Synthetic Dipeptide Research Articles

The Smallest Vertebrate, Teleost Fish, Can Utilize Synthetic Dipeptide-Based Diets

In vitro studies of brush-border intestinal transport of dipeptides and cytoplasmic hydrolysis in fish suggest that these processes could be key mechanisms in the absorption and utilization of nutrients for growth. However, in vivo experimentation to study the nutritional importance of these processes was needed. We compared three dietary formulations based on free, peptide and protein sources of amino acids. Our results were the first to show that a synthetic dipeptide (PP)-based diet could support growth in the early stages of ontogenesis of a teleost fish, rainbow trout (Oncorhynchus mykiss), whereas a free amino acid (FAA)-based diet failed. We found that fish fed an FAA-based diet had an increased rate of ammonia excretion [1.78 +/- 0.19 mmol NH(3)-N/(kg body wt.h)], compared with fish fed a PP-based diet [1.25 +/- 0.07 mmol NH(3)-N/(kg body wt.h)], suggesting that deamination is involved in the metabolism of dietary FAA. Teleost fish are known to obtain a high proportion of total energy from protein, compared with higher vertebrates. However, we found that feeding trout alevins a PP-based diet increased postprandial oxygen consumption for 2 to 24 h, whereas other treatments decreased 24-h postprandial metabolism. This may indicate that peptide metabolism is less efficient than protein metabolism. Juvenile rainbow trout differed from alevins in their response to FAA- and PP-based diets. These observations strongly suggest that intestinal dietary peptide transport and hydrolysis could support protein synthesis and growth in vertebrates that respond poorly to FAA-based diets. We conclude that nutrient administration may be improved by manipulating dietary peptide composition and peptide/protein ratios, leading to better utilization of synthetic peptides, with nutritional and therapeutic implications for all vertebrates.

Host Recognition of Bacterial Muramyl Dipeptide Mediated through NOD2: IMPLICATIONS FOR CROHN′S DISEASE

NOD2, a protein associated with susceptibility to Crohn's disease, confers responsiveness to bacterial preparations of lipopolysaccharide and peptidoglycan, but the precise moiety recognized remains elusive. Biochemical and functional analyses identified muramyl dipeptide (MurNAc-L-Ala-D-isoGln) derived from peptidoglycan as the essential structure in bacteria recognized by NOD2. Replacement of L-Ala for D-Ala or D-isoGln for L-isoGln eliminated the ability of muramyl dipeptide to stimulate NOD2, indicating stereoselective recognition. Muramyl dipeptide was recognized by NOD2 but not by TLR2 or co-expression of TLR2 with TLR1 or TLR6. NOD2 mutants associated with susceptibility to Crohn's disease were deficient in their recognition of muramyl dipeptide. Notably, peripheral blood mononuclear cells from individuals homozygous for the major disease-associated L1007fsinsC NOD2 mutation responded to lipopolysaccharide but not to synthetic muramyl dipeptide. Thus, NOD2 mediates the host response to bacterial muropeptides derived from peptidoglycan, an activity that is important for protection against Crohn's disease. Because muramyl dipeptide is the essential structure of peptidoglycan required for adjuvant activity, these results also have implications for understanding adjuvant function and effective vaccine development.

The role of amino acids and synthetic dipeptides

reports. The likely requirements for intravenous amino acids were clearly spelt out and numerous commercial preparations (mostly for adults) became available in Europe. The composition of these preparations, however, was mainly based on oral requirement data in healthy man. The major nutrition efforts were directed to improve tolerance of the nitrogen load in the presence of illness rather than to provide specific nutrients for individual organs or tissues. At most instances, the efficacy of an amino acid therapy was monitored in blood (plasma). However, as shown over the past 20 years, changes in plasma-free amino acid concentrations may parallel those in intracellular compartment, but they frequently differ either quantitatively or qualitatively. The importance of this area was recognised early in the history of ESPEN when in the 1984 Arvid

CPG70 Is a Novel Basic Metallocarboxypeptidase with C-terminal Polycystic Kidney Disease Domains from Porphyromonas gingivalis

