Specificity of Proteinase K from Tritirachium album Limber for Synthetic Peptides

The specificity of proteinase K from Tritirachium album Limber was determined using various synthetic peptide substrates. The esterase activity against N-acylated amino acid esters indicated that the enzyme is primarily specific against aromatic or hydrophobic amino acid residues at the carboxyl side of the splitting point. Secondary interaction for hydrolysis was also studied using peptide esters or others, which showed that the enzyme activity is markedly promoted by elongating the peptide chain to the N-terminal from the splitting point. Thus, peptide chloromethyl ketone derivatives such as Cbz-Ala-Gly-PheCH₂Cl inactivated the enzyme activity markedly.     

Enzymatic methylation of band 3 anion transporter in intact human erythrocytes

Band 3, the anion transport protein of erythrocyte membranes, is a major methyl-accepting substrate of the intracellular erythrocyte protein carboxyl methyltransferase (S-adenosyl-L-methionine: protein-D-aspartate O-methyltransferase; EC 2.1.1.77) [Freitag, C., & Clarke, S. (1981) J. Biol. Chem. 256, 6102-6108]. The localization of methylation sites in intact cells by analysis of proteolytic fragments indicated that sites were present in the cytoplasmic N-terminal domain as well as the membranous C-terminal portion of the polypeptide. The amino acid residues that serve as carboxyl methylation sites of the erythrocyte anion transporter were also investigated. 3H-Methylated band 3 was purified from intact erythrocytes incubated with L-[methyl-3H]methionine and from trypsinized and lysed erythrocytes incubated with S-adenosyl-L-[methyl-3H]methionine. After proteolytic digestion with carboxypeptidase Y, D-aspartic acid beta-[3H]methyl ester was isolated in low yields (9% and 1%, respectively) from each preparation. The bulk of the radioactivity was recovered as [3H]methanol, and the amino acid residue(s) originally associated with these methyl groups could not be determined. No L-aspartic acid beta-[3H]methyl ester or glutamyl gamma-[3H]methyl ester was detected. The formation of D-aspartic acid beta-[3H]methyl esters in this protein in intact cells resulted from protein carboxyl methyltransferase activity since it was inhibited by adenosine and homocysteine thiolactone, which increases the intracellular concentration of the potent product inhibitor S-adenosylhomocysteine, and cycloleucine, which prevents the formation of the substrate S-adenosyl-L-[methyl-3H]methionine.     

In Vacuo Esterification of Carboxyl Groups in Lyophilized Proteins

A new method is described for the esterification of carboxyl groups in proteins by reaction of the lyophilized protein in vacuo with gaseous alcohol and HCI catalyst. Carboxyl groups are rapidly esterified with no protein degradation. 13C-Methyl or 13C-ethyl esters of the alpha-, gamma- and delta-carboxyl groups could be distinguished by the distinct chemical shifts of their resonances. Within the class of gamma- or delta-esters, the chemical shifts have little variation; however, the chemical shift of a C-terminal esterified alpha-carboxyl group shows a strong dependence on the nature of the C-terminal amino acid and sequence. Iodomethane reacts with deprotonated carboxyl groups in lyophilized proteins to form methyl esters, but unlike the reaction with gaseous methanol/HCI, it does not selectively methylate carboxyl groups. The procedure permits the cost-effective incorporation of isotopic labels and provides a new approach using 13C-NMR spectroscopy for determining the number of different C-termini present in a protein preparation.     

Deprotonation of hydrochloride salts of amino acid esters and peptide esters using commercial zinc dust.

The deprotonation of hydrochloride salts of ethyl and methyl esters of amino acids and peptides is accomplished using activated zinc dust. The reaction is neat and quantitative. Thus, the free amino acid esters and peptide esters have been isolated in good yield and purity.     

