Carboxyl Group Of Glutamic Acid Research Articles

Liquid chromatography of short chain carboxylic acids using a glutamic acid surfactant coated C18 stationary phase

A C18 column was modified with the anionic amino acid surfactant lauroyl-l-glutamate (LLG) to facilitate the separation of ten short-chain aliphatic carboxylic acids (oxalic, tartaric, malic, malonic, lactic, acetic, maleic, citric, fumaric, and succinic). The developed method was proven to be fast, versatile, and environmentally friendly. After the coating of the column using 1% LLG solution and optimizing chromatographic conditions such as pH and temperature, near baseline resolution of the ten carboxylic acids within 4 min with excellent peak shape at pH = 1.8 using 100% H2O acidified with sulfuric acid was possible. Although the design of this stationary phase, with the hydrophilic group at the end of the alkyl chain, seems to be in contrast to such columns designed for a totally aqueous mobile phase that have a polar (often amide) group embedded near the silica surface, no evidence of phase collapse was noted. Linear relationships of ln retention factor (k) versus 1/Temperature (T) (van't Hoff plots) were generated for all the acids indicating a single retention mechanism was likely. As the pH of the mobile phase decreased, the analyte retention factors increased due to the increase of the fraction of the analyte with neutral charge (alpha zero). The surfactant amide linkage, being electron donating, increased the pKa of the more acidic carboxyl group of glutamic acid so both carboxyl groups were protonated (neutral) at pH 1.8. The exact nature of the retention mechanism is uncertain but there certainly seems to be a pronounced hydrophobic component due to the large difference in retention of fumaric acid and methyl fumarate at pH 1.8. In addition, eleven beverage samples were analyzed for their aliphatic carboxylic acid contents. The results showed that malic, fumaric, and citric acids were the most common carboxylic acids in natural beverages with concentrations as high as 6432 ppm of malic acid in organic apple juice, 64 ppm of fumaric acid in organic concord juice, and 6543 ppm citric acid in strawberry lemonade juice.

Glutamyl Endopeptidases: The Puzzle of Substrate Specificity

Glutamyl endopeptidases (GEPases) are chymotrypsin-like enzymes that preferentially cleave the peptide bonds of the α-carboxyl groups of glutamic acid. Despite the many years of research, the structural determinants underlying the strong substrate specificity of GEPases still remain unclear. In this review, data concerning the molecular mechanisms that determine the substrate preference of GEPases is generalized. In addition, the biological functions of and modern trends in the research into these enzymes are outlined.

Acylpeptides, the inhibitors of cyclic adenosine 3',5'-monophosphate phosphodiesterase. III. Inhibition of cyclic AMP phosphodiesterase.

Acylpeptides, APD-I, -II and -III, were inhibitors of cyclic adenosine 3',5'-monophosphate (cAMP) phosphodiesterase, and their inhibition types were non-competitive. The inhibitory activity of APD-II was the most potent among them. Opening of the lactone linkage reduced the inhibitory activity to about half. The activity almost disappeared when an inhibitor or a derivative with opened lactone linkage was methylated with diazomethane. The activity was, however, restored by the addition of metal ions such as Ca2+, Mn2+, Fe2+, and Co2+. This suggests that the inhibition may be caused by a chelating action of the free carboxyl groups of glutamic acid and aspartic acid in the peptide.

Covalent modification of fourth component of human complement with primary amines. Binding studies with metastable fragment C4b and with C4.

A C4-dependent hemolytic complement assay and competitive binding assays with [1,4(n)-3H]putrescine or [14C]methylamine were used to determine the structural preferences of C4 and of metastable C4 for covalent modification with a series of alkyl primary amines. The pKa values of individual amines did not correspond with their ability to inactivate the hemolytic function of C4. The rank order of effectiveness did correlate with the molecular weight and conformation of the organic amines tested. In contrast to results with C4, metastable C4b displays a general increased susceptibility for modification by C3 and C4 alkyl amines. However, C4b exhibits a distinct preference for diamines, putrescine and 1,3-diaminopropane, over monoamines of the same alkyl chain length, s-butylamine and n-propylamine. Taken together, these studies provide the first direct evidence for a conformational change in the thioester region of C4 upon proteolytic activation to metastable C4b. A model is proposed to explain the results of competitive binding experiments with metastable C4b in terms of two binding sites for amines on C4b, the presumptive thioester and a second site such as the free side chain carboxyl group of glutamic acid. It is suggested that all amines except methylamine bind preferentially to the latter site; once bound, only diamines would still be capable of mounting a nucleophilic attack on the thioester bond with the second amino group.

Action of glutamate and aspartate analogues on rod horizontal and bipolar cells.

Electrophysiological evidence indicates that transmitter release from retinal rod terminals occurs at a high rate in the dark and is reduced by light1–6. The rod transmitter closes ionic channels (mainly sodium channels) in the subsynaptic membrane of a class of bipolar cells (on-centre bipolar cells) which depolarize in response to light4,7–9. However, the transmitter opens sodium channels in the subsynaptic membrane of horizontal cells4–6,9,10. We report here that several compounds, chemically related to the amino acids aspartate and glutamate, have similar post-synaptic actions to that of the rod neurotransmitter. Kainic acid11, in micromolar concentration, hyperpolarizes rod on-centre bipolar cells, increasing their membrane resistance while depolarizing rod horizontal cells. Experiments with 2-amino-4-phosphonobutyric acid (APB), in which a phosphono group is substituted for a carboxyl group of glutamic acid, have further distinguished between different binding sites on the cells which make synaptic contact with rods. APB is an agonist at the on-centre bipolar cells, closing the same ionic channels as the rod neurotransmitter, but is without effect on rod horizontal cells. The evidence presented here suggest that the widely held view that glutamate or aspartate is the rod neurotransmitter needs to be re-examined.

