Measurement of phenylalanine hydroxylase turnover in cultured hepatoma cells.

A substantially new method has been developed to measure protein turnover. Its basis is the notion that in labeling experiments a secreted protein can be used to determine the specific radioactivity of the intracellular amino acid precursor pool. To measure protein turnover in the Reuber hepatoma H4 cell line, cultures were labeled with [3H]leucine for specified periods after which phenylalanine hydroxylase was isolated and its leucine specific radioactivity determined. Serum albumin secreted by the cultures was also isolated and used to estimate the leucine precursor pool specific radioactivity. The protein half-life of phenylalanine hydroxylase could them be calculated. Experiments performed at long and short labeling times and with high and low concentrations of leucine in the medium yielded equivalent results. Phenylalanine hydroxylase half-life in the H4 cells was investigated under both normal and hydrocortisone-induced growth conditions. Average half-lives of 7.4 and 8.2 h were found for induced and uninduced cultures, respectively. Although these measured enzyme half-lives were not essentially different, the steady state level of phenylalanine hydroxylase was increased 6.2-fold upon hydrocortisone induction, from 0.076 to 0.47 microgram/10(6) cells. The results demonstrated that hydrocortisone induces phenylalanine hydroxylase in the H4 cells by causing an increase in the rate of enzyme synthesis.     

A possibility to recognize chirality by an excitable artificial liquid membrane

Studies were made on oscillations across a liquid membrane consisting of an oil layer, nitrobenzene containing picric acid, between two aqueous layers: a solution of 1.5 M ethanol and 5 mM optically-active cationic detergent, the D- or L-form of N-alpha-methylbenzyl-N,N-dimethylmyristylammonium bromide, on the left and 0.1 M D- or L-form of various ligands, such as glucose, arabinose, alanine, glutamic acid, threonine, leucine, proline, or phenylalanine on the right. This system showed sustained rhythmic oscillations of electrical potential of 200-300 mV with intervals of the order of 1 min. The frequency of oscillations depended on the combination of chiralities of the detergent and ligand. This means that the two forms (D and L) of chiral ligands can be distinguished by differences in the electrical response of the liquid membrane.     

一种适合14C-尿素呼气试验的环境友好型闪烁液的效果评价

目的评价环境友好型闪烁液应用于14C-尿素呼气试验诊断幽门螺杆菌感染的效果。方法149例患者接受14C-尿素呼气试验,均分别采用环境友好型和传统液体闪烁液进行检测,比较两者结果。结果应用环境友好型闪烁液进行14C-尿素呼气试验诊断幽门螺杆菌感染,与应用传统型闪烁液相比,两者差异无显著性,结果符合率为97.99%。结论环境友好型闪烁液对14C-尿素呼气试验结果无明显负面影响,可替代传统闪烁液。     

Human polymorphonuclear leukocytes aminopeptidases

In human polymorphonuclear leukocytes a methionine, leucine, arginine, phenylalanine and alanine aminopeptidase activities were detected, both in cytosol and secondary granules. All activities were EDTA sensitive and their pH optima were in the range of pH 6.5 to 8.6. In the cytosol two enzymes could be distinguished, broad substrate specificity aminopeptidase of pH 4.7-4.9 and a chloride dependent arginine aminopeptidase of pI 5.3-5.5. The granules contain aminopeptidase of pI 4.0-4.6 and of pI 9.8-10.2, different from those in the cytosol. Among them broad specificity aminopeptidases and possibly specific methionine and leucine aminopeptidases could be discerned.     

Identification of the LIV-I/LS system as the third phenylalanine transporter in Escherichia coli K-12

