Rate Of Peptide Hydrolysis Research Articles

The effect of chemical structure on hydrolysis pathways of small peptides in coastal seawater

The hydrolysis pathway of a given peptide or protein in seawater, or how it is hydrolyzed by extracellular enzymes, may depend on its chemical structure or specific amino acid composition. This knowledge is important for a better understanding of the first steps on decomposition of labile organic matter that is often dominated by proteins. The hydrolysis pathway can be estimated from the hydrolyzed fragments of small peptides through incubation experiments. Here we incubated a group of small peptides with different amino acid compositions in coastal seawater, including tetrapeptides alanine-valine-phenylalanine-alanine (AVFA) and its modifications of the N terminal A with valine (V), serine (S), arginine (R) and aspartic acid (D) (i.e., peptides VVFA, SVFA, RVFA, DVFA). Through these incubations, we evaluated the effect of chemical structure or specific amino acid composition on peptide hydrolysis pathways and the roles of different types of peptidases. Our results showed that aminopeptidases preferentially hydrolyzed tetrapeptides with N terminal A and R (29–100% of the total peptidases), followed by V (30–90%) and S (20–51%), but were ineffective with acidic amino acid D (0–14%). This pattern indicates that the N-terminal amino acid affects how a peptide is hydrolyzed in a given environment. For N-terminal amino acids with uncharged side chains, aminopeptidases preferred hydrophobic A, V to polar uncharged S, and for those with charged side chains, they preferred positively-charged R to negatively-charged D. Our results also suggest that if aminopeptidases could not access the N-terminal amino acid of a peptide, carboxypeptidases and/or endopeptidases will hydrolyze it, leading to similar peptide hydrolysis rates among different peptides although their hydrolysis pathways may differ. The effect of amino acid composition on peptide hydrolysis pathways is further demonstrated by comparing trialanine (AAA), AVF, AVFA, VFA, phenylalanine-alanine-serine-tryptophan-glycine-alanine (FASWGA) and SWGA. Overall, results from this study show that the chemical structure of a peptide affects its hydrolysis pathway in coastal seawater, and shed new lights on the early steps of organic nitrogen degradation and further nitrogen cycling in marine environments.

Structure and function of high Arctic pelagic, particle-associated and benthic bacterial communities.

Arctic marine microbes are affected by environmental changes that may ultimately influence their functions in carbon cycling. Here, we investigated in concert the structure and enzymatic activities of pelagic, particle-associated and benthic bacterial communities in the central Arctic Ocean, and used these data to evaluate microbial structure-function relationships. Our findings showed influences of hydrographic conditions and particle association on community composition, and sharp pelagic-benthic contrasts. In addition to community compositional differences, regional and depth-related patterns in enzymatic activities were observed. Peptide hydrolysis rates were highest in surface waters, especially at ice-free and first year ice-covered regions, and decreased with depth. While the range of hydrolysed polysaccharides showed varying geographic patterns, particles often showed a wider spectrum of polysaccharide hydrolase activities. Summed benthic peptidase rates differed across stations but showed similar proportions of individual enzyme activities. Analysing for potential linkages between structure and function after subtracting the effect of environmental conditions revealed no direct link, indicating functional redundancy to carry out peptide hydrolysis among pelagic microbes. Thus, while community composition and activities are influenced by environmental conditions, bacterial functional redundancy suggests that compositional shifts - in response to the changing Arctic - may have complex and less predictable functional consequences than previously anticipated. © 2018 Society for Applied Microbiology and John Wiley & Sons Ltd.

