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PubMed2003

Determination of free proline and monosaccharides in wine samples by high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD).

A sensitive and selective analytical method for the simultaneous separation and quantitative determination of proline and free monosaccharides in wine samples by high-performance anion-exchange chromatography coupled with pulsed amperometric detection is described. Under optimized experimental conditions, a complete separation was obtained in less than 30 min, using an isocratic elution with 10 mM NaOH and 1 mM Ba(OAc)(2). No postcolumn addition of strong bases to the eluent for enhancing detection sensitivity was needed. Upon 25-fold sample dilution and purification to avoid interference of tannins, pigments, and phenolic compounds, the fingerprinting of common monosaccharides (i.e., arabinose, glucose, fructose, galactose, and xylose) and proline in wines, musts, and vinegars can be easily accomplished. The method allows high recovery and satisfies the necessary requirements for accuracy, repeatability, and sensitivity. Values obtained for proline content ranged from 470 to 1190 mg/L in "Aglianico" red wines (mean value, 870 +/- 192 mg/L, n = 21) and from 168 to 286 mg/L in white wines (mean value, 208 +/- 32 mg/L, n = 11). Lower levels were found in musts of red and white grapes, 550 and 87 mg/L, respectively. The lowest content of proline, ca. 10 mg/L, was found both in white and red vinegars.

Acetic Acid↗
PubMed1990

(Phosphinyloxy)acyl amino acid inhibitors of angiotensin converting enzyme. 2. Terminal amino acid analogues of (S)-1-[6-amino-2 [[hydroxy(4-phenylbutyl)phosphinyl]oxy]-1-oxohexyl]-L-proline.

Analogues of (S)-1-[6-amino-2[[hydroxy(4-phenylbutyl)phosphinyl] oxy]-1-oxohexyl]-L-proline (1, SQ 29,852) in which the terminal proline residue has been replaced by a variety of substituted and heteroatom-substituted prolines, N-arylglycines, N-cycloalkylglycines, and bicyclic amino acids have been synthesized and evaluated as inhibitors of angiotensin converting enzyme in vitro and in vivo. In general, the addition of lipophilic substituents to the 4-position of proline of the parent phosphonate 1 resulted in substantial increases in in vitro activity. The largest improvements were observed in the case of cis-benzyl (36-fold) and dithioketal (24-fold) analogues 2r and 2x, respectively. These enhancements of in vitro activity were accompanied by modest increases (2-3.5-fold) in in vivo (iv) activity. Among the various terminal amino acid replacements examined in this study, the indoline-based analogue 2i was by far the most potent compound on iv administration in the normotensive rat.

Angiotensin-Converting Enzyme Inhibitors↗
PubMed1983

Design, synthesis, and testing of antisickling agents. 2. Proline derivatives designed for the donor site.

We have used a three-dimensional model of deoxyhemoglobin to design potential antisickling agents with an intended binding site in the vicinity of the beta-6 mutation (donor site). Two proline derivatives, (4S)-1-butyryl-4-[(carboxymethyl)amino]-L-proline (9a) and its 1-benzoyl analogue (9b), were designed to interact, via ionic or hydrogen bonds, with polar residues beta His-2, beta Thr-4, and beta Lys-132 of hemoglobin S (HbS). Two other proline derivatives containing a salicylate leaving group, (4S)-1-butyryl-4-[(carboxymethyl)methylamino]-L-proline, 2-ester with salicyclic acid (14a), and its 1-benzoyl analogue (14b), were designed to bind covalently to beta Lys-132, as well as to interact with beta His-2 and beta Thr-4 via ionic and hydrogen bonds. This paper describes the synthesis of these agents, beginning with natural L-hydroxyproline methyl ester, and the testing of their ability to increase or decrease the solubility of dHbS by using a standard solubility assay. The covalent derivatives 14a,b were found to be inactive, while the noncovalent compounds 9a,b showed weak antigelling activity, below that observed for phenylalanine. The presence of only weak activity does not invalidate this approach, since only one structural parameter has been investigated.

Antisickling Agents↗
PubMed2005

Puckering transition of 4-substituted proline residues.

