l-Phenylalanyl-l-Glutamate-Stimulated, Chloride-Dependent Glutamate Binding Represents Glutamate Sequestration Mediated by an Exchange System

Stimulation of glutamate binding by the dipeptide L-phenylalanyl-L-glutamate (Phe-Glu) was inhibited by the peptidase inhibitor bestatin, suggesting that the stimulation was caused by glutamate liberated from the dipeptide and not by the dipeptide itself. It further suggests that this form of glutamate binding should be reinterpreted as glutamate sequestration and that stimulation of binding both by dipeptides and after preincubation with high concentrations of glutamate is likely to be due to counterflow accumulation. Several other criteria indicate that most of glutamate binding stimulated by chloride represents glutamate sequestration: Binding is reduced when the osmolarity of the incubation medium is increased, when membranes incubated with [3H]glutamate are lysed before filtration, and when membranes are made permeable by transient exposure to saponin. Moreover, dissociation of bound glutamate after a 100-fold dilution of the incubation medium is accelerated about 50 times by the addition of glutamate to the dilution medium. This result would be anomalous if glutamate were bound to a receptor site; it suggests instead that glutamate is transported in and out of membrane vesicles by a transport system that preferentially mediates exchange between internal and external glutamate. Glutamate binding contains a component of glutamate sequestration even when measured in the absence of chloride. Sequestration is adequately abolished only after treating membranes with detergents; even extensive lysis, sonication, and freezing/thawing may be insufficient.     

Identification and Characterization of γ-Glutamylamine Cyclotransferase,an Enzyme Responsible for γ-Glutamyl-?-lysine Catabolism

γ-Glutamylamine cyclotransferase (GGACT) is an enzyme that converts γ-glutamylamines to free amines and 5-oxoproline. GGACT shows high activity toward γ-glutamyl-ϵ-lysine, derived from the breakdown of fibrin and other proteins cross-linked by transglutaminases. The enzyme adopts the newly identified cyclotransferase fold, observed in γ-glutamylcyclotransferase (GGCT), an enzyme with activity toward γ-glutamyl-α-amino acids (Oakley, A. J., Yamada, T., Liu, D., Coggan, M., Clark, A. G., and Board, P. G. (2008) J. Biol. Chem. 283, 22031–22042). Despite the absence of significant sequence identity, several residues are conserved in the active sites of GGCT and GGACT, including a putative catalytic acid/base residue (GGACT Glu⁸²). The structure of GGACT in complex with the reaction product 5-oxoproline provides evidence for a common catalytic mechanism in both enzymes. The proposed mechanism, combined with the three-dimensional structures, also explains the different substrate specificities of these enzymes. Despite significant sequence divergence, there are at least three subfamilies in prokaryotes and eukaryotes that have conserved the GGCT fold and GGCT enzymatic activity.     

Dipeptides as Leaving Group in the Enzyme‐Catalyzed DNA Synthesis

Conjugates of 2′‐deoxyadenosine monophosphate with dipeptides have been synthesized and tested as substrates for several polymerases. Although the incorporation efficiency is not very high, it demonstrates that some of these dipeptides can be accommodated in the active site of polymerases and function as leaving groups in the enzymatic synthesis of DNA.     

Reverse structure of carnosine-induced sedative and hypnotic effects in the chick under acute stress

In the central nervous system, beta-alanine is thought to act as an inhibitory neurotransmitter, but the role or precise mechanism of beta-alanine in the brain has not been clearly defined. beta-Alanine is found in high levels in the chicken brain as a component of the dipeptides carnosine (beta-alanyl-L-histidine) and anserine, or as a free amino acid. We focused on the position of beta-alanine, i.e., at the carboxyl terminus. In Experiment 1, the central effects of glycyl-beta-alanine, L-histidyl-beta-alanine and L-valyl-beta-alanine were compared with a saline control in chicks. L-Histidyl-beta-alanine significantly induced sedative and hypnotic effects. In Experiment 2, the effects of carnosine, its reverse (L-histidyl-beta-alanine), and their combination were investigated. Central carnosine-induced hyperactivity while reverse carnosine-induced hypoactivity, and the behaviors were intermediate following the combination of the two peptides. Finally, the central effect of reverse carnosine was compared with beta-alanine alone and L-seryl-beta-alanine in Experiment 3. Reverse carnosine showed similar effects to beta-alanine. In conclusion, L-histidyl-beta-alanine not only has the reverse structure of carnosine, but also reverse function. Thus, we propose to name reverse carnosine (L-histidyl-beta-alanine) rev-carnosine.     

