Separation of peptide transport and hydrolysis in trimethionine uptake by Saccharomyces cerevisiae.

Intact cells of Saccharomyces cerevisiae 139 hydrolyzed amino acid-p-nitroanilide by an activity similar to that of aminopeptidase II, as well-characterized external peptidase in yeast. In contrast, trimethionine, a model peptide used in transport assays, was not hydrolyzed by this aminopeptidase II-like activity, and the peptidase activity toward this substrate was localized in the soluble fraction of the yeast. We conclude that this tripeptide is taken up by S. cerevisiae intact and rapidly hydrolyzed inside the cell.     

Photoinactivation of the thiamin transport system in Saccharomyces cerevisiae with azidobenzoyl derivatives of thiamin

In an attempt to obtain a potent inhibitor for thiamin transport of Saccharomyces cerivisiae three novel thiamin derivatives having an arylazido substituent in the thiazole moiety have been synthesized. The derivatives prepared were 4-azidobenzoylthiamin (ABT), 4-azidobenzoylthiamin disulfide (ABTD), and 4-azido-2-nitrobenzoylthiamin disulfide (ANBTD). Among the newly prepared photoreactive azidobenzoyl derivatives of thiamin, ANBTD showed the strongest competitive inhibition with an apparent Ki of 7.9 nM against thiamin uptake by S. cerevisiae IFO-2375. The Ki values for ABT, 4-azido-2-nitrobenzoylthiamin (ANBT), and ABTD were 187 nM, 83 nM, and 15 nM, respectively. When exposed to visible light, ANBTD inactivated in a time- and concentration-dependent manner the uptake of [14C]thiamin by yeast protoplasts as well as intact cells. Remaining activities of the thiamin uptake by the intact cells were 71.9%, 27.3%, 40.1%, and 15.0% after visible light irradiation for 15 min in the presence of 1 microM ABT, ANBT, ABTD, and ANBTD, respectively. The inactivation by ANBTD (0.05 microM) was partially prevented by previous addition of an excessive amount of thiamin (5 microM). Furthermore, it was found that ANBTD (0.5 microM) irreversibly inactivated 70.6% of the thiamin-binding activity of the membrane fraction from S. cerevisiae IFO-2375. These results suggest that ANBTD can inhibit yeast thiamin transport by photoinactivation of membrane-bound thiamin-binding protein in the plasma membrane which may be a functional component involved in the thiamin transport system of S. cerevisiae.     

Possible site-specific reagent for the general amino acid transport system of Saccharomyces cerevisiae.

The general amino acid transport system of Saccharomyces cerevisiae functions in the uptake of neutral, basic, and acidic amino acids. The amino acid analogue N-delta-chloroacetyl-L-ornithine (NCAO) has been tested as potential site specific reagent for this system. L-Tryptophan, which is transported exclusively by the general transport system, was used as a substrate. In the presence of glucose as an energy source, NCAO inhibited tryptophan transport competitively (Ki = 80 micrometer) during short time intervals (1-2 min), but adding 100 micrometer NCAO to a yeast cell suspension resulted in a time-dependent activation of tryptophan transport during the first 15 min of treatment. Following the activation a time-dependent decay of tryptophan transport activity occurred. Approximately 80% inactivation of the system was observed after 90 min. When a yeast cell suspension was treated with NCAO in the absence of an energy source, an 80% inactivation of tryptophan transport occurred in 90 min. The inactivation was noncompetitive (Ki congruent to 60 micrometer) and could not be reversed by the removal of the NCAO. Addition of a five-fold excess of L-lysine during NCAO treatment or prevented inactivation of tryptophan transport. Under parallel conditions of incubation, other closely related transport systems were not inhibited by NCAO.     