In a search for a basic carboxypeptidase that might work in concert with the major virulence factors, the Arg- and Lys-specific cysteine endoproteinases of Porphyromonas gingivalis, a novel 69.8-kDa metallocarboxypeptidase CPG70 was purified to apparent homogeneity from the culture fluid of P. gingivalis HG66. Carboxypeptidase activity was measured by matrix-assisted laser desorption ionization-mass spectrometry using peptide substrates derived from a tryptic digest of hemoglobin. CPG70 exhibited activity with peptides containing C-terminal Lys and Arg residues. The k(cat)/K(m) values for the hydrolysis of the synthetic dipeptides FA-Ala-Lys and FA-Ala-Arg by CPG70 were 99 and 56 mm(-1)s(-1), respectively. The enzyme activity was strongly inhibited by the Arg analog (2-guanidinoethylmercapto)succinic acid and 1,10-phenanthroline. High resolution inductively coupled plasma-mass spectrometry demonstrated that 1 mol of CPG70 was associated with 0.6 mol of zinc, 0.2 mol of nickel, and 0.2 mol of copper. A search of the P. gingivalis W83 genomic data base (TIGR) with the N-terminal amino acid sequence determined for CPG70 revealed that the enzyme is an N- and C-terminally truncated form of a predicted 91.5-kDa protein (PG0232). Analysis of the deduced amino acid sequence of the full-length protein revealed an N-terminal signal sequence followed by a pro-segment, a metallocarboxypeptidase catalytic domain, three tandem polycystic kidney disease domains, and an 88-residue C-terminal segment. The catalytic domain exhibited the highest sequence identity with the duck metallocarboxypeptidase D domain II. Insertional inactivation of the gene encoding CPG70 resulted in a P. gingivalis isogenic mutant that was avirulent in the murine lesion model under the conditions tested.

A novel binding assay for metabotropic glutamate receptors using [3H] L-quisqualic acid and recombinant receptors.

We established a methodology to analyze radioligand binding to the recombinant type la metabotropic glutamate receptor (mGluRla). A full-length cDNA encoding mGluR1a, which was isolated from a lambda gt 11 cDNA library of human cerebellar origin, was expressed in a baculovirus/Sf9 insect cell system. Membrane fractions with recombinant receptor expression were analyzed for the binding of [3H]L-quisqualic acid (L-QA), which is known to be a potent agonist of mGluRla. Efficient binding of the radioligand to the human receptor was observed in a saturable manner, giving an apparent Kd= 0.091 microM. [3H]L-QA bound to the human mGluR1a was displaced by known ligands such as L-QA, L-Glu, t-ACPD ((+/-)-1-aminocyclopentane-trans-1,3-dicarboxylic acid) with IC50s = 0.056, 0.97 and 4.0 microM, respectively. MCPG (alpha-methyl-4-carboxyphenylglycine) displaced the radioligand binding with lower potency. Using this binding protocol, we then evaluated the ligand ability of synthetic dipeptides. Among peptides tested, only Glu-containing dipeptides inhibited the radioligand binding, e.g. IC50 of L-Met-L-Glu was 4.3 microM. When phosphatidyl inositol turnover was assayed in mGluR1a-expressing CHO cells, L-Met-L-Glu was partially agonistic. We further expanded this [3H]L-QA binding protocol to type 5a mGluR, another member of group I mGluRs, as well as to AMPA receptor, a member of ionotropic glutamate receptors, since L-QA is also known to be a potent ligand for these receptors. Data shown here will provide a novel system not only to search for ligands for the glutamate receptors, but also to biochemically analyze the interaction modes between glutamate receptors and their ligands.

Stereo-isomer specific induction of renal cell apoptosis by synthetic muramyl dipeptide (N-acetylmuramyl-L-alanyl-D-isoglutamine).