Density functional calculations of 15N chemical shifts in solvated dipeptides

We performed density functional calculations to examine the effects of solvation, hydrogen bonding, backbone conformation, and the side chain on 15N chemical shielding in proteins. We used N-methylacetamide (NMA) and N-formyl-alanyl-X (with X being one of the 19 naturally occurring amino acids excluding proline) as model systems. In addition, calculations were performed for selected fragments from protein GB3. The conducting polarizable continuum model was employed to include the effect of solvent in the density functional calculations. Our calculations for NMA show that the augmentation of the polarizable continuum model with the explicit water molecules in the first solvation shell has a significant influence on isotropic 15N chemical shift but not as much on the chemical shift anisotropy. The difference in the isotropic chemical shift between the standard beta-sheet and alpha-helical conformations ranges from 0.8 to 6.2 ppm depending on the residue type, with the mean of 2.7 ppm. This is in good agreement with the experimental chemical shifts averaged over a database of 36 proteins containing >6100 amino acid residues. The orientation of the 15N chemical shielding tensor as well as its anisotropy and asymmetry are also in the range of values experimentally observed for peptides and proteins.     

Cytotoxicity of boron containing dipeptide analogs

Summary This work is an extension of our previous work (Hall et al., 1993) on the synthesis and cytotoxic activity of boronated peptides. The aim of this work was to carry out structural modifications of the amine terminal in compounds1 and2, to increase water solubility, and its effect on the cytotoxicity to tumor cell lines. Surprisingly, only compounds4,7 and8 were more water soluble than the parent compounds. With the exception of compound4, the new derivatives were generally less effective than the parent compounds (1 and2). There was no apparent correlation between structure and activity. Cytotoxic effect was more pronounced in single cell suspended cells. The growth of solid tumor cell lines was not significantly reduced. The most active derivative, (methanamine)dihydro[[[1-(phenylmethyl)-2-methylamino-2-oxoethyl]amino]carboxy]boron (4), inhibited DNA, RNA, and protein synthesis in Tmolt₃ cells. Enzymatic activities, e.g., DNA polymerase, m-RNA polymerase, PRPP amidotransferase, carbamyl phosphate synthetase, TMP-kinase, TDP-kinase, dihydrofolate reductase, and ribonucleoside reductase were reduced after 60 min incubation with4. d(TTP) and d(CTP) pool levels were also reduced by 60 min incubation with4.     

Influence of dipeptides on precipitated morphine withdrawal in the mouse

Effects of various dipeptides on naloxone-precipitated morphine withdrawal were studied in the mouse. Mice were rendered dependent on morphine by implantation of morphine pellets and the withdrawal syndrome was measured by the latency of the onset of stereotyped jumpings. In accordance with previous data, subcutaneous injection of Z-prolyl-D-leucine significantly delayed the onset of morphine withdrawal. The all-L enantiomer of the dipeptide (Z-L-prolyl-L-leucine) did not affect morphine withdrawal in the dose studied. Replacement of L-proline by L-glutamate or L-pyroglutamate (Z-L-glutamyl-L-leucine and L-pyroglutamyl-L-leucine) resulted in dipeptides which were more potent towards morphine withdrawal than Z-prolyl-D-leucine. Z-L-glycyl-L-proline attenuated the morphine withdrawal syndrome more effectively than Z-L-prolyl-D-leucine, but Z-L-leucyl-L-glycine was ineffective in this respect. The data reveal that certain dipeptides—which in their nonprotected forms are normal sequences of endogenous peptides—affect morphine withdrawal more potently than Z-prolyl-D-leucine, a synthetic dipeptide known to attenuate morphine dependence.     

Dipeptidyl-alpha,beta-epoxyesters as potent irreversible inhibitors of the cysteine proteases cruzain and rhodesain

The dipeptidyl epoxyesters 3 and 4 are potent, irreversible inhibitors of cruzain and rhodesain.     

The renal type H+/peptide symporter PEPT2: structure-affinity relationships

Summary. The H⁺/peptide cotransporter PEPT2 is expressed in a variety of organs including kidney, lung, brain, mammary gland, and eye. PEPT2 substrates are di- and tripeptides as well as peptidomimetics, such as β-lactam antibiotics. Due to the presence of PEPT2 at the bronchial epithelium, the aerosolic administration of peptide-like drugs might play a major role in future treatment of various pulmonary and systemic diseases. Moreover, PEPT2 has a significant influence on the in vivo disposition and half-life time of peptide-like drugs within the body, particularly in kidney and brain. PEPT2 is known to have similar but not identical structural requirements for substrate recognition and transport compared to PEPT1, its intestinal counterpart. In this review we compiled available affinity constants of 352 compounds, measured at different mammalian tissues and expression systems and compare the data whenever possible with those of PEPT1.