Peptide hydrolysis and uptake of peptide hydrolysis products in the James River estuary and lower Chesapeake Bay

Abstract The lability of small peptides was investigated along the salinity gradient of the James River estuary and lower Chesapeake Bay using two fluorescent analogs, Lucifer Yellow Anhydride-alanine-valine-phenylalanine-alanine (LYA-AVFA) and LYA-tetraalanine (LYA-AlA4). Hydrolysis rates of these compounds were compared with each other, and with uptake rates of potential hydrolysis products and other smaller derivatives (e.g., glutamic acid and dialanine). Results suggest that rates of peptide hydrolysis and uptake of hydrolysis products were not always correlated with each other along the salinity gradient, or to other environmental parameters measured in the James River and lower Chesapeake Bay. This may be because diverse input and removal processes can influence both peptide hydrolysis and uptake, but not necessarily simultaneously. Rates of both peptide hydrolysis and free amino acid uptake were strongly associated with particles, particularly those freshly produced. This suggests more rapid turnover of enzymatically available organic nitrogen in regions with elevated phytoplankton biomass. Changes in the abundance and composition of dissolved amino acids in these waters were also examined. Dissolved amino acid compositions, but not concentrations, varied with salinity along a gradient from the tidal fresh James River to the mouth of Chesapeake Bay. These compositional variations demonstrate the mixing of terrestrial organic nitrogen and in-situ production along the salinity transect.

A new method to measure small peptides amended in seawater using high performance liquid chromatography coupled with mass spectrometry

Quantifying peptide decomposition rate is crucial in understanding marine carbon and nitrogen cycling, because proteins and peptides constitute a major fraction of labile organic matter. However, analytical techniques of detecting small peptides in nanomolar levels in seawater are limited. A new method was developed to measure low concentrations of small peptides amended in seawater, using high performance liquid chromatography coupled with mass spectrometry (HPLC–MS). This technique reduces the detection limit of small peptides by two orders of magnitude relative to the common ultraviolet (UV) detection. A 6-way valve was added before the MS and the valve was programmed to guide the salt peak to waste before the peptide peak was introduced to the MS. Therefore, peptides amended in seawater were injected directly to the HPLC–MS without desalting pretreatment. This new method can detect as low as 0.23pmol of tetrapeptide alanine–valine–phenylalanine–alanine (AVFA), a peptide fragment of Ribulose-1,5-bisphosphate carboxylase oxygenase (RuBisCO), with less than 5% precision (relative standard deviation). This method was applied successfully to determine decomposition rates of two small peptides, AVFA and serine–tryptophan–glycine–alanine (SWGA), in coastal oceans, and first data of peptide hydrolysis rates using small plain peptides at lower than micromolar concentrations were obtained. Hydrolysis of AVFA and its fluorescent analog was also compared at such low concentration levels. This analytical method broadens our capability to examine the biogeochemical behavior of small peptides, including their hydrolysis, decomposition, and other possible transformation processes in aquatic environments.

Variably modulated gating of the 26S proteasome by ATP and polyubiquitin

The 26S proteasome is a 2500 kDa protease complex that degrades polyubiquitylated proteins by a mechanism that requires ATP hydrolysis. It also degrades short non-ubiquitylated peptides and certain unstructured proteins by an energy-independent mechanism that requires bound ATP to maintain its component subcomplexes, the 20S proteasome and PA700, in a functionally assembled state. Proteolysis of both types of substrate requires PA700-induced opening of reversible gates at substrate-access pores of the 20S proteasome. In the present study we demonstrate that the rate of peptide substrate hydrolysis, a functional monitor of gate opening, is regulated variably by multiple effectors. ATPgammaS (adenosine 5'-[gamma-thio]triphosphate) and other non-hydrolysable ATP analogues increased peptide substrate hydrolysis by intact 26S proteasomes. Thus nucleotides that maintained 26S proteasome structure, but did not support ATP hydrolysis or the degradation of polyubiquitylated proteins, promoted enhanced rates of peptide hydrolysis. Polyubiquitin and a peptoid that binds selectively to a single ATPase subunit of PA700 also increased rates of peptide hydrolysis but had disparate effects on rates of ATP hydrolysis. The effect of polyubiquitin was specific for ubiquitin-ubiquitin linkages that supported proteolysis of protein substrates. These results indicate that gating of the 26S proteasome is not a simple two-state process but can be variably modulated. Our results suggest that modulated gating of the proteasome may be an important element of the mechanism of proteolysis of polyubiquitylated proteins.