The cytotoxicity of bacterial cell wall components, muramyl dipeptide (synthetic N-acetylmuramyl-L-alanyl-D-isoglutamine; L,D-MDP) and lipopolysaccharide (LPS), was investigated in several kidney cell lines. MDP and LPS were toxic to rabbit and monkey kidney cells, MDP was toxic to canine kidney cells, but not to human or porcine kidney cells. Notably, L,D-MDP was >100-fold more cytotoxic/microg than the D,D-MDP and L,L-MDP, as well as LPS. L,D-MDP and analogs containing L,D-MDP were the most widely cytotoxic of the MDP tested. The MDP-induced cytotoxicity was characterized as apoptosis by DAPI staining and DNA laddering. The acute rabbit kidney (RK13) cell apoptosis (cell death in < 5 h) induced by apical or basal application of MDP was associated with glutamate (Glu) release, decreased gamma-glutamyltranspeptidase (GGT) and acidosis and was suppressed by Indomethacin, Naproxen and Curcumin. The cytotoxic activity of L,D-MDP was decreased significantly by 24 h incubation in human sera. Aged (> 2 year-old) rabbits that apparently failed to quickly clear and excrete a uveitogenic dose of MDP within 24 h died in I week. The results indicate that minute amounts (5 ng/ml) of MDP containing L-alanyl-D-isoglutamine can induce renal cell apoptosis in vitro and support MDP-induced kidney cytotoxicity in rabbits. Also, the results indicate that MDP in sera can be detected utilizing the RK13 cell bioassay and that failure to rapidly clear and excrete L,D-MDP is associated with uveitis and death in aged rabbits.

New Developments in Glutamine Delivery

Numerous studies demonstrate that free glutamine can be added to commercially available crystalline amino acid-based preparations before their administration. Instability during heat sterilization and prolonged storage and limited solubility (35 g/L at 20 degrees C) hamper the use of free glutamine in the routine clinical setting. Indeed, there are many well-controlled and valuable trials with free glutamine, yet its use is restricted to clinical research. The obvious limitations of using free glutamine initiated an intensive search for alternative substrates. Synthetic glutamine dipeptides are stable under heat sterilization and highly soluble; these properties qualify the dipeptides as suitable constituents of nutritional preparations. Industrial production of these dipeptides at a reasonable price is an essential prerequisite for implications of dipeptide-containing solutions in clinical practice. Recent development of novel synthesis procedures allows increased capacity in industrial-scale production. Basic studies with synthetic glutamine-containing short-chain peptides provide convincing evidence that these new substrates are cleared rapidly from plasma after parenteral administration, without being accumulated in tissues and with negligible loss in urine. The presence of membrane-bound as well as tissue-free extracellular hydrolase activity facilitates a prompt and quantitative peptide hydrolysis, the liberated amino acids being available for protein synthesis and/or generation of energy. In the clinical setting, glutamine dipeptide nutrition beneficially influences outcome (nitrogen balance, immunity, gut integrity, hospital stay, morbidity and mortality). The provision of conditionally indispensable glutamine should be considered a necessary replacement of a deficiency rather than a supplementation. The beneficial effects observed with glutamine dipeptide nutrition should be seen simply as a correction of disadvantages produced by the inadequacy of conventional clinical nutrition. The availability of stable dipeptide preparations certainly facilitates, for the first time, adequate amino acid nutrition of critically ill, malnourished or stressed patients in the routine clinical setting and, thus, represents a new dimension in artificial nutrition.

Variations among Unbred Heifers in the Activities of Polymorphonuclear Leucocytes from the Mammary Gland and Blood

The phenotypic characteristics are described for the activity of polymorphonuclear leucocytes NMN) obtained by either lavage of the cavity system of juvenile mammary glands stimulated with a synthetic muramyl dipeptide analogue or isolation from the peripheral blood. Attention was paid to the variability of characteristics and its sources, and to correlations among them. The following characteristics were investigated in 27 clinically healthy, unbred Bohemian Red Pied x Holstein heifers: migration activity in situ, number of phagocytosing PMN, phagocytotic index, bactericidal activity of PMN and unstimulated and zymosan-stimulated luminol-dependent chemiluminescence. Considerable individual variation was found in the characteristics. Significant differences between blood PMN and PMN from lavages after influx induction were found for bactericidal activity (P < 0.05) and chemiluminescence (P < 0.01). A significant correlation between blood PMN and mammary gland PMN was found only for the number of phagocytosing cells (r = 0.329; P < 0.01). Highly significant positive correlations (P < 0.01) were demonstrated between the number of phagocytosing PMN [a], phagocytotic index [b], and bactericidal activity [c] in both blood PMN (r(ab) = 0.602; r(ac) = 0.565; r(bc) = 0.529) and mammary gland PMN (r(ab) = 0.730, r(ac) = 0.618, r(bc) = 0.589). No significant correlation was demonstrated for non-stimulated (NS), zymosan-stimulated (ZS), or opsonized zymosan-stimulated (OZS) chemiluminescence with any of the other characteristics of phagocytotic activity, in either blood PMN or mammary gland PMN (P > 0.05). The animal was a highly significant source of variability for all the phagocytotic activity characteristics (P < 0.01). Udder quarter was a non-significant source of variability for all the characteristics of phagocytotic activity except for NS chemiluminescence (P < 0.05) and ZS or OZS chemiluminescence (P < 0.01). However, udder quarter was a non-significant source of variability of chemiluminescence indices ZS/NS and OZS/NS (P > 0.05). It has been demonstrated that in situ migration activity, the number of phagocytosing PMN, phagocytotic index, bactericidal activity of PMN and chemiluminescence indices of PMN collected from juvenile mammary glands of unbred heifers after influx induction can be regarded as candidate early markers of resistance to mammary infections.