In Escherichia coli, the active transport of phenylalanine is considered to be performed by two different systems, AroP and PheP. However, a low level of accumulation of phenylalanine was observed in an aromatic amino acid transporter-deficient E. coli strain (DeltaaroP DeltapheP Deltamtr Deltatna DeltatyrP). The uptake of phenylalanine by this strain was significantly inhibited in the presence of branched-chain amino acids. Genetic analysis and transport studies revealed that the LIV-I/LS system, which is a branched-chain amino acid transporter consisting of two periplasmic binding proteins, the LIV-binding protein (LIV-I system) and LS-binding protein (LS system), and membrane components, LivHMGF, is involved in phenylalanine accumulation in E. coli cells. The K(m) values for phenylalanine in the LIV-I and LS systems were determined to be 19 and 30 micro M, respectively. Competitive inhibition of phenylalanine uptake by isoleucine, leucine, and valine was observed for the LIV-I system and, surprisingly, also for the LS system, which has been assumed to be leucine specific on the basis of the results of binding studies with the purified LS-binding protein. We found that the LS system is capable of transporting isoleucine and valine with affinity comparable to that for leucine and that the LIV-I system is able to transport tyrosine with affinity lower than that seen with other substrates. The physiological importance of the LIV-I/LS system for phenylalanine accumulation was revealed in the growth of phenylalanine-auxotrophic E. coli strains under various conditions.     

The role of leucine in ketogenesis in starved rats.

The quantitative significance of the conversion in vivo of L-[U-14C]leucine to ketone bodies was determined in rats starved for 3 or 48 h. In animals starved for 3 h, 4.4% of ketone-body carbon is derived from the metabolism of leucine, and in rats starved for 48 h the corresponding value is 2.3%. This conversion occurs rapidly, and the specific radioactivity of ketone bodies in blood is maximal at 2 min after the intravenous injection of labelled leucine for both periods of starvation. The flux of leucine in the blood is 1.01 and 1.04 mumol/min per 100 g body wt. respectively for animals starved for 3 and 48 h. The specific radioactivity of blood ketone bodies was compared at 2 min after the injection of labelled leucine, lysine and phenylalanine. The specific radioactivity was 4-5 fold higher with leucine than with lysine or phenylalanine.     

A high-performance liquid chromatography-based radiometric assay for acyl-CoA:alcohol transacylase from jojoba.

Acyl-CoA:alcohol transacylase catalyzes the final step in the biosynthesis of storage liquid wax esters from acyl-CoA fatty acids and fatty alcohols in a limited number of microbes, algae, and Simmondsia chinensis Link (jojoba). An improved and automated method of enzyme assay for this catalyst from cotyledons of jojoba is described. The assay method uses reversed-phase C18 high performance liquid chromatography (HPLC) to separate the labeled C30:1 liquid wax product, [14C]-dodecanyl-octadecenoate, from the unreacted substrate, [14C]octadecenoyl-CoA (oleyl-CoA), and other components produced from enzymes present in the crude homogenate of jojoba cotyledons, including [14C]-octadecenoic acid (oleic acid) and [14C]octadecenol (oleyol). Methods are also described for microscale chemical synthesis in one vessel of 14C-radiolabeled substrates and products for the transacylase. These labeled reagents are required to confirm the HPLC separations of reaction products. The radioactive components are quantitated using an on-line flow-through scintillation detector enabling sensitive and precise analysis of the reaction products.     

Enantiomeric separation of five amino acids by high-performance liquid chromatography on a chiral crown ether column

A chiral liquid chromatographic method was validated to analyze the D-and L-enantiomers of five amino acids contained in a commercial solution: aspartic acid, leucine, lysine, phenylalanine, and valine. These 10 compounds were separated on a chiral crown ether column with a mobile phase composed of water adjusted to pH 1.5 with perchloric acid, with ultraviolet detection at 220 nm. The method was applied to the commercial amino acid solution before and after sterilization by 5 kGy irradiation; no stereoconversion was observed following sterilization. Chirality 9:150–152, 1997. © 1997 Wiley-Liss, Inc.     

Source of amino acids for tRNA acylation in growing chicks

Summary Specific radioactivity in three amino acid compartments was examined in broiler chicks following a flooding dose of leucine or phenylalanine. In general, specific radioactivity of leucine and phenylalanine in deproteinated plasma (SA_e) and tissue (SA_i) compartments, exceeded that in acylated-tRNA (SA_t). In most tissues, SA_e and SA_i rapidly reached a similar peak level by 5 min followed by a slow decline for the next 30 minutes. Many tissues (eg. GI tract, liver, skin, and thigh) failed to maintain equilibrium between SA_e and SA_i over time. More metabolically active tissues, such as GI and liver had the greatest differences between these compartments. The difference between SA_e and SA_i for both leucine and phenylalanine were due to SA_i decreasing faster than SA_e, indicating dilution with unlabelled amino acids from proteolysis. Plasma and tissue specific radioactivity overestimated tRNA specific radioactivity by as much as 5 and 2.8 fold using leucine or 2.7 and 1.4 fold using phenylalanine, respectively. These data suggest that intracellular compartmentation of protein metabolism and the coupling of protein degradation and synthesis occur, in vivo.     