The puckering transition of 4-substituted proline residues by electron-withdrawing groups, i.e., 4(R)-hydroxy-L-proline (Hyp) and 4(R)-fluoro-L-proline (Flp) residues, with trans and cis prolyl peptide bonds was studied by adiabatic optimizations along the torsion angle chi1 of the prolyl ring at the HF/6-31+G(d) level. By analyzing the potential energy surface and local minima, it is observed that the puckering transition of the prolyl ring for Hyp and Flp residues proceeds from a down-puckered conformation to an up-puckered one through the transition state with an envelope form having the N atom at the top of envelope and not a planar one for both trans and cis conformers, which is the same as found for the unsubstituted proline residue. At HF/6-31+G(d) and B3LYP/6-311++G(d,p) levels, the structures of the backbone and prolyl ring for local minima of Ac-Hyp-NHMe and Ac-Flp-NHMe are quite similar to those of Ac-Pro-NHMe. However, the relative stability of the up-puckered conformation to the down-puckered one is increased for Ac-Hyp-NHMe with the cis imide bond and for Ac-Flp-NHMe with the trans and cis imide bonds. In particular, the 4(R)-substitution by hydroxy and fluorine groups has brought some structural changes in the prolyl ring of the transition states and the changes in barriers for the puckering transition. The puckering transitions for Ac-Hyp-NHMe and Ac-Flp-NHMe are proven to be predominantly electronically driven by analyzing the electronic and enthalpic contributions to the barriers, as seen for Ac-Pro-NHMe.

Models, Molecular↗
PubMed2005

Structures of cationized proline analogues: evidence for the zwitterionic form.

The structures of lithiated and sodiated alpha-methyl-proline (alpha-Me-Pro) and structural isomers, both with and without a water molecule, are investigated using blackbody infrared radiative dissociation (BIRD) and density functional theory. From the BIRD kinetic data measured as a function of temperature, combined with master equation modeling of these data, threshold dissociation energies for the loss of a water molecule from these clusters are obtained. These energies are 77.5 +/- 0.5 and 53 +/- 1 kJ/mol for lithiated and sodiated alpha-Me-Pro, respectively. For the nonzwitterionic isomer, proline methyl ester, these values are 3.0-4.5 kJ/mol higher. These results provide compelling experimental evidence that alpha-Me-Pro is zwitterionic in these clusters. Theory at the temperature corrected B3LYP/6-311++G**//B3LYP/6-31++G** level indicates that the salt-bridge or zwitterionic forms of lithiated and sodiated alpha-Me-Pro are between 17 and 23 kJ/mol lower in energy than the nonzwitterionic or charge-solvated forms and that attachment of a single water molecule does not significantly change the structure or the relative energies of these clusters. The proton affinity of proline is 8 kJ/mol higher than that of alpha-Me-Pro, indicating that lithiated and sodiated singly hydrated proline should also be zwitterionic.

Anions↗
PubMedDate not supplied

Prolidase, a potential enzyme target for melanoma: design of proline-containing dipeptide-like prodrugs.

Bioinformatics tools such as Perl, Visual Basic, Cluster, and TreeView were used to analyze public gene expression databases in order to identify potential enzyme targets for prodrug strategies. The analyses indicated that prolidase might be a desirable enzyme target based on its differential expression in melanoma cancer cell lines and its high substrate specificity for dipeptides containing proline at the carboxy terminus. RT-PCR expression of prolidase and hydrolytic activity against N-glycyl-l-proline (GLY-PRO), a standard substrate of prolidase, determined in tumor cell lines, exhibited a high correlation (r(2) = 0.95). These results suggest the possibility of targeting prolidase with prodrugs of anticancer agents for enhanced selectivity. The feasibility of such a scenario was tested by (a) synthesizing prodrugs of melphalan that comprised linkage of the carboxy terminus of the l-phenylalanine moiety of melphalan to the N-terminus of l and d stereoisomers of proline and (b) determining their bioconversion and antiproliferative activities in SK-MEL-5 cells, a melanoma cancer cell line with high expression levels of prolidase. The results of hydrolysis studies of the l- and d-proline prodrugs of melphalan, designated as prophalan-l and prophalan-d, respectively, indicated a approximately 7-fold higher rate of activation of prophalan-l compared to prophalan-d in SK-MEL-5 cell homogenates. Prophalan-l exhibited cytotoxicity (GI(50) = 74.8 microM) comparable to that of melphalan (GI(50) = 57.0 microM) in SK-MEL-5 cells while prophalan-d was ineffective, suggesting that prolidase-specific activation to the parent drug may be essential for cytotoxic action. Thus, melphalan prodrugs such as prophalan-l that are cleavable by prolidase offer the potential for enhanced selectivity by facilitating cytotoxic activity only in cells overexpressing prolidase.

Amino Acid Sequence↗
PubMed2003

Origins of opposite absolute stereoselectivities in proline-catalyzed direct Mannich and aldol reactions.