Préparation enzymatique de peptides du type (L)meso-diaminopimélyl (L)-D-Ala[ 14C] : par réactions d'échange entre les peptides correspondants non radioactifs et la D-alanine [ 14C

Membrane bound LD-transpeptidase of Streptococcus faecalis ATCC 9790 was used to catalyse transpeptidation reactions between non radioactive peptide donors of the general type: L-Ala-D-Glu (L)meso-A2pm(L)-D-Ala and D-(14C) alanine acceptor. The presence of one or two amide residues on the carboxyl groups of glutamic acid and meso-diaminopimelic acid increases the transfer reactions and subsequently the yield in radioactive peptides L-Ala-D-Glu (L)meso-A2pm(L)-D-(14C) Ala.

A re-examination of the conformation of secretin in water.

An examination of a series of peptides corresponding to partial sequences of secretin and the application of empirical conformational parameters to the sequence has reopened the question about the position of the short helical stretch in the folded chain. In earlier studies, this was tentatively placed near the N-terminus, while newly accumulated evidence points to the C-terminal area. The possible role of ion pairs in the stabilization of the folding was investigated by the preparation and examination of synthetic analogues of the C-terminal tricosapeptide part of secretin. The carboxyl groups of glutamic acid (in position 9) and of aspartic acid (in position 15) were replaced by carboxamides. The 9-glutamine and the 15-asparagine analogues show a significant decrease in helical character. This loss of "structure" is even more pronounced in the 9-glutamine-15-asparagine tricosapeptide. Thus, ion-pair formation is indeed implicated as one of the forces which stabilize the folded conformation of the chain. Possible correlations between biological activity and secretin-like architecture were studied on several smooth muscle preparations.

Chemical evidence for a functional carboxyl group in carboxypeptidase A

Modification of carboxypeptidase A with 1-cyclohexyl-3-(2-morpholinoethyl)-carbodiimide metho-p-toluene sulfonate at pH 6.0 simultaneously abolishes both esterase and peptidase activities. Inactivation is due to a specific reaction with a single residue in the active center of the enzyme most probably the carboxyl group of glutamic acid 270, previously implicated in the catalytic mechanism by X-ray crystallography.

THE METABOLISM OF PROPIONATE BY WHEAT STEM RUST UREDOSPORES

When uredospores of wheat stem rust were shaken for 3 hours with phosphate buffer (pH 6.2) containing propionate-1-C14, -2-C14, or -3-C14, about 55% of the carbon-14 was removed from the solution. With propionate-1-C14, most of the carbon-14 taken up was released as carbon dioxide-C14, whereas about 20% and 31% of propionate carbon 2 and carbon 3, respectively, was incorporated into the spores. The specific activity of a fraction consisting of the free amino acids of a hot-alcohol and hot-water extract of the spores increased markedly with increase in the position number of propionate in which the carbon-14 was located. A similar relation was observed for other fractions such as soluble carbohydrates, ether-soluble material, organic acids, and insoluble residue from spores. The most active amino acids isolated were glutamic acid, γ-aminobutyric acid, and alanine. Partial degradations showed that with propionate-2-C14the carboxyl groups of glutamic acid were especially radioactive, whereas with propionate-3-C14the internal carbons were most radioactive.It is concluded that propionate metabolism in the rust spores involved conversion of carbon 1 to carbon dioxide, and utilization of carbons 2 and 3 as acetate with carbon 2 behaving as the carboxyl carbon.

THE METABOLISM OF PROPIONATE BY WHEAT STEM RUST UREDOSPORES

When uredospores of wheat stem rust were shaken for 3 hours with phosphate buffer (pH 6.2) containing propionate-1-C14, -2-C14, or -3-C14, about 55% of the carbon-14 was removed from the solution. With propionate-1-C14, most of the carbon-14 taken up was released as carbon dioxide-C14, whereas about 20% and 31% of propionate carbon 2 and carbon 3, respectively, was incorporated into the spores. The specific activity of a fraction consisting of the free amino acids of a hot-alcohol and hot-water extract of the spores increased markedly with increase in the position number of propionate in which the carbon-14 was located. A similar relation was observed for other fractions such as soluble carbohydrates, ether-soluble material, organic acids, and insoluble residue from spores. The most active amino acids isolated were glutamic acid, γ-aminobutyric acid, and alanine. Partial degradations showed that with propionate-2-C14the carboxyl groups of glutamic acid were especially radioactive, whereas with propionate-3-C14the internal carbons were most radioactive.It is concluded that propionate metabolism in the rust spores involved conversion of carbon 1 to carbon dioxide, and utilization of carbons 2 and 3 as acetate with carbon 2 behaving as the carboxyl carbon.

Pepsinogen and Pepsin

Evidence relating to the structure and properties of swine pepsinogen and pepsin has been reviewed and used to suggest a tentative two dimensional picture of the skeleton of these two proteins. When pepsinogen, a folded single peptide chain, is converted to pepsin, there is a profound change in the physical and chemical properties of the protein. In an as yet unknown manner, except that it is initiated by a peptic cleavage of the protein chain, a single enzymic site is formed. This site is made up, quite probably, of the secondary carboxyl group of glutamic acid or of aspartic acid and a tyrosine phenol group in close proximity so that they can form hydrogen or hydrophobic bonds with the substrate in some unique manner that permits hydrolysis to occur at an accelerated rate.