Regulation of RNA degradation in cultured rat hepatocytes: Effects of specific amino acids and insulin

The regulation of RNA degradation by specific amino acids and insulin was investigated in cultured rat hepatocytes from fed rats previously injected in vivo with [6-¹⁴C]orotic acid. The effects of three groups of amino acids were compared to those of a complete amino acid mixture. The first one consisted of the eight amino acids (leucine, proline, glutamine, histidine, phenylalanine, tyrosine, methionine, tryptophan) previously found to be particularly effective in the control of proteolysis. The two other groups were defined from our study with single additions of amino acids, one consisting of proline, asparagine, glutamine, alanine, phenylalanine, and leucine and the other including the latter group with serine, histidine, and tyrosine. The results showed that these three groups were able to strongly inhibit deprivation-induced RNA breakdown at one and ten times normal plasma concentrations but to a lower extent than the complete amino acid mixture. Six amino acids (proline, asparagine, glutamine, alanine, phenylalanine, leucine) inhibited individually RNA degradation by more than 20%. However, the deletions of proline, asparagine, glutamine, or alanine from the group of these six amino acids were not followed by a loss of inhibitory effect. On the contrary, an important loss of inhibition was observed when leucine and phenylalanine were deleted. Furthermore, only these two amino acids exhibited an additive inhibitory effect. Thus leucine and phenylalanine could be considered as important inhibitors of RNA breakdown in cultured rat hepatocytes. Finally, insulin which had no significant effect on RNA degradation in the absence of amino acids, was able to potentiate the inhibitory effect of different amino acid groups. © 1993 Wiley-Liss, Inc.     

Misreading of the argI message in Escherichia coli

It has previously been shown that either phenylalanine codon, UUU or UUC, could be misread as leucine during phenylalanine starvation, if the codons encoded residue 8 of the Escherichia coli argI gene product, ornithine transcarbamylase (OTC). However, no leucine misincorporation was detected when either of these same codons encoded residue 3. Here we report that leucine misincorporation can be directed by a UUU codon for residue 3 of OTC during phenylalanine starvation, if the argI gene has been mutated so that the codon preceding the UUU has been changed from the rarely used glycine codon GGG to the more commonly used GGC.     

THE EFFECTS OF PHENYLALANINE ON AMINO ACID METABOLISM AND PROTEIN SYNTHESIS IN BRAIN CELLS IN VITRO1

Incubation of brain cell suspensions with 14 mM-phenylalanine resulted in rapid alterations of amino acid metabolism and protein synthesis. Both thc rate of uptake and the final intracellular concentration of several radioactively-labelled amino acids were decreased by high concentrations oi phenylalanine. By prelabelling cells with radioactive amino acids, phenylalanine was also shown to effect a rapid loss of the labelled amino acids from brain cells. Amino acid analysis after the incubation of the cells with phenylalanine indicated that several amino acids were decreased in their intracellular concentrations with effects similar to those measured with radioisotopic experiments (large neutral > small and large basic > small neutral > acidic amino acids). Although amino acid uptake and efflux were altered by the presence of 14 mwphenylalanine, little or no alteration was detected in the resulting specific activity of the intracellular amino acids. High levels of phenylalanine did not significantly altcr cellular catabolism of either alanine, lysine, leucine or isoleucine. As determined by the isolation of labcllcd aminoacyl-tRNA from cells incubated with and without phenylalanine, there was little or no alteration in the level of this precursor for radioactive alanine and lysine. There was, however, a detectable decrease in thc labelling of aminoacyl-tRNA for leucine and isoleucine. Only aftcr correcting for the changes of the specific activity of the precursors and thcir availability to translational events, could the effects of phenylalanine on protein synthesis be established. An inhibition of the incorporation into protein for each amino acid was approximately 20%.     