[structure: see text] The first computational studies to elucidate the stereoselectivity of the proline-catalyzed direct Mannich reaction have been performed using density functional theory (B3LYP/6-31G*). The transition states for the proline-catalyzed direct Mannich reaction of the proline enamine of acetone with the N-phenyl imine of acetaldehyde are reported here. The computed transition states provide a general model that explains the origin of the opposite stereoselectivities of proline-catalyzed Mannich and aldol reactions.

Acetaldehyde↗
PubMed2000

Bridging the gap: A set of selective 1H-15N-correlations to link sequential neighbors of prolines.

Triple-resonance experiments are standard in the assignment of protein spectra. Conventional assignment strategies use 1H-15N-correlations as a starting point and therefore have problems when proline appears in the amino acid sequence, which lacks a signal in these correlations. Here we present a set of amino acid selective pulse sequences which provide the information to link the amino acid on either side of proline residues and thus complete the sequential assignment. The experiments yield amino acid type selective 1H-15N-correlations which contain signals from the amino protons of the residues either preceding or following proline in the amino acid sequence. These protons are correlated with their own nitrogen or with that of the proline. The new experiments are recorded as two-dimensional experiments and their performance is demonstrated by application to a 115-residue protein domain.

Amino Acids↗
PubMed2001

DcAGP1, a secreted arabinogalactan protein, is related to a family of basic proline-rich proteins.

A cDNA corresponding to the core protein of an immunoaffinity-purified arabinogalactan protein (AGP) secreted aucus carota (carrot) cells in liquid culture was isolated. This cDNA, DcAGP1, encodes a new class of non-classical' AGP with strong similarity to a family of basic proline-rich proteins. The protein is rich in proline (17%), alanine (10%) and lysine (11%) and contains four distinct domains: a signal peptide, a proline-rich domain, a histidine-rich basic domain and a cysteine-containing 'PAC' domain that is found in a range of other cell wall proteins. The protein contains several sequence motifs found in otherwise unrelated cell wall proteins, but also displays some unique features. Northern blot analyses show that while the DcAGP1 transcript is abundant in the suspension-culture cells from which the AGP was obtained; in carrot seedlings the gene is only expressed at low levels in the roots and it is neither wound- nor stress-inducible. Furthermore, northern and western blot analyses demonstrate that the core polypeptide of DcAGP1 is differentially glycosylated in two different carrot suspension cultures. The unusual features of the protein sequence suggest that the DcAGP1 protein is a member of a family of basic proline-rich proteins defined by the C-terminal PAC domain, and the possible function(s) of the DcAGP1 protein is considered in the light of current views on AGP structure and function.

Amino Acid Sequence↗
PubMed2001

Inhibition of Na+,K+-ATPase activity from rat hippocampus by proline.

Na+,K+-ATPase and Mg2+-ATPase activities were determined in the synaptic plasma membranes from hippocampus of rats subjected to chronic and acute proline administration. Na+,K+-ATPase activity was significantly reduced in chronic and acute treatment by 33% and 40%, respectively. Mg2+-ATPase activity was not altered by any treatment. In another set of experiments, synaptic plasma membranes were prepared from hippocampus and incubated with proline or glutamate at final concentrations ranging from 0.2 to 2.0 mM. Na+,K+-ATPase, but not Mg2+-ATPase was inhibited (30%) by the two amino acids. In addition, competition between proline and glutamate for the enzyme activity was observed, suggesting a common binding site for these amino acids. Considering that Na+,K+-ATPase activity is critical for normal brain function, the results of the present study showing a marked inhibition of this enzyme by proline may be associated with the neurological dysfunction found in patients affected by type II hyperprolinemia.

Animals↗
PubMed1999

Profilin binds proline-rich ligands in two distinct amide backbone orientations.

The actin regulatory protein profilin is targeted to specific cellular regions through interactions with highly proline-rich motifs embedded within its binding partners. New X-ray crystallographic results demonstrate that profilin, like SH3 domains, can bind proline-rich ligands in two distinct amide backbone orientations. By further analogy with SH3 domains, these data suggest that non-proline residues in profilin ligands may dictate the polarity and register of binding, and the detailed organization of the assemblies involving profilin. This degeneracy may be a general feature of modules that bind proline-rich ligands, including WW and EVH1 domains, and has implications for the assembly and activity of macromolecular complexes involved in signaling and the regulation of the actin cytoskeleton.

Amino Acid Sequence↗
PubMed2003

Structure of the proline dehydrogenase domain of the multifunctional PutA flavoprotein.