Effect of synthetic thymic and pineal peptides on biomarkers of ageing, survival and spontaneous tumour incidence in female CBA mice

Fifty female CBA mice were injected s.c. either with 0.1 ml saline, or with synthetic thymic dipeptide Lys–Glu or with synthetic pineal tetrapeptide Ala–Glu–Asp–Gly both in a single dose of 0.1 μg/animal monthly for five consecutive days from the age of 6 months until natural death. Lys–Glu did not significantly influence the body weight and food consumption, free radical processes and estrus function in mice and did increase their physical activity with the subsequent decrease in spontaneous lung adenomas incidence. The pineal peptide treatment was failed to modify the food consumption and physical strength of mice, and was followed by the increase in the body weight, mean survival (by 5.3%, P < 0.05) and maximum (by 10 months), by slow down of the ageing of estrus function, by the decrease in body temperature, physical activity, free radical processes and spontaneous tumor incidence (mainly, lung adenomas) in mice. These data suggest the geroprotector potential of the pineal peptide.

Induction of tumor necrosis factor-α in solid tumor region by the orally administered synthetic muramyl dipeptide analogue, romurtide

The novel applications of a muramyl dipeptide analogue, romurtide, were investigated. When mice were intravenously injected with 10–1000 μg/mouse of romurtide as a primer and 3 KE/mouse of OK-432 as a trigger, systemic tumor necrosis factor-α (TNF-α) activity was increased. Similar effects were also detected by oral administration of romurtide at 1000 to 10,000 μg/mouse. These effects reached maximum at 3 h (intravenous injection, i.v.) or 6 h (oral administration, p.o.) after administration and were sustained for 48 h. When romurtide was administered to MM46 tumor-bearing mice orally, the significant augmentation of TNF-α production could be detected in the solid tumor and the liver. When romurtide was intravenously injected to the tumor bearer, the TNF-α production was significantly augmented in the spleen. These findings suggest that romurtide can augment the host defense system by both the parenteral and oral route, and indicate the possibility of a new type of anti-tumor therapy to induce TNF-α in the tumor region.

The peptide (Z)-Pro–Leuol

The structure of the synthetic protected dipeptide (Z)-Pro–Leuol [systematic name: benzyl 2-(1-hydroxy­methyl-3-methyl­butyl­amino­carbonyl)­pyrrolidine-1-carboxyl­ate], C19H28N2O4, was determined by X-ray crystallography. The peptide adopts a novel backbone conformation compared with other longer oligopeptides containing Pro–Leuol.

Cost containment through L-alanyl-L-glutamine supplemented total parenteral nutrition after major abdominal surgery: a prospective randomized double-blind controlled study