Regulation of uptake of purines, pyrimidines and amino acids by Candida utilis

Uptake of uracil by Candida utilis is increased by addition of leucine to a minimal medium in which organisms are growing. This response requires protein synthesis and has kinetics consistent with the induction of additional uracil transport by the amino acid or a derivative. Consequently, the contribution of exogenous radioactive uracil to the pyrimidine nucleotide pools increases so that RNA made after the amino acid is added is of greater specific radioactivity. Some other amino acids are as effective as leucine in increasing the incorporation of uracil into RNA. Growth with leucine present also increases to different extents the initial rates of uptake of adenine, cytosine, uridine, lysine, histidine, threonine, phenylalanine, aspartic acid and leucine itself. The action of leucine on lysine transport appears to involve induction. These effects are not restricted to leucine; growth with aspartic acid or phenylalanine in the medium gives similar results. Lysine, on the other hand, is without action on the uptake of leucine, aspartic acid, phenylalanine, threonine or uracil but decreases the initial rates of uptake of both histidine and lysine. We suggest that lysine represses its own transport. Similarly, there is a specific decrease in uracil uptake caused by growth with this pyrimidine. Thus in C. utilis there are complex interrelationships in the uptake of nitrogen-containing compounds.     

Competition between leucine and phenylalanine and its relation to p-fluorophenylalanine resistant mutations in Aspergillus nidulans

Summary Auxanography and growth kinetics of a leucine and phenylalaninerequiring strain of Aspergillus nidulans reveals that (a) a phenylalanine-requiring strain is competitively inhibited by leucine but a leucine-requiring strain is not inhibited by phenylalanine, (b) the molar ratio of the two amino acids is critical for inhibition, and (c) leucine is specific for the possible replacement of phenylalanine.Failure to isolate a leucine-resistant phenylalanine-auxotroph suggests that the competition between these two amino acids does not take place at the coding level. By mitotic and meiotic analysis the mutant fpaB37 has been located on the left arm of linkage group I and has been found to be distinct and different from the locus trypB.Interactions between p-fluorophenylllanine-resistance and amino acid requirements and uptake experiments indicate that there are at least two sites for which leucine competes with phenylalanine-one of them being the site of entry of these essential amino acids into the mycelium. Both of these interaction sites are common for leucine, phenylalanine and p-fluorophenylalanine.     

A radiochemical-high-performance liquid chromatographic assay for urate oxidase in human plasma

Polyethylene glycol-modified urate oxidase (PEG-uricase) holds promise as a hypouricemic agent for treating gout and as an adjunct to cytolytic therapy of hematologic malignancies. Spectrophotometric assays of urate oxidase are not sensitive enough for pharmacokinetic evaluation of PEG-uricase in clinical trials. We have therefore developed a more sensitive radiochemical-HPLC assay for urate oxidase activity in untreated plasma, in which 14C in urate and in the reaction product, allantoin, is monitored in the uv detector effluent with a flow-through scintillation counter. The assay is linear with amount of enzyme and time of incubation and can detect less than 1 x 10(-5) U/ml uricase in plasma. The assay accounts for plasma samples of widely differing urate content.     

Enzymatic mass determination of high-specific-radioactivity 3H- and 14C-labeled polyunsaturated fatty acids

The soybean lipoxygenase reaction has been applied to calculating the specific activities of 3H- and 14C-labeled polyunsaturated fatty acids of the omega 3 and omega 6 families. The extent of the soybean lipoxygenase reaction with polyunsaturated fatty acids was determined by measuring the increase in absorbance at 234 nm. A salient feature of this application of of the soybean lipoxygenase assay involves the use of a solution which contains highly purified [1-14C]arachidonic acid of known specific radioactivity as a convenient and versatile standard. Because the amount of arachidonic acid in this standard can be easily measured by liquid scintillation counting, the problems of accurately weighing liquid fatty acid standards are avoided.     

Primary site and second site revertants of missense mutants of the evolutionarily invariant tryptophan 64 in iso-1-cytochrome c from yeast.