A novel regulatory mechanism for amino acid absorption in lepidopteran larval midgut

A number of methyl and ethyl esters of naturally occurring amino acids exert a potent stimulatory effect on the cotransport system responsible for the absorption of most essential amino acids along the midgut of the silkworm Bombyx mori. L-Leucine methyl ester (Leu-OMe), one of the most effective activators, induces a large increase of the initial rate of leucine uptake in midgut brush border membrane vesicles (BBMV) from the anterior-middle (AM) region, and a small effect in BBMV from the posterior (P) region. Nonetheless, the methyl ester causes in both regions a relevant K(+)-, Deltapsi- and pH-independent increase of the intravesicular accumulation of the amino acid. The activation by Leu-OMe proves that amino acid absorption can be modulated all along the B. mori larval midgut and that the AM region, where the ability to transport and concentrate the substrate is very low, is more susceptible than the P region. Leucine uptake in AM-BMMV can be activated by amino acid methyl esters with definite structural requisites, with the following order of potency: L-leucine>L-phenylglycine>L-methionine>L-phenylalanine>L-norleucinez.Gt;L-isoleucine. The activation is stereospecific and occurs also with some ethyl esters (e.g. leucine and phenylalanine). No activation was observed with esters of amino acids with short hydrophobic or polar side-chains. The activation mechanism here described plays a fundamental role in larval growth since silkworms reared on artificial diets supplemented with leucine or methionine methyl esters reach maximum body weight 12-18 h before control larvae and spin cocoons with a larger shell weight. This novel regulatory mechanism of an amino acid transport protein appears to be widespread among lepidopteran larvae.     

Specificity of alpha-chymotrypsin with exposed carboxyl groups blocked.

The 15 exposed carboxyl groups of alpha-chymotrypsin were modified with glycine ethyl ester at low pH using barbodiimide reagent. The specificity of the modified enzyme (Chy-15) was studied over the pH range of 4 to 9 with both N-acylated and non-N-acylated amino acid esters. The modified enzyme had lower reactivity toward N-acylated esters than non-N-acylated esters compared to the native enzyme. Typical substances such as acetyl- and benzoyl-L-tyrosine ethyl esters retained 4 and 9% activity, whereas phenylalanine ethyl ester was slightly more reactive with the modified than with the native enzyme. The pH-rate profiles of acetyl-L-phenylalanine ethyl ester and tryptophan ethyl and benzyl esters were investigated in detail. Analysis of these profiles revealed three pKa values of approximately 5, 7, and 9 related to a functional carboxyl, imidazoyl, and an amino group, respectively. Since similar pKa values occur for the native enzyme, modification did not block the carboxyl corresponding to pKa 5. A mechanism is proposed for catalysis which includes both the protonated and unprotonated form of the imidazoyl (His-57) and utilizes water rather than a carboxyl (Asp-102) as the proton sink.     

Amino acid sequencing by gas chromatography-mass spectrometry using perfluoro-dideuteroalkylated peptide derivatives: B. Interpretation of the mass spectra

The mass spectra of the O-trimethylsilylated trifluoro-dideuteroethyl polyamino alcohols, produced by LiAlD₄-reduction and O-trimethylsilylation of N-trifluororacetyl oligopeptide methyl esters, are evaluated. Characteristic mass spectra of derivatives are shown which are derived from peptides containing all protein amino acids including Arg, His, Trp, Gln, Asn and carboxyl terminal amides as well as modified Cys-residues. The mass spectra of these derivatives can be easily interpreted in terms of the amino acid sequence of the original peptides since they contain abundant and intensity-balanced sequence-determining ions.     

Uptake of basic amino acids and peptides into liposomes in response to transmembrane pH gradients.

The uptake of derivatives of lysine and a pentapeptide (ala-met-leu-trp-ala) into large unilamellar vesicle (LUV) systems in response to transmembrane pH gradients has been examined. In these derivatives, the C-terminal carboxyl functions have been converted to methyl esters or amides. It is shown that the presence of a pH gradient (interior acidic) results in the rapid and efficient accumulation of these weak base amino acid and peptide derivatives into LUVs in a manner consistent with permeation of the neutral (deprotonated) form. It is suggested that this property may have general implications for mechanisms of transbilayer translocation of peptides, such as signal sequences, which exhibit weak base characteristics.     