Peptide hydrolysis and the uptake of dipeptides by phytoplankton

Rates of peptide hydrolysis (using the fluorescent substrate, lucifer yellow anhydride‐labeled tetra‐alanine) and dipeptide uptake (using dually labeled, 15N and 13C, dialanine) were measured in phytoplankton cultures and in natural populations during algal blooms dominated by one or two taxa. During most sampling events, both peptide hydrolysis and dipeptide uptake were greatest in the size fraction containing the dominant phytoplankter, suggesting that phytoplankton contribute substantially to or may even dominate observed extracellular peptide hydrolysis and dipeptide uptake in the environment. These are the first data suggesting that dipeptides may be taken up directly by phytoplankton and this may represent a previously unaccounted‐for nitrogen source in aquatic systems. Like many other processes in phytoplankton, peptide hydrolysis appears sensitive to the diel light cycle and the nutrient environment, with rates varying depending on the dominant N source, but with no clear pattern. Uptake of dialanine, the dominant product of the hydrolysis of the peptide tetra‐alanine, also varied depending on the dominant taxa and the nutrient regime. Most of the time, it appeared that low production of dialanine by tetra‐alanine hydrolysis limited the uptake of the dipeptide. Close coupling between peptide hydrolysis and dipeptide uptake may also help explain the absence of correlations between rates of peptide hydrolysis and the concentration and composition of the free amino acid pool.

Peptide Synthesis in Early Earth Hydrothermal Systems

We report here results from experiments and thermodynamic calculations that demonstrate a rapid, temperature-enhanced synthesis of oligopeptides from the condensation of aqueous glycine. Experiments were conducted in custom-made hydrothermal reactors, and organic compounds were characterized with ultraviolet-visible procedures. A comparison of peptide yields at 260 degrees C with those obtained at more moderate temperatures (160 degrees C) gives evidence of a significant (13 kJ . mol(-1)) exergonic shift. In contrast to previous hydrothermal studies, we demonstrate that peptide synthesis is favored in hydrothermal fluids and that rates of peptide hydrolysis are controlled by the stability of the parent amino acid, with a critical dependence on reactor surface composition. From our study, we predict that rapid recycling of product peptides from cool into near-supercritical fluids in mid-ocean ridge hydrothermal systems will enhance peptide chain elongation. It is anticipated that the abundant hydrothermal systems on early Earth could have provided a substantial source of biomolecules required for the origin of life.

Microbial degradation rates of small peptides and amino acids in the oxygen minimum zone of Chilean coastal waters

Abstract We found similar microbial degradation rates of labile dissolved organic matter in oxic and suboxic waters off northern Chile. Rates of peptide hydrolysis and amino acid uptake in unconcentrated water samples were not low in the water column where oxygen concentration was depleted. Hydrolysis rates ranged from 65 to 160 nmol peptide L −1 h −1 in the top 20 m, 8–28 nmol peptide L −1 h −1 between 100 and 300 m (O 2 -depleted zone), and 14–19 nmol peptide L −1 h −1 between 600 and 800 m. Dissolved free amino acid uptake rates were 9–26, 3–17, and 6 nmol L −1 h −1 at similar depth intervals. Since these findings are consistent with a model of comparable potential activity of microbes in degrading labile substrates of planktonic origin, we suggest, as do other authors, that differences in decomposition rates with high and low oxygen concentrations may be a matter of substrate lability. The comparison between hydrolysis and uptake rates indicates that microbial peptide hydrolysis occurs at similar or faster rates than amino acid uptake in the water column, and that the hydrolysis of peptides is not a rate-limiting step for the complete remineralization of labile macromolecules. Low O 2 waters process about 10 tons of peptide carbon per h, double the amount processed in surface-oxygenated water. In the oxygen minimum zone, we suggest that the C balance may be affected by the low lability of the dissolved organic matter when this is upwelled to the surface. An important fraction of dissolved organic matter is processed in the oxygen minimum layer, a prominent feature of the coastal ocean in the highly productive Humboldt Current System.