The PutA flavoprotein from Escherichia coli plays multiple roles in proline catabolism by functioning as a membrane-associated bi-functional enzyme and a transcriptional repressor of proline utilization genes. The human homolog of the PutA proline dehydrogenase (PRODH) domain is critical in p53-mediated apoptosis and schizophrenia. Here we report the crystal structure of a 669-residue truncated form of PutA that shows both PRODH and DNA-binding activities, representing the first structure of a PutA protein and a PRODH enzyme from any organism. The structure is a domain-swapped dimer with each subunit comprising three domains: a helical dimerization arm, a 120-residue domain containing a three-helix bundle similar to that in the helix-turn-helix superfamily of DNA-binding proteins and a beta/alpha-barrel PRODH domain with a bound lactate inhibitor. Analysis of the structure provides insight into the mechanism of proline oxidation to pyrroline-5-carboxylate, and functional studies of a mutant protein suggest that the DNA-binding domain is located within the N-terminal 261 residues of E. coli PutA.

Bacterial Proteins↗
PubMed2005

Preferred stereoselective transport of the D-isomer of cis-4-[18F]fluoro-proline at the blood-brain barrier.

Generally, L-amino acids are preferably transported into mammalian cells compared with their D-isomers, and only L-amino acids are incorporated into proteins. Former studies, however, indicated that D-[H]proline is accumulated in the brain of mice after injection, while L-[3H]proline is not. We investigated the differential cerebral uptake of the D- and L-isomers of the PET tracer cis-4-[18F]fluoroproline (D-/L-cis-FPro) and of D-/L-[3H]proline (D-/L-Pro) in rats by dual tracer autoradiography and the uptake of D-cis-FPro in two human subjects by PET. The standardized uptake value (SUV) of D-cis-FPro in the cerebral cortex of rats 2 h p.i. was 3.05+/-1.18 (n=9) versus 0.06+/-0.01 (n=4) for L-cis-FPro (P<0.001) and 1.29+/-0.27 (n=4) for D-Pro versus 0.30+/-0.14 (n=9) for L-Pro (P<0.001). Analysis of the rat brain tissue after injection of D-cis-FPro (n=3) revealed no radioactivity in the proteins but a relevant part in the form of L-trans-FPro. The PET studies yielded a four- to five-fold higher SUV and influx rate constant in the human cortex for D-cis-FPro than for L-cis-FPro. We conclude that D-cis-FPro and D-Pro are preferably transported at the blood-brain barrier compared with their L-isomers and isomerized to the L-form within the brain. Thus, D-Pro in the plasma might be a source of intracerebral L-proline, which has been shown to act as a modulator of excitatory neurotransmission.

Animals↗
PubMed2003

Characterisation of the active site of a newly-discovered and potentially significant post-proline cleaving endopeptidase called ZIP using LC-UV-MS.

There are enzymes that specifically recognise the amino acid proline within peptides and proteins that are called post-proline cleaving enzymes. Many of them are implicated in neurodegenerative disorders and psychiatric diseases. ZIP is one such newly-discovered peptidase. In this work, it has been purified from bovine serum and subjected to various analytical studies in order to characterise it. A series of reactions between synthesised peptides and ZIP were carried out in order to elucidate the size and specificity of the active site of the enzyme. On-line LC-MS was carried out on samples before and after incubation and the results obtained allowed us to detect if cleavage of the peptides was taking place, and if so, where in the peptide chain. It was found that the enzyme has a preference for another larger, bulky amino acid to follow the proline and that little or no cleavage was observed when polar acidic or other small amino acids were in that location. In terms of the size of the active site, the endopeptidase was found to have optimum activity when there were two more amino acids after the proline, with a fall-off in activity detected for the longer peptides. Data from kinetic studies confirmed the LC-MS results. The methodology described in this paper, which is a combination of LC separation and UV-MS detection, is required for the accurate monitoring of the reactions between the peptidase and its peptide substrates and for analysis of the products of such enzyme-peptide reactions. This work will assist in the design of site-directed inhibitors for new drug therapies.

Carrier Proteins↗
PubMed1974

Age-related variations in hydroxylation of lysine and proline in collagen.