9-Fluorenylmethyl esters

N alpha-Protected amino acid 9-fluorenylmethyl esters (Fm esters) were prepared by imidazole-catalyzed transesterification of active esters with 9-fluorenylmethanol (9-hydroxymethylfluorene). The new carboxyl protection is unaffected by acids, but is efficiently removed by beta-elimination under the influence of secondary and tertiary amines. Primary amines and ammonia can cause slight amide formation. Deblocking was achieved also by catalytic hydrogenation.     

Amplification and detection of substrates for protein carboxyl methyltransferases in PC12 cells.

A strategy that facilitates the identification of substrates for protein carboxyl methyltransferases that form "stable" methyl esters, i.e., those that remain largely intact during conventional polyacrylamide gel electrophoresis is described. Rat PC12 cells were cultured in the presence of adenosine dialdehyde (a methylation inhibitor) to promote the accumulation of hypomethylated proteins. Nonidet P-40 cell extracts were then incubated in the presence of S-[methyl-3H]adenosyl-L-methionine to label methyl-accepting sites via endogenous methyltransferases. After labeled proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, gel slices were incubated in 4 N methanesulfonic acid or 6 N HCl to hydrolyze methyl esters. The resulting [3H]methanol was detected by trapping in liquid scintillation fluid. Seven carboxyl methylated proteins were observed with masses ranging from 18 to 96 kDa. Detection of five of these proteins required prior treatment of cells with adenosine dialdehyde, while methyl incorporation into one protein at 18 kDa was substantially enhanced by the treatment. The use of acidic conditions for methyl ester hydrolysis has an important advantage over assays that utilize alkaline hydrolysis conditions. In PC12 cells, and possibly other cell types where there are significant levels of arginine methylation, the methanol signal becomes obscured by high levels of volatile methylamines generated under the alkaline conditions. Carrying out diffusion assays under acidic conditions eliminates this interference. Adenosine dialdehyde, by virtue of increasing the methyl-accepting capacity of substrates for protein carboxyl methyltransferases, in combination with a more selective assay for carboxyl methylation, should prove useful in the isolation and characterization of new protein carboxyl methyltransferases and their substrates.     

The carboxylic acid groups of bovine luteinizing hormone. The effects of their modification on receptor site binding and subunit-subunit interaction.

The modification of the carboxyl groups of the subunits of bovine luteinizing hormone to neutral derivatives by carbodiimide-mediated coupling with glycine methyl ester has been studied. The modified alpha subunit, which has 8 residues of glycine methyl ester incorporated, will no longer recombine with native beta (hormone-specific) subunit, but the modified beta subunit, with 6 to 7 glycine methyl esters incorporated, will recombine with native alpha to yield a partially active hormone. Derivatization of the intact hormone results in dissociation to subunits together with formation of a major side product which is covalently cross-linked. Significant cross-linked product was not obtained during modification of individual subunits, thus indicating an orientation between an activated carboxyl group(s) and a nucleophile(s) in the intact hormone which favors coupling. Separation of subunits from the derivatized, noncross-linked fraction by countercurrent distribution reveals a heterogeneous preparation of the modified alpha subunit which also will not recombine with either a native or modified beta subunit. The beta subunit from the modified intact hormone was indistinguishable from the modified isolated beta subunit in amino acid composition and in ability to recombine with native alpha subunit. The results are consonant with data from this and other laboratories in which various modifications of the alpha chain, the subunit common to the glycoproteins, more seriously affect recombination than similar modifications of the beta subunits. The number of carboxyl groups modified in each subunit is compatible with but not in total agreement with assignments of amides reported from sequence studies.     

Synthesis and solid state 13C and 1H NMR analysis of new oxamide derivatives of methyl 2-amino-2-deoxy-alpha-D-glucopyranoside and ester of amino acids or dipeptides

The syntheses of new oxamide derivatives of methyl 2-amino-2-deoxy-alpha-D-glucopyranoside and amino acid or peptide esters are presented. The reaction of methyl 3,4,6-tri-O-acetyl-2-acetamido-2-deoxy-alpha-D-glucopyranoside and oxalyl chloride gave N-(methyl 3,4,6-tri-O-acetyl-2-deoxy-alpha-D-glucopyranosid-2-yl) oxamic acid chloride which on reaction with the ester of Gly, L-Ala, L-Phe, GlyGly, Gly-L-Phe and Gly-L-Ala afforded N-(methyl 3,4,6-tri-O-acetyl-2-deoxy-alpha-D-glucopyranosid-2-yl), N'-oxalyl-amino acid or dipeptide esters. The structure of the oxamides was studied using 1H, 13C NMR in solution and solid state.     