Proteasome Channel Opening as a Rate‐Limiting Step in the Ubiquitin‐Proteasome System

AbstractThe 26S proteasome eliminates multiubiquitinated proteins in cytosol and nucleus, and from the secretory pathway by a mechanism known as ER‐associated degradation (ERAD). Access to the proteasomal 20S catalytic core particle is hindered by conserved N‐terminal tails of α‐subunits that form a gated pore into the central channel. Hence, the isolated 20S core particle possesses slower peptide hydrolysis rates and cannot degrade multiubiquitinated proteins. Purified catalytic particles from an α3α7ΔN open channel double mutant, in which the N‐terminal tails of α‐subunits from opposite sites of the α ring are deleted, showed significantly enhanced peptidase activity and proteolytic properties. Here we show that also in vivo the access of substrates to the proteasomal catalytic chamber partially limits the overall rate of protein elimination. This regulation applies to unstable cytosolic proteins of the N‐end rule and ubiquitin fusion degradation (UFD) pathways, as well as to ERAD substrates that must dislocate from the ER back to the cytosol in order to become ubiquitinated and degraded by the proteasome. Hence, even for a complicated multistep process such as ERAD, traffic through the proteasome itself is partially rate limiting for the entire proteolytic process. However, proteasome gating can be added to a growing list of phenomena that distinguish membrane ERAD substrates from lumenal ones because while gating hinders access of lumenal substrates, it is less effective in controlling the entry of membrane substrates. The open channel mutant is a new class of proteasome mutant, which is unrelated to the catalytic protease active sites or to the “classical” regulatory particle mutants. Its improved performance at high temperatures is in stark contrast to the behavior of the “classical” mutants, suggesting that the α3α7ΔN mutant adapts better to mild stress conditions.

Synthesis and Application of Poly(phenylene Ethynylene)s for Bioconjugation: A Conjugated Polymer-Based Fluorogenic Probe for Proteases

A set of carboxylate-functionalized poly(phenylene ethynylene)s (PPEs) has been synthesized in which the carboxylic acid groups are separated from the polymer backbone by oligo(ethylene glycol) spacer units. These polymers are soluble in water and organic solvents and have photophysical properties that are sensitive to solvent conditions, with high salt content and the absence of surfactant promoting the formation of aggregates of relatively low quantum yield and long fluorescence lifetime. Quenching of these materials by the dinitrophenyl (DNP) chromophore (K(SV) approximately 10(4)) is also highly solvent-dependent. The presence of carboxylate groups far from the polymer backbone appended to each repeating unit allows for the postpolymerization modification of these PPEs with peptides by methods analogous to those described for carboxylate-functionalized small-molecule dyes. Covalent attachment of the fluorescence-quenching 14-mer Lys(DNP)-GPLGMRGLGGGGK to the PPE results in a nonemissive substrate whose fluorescence is restored upon treatment with trypsin. The rate of fluorescence turn-on in this case is increased 3-fold by the presence of surfactant, though the actual rate of peptide hydrolysis remains the same. A small-molecule mimic of the polymer-peptide system shows a smaller fluorescence enhancement upon treatment with trypsin, illustrating the value of polymer-based amplification in this sensory scheme.

Peptide hydrolysis, amino acid oxidation, and nitrogen uptake in communities seasonally dominated by Aureococcus anophagefferens

Elevated levels of dissolved organic nitrogen (DON) and dissolved inorganic nitrogen (DIN) are among the factors implicated in the initiation of algal blooms. However, the degree to which phytoplankton augment their autotrophic metabolism with heterotrophic uptake of organic carbon that is associated with DON is unknown. We evaluated the relative importance of peptide hydrolysis, amino acid oxidation, and amino acid uptake over a seasonal cycle in an embayment on Long Island, New York, that had high concentrations of dissolved organic matter (DOM) and a bloom of the brown tide pelagophyte, Aureococcus anophagefferens. Amino acids were a significant component (up to 50%) of the total N uptake, particularly during the late summer. About half of the associated amino acid C was also taken up. Amino acid oxidation rates were an order of magnitude lower than free amino acid uptake rates, but still supplied up to 32.5% of the NH taken up. Up to 75% of the amino acid oxidation was in the bacterial size fraction (‹1.2 μm), and rates were significantly correlated with bacterial densities. Peptide hydrolysis rates were high, and most (up to 72%) occurred in the brown tide size fraction (1.2–5 μm). The high rates of peptide hydrolysis and amino acid uptake measured in cultures of A. anophagefferens confirm that this species can readily hydrolyze peptides and take up N and C from amino acids. Laboratory findings and size‐fractionation studies in the field suggest that A. anophagefferens plays a major role in consumption of both C and N from DOM.