Affinity labeling of a glutamyl peptide in the coenzyme binding site of NADP+-specific glutamate dehydrogenase of Salmonella typhimurium by 2-[(4-bromo-2,3-dioxobutyl)thio]-1,N6-ethenoadenosine 2',5'-bisphosphate

NADP+-specific glutamate dehydrogenase from Salmonella typhimurium, cloned and expressed in Escherichia coli, has been purified to homogeneity. The nucleotide sequence of S. typhimurium gdhA was determined and the amino acid sequence derived. The nucleotide analogue 2-[(4-bromo-2,3-dioxobutyl)thio]-1,N6-ethenoadenosine 2',5'-bisphosphate (2-BDB-T epsilon A-2',5'-DP) reacts irreversibly with the enzyme to yield a partially inactive enzyme. After about 60% loss of activity, no further inactivation is observed. The rate of inactivation exhibits a nonlinear dependence on 2-BDB-T epsilon A-2',5'-DP concentration with kmax = 0.160 min-1 and KI = 300 microM. Reaction of 200 microM 2-BDB-T epsilon A-2',5'-DP with glutamate dehydrogenase for 120 min results in the incorporation of 0.94 mol of reagent/mol of enzyme subunit. The coenzymes, NADPH and NADP+, completely protect the enzyme against inactivation by the reagent and decrease the reagent incorporation from 0.94 to 0.5 mol of reagent/mol enzyme subunit, while the substrate alpha-ketoglutarate offers only partial protection. These results indicate that 2-BDB-T epsilon A-2',5'-DP functions as an affinity label of the coenzyme binding site and that specific reaction occurs at only about 0.5 sites/enzyme subunit or 3 sites/hexamer. Glutamate dehydrogenase modified with 200 microM 2-BDB-T epsilon A-2',5'-DP in the absence and presence of coenzyme was reduced with NaB3H4, carboxymethylated, and digested with trypsin. Labeled peptides were purified by high performance liquid chromatography and characterized by gas phase sequencing. Two peptides modified by the reagent were isolated and identified as follows: Phe-Cys(CM)-Gln-Ala-Leu-Met-Thr-Glu-Leu-Tyr-Arg and Leu-Cys(CM)-Glu-Ile-Lys. These two peptides were located within the derived amino acid sequence as residues 146-156 and 282-286. In the presence of NADPH, which completely prevents inactivation, only peptide 146-156 was labeled. This result indicates that modification of the pentapeptide causes loss of activity. Glutamate 284 in this peptide is the probable reaction target and is located within the coenzyme binding site.     

N-arylazido-beta-alanyl-NAD+, a new NAD+ photoaffinity analogue. Synthesis and labeling of mitochondrial NADH dehydrogenase

N-Arylazido-beta-alanyl-NAD+ [N3'-O-(3-[N-(4-azido-2-nitrophenyl)amino]propionyl)NAD+] has been prepared by alkaline phosphatase treatment of arylazido-beta-alanyl-NADP+ [N3'-O-(3-[N-(4-azido-2-nitrophenyl)amino]propionyl)NADP+]. This NAD+ analogue was found to be a potent competitive inhibitor (Ki = 1.45 microM) with respect to NADH for the purified bovine heart mitochondrial NADH dehydrogenase (EC 1.6.99.3). The enzyme was irreversibly inhibited as well as covalently labeled by this analogue upon photoirradiation. A stoichiometry of 1.15 mol of N-arylazido-beta-alanyl-NAD+ bound/mol of enzyme, at 100% inactivation, was determined from incorporation studies using tritium-labeled analogue. Among the three subunits, 0.85 mol of the analogue was bound to the Mr = 51,000 subunit, and each of the two smaller subunits contained 0.15 mol of the analogue when the dehydrogenase was completely inhibited upon photolysis. Both the irreversible inactivation and the covalent incorporation could be prevented by the presence of NADH during photolysis. These results indicate that N-arylazido-beta-alanyl-NAD+ is an active-site-directed photoaffinity label for the mitochondrial NADH dehydrogenase, and are further evidence that the Mr = 51,000 subunit contains the NADH binding site. Previous studies using A-arylazido-beta-alanyl-NAD+ [A3'-O-(3-[N-(4-azido-2-nitrophenyl)amino]propionyl)NAD+] demonstrated that the NADH binding site is on the Mr = 51,000 subunit [Chen, S., & Guillory, R. J. (1981) J. Biol. Chem. 256, 8318-8323]. Results are also presented to show that N-arylazido-beta-alanyl-NAD+ binds the dehydrogenase in a more effective manner than A-arylazido-beta-alanyl-NAD+.     