Background & Aims: Glutamine is recognized as a conditionally essential amino acid. Recent studies indicate that glutamine-containing total parenteral nutrition improves nitrogen economy, enhances gastrointestinal and immune functions and shortens hospital stay.Methods: Thirty-seven patients (19 w and 18 m; age 61.4±10.4 years; BMI 23.7±2.8 kg/m2) following major abdominal surgery receiving an isonitrogenous isoenergetic TPN with or without alanyl-glutamine supplementation (0.5 g/kg BW/day), were evaluated in a double-blind, randomized, controlled trial over a five-day period by measuring nitrogen balance, selected biochemical parameters and length of hospital stay.Results: Supplemental alanyl-glutamine improved the overall mean (−3.5±1.6 vs. −5.5±1.4 g N;P<0.05) and cumulative nitrogen balance (−14.1±9.1 vs. −21.7±11.4 g N;P<0.05) compared with the isonitrogenous, isoenergetic standard regimen. Alanyl-glutamine normalized plasma glutamine concentration and reduced the length of hospital stay (12.8±2.6 vs. 17.5±6.4 days;P<0.05).Conclusions: The results of the study confirm that supplementation with synthetic alanyl-glutamine dipeptide is associated with cost containment due to shortened hospitalization and improved nitrogen economy.

Ribosomal protein L2 is involved in the association of the ribosomal subunits, tRNA binding to A and P sites and peptidyl transfer.

Ribosomal proteins L2, L3 and L4, together with the 23S RNA, are the main candidates for catalyzing peptide bond formation on the 50S subunit. That L2 is evolutionarily highly conserved led us to perform a thorough functional analysis with reconstituted 50S particles either lacking L2 or harboring a mutated L2. L2 does not play a dominant role in the assembly of the 50S subunit or in the fixation of the 3'-ends of the tRNAs at the peptidyl-transferase center. However, it is absolutely required for the association of 30S and 50S subunits and is strongly involved in tRNA binding to both A and P sites, possibly at the elbow region of the tRNAs. Furthermore, while the conserved histidyl residue 229 is extremely important for peptidyl-transferase activity, it is apparently not involved in other measured functions. None of the other mutagenized amino acids (H14, D83, S177, D228, H231) showed this strong and exclusive participation in peptide bond formation. These results are used to examine critically the proposed direct involvement of His229 in catalysis of peptide synthesis.

Cyclopeptide alkaloids from Paliurus ramossisimus.

Seven zizyphine A-type cyclopeptide alkaloids were isolated from the roots of Paliurus ramossisimus by the combination of centrifugal partition chromatography and conventional separation methods. The novel structures of paliurines A-F (1-6) were characterized and established on the basis of MS and elaborate NMR spectral analyses. Terminal dipeptide stereochemistry was confirmed by correlation with the synthetic dipeptides via comparison of their (13)C NMR data.

Intrapleural therapy with MDP-Lys (L18), a synthetic derivative of muramyl dipeptide, against malignant pleurisy associated with lung cancer

N 2-[( N-acetylmuramoyl)- l-alanyl- d-isoglutaminyl]- N 6-stearoyl- l-lysine (MDP-Lys (L18), romurtide) is a synthetic muramyl dipeptide derivative, and has immunomodulating activities including activation of cells of monocyte-macrophage lineage. We examined the effect of intrapleural instillation of MDP-Lys (L18) against malignant pleurisy associated with lung cancer. Six patients with cytologically-positive malignant pleural effusion (four with adenocarcinoma, one with small cell carcinoma and one with large cell carcinoma) were treated with single intrapleural instillation of MDP-Lys (L18) of 200 μg. Clinically, no reaccumulation of pleural effusion for at least 4 weeks was observed in four patients. No major side effects were observed. Total cell number elevated remarkably 4 h after instillation, and main increased population was that of neutrophils. Levels of chemotactic cytokines, such as interleukin (IL)-8, and monocyte chemotactic protein (MCP)-1 and levels of pro-inflammatory cytokines, such as tumor necrosis factor (TNF)-α, interleukin (IL)-1β and IL-6, elevated in pleural effusion, and peak IL-1β and IL-6 levels tended to be higher in clinical responders than non-responders. These results suggest MDP-Lys (L18) instilled by intrapleural route had a potential local immunomodulatory activity. Further study is warranted to further determine the critical factors which correlate with the clinical response.

Immunomodulatory synthetic dipeptide L-Glu-L-Trp slows down aging and inhibits spontaneous carcinogenesis in rats.