Peptide decomposition by extracellular hydrolysis in coastal seawater and salt marsh sediment

Abstract Extracellular peptide hydrolysis rates were measured in seawater and sediment from Flax Pond salt marsh using peptide analogs (LYA-peptides) as substrates. This technique allows the direct measurement of specific hydrolysis products and thus provides insights into enzymatic hydrolysis pathways. In sediments, hydrolysis rate constants of LYA-peptides varied seasonally and with depth. Highest activity was found in spring and summer, and most cores exhibited a subsurface maximum. Calculations using the concentrations of chemically-measured peptides suggested that extracellular hydrolysis of peptides is faster than the rate of free amino acids uptake. However, not all peptides may be available for enzymatic hydrolysis. In both seawater and sediment, extracellular hydrolysis of peptides of up to 8 amino acids yielded smaller peptides and free amino acids. Hydrolysis rates depended on size of the peptide substrate, although a clear relationship with number of amino acid constituents was not evident. Peptides containing >2 amino acids were hydrolyzed 10–400 times faster than dipeptides or the fluorogenic substrate Leucine-MCA. Thus, dipeptidases are either uncommon in nature, or hydrolysis is carried out by nonspecific hydrolases that with a low affinity for dipeptides. This is also suggested by the presence of a lag time before dipeptide hydrolysis begins, and the absence of dipeptide hydrolysis in 0.2-μm-filtered. One implication of this finding is that measurements of hydrolysis rates using substrates like Leu-MCA may not accurately predict the magnitude of hydrolysis rates of macromolecules in the marine environment. Even though dipeptide hydrolysis is slow compared to that of larger peptides, LYA-dipeptides are preferentially produced from the hydrolysis of larger substrates. LYA-dipeptides do not penetrate cell membranes of microorganisms because of their size, but natural dipeptides are smaller and can be transported across the cell membrane. Since dipeptides do not appear to accumulate in natural waters, they must be rapidly removed by microorganisms.

Studies on the activation by ATP of the 26 S proteasome complex from rat skeletal muscle

The 26 S proteasome complex is thought to catalyse the breakdown of ubiquitinated proteins within eukaryotic cells. In addition it has been found that the complex also degrades short-lived proteins such as ornithine decarboxylase in a ubiquitin-independent manner. Both proteolytic processes are paralleled by the hydrolysis of ATP. Here we show that ATP also affects the hydrolytic activity towards fluorigenic peptide substrates by the 26 S proteasome complex from rat skeletal muscle tissue. Low concentrations of ATP (about 25 microM) optimally activate the so-called chymotryptic and tryptic activity by increasing the rate of peptide hydrolysis but not peptidylglutamylpeptide hydrolysis. Activation of the enzyme by ATP is transient but this effect can be enhanced and prolonged by including in the assay an ATP-regenerating system, indicating that ATP is hydrolysed by the 26 S proteasome complex. Although ATP cannot be substituted for by adenosine 5'-[beta,gamma-methylene]triphosphate or AMP, hydrolysis of the phosphoanhydride bond of ATP seems not to be necessary for the activation process of the proteasome complex, a conclusion drawn from the findings that ATP analogues such as adenosine 5'-[beta,gamma-imido]triphosphate, adenosine 5'-O-[gamma-thio]triphosphate, adenosine 5'-O-[beta-thio]-diphosphate and adenosine 5'-[alpha,beta-methylene]triphosphate give the same effect as ATP, and vanadate does not prevent ATP activation. These effects are independent of the presence of Mg2+. Thus, ATP and other nucleotides may act as allosteric activators of peptide-hydrolysing activities of the 26 S proteasome complex as has also been found with the lon protease from Escherichia coli.