The effect of age on the extent of hydroxylation of lysine and proline both generally and at certain specific sites in collagens from bone, skin and tendon was examined in the chick from the 14-day embryo to the 18-month-old adult. For all collagens there was a marked fall in the overall extent of hydroxylation of lysine with increasing age in both alpha(1) and alpha(2) chains, this fall occurring mostly in a relatively short period immediately after hatching. Hydroxylation of lysine declined to a constant value which, as expected, differed appreciably for each collagen and was considered to be characteristic of the collagen according to its tissue of origin. Hydroxylation of lysine in the N-terminal, non-helical telopeptide region of both alpha(1) and alpha(2) chains, which is important with regard to cross-linking, was relatively high in embryonic collagens. There was, however, a rapid loss of hydroxylation at these sites in skin collagen, occurring both during development of the embryo and in the period immediately after hatching. In contrast some hydroxylation at these sites persisted in bone and tendon collagens and, as judged by examination of peptide alpha(1)-CB1, appeared to reach a constant value in time of about 33% in bone and about 15% in tendon collagen. The actual extent of hydroxylation of lysine in the N-terminal telopeptides and the size of the changes in these values with age appeared to be unrelated to the corresponding whole-chain values, and it is suggested therefore that hydroxylation of telopeptidyl lysine may be under separate enzymic control. The increased hydroxylation of lysine in the embryo was accompanied by only minimal changes in proline hydroxylation, which was very slightly increased in embryonic bone and tendon collagens. Increased hydroxylation of proline in the embryo was, however, readily observed in peptide alpha(1)-CB2 from the helical region of tendon collagen. This hydroxylation was close to the theoretical maximum, in contrast with that observed in post-embryonic tendon, where hydroxylation was incomplete, as in rat tendon (Bornstein, 1967), only four on average, of the six susceptible proline residues being hydroxylated.

Age Factors↗
PubMed1975

Synthesis of proline and hydroxyproline in human lung (WI-38) fibroblasts.

Human lung fibroblasts (WI-38) in late exponential phase of growth, in stationary phase after confluency was reached, and at high or low number of population doublings were used to investigate the synthesis of proline and hydroxyproline from glutamate or arginine. Glutamate was from two to five times as effective a precursor as arginine; glutamine did not seem to be involved in these metabolic pathways. Accumulation of protein-bound hydroxyproline in cell layers was observed only after confluency. Confluent cells synthesized more proline from glutamate than did cells in late exponential growth. Conversion of glutamate into intracellular free proline was conducted also to a greater extent in confluent cells at a high number of population doublings. Conversion of glutamate into proline or hydroxyproline in cell-layer protein was not affected significantly by the number of population doublings. Less total protein as well as less hydroxyproline accumulated with cells at a high number of population doublings.

Arginine↗
PubMed1979

The nature of the hydroxyapatite-binding site in salivary acidic proline-rich proteins.

Protein A and C, which are major components of the acidic proline-rich proteins in human saliva, were digested, before or after adsorption to hydroxyapatite, with alkaline phosphatase, trypsin, thermolysin and a proteinase preparation from salivary sediment. The results demonstrate that the binding site is located in the proline-poor N-terminal part of the protein, possibly between residues 3 and 25. Phosphoserine is necessary for maximal adsorption of the proteins to hydroxyapatite. When proteins A and C are adsorbed to hydroxyapatite before proteolytic digestion there is a protection of some of the susceptible bonds in the N-terminal part of the proteins and a gradual removal of the proline-rich C-terminal part. Thermolysin can cleave susceptible bonds in the part of the protein that remains bound to hydroxyapatite, but at least some of the resulting peptides are retained on the mineral. Since the ability of the proteins to inhibit hydroxyapatite formation and to bind calcium is located in the N-terminal proline-poor part, it is possible that these activities are retained after proteolytic digestion of the adsorbed proteins.

Adsorption↗
PubMed1983

The F1F0-ATPase of Escherichia coli. Substitution of proline by leucine at position 64 in the c-subunit causes loss of oxidative phosphorylation.

The uncE410 allele differs from the normal uncE gene in that C leads to T base changes occur at nucleotides 190 and 191, resulting in proline at position 64 in the c-subunit of the F1F0-ATPase being replaced by leucine. Two partial-revertant strains were isolated in which alanine-20 of the c-subunit was replaced by proline, owing to a G leads to C base change at nucleotide 58. These c-subunits, coded for by the uncE501 and uncE502 alleles, therefore contained two amino acid changes, namely proline-64 leads to leucine, and alanine-20 leads to proline. Membranes prepared from the partial-revertant strains lacked ATP-dependent atebrin-fluorescence-quenching activity but were able to carry out oxidative phosphorylation. The ATPase activity of the F1-ATPase was inhibited when bound to membranes from strains carrying the uncE410, uncE501 and uncE502 alleles. It is concluded that a bend in the helix axis in one of the arms of the c-subunit hairpin structure is required for integration of the c-subunit into a functional F1F0-ATPase.

Adenosine Triphosphatases↗