Immunomodulatory molecule L-Glu-L-Trp was isolated from natural calf thymic peptide complex Thymalin by reverse-phase high performance liquid chromatography. On the basis of the synthesized dipeptide a pharmaceutical was designed containing this compound, which later receives the brand name Thymogen. The agent activated T-cell differentiation, T-cell recognition of peptide-MHC complexes, induced changes in intracellular composition of cyclic nucleotides, and activated neutrophilic chemotaxis and phagocytosis. The effect of dipeptide on survival, life span and spontaneous tumor development was studied in female rats. Seventy-six, five-month-old outbred female rats were randomly subdivided into two groups and were subcutaneously injected with 0.2 ml of normal saline (controls, 32 rats) or with 5 micrograms/rat of the dipeptide L-Glu-L-Trp, dissolved in 0.2 ml of saline (44 rats), 5 times per week for 12 months. Animals were monitored up to their natural death and all the tumors discovered were studied microscopically. Mean life span of rats in both groups was similar but that of 10% maximum survived control rats constituted 949 +/- 16.1 days, whereas in the dipeptide-treated rats this value was 1048 +/- 21.1 days (P < 0.001). Six out of 44 rats treated with the drug survived over the maximum life span of control rats (965 days). The aging rate indicated as alpha in the Gompertz equation, was 0.0071 days-1 in controls and 0.0041 days-1 in rats exposed to L-Glu-L-Trp. Total tumor incidence was 1.5 times lower (P < 0.01), malignant tumor incidence 1.7 times lower (P < 0.01), and hematopoietic malignancies (leukemias and lymphomas) 3.4 times lower (P < 0.02) in rats exposed to the dipeptide in comparison with controls. Thus, treatment with L-Glu-L-Trp delayed aging rate and decreased spontaneous tumor incidence in rats.

Basis Set, Level, and Continuum Solvation Effects on the Stability of a Synthetic Dipeptide: PIDOTIMOD

The conformational preferences of PIDOTIMOD, namely 3-(5-oxo-l-propyl)-l-thiazolidin-4-carboxyl acid, a synthetic dipeptide showing a beneficial influence on some immune functions both in mammalians and in humans, have been searched by varying two of its rotatable dihedral angles. Preliminary results in vacuo on the conjugated basis used the STO-3G* basis set. At that level, the φ1, φ3 potential energy surfaces for φ2 equal either to 0° or 180° were also explored in the flexible rotor approximation. When φ2 is equal to 180°, however, for 120° ≤ φ1 ≤ 260° the nitrogen proton shuttling to the adjacent carboxyl anion was observed; therefore, a constraint had to be imposed on the N−H bond distance to prevent this artifact. In the φ2 = 180° potential energy surface computed employing the 6-31G* basis set, in fact, the proton remained on the N atom everywhere. The proton transfer was then studied in vacuo at the HF/6-31G* and 6-31+G* levels and, in contrast to the STO-3G* spontaneous process, the energy barrier...

γ-L-glutamyl-L-DOPA inhibits Na+-phosphate cotransport across renal brush border membranes and increases renal excretion of phosphate

For treatment of phosphate (Pi) overload in various pathophysiological states, an agent that selectively increases renal Pi excretion would be of major value. Previously, we have shown that dopamine (DA) inhibits Na(+)-Pi cotransport in renal epithelia. However, the administration of DA or its immediate precursor L-DOPA increases DA in multiple tissues. Synthetic dipeptide gamma-L-glutamyl-L-DOPA (gludopa) can serve as an inactive precursor (pro-pro-drug) of DA. This study tested the hypothesis that, because of the unique colocalization of gamma-glutamyltransferase (gamma-GT), aromatic amino acid decarboxylase, Na(+)-Pi cotransporter, and Na(+)-L-DOPA cotransporter in brush border membrane (BBM) of proximal tubular cells, gludopa may elicit phosphaturia by action of DA generated within the kidney. Thyroparathyrectomized rats were given placebo, or gludopa, or gludopa + gamma-GT inhibitor acivicin. Urinary excretion of Pi, Ca2+, Na+, K+, DA, cAMP, and cGMP was determined, and Na(+)-Pi cotransport was measured in BBM prepared from kidneys of rats at the end of the experiment. The administration of gludopa resulted in: (a) an inhibition of Na(+)-Pi cotransport, but not cotransport of Na(+)-proline and Na(+)-alanine in BBM; (b) an increase (+300%) of fractional excretion (FE) of Pi and a drop (-35%) of plasma Pi, whereas the plasma levels and FEs of Ca2+, Na+, and K+ were unchanged; (c) an increase in urinary excretion of cAMP. but not cGMP; (d) a 1000-fold increase of urinary excretion of DA, without a change in excretion of norepinephrine; and (e) an incubation of gludopa with BBM in vitro, which caused a release of L-DOPA, and the in vivo administration of acivicin, which blocked actions of gludopa to inhibit Na(+)-Pi cotransport and to increase urinary excretions of Pi and DA. We conclude that colocalization of enzymes of biotransformation, BBM transporters, and the autocrine/paracrine DA system in cells of proximal tubules constitutes a cellular basis for the potent and specific phosphaturic action of gludopa.