Glucocorticoids do not alter peptidase expression on a human bronchial epithelial cell line.

Respiratory epithelial cell surface neutral endopeptidase 24.11 (NEP-24.11) degrades proinflammatory peptides, and it has been suggested that glucocorticoids may reduce airway inflammation, in part, by upregulation of NEP-24.11. Despite the potential importance of the epithelium as a metabolic barrier, little is known regarding what other peptidases may be present on the epithelial cell surface. Using an immortalized bronchial epithelial cell line (BEAS-2B), we have shown that human epithelial cells express no detectable angiotensin-converting enzyme, carboxypeptidase N, or dipeptidyl(amino)peptidase IV, but express significant levels of aminopeptidase M (AmM), as well as NEP-24.11. The presence of these enzymes was demonstrated via their degradation of biologically active peptides and by flow cytometry. Exposure of cells to the glucocorticoid budesonide (10(-7) M) for up to 5 days did not markedly alter the expression of NEP-24.11 or AmM, as assessed by flow cytometry, nor did glucocorticoid treatment modify rates of peptide hydrolysis by NEP-24.11 or AmM. Thus, BEAS-2B cells have both AmM and NEP-24.11 on their surface, and expression of these enzymes is not altered by glucocorticoids.

Enzyme reaction engineering: Design of peptide synthesis by stabilized trypsin

By using very active and very stable trypsin agarose derivatives, we have optimized the design of the synthesis of a model dipeptide, benzoylarginine leucinamide, by two different strategies: (i) kinetically controlled synthesis (KCS), by using benzoyl arginine ethyl ester and leucinamide as substrates, and (ii) thermodynamically controlled synthesis (TCS), by using benzoyl arginine and leucinamide as substrates. In each strategy, we have studied the integrated effect of a number of variables that define the reaction medium on different parameters of industrial interest, e.g. time course of peptide synthesis, higher synthetic yields, and stability of the catalyst, as well as aminolysis/hydrolysis ratios and rate of peptide hydrolysis in the case of KCS. Both synthetic approaches were carried out in monophasic water or water-organic cosolvent systems. We have mainly tested a number of variables, e.g. temperature, polarity of the reaction medium (presence of cosolvents, presence of ammonium sulfate), and exact structure of the trypsin derivatives. Optimal experimental conditions for these synthetic approaches were established in order to simultaneously obtain good values for all industrial parameters. The use of previously stabilized trypsin derivatives greatly improves the design of these synthetic approaches (e.g. by using drastic experimental conditions: 1 m ammonium sulfate (KCS) or 90% organic cosolvents (TCS)). In these conditions, our derivatives preserve more than 95% of activity after 2 months and we have been able to reach synthetic productivities of 180 (KCS) and 1 (TCS) tons of dipeptide per year per liter of catalyst.

Utilisation of protein by human gut bacteria

Mixed populations of human gut bacteria degraded cas casein by producing a variety of cell-bound and extracellular proteolytic enzymes. Casein was initially hydrolysed to TCA soluble peptides which were subsequently broken down to volatile fatty acids, ammonia, dicarboxylic acids and a range of phenolic compounds. Amino acids did not accumulate to any extent during casein breakdown, suggesting that the rate of peptide hydrolysis was the limiting step in protein utilisation by these bacteria. Similar fermentation products were produced from bovine serum albumin, however, the insoluble protein collagen was considerably more resistant to degradation by the colonic microflora, as evidenced by the reduced quantities of fermentation end-products formed.

Utilisation of protein by human gut bacteria

Mixed populations of human gut bacteria degraded cas casein by producing a variety of cell-bound and extracellular proteolytic enzymes. Casein was initially hydrolysed to TCA soluble peptides which were subsequently broken down to volatile fatty acids, ammonia, dicarboxylic acids and a range of phenolic compounds. Amino acids did not accumulate to any extent during casein breakdown, suggesting that the rate of peptide hydrolysis was the limiting step in protein utilisation by these bacteria. Similar fermentation products were produced from bovine serum albumin, however, the insoluble protein collagen was considerably more resistant to degradation by the colonic microflora, as evidenced by the reduced quantities of fermentation end-products formed.