Transport and metabolism of bacilysin and other peptides by suspensions of Staphylococcus aureus

L-Alanyl-L-tyrosine and glycyl-L-phenylalanine labelled with 14C competed with each other and with the dipeptide antibiotic bacilysin for transport into Staphylococcus aureus NCTC 6571 in a medium which did not support growth. They also competed with other dipeptides and several tripeptides. The fast initial transport ofthe two labelled peptides appeared to show Michaelis-Menten kinetics. Neither was transported into a bacilysin-resistant mutant of S. aureus NCTC 6571, although tyrosine was taken up by the mutant as readily as it was by the parent strain. Uptake of alanyltyrosine or glycylphenylalanine was followed by rapid hydrolysis of the peptide and the excretion of tyrosine or phenylalanine. Glycine liberated from glycylphenylalanine was partly degraded and partly incorporated into the bacterial wall. The behaviour of these dipeptides paralleled the inactivation of bacilysin by suspensions of S. aureus and the appearance of its C-terminal amino acid, anticapsin, in the extracellular fluid.     

Neutral amino acid transport at the human blood-brain barrier

The kinetics of human blood-brain barrier neutral amino acid transport sites are described using isolated human brain capillaries as an in vitro model of the human blood-brain barrier. Kinetic parameters of transport (Km, Vmax, and KD) were determined for eight large neutral amino acids. Km values ranged from 0.30 +/- 0.08 microM for phenylalanine to 8.8 +/- 4.6 microM for valine. The amino acid analogs N-methylaminoisobutyric acid and 2-aminobicyclo[2.2.1]heptane-2-carboxylic acid were used as model substrates of the alanine- and leucine-preferring transport systems, respectively. Phenylalanine is transported solely by the L-system (which is sensitive to 2-aminobicyclo[2.2.1]heptane-2-carboxylic acid), and leucine is transported equally by the L- and ASC-system (which is sodium-dependent and N-methylaminoisobutyric acid-independent). Dose-dependent inhibition of the high affinity transport system by p-chloromercuribenzenesulfonic acid is demonstrated for phenylalanine, similar to the known sensitivity of blood-brain barrier transport in vivo. The Km values for the human brain capillary in vitro correlate significantly (r = 0.83, p less than 0.01) with the Km values for the rat brain capillary in vivo. The results show that the affinity of human blood-brain barrier neutral amino acid transport is very high, i.e. very low Km compared to plasma amino acid concentrations. This provides a physical basis for the selective vulnerability of the human brain to derangements in amino acid availability caused by a selective hyperaminoacidemia, e.g. hyperphenylalaninemia.     

Peptidase activity in the hypothalamus of the rat: utilization of leucine-p-nitroanilide to monitor the activity degrading luteinizing hormone releasing hormone

Previous studies by other investigators have shown that luteinizing hormone releasing hormone (LHRH) and amino acid derivatives of p-nitroanilide are probably degraded by a common enzymatic activity; however, most of these studies are inferential in that they are largely based upon kinetic inhibition data derived from relatively crude tissue preparations. The purpose of this work was to determine whether the synthetic substrate leucine-p- nitroanilide (Leu-p-NA) and LHRH were degraded by the same peptidase activity. Supernatants (10,000 X g) from homogenates of rat hypothalami were eluted from Sephadex G-200, and the resultant fractions were assayed for degrading activity toward LHRH and Leu-p-NA. Radioimmunoassay (RIA) indicated that loss of immunologically active LHRH occurred in the same fractions in which maximal Leu-p-NA degrading activity eluted. Kinetically, exogenous LHRH inhibited degradation of Leu-p-NA in a concentration-dependent manner. When fractions evidencing Leu-p-NA degrading activity were incubated with 125I-LHRH, polyacrylamide gel electrophoresis (PAGE) indicated a time-dependent loss of LHRH with the concomitant production of a radioactive peptide fragment. High-performance liquid chromatography (HPLC) analysis of unlabeled LHRH incubations revealed, within the Leu-p-NA degrading fractions, the formation of two peptide fragments. These studies have further substantiated the likelihood that LHRH and Leu-p-NA are degraded by a common enzyme activity as indicated not only by kinetic inhibition data, but also by cofractionation of activity toward both substrates and by two analytical methods capable of detecting LHRH fragmentation (PAGE and HPLC).     