The three missense mutants cyc1-132, cyc1-166 and cyc1-189 in the yeast Saccharomyces cerevisiae contain nonfunctional and thermolabile iso-1-cytochromes c and have different replacements of the tryptophan at position 64 which corresponds to the invariant tryptophan residue found in cytochromes c from all eukaryotic species. The cyc1-166 and cyc1-189 mutants contain single replacements of, respectively, serine 64 and cysteine 64, while the cyc1-132 mutant contains a double replacement of glycine 64 and alanine 65 instead of the normal tryptophan 64 and aspartic acid 65. Twenty-three intragenic revertants having at least partially functional iso-1-cytochromes c arose from these three missense mutants by single amino acid replacements of either tryptophan, phenylalanine, tyrosine or leucine at position 64, or by second-site replacements in which the mutant residues at position 64 are retained and the normal serine 45 is replaced by phenylalanine 45. Specific activities of the iso-1-cytochromes c were estimated by growth of strains on lactate medium and are as follows, in terms of the normal, for iso-1-cytochromes c altered specifically in the ways shown: 100% for phenylalanine 64; 25% for tyrosine 64; between 0 and 25% for leucine 64; 100% for phenylalanine 45, cysteine 64; 25% for phenylalanine 45, serine 64; between 0 and 25% for phenylalanine 45, glycine 64, alanine 65; and 0% for serine 64, for cysteine 64, and for glycine 64, alanine 65 iso-1-cytochromes c. The results demonstrate that small residues of glycine, serine, and cysteine at position 64 are incompatible with function; they imply that many of the 10 amino acids accessible by single base-pair substitution but not observed in primary site revertants also are incompatible with function; and they show that large hydrophobic residues of phenylalanine, leucine, and tyrosine at position 64 are capable of restoring at least partial function. The second site revertants indicate that deleterious effects of the three missense mutants can be compensated by the introduction of phenylalanine 45, which may occupy space normally filled by tryptophan 64. Altered shapes of Calpha-band spectra and at least partial instability were characteristics of all iso-1-cytochromes c found lacking tryptophan 64. Apparently, the principal role of the invariant tryptophan is stabilization of the active protein structure, by providing a large hydrophobic group at the proper location.     

Transport of amino acids in Lactobacillus casei by proton-motive-force-dependent and non-proton-motive-force-dependent mechanisms.

Lactobacillus casei 393 cells which were energized with glucose (pH 6.0) took up glutamine, asparagine, glutamate, aspartate, leucine, and phenylalanine. Little or no uptake of several essential amino acids (valine, isoleucine, arginine, cysteine, tyrosine, and tryptophan) was observed. Inhibition studies indicated that there were at least five amino acid carriers, for glutamine, asparagine, glutamate/aspartate, phenylalanine, or branched-chain amino acids. Transport activities had pH optima between 5.5 and 6.0, but all amino acid carriers showed significant activity even at pH 4.0. Leucine and phenylalanine transport decreased markedly when the pH was increased to 7.5. Inhibitors which decreased proton motive force (delta p) nearly eliminated leucine and phenylalanine uptake, and studies with de-energized cells and membrane vesicles showed that an artificial electrical potential (delta psi) of at least -100 mV was needed for rapid uptake. An artificial delta p was unable to drive glutamine, asparagine, or glutamate uptake, and transport of these amino acids was sensitive to a decline in intracellular pH. When intracellular pH was greater than 7.7, glutamine, asparagine, or glutamate was transported rapidly even though the proton motive force had been abolished by inhibitors.     

A thermostable cocktail for the liquid scintillation counting of heterogeneous media

Considerable analytical errors arise in the liquid scintillation counting of heterogeneous media as a consequence of gel instability. With large sample numbers, a major causative factor of this instability is temperature changes during the counting period. An emulsifier-scintillation cocktail has been designed to provide stable counting conditions for heterogeneous media over a temperature range of 10–30°C, i.e., the wide range of temperature likely to be encountered in liquid scintillation counters lacking sample cooling facilities. A comparison was made with a conventional commercially available emulsifier-scintillator.     

Measurement of 131 I and 125 I by liquid scintillation counting

When aqueous samples are made miscible with a toluene scintillation solution by means of Bio-Solv BBS-3, high ¹²⁵I and ¹³¹I efficiencies can be achieved over a considerable range of “impurity” quenching, and adequate isotope separations can be achieved using liquid scintillation counters with “linear,” “pseudologarithmic,” or “logarithmic” amplification. Using an example of each sort of counter, we have graphically outlined the two somewhat different procedures for choosing the best instrument settings for single- and double-isotope counting. “Linear” instruments, despite a slightly more complex procedure for selection of settings, may offer the advantage, in double-isotope counting, of somewhat greater isotope separations because of the greater attenuation of photo-electron spectra.