Assimilation of alpha-glutamyl-peptides by human erythrocytes. A possible means of glutamate supply for glutathione synthesis.

Human erythrocytes are essentially impermeable to glutamate and yet there is a continual requirement for the amino acid for glutathione synthesis. In addition, the intracellular glutamate concentration is approximately five times that of plasma. We present evidence that glutamate enters the red cell as small peptides which are rapidly hydrolysed by cytoplasmic peptidase(s) and that with the estimated physiological levels of plasma glutamyl-peptides the rate of inward flux would be adequate to maintain the glutamate pool at its observed level. Experimentally, we used 1H spin-echo n.m.r. spectroscopy to follow peptide hydrolysis, since peptide spectra are different from those of the free amino acids and the spin-echo sequence enables the monitoring of reactions in concentrated lysates and whole cell suspensions. Thus, the system was studied under near-physiological conditions. Weighted non-linear regression analysis of progress curves using the integrated Michaelis-Menten equation was used to obtain estimates of Km and Vmax. for the hydrolysis of alpha-L-glutamyl-L-alanine and L-alanyl-alpha-L-glutamate in lysates and whole cell suspensions; the values for lysates were Km = 3.60 +/- 0.29 and 5.4 +/- 0.4 mmol/l and Vmax. = 120 +/- 4 and 46.7 +/- 1.7 mmol/h per 1 of packed cells respectively. In whole cell suspensions the rate of peptide hydrolysis was much slower and dominated by the transmembrane flux-rate. The estimates of the steady-state kinetic parameters for the transport were Kt = 2.35 +/- 0.41 and 11.2 +/- 1.0 mmol/l and Vmax. = 3.26 +/- 0.13 and 19.7 +/- 0.7 mmol/h per 1 of packed cells respectively for the previously mentioned peptides. Using the n.m.r. procedure we failed to detect any glutaminase activity in whole cells or lysates; thus, we exclude the possibility that glutamate gains entry to the cell as glutamine which is subsequently hydrolysed by glutaminase.

Rates of peptide bond hydrolysis by cobalt(III) complexes: observation by rate of amino acid release

The rates of hydrolysis of eleven dipeptides (Gly-Gly; Gly- l-Leu; Gly- d,l-Val; Gly- l-Phe: d,l-Leu-Gly; l-Leu- l-Tyr; l-Pro- l-Tyr; l-Ala- l-Phe; l-Val-Gly; l-Val- l-Ile; l-Ile- l-Val) by cis-β-Co(trien)(OH)(H 2O) 2+ under pseudo first order conditions (excess Co(III)) are reported. The rates are determined by the rate of amino acid release by liquid chromatography. The range of rates observed for the eleven dipeptides is 10.1. Two of the dipeptides are hydrolyzed more rapidly than Gly-Gly, the remaining six more slowly. Phosphate does not have any significant effect on the rate of peptide hydrolysis. Aquohydroxy-1,8-diamino-3,6-dithiaoctanecobalt(III) 2+ did not hydrolyze Gly-Gly at 25°, pH8, and aquohydroxy-1,6-bis-(α-pyridyl)-2,5-diazahexanecobalt(III) 2+ did not hydrolyze Gly-Gly at 65°, pH 8, to any significant extent.

Fluorimetric assay for human intestinal glycine peptidases

A sensitive but simple fluorimetric assay for intestinal enzymes capable of hydrolysing certain glycine-containing peptides is described. In the standard assay, 1 nanomole of glycine can be accurately estimated in the presence of a 2000-fold molar excess of unhydrolysed peptide. Initial rates of peptide hydrolysis can be measured on the density gradient fractions obtained by analytical subcellular fractionation of extracts from milligram quantities of human jejunal tissue. Differences in the subcellular localisation of activity against two peptides are illustrated. The implications of these findings in relation to the study of intestinal peptidase deficiency in disease states are discussed.