Peptide and amino acid transport in Streptococcus bovis

Summary In amino acid transport studies with Streptococcus bovis using ¹⁴C-labelled amino acids, it has been shown that between 87% and 95% of cell-associated radioactivity was located in the cytosol. In similar studies with unlabelled peptides, most test peptide associated with S. bovis was truly intracellular. Using sodium dodecyl sulphate-polyacrylamide gel electrophoresis, the proteolytic activity in S. bovis was found to be largely cell-associated and of the serine-protease type, but stimulated by dithiothreitol. A wide range of extracellular peptide hydrolysing activities was demonstrated against the pentapeptide Leu-Trp-Met-Arg-Phe, which was completely hydrolysed to eight products after 10 min incubation. Some of this pentapeptide was transported intact, indicating the existence of mechanisms for the transport of peptides up to 751 Da. In studies with Arg-Phe-Ala, only Phe (F) and Ala (A), and to a much lesser extent Phe-Ala (FA) were transported after extracellular hydrolysis to FA, Arg (R), F and A. In this case, amino acid transport was much more predominant than peptide transport. The extent and nature of peptide transport was affected by the addition of protease inhibitors. Offprint requests to: R. I. Mackie     

Dual system for potassium transport in Saccharomyces cerevisiae.

In a newly formulated growth medium lacking Na+ and NH4+, Saccharomyces cerevisiae grew maximally at 5 microM K+. Cells grown under these conditions transported K+ with an apparent Km of 24 microM, whereas cells grown in customary high-K+ medium had a significantly higher Km (2 mM K+). The two types of transport also differed in carbonyl cyanide-m-chlorophenyl hydrazone sensitivity, response to ATP depletion, and temperature dependence. The results can be accounted for either by two transport systems or by one system operating in two different ways.     

Transport of 2-methyl-4-amino-5-hydroxymethylpyrimidine in Saccharomyces cerevisiae

The transport of 2-methyl-4-amino-5-hydroxymethylpyrimidine (hydroxymethylpyrimidine) was studied in resting cells of Saccharomyces cerevisiae. Hydroxymethylpyrimidine uptake was an energy- and temperature-dependent process which has an optimal pH at 4.5. The apparent Km for hydroxymethylpyrimidine uptake was 0.37 microM, and the uptake was inhibited by 2-methyl-4-amino-5-aminomethylpyrimidine, thiamin and pyrithiamin. Furthermore, hydroxymethylpyrimidine uptake was inhibited by 4-azido-2-nitrobenzoylthiamin, a specific and irreversible inhibitor of the yeast thiamin transport system and it was greatly impaired in the thiamin transport mutant of S. cerevisiae. Thus, hydroxymethylpyrimidine is taken up by a common transport system with thiamin in S. cerevisiae, but in contrast to thiamin transport, accumulated hydroxymethylpyrimidine is released from yeast cells showing an overshoot phenomenon.     

Multiplicity and regulation of genes encoding peptide transporters in Saccharomyces cerevisiae

The model eukaryote Saccharomyces cerevisiae has two distinct peptide transport mechanisms, one for di-/tripeptides (the PTR system) and another for tetra-/pentapeptides (the OPT system). The PTR system consists of three genes, PTR1, PTR2 and PTR3. The transporter (Ptr2p), encoded by the gene PTR2, is a 12 transmembrane domain (TMD) integral membrane protein that translocates di-/tripeptides. Homologues to Ptr2p have been identified in virtually all organisms examined to date and comprise the PTR family of transport proteins. In S. cerevisiae, the expression of PTR2 is highly regulated at the cellular level by complex interactions of many genes, including PTR1, PTR3, CUP9 and SSY1. Oligopeptides, consisting of four to five amino acids, are transported by the 12-14 TMD integral membrane protein Opt1p. Unlike Ptr2p, distribution of this protein appears limited to fungi and plants, and there appears to be three paralogues in S. cerevisiae. This transporter has an affinity for enkephalin, an endogenous mammalian pentapeptide, as well as for glutathione. Although it is known that OPT1 is normally expressed only during sporulation, to date little is known about the genes and proteins involved in the regulation of OPT1 expression.     