Purification and characterization of a cobalt-activated carboxypeptidase from the hyperthermophilic archaeon Pyrococcus furiosus.

A novel metallocarboxypeptidase (PfuCP) has been purified to homogeneity from the hyperthermophilic archaeon, Pyrococcus furiosus, with its intended use in C-terminal ladder sequencing of proteins and peptides at elevated temperatures. PfuCP was purified in its inactive state by the addition of ethylenediaminetetraacetic acid (EDTA) and dithiothreitol (DTT) to purification buffers, and the activity was restored by the addition of divalent cobalt (K, = 24 +/- 4 microM at 80 degrees C). The serine protease inhibitor phenylmethylsulfonyl fluoride (PMSF) had no effect on the activity. The molecular mass of monomeric PfuCP is 59 kDa as determined by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) and 58 kDa by SDS-PAGE analysis. In solution, PfuCP exists as a homodimer of approximately 128 kDa as determined by gel filtration chromatography. The activity of PfuCP exhibits a temperature optimum exceeding 90 degrees C under ambient pressure, and a narrow pH optimum of 6.2-6.6. Addition of Co2+ to the apoPfuCP at room temperature does not alter its far-UV circular dichroism (CD) or its intrinsic fluorescence spectrum. Even when the CoPfuCP is heated to 80 degrees C, its far-UV CD shows a minimal change in the global conformation and the intrinsic fluorescence of aromatic residues shows only a partial quenching. Changes in the intrinsic fluorescence appear essentially reversible with temperature. Finally, the far-UV CD and intrinsic fluorescence data suggest that the overall structure of the holoenzyme is extremely thermostable. However, the activities of both the apo and holo enzyme exhibit a similar second-order decay over time, with 50% activity remaining after approximately 40 min at 80 degrees C. The N-blocked synthetic dipeptide, N-carbobenzoxy-Ala-Arg (ZAR), was used in the purification assay. The kinetic parameters at 80 degrees C with 0.4 mM CoCl2 were: Km, 0.9 +/- 0.1 mM; Vmax, 2,300 +/- 70 U mg(-1); and turn over number, 600 +/- 20 s(-1). Activity against other ZAX substrates (X = V, L, I, M, W, Y, F, N, A, S, H, K) revealed a broad specificity for neutral, aromatic, polar, and basic C-terminal residues. This broad specificity was confirmed by the C-terminal ladder sequencing of several synthetic and natural peptides, including porcine N-acetyl-renin substrate, for which we have observed (by MALDI-TOF MS) stepwise hydrolysis by PfuCP of up to seven residues from the C-terminus: Ac-Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu-Leu-Val-Tyr-Ser.

Protease-catalyzed condensation of peptides as a potential means to reduce the bitter taste of hydrophobic peptides found in protein hydrolysates

Synthetic dipeptides of varying hydrophobicity were used as a model system to investigate the feasibility of reversed protease action as a means of improving the flavor of bitter peptides found in protein hydrolysates. The four different proteases tested were able to convert hydrophobic dipeptides into an insoluble precipitate, but with different efficiencies. Hydrophilic dipeptides did not give detectable condensation or precipitation. Solid-state and solution NMR were used to monitor peptide bond formation in 13C-labelled peptides. NMR and MS analyses showed that the products consisted of higher oligomers of the original peptide. Bitterness decreased in the order starting with dipeptides > whole reaction mixtures ⪢ isolated precipitates. Taste improvement correlated with peptide bond formation and the formation of insoluble material. These observations indicated that the condensation reaction was driven by precipitation of insoluble reaction products and that this process is a potential means to improve the flavor of bitter peptides extracted from protein hydrolysates.