation of the multilamellar cylindrical tubes with varying field-strength, frequency and tube size. It has been observed that both l- and dl-DPPC tubes are similar in the profiles of field-strength dependence and frequency dependence on the curvature deformation, but different in the deformed curvatures. dl-DPPC tubes deform largely as compared with l-DPPC tubes. The square of the deformed curvature of dl-DPPC tubes is larger than that of l-DPPC by about 37% on average. The result indicates that the racemic membrane is responsive to the electric field as compared with the l-enantiomer membrane. This suggests that a hybrid arrangement of head groups of the racemic lipid leads an effective response of the membrane due to the head group orientation. 相似文献