Ammonia regulation of amino acid permeases in Saccharomyces cerevisiae.

The activities of the proline-specific permease (PUT4) and the general amino acid permease (GAP1) of Saccharomyces cerevisiae vary 70- to 140-fold in response to the nitrogen source of the growth medium. The PUT4 and GAP1 permease activities are regulated by control of synthesis and control of activity. These permeases are irreversibly inactivated by addition of ammonia or glutamine, lowering the activity to that found during steady-state growth on these nitrogen sources. Mutants altered in the regulation of the PUT4 permease (Per-) have been isolated. The mutations in these strains are pleiotropic and affect many other permeases, but have no direct effect on various cytoplasmic enzymes involved in nitrogen assimilation. In strains having one class of mutations (per1), ammonia inactivation of the PUT4 and GAP1 permeases did not occur, whereas glutamate and glutamine inactivation did. Thus, there appear to be two independent inactivation systems, one responding to ammonia and one responding to glutamate (or a metabolite of glutamate). The mutations were found to be nuclear and recessive. The inactivation systems are constitutive and do not require transport of the effector molecules per se, apparently operating on the inside of the cytoplasmic membrane. The ammonia inactivation was found not to require a functional glutamate dehydrogenase (NADP). These mutants were used to show that ammonia exerts control of arginase synthesis largely by inducer exclusion. This may be the primary mode of nitrogen regulation for most nitrogen-regulated enzymes of S. cerevisiae.     

Absence of derepression of amino acids transport in Candida

The transport of glycine, L-alanine, L-proline, L-leucine, L-lysine, L-phenylalanine and L-glutamic acid did not enhance in various strains of Candida cells, when they were grown in proline containing medium or preincubated with proline. However, under similar conditions, a significant enhancement in the level of accumulation of amino acids (derepression) was observed in Saccharomyces cerevisiae X-2180-A2 (GAP+) cells, which was sensitive to ammonium ions (NH4+). As expected, the derepression was absent in GAP- cells of S. cerevisiae X-2180 (GAP- mutant). In contrast to S. cerevisiae (GAP+) cells, the increase in few amino acids uptake in different Candida strains, grown in proline or preincubated in proline, could not be inhibited by cycloheximide, NH4+ or their D-stereoisomers. It appears that derepression of amino acids transport, a well known phenomenon in S. cerevisiae, may not exist in Candida species.     

Multiplicity of aspartate transport in thin wastewater biofilms

This research documents the multiplicity of L-aspartate transport in thin wastewater biofilms. A Line-weaver-Burk analysis of incorporation produced a curvilinear plot (concave down) that suggested active transport by two distinct systems (1 and 2). The inactivation of system 2 with AsO4 or osmotic shock resolved system 1, which was a high-affinity, low-capacity system with an apparent Kt (Michaelis-Menten constant) of 4.3 microM (AsO4) or 4.6 microM (osmotic shock). The inactivation of system 1 with dinitrophenol resolved system 2, which was a low-affinity, high-capacity system with an apparent Kt of 116.7 microM. System 1 was more specific for aspartate than system 2 in the presence of aspartate analogs. Sodium had no discernible effect on the incorporation velocities by either system. These results indicate that system 1 is a membrane-bound proton symport coupled to the proton gradient component of the proton motive force and that system 2 is a binding protein-mediated system coupled to phosphate bond energy. Analyses of diffusional limitations on the derived transport constants indicated that internal resistances were present but that the apparent constants were close to the intrinsic values, especially for system 1. Metabolic inactivation of the biofilm with dinitrophenol and AsO4 did not completely inactivate aspartate incorporation, which indicated that some simple adsorption of the aspartate anion by the biofilm had occurred. These results show that aspartate is transported by wastewater biofilm bacteria via systems with different affinities, specificities, and mechanisms of energy coupling.     

Multiplicity of aspartate transport in thin wastewater biofilms.

This research documents the multiplicity of L-aspartate transport in thin wastewater biofilms. A Line-weaver-Burk analysis of incorporation produced a curvilinear plot (concave down) that suggested active transport by two distinct systems (1 and 2). The inactivation of system 2 with AsO4 or osmotic shock resolved system 1, which was a high-affinity, low-capacity system with an apparent Kt (Michaelis-Menten constant) of 4.3 microM (AsO4) or 4.6 microM (osmotic shock). The inactivation of system 1 with dinitrophenol resolved system 2, which was a low-affinity, high-capacity system with an apparent Kt of 116.7 microM. System 1 was more specific for aspartate than system 2 in the presence of aspartate analogs. Sodium had no discernible effect on the incorporation velocities by either system. These results indicate that system 1 is a membrane-bound proton symport coupled to the proton gradient component of the proton motive force and that system 2 is a binding protein-mediated system coupled to phosphate bond energy. Analyses of diffusional limitations on the derived transport constants indicated that internal resistances were present but that the apparent constants were close to the intrinsic values, especially for system 1. Metabolic inactivation of the biofilm with dinitrophenol and AsO4 did not completely inactivate aspartate incorporation, which indicated that some simple adsorption of the aspartate anion by the biofilm had occurred. These results show that aspartate is transported by wastewater biofilm bacteria via systems with different affinities, specificities, and mechanisms of energy coupling.     

Differential regulation and substrate preferences in two peptide transporters of Saccharomyces cerevisiae

Dal5p has been shown previously to act as an allantoate/ureidosuccinate permease and to play a role in the utilization of certain dipeptides as a nitrogen source in Saccharomyces cerevisiae. Here, we provide direct evidence that dipeptides are transported by Dal5p, although the affinity of Dal5p for allantoate and ureidosuccinate is higher than that for dipeptides. Allantoate, ureidosuccinate, and to a lesser extent allantoin competed with dipeptide transport by reducing the toxicity of the peptide Ala-Eth and decreasing the accumulation of [(14)C]Gly-Leu. In contrast to the well-studied di/tripeptide transporter Ptr2p, whose substrate specificity is very broad, Dal5p preferred to transport non-N-end rule dipeptides. S. cerevisiae W303 was sensitive to the toxic peptide Ala-Eth (non-N-end rule peptide) but not Leu-Eth (N-end rule peptide). Non-N-end rule dipeptides showed better competition with the uptake of [(14)C]Gly-Leu than N-end rule dipeptides. Similar to the regulation of PTR2, DAL5 expression was influenced by the addition of Leu and by the CUP9 gene. However, DAL5 expression was downregulated in the presence of leucine and the absence of CUP9, whereas PTR2 was upregulated. Toxic dipeptide and uptake assays indicated that either Ptr2p or Dal5p was predominantly used for dipeptide transport in the common laboratory strains S288c and W303, respectively. These studies highlight the complementary activities of two dipeptide transport systems under different regulatory controls in common laboratory yeast strains, suggesting that dipeptide transport pathways evolved to respond to different environmental conditions.     

Multiplicity of oligopeptide transport systems in Escherichia coli.

The ability of Escherichia coli K-12 4212 to utilize a variety of oligopeptides as sources of required amino acids was examined. Triornithine-resistant mutants of this strain were oligopeptide permease deficient (Opp-) as judged by their inability to utilize (Lys)3 and (Lys)4 as sources of lysine and their resistance to the toxic tripeptide (Val)3. These same mutants were able to grow when Met-Met-Met, Met-Gly-Met, Met-Gly-Gly, Gly-Met-Gly, Gly-Gly-Met, Gly-Met-Met, Met-Met-Gly, or Leu-Leu-Leu were supplied in place of the requisite amino acid. The system mediating the uptake of these peptides, herein designated Opr I, was not able to transport N-alpha-acetylated peptides, nor the tetrapeptides Met-Gly-Met-Met, Met-Met-Gly-Met, or Met-Met-Met-Gly. Competition experiments indicated that trimethionine and trileucine enter E. coli K-12 via either Opp or Opr I. Analogous results were found using the methionine, leucine-requiring auxotroph E. coli B163. It appears that more than one oligopeptide transport system exists in E. coli and that the system mediating peptide uptake is complex.