Identification of ornithine and arginine conjugates of cholic acid by mass spectrometry.

Nalpha-Cholylornithine, -arginine, and -histidine were prepared according to a method previously employed for the chemical synthesis of the monoamino acid conjugates of bile acids. The products were shown to involve the alpha amino group of the dibasic amino acids by examination of the mass spectra of the original compounds, their lactams, their methyl esters and the methyl ester acetates. Only the methyl ester acetates gave detectable amounts of molecular ion. The free acids and the methyl esters of Nalpha-cholylornithine and -arginine gave identical lactams upon sublimation from the direct insertion probe. The synthetic Nalpha-cholylarginine was shown to yield a mass spectrum identical to that of an arginocholic acid recovered from the bile of an isolated perfused rat liver.     

Synthesis and pharmacological activity of amides and ozonolysis product of maleopimaric acid

The synthesis of a new group of maleopimaric acid amides containing fragments of the methyl esters of amino acids, aliphatic amines, imidazole and N-methylpiperazine was carried out. Ozonolysis of methyl maleopimarate flows through the cleavage of double bond C18(19) and the disclosure of anhydrous cycle with formation of secotriacid. As a result of screening of anti-inflammatory and antiulcer activity of maleopimaric acid derivatives new effective compounds such as methyl esters of maleopimaric acid and product of ozonolysis - diterpenic secotriacid, maleopimaric acid amide with L-leucine were revealed. An important advantage of the compounds studied is the low toxicity and the presence of bidirectional activity in the absence of adverse effects on the animal.     

Novel S-adenosyl-L-methionine:salicylic acid carboxyl methyltransferase,an enzyme responsible for biosynthesis of methyl salicylate and methyl benzoate,is not involved in floral scent production in snapdragon flowers

Using a functional genomic approach we have isolated and characterized a cDNA that encodes a salicylic acid carboxyl methyltransferase (SAMT) from Antirrhinum majus. The sequence of the protein encoded by SAMT has higher amino acid identity to Clarkia breweri SAMT than to snapdragon benzoic acid carboxyl methyltransferase (BAMT) (55 and 40% amino acid identity, respectively). Escherichia coli-expressed SAMT protein catalyzes the formation of the volatile ester methyl salicylate from salicylic acid with a K(m) value of 83 microM. It can also methylate benzoic acid to form methyl benzoate, but its K(m) value for benzoic acid is 1.72 mM. Snapdragon flowers do not emit methyl salicylate. The potential involvement of SAMT in production and emission of methyl benzoate in snapdragon flowers was analyzed by RNA gel blot analysis. SAMT mRNA was not detected in floral tissues by RNA blot hybridization, but low levels of SAMT gene expression were detected after real-time RT-PCR in the presence of SAMT-specific primers, indicating that this gene does not contribute significantly, if at all, in methyl benzoate production and emission in snapdragon flowers. Expression of SAMT in petal tissue was found to be induced by salicylic and jasmonic acid treatments.     

Synthesis and Immunomodulating Activity of New Amino Acid Derivatives of Glycyrrhizic Acid and Its Methyl Ester

New amino acid derivatives of glycyrrhizic acid and its methyl ester were selectively synthesized using active N-succinimide esters. The compounds with residues of glycine ethyl ester and alanine methyl and butyl esters increased the level of agglutinins and hemolysins in blood serum of mice two- to threefold in comparison with the control upon parenteral administration at a dose of 2 mg/kg for 14 days.     

Interaction of cartilage proteoglycans with hyaluronic acid. The role of the hyaluronic acid carboxyl groups.

Hyaluronic acid-derived oligomers of five to fifteen repeat dissaccharides effectively bind to bovine nasal-cartilage proteoglycan and inhibit the interaction between proteoglycans and high-molecular-weight hyaluronic acid. If, however, the hyaluronic acid oligosaccharides are modified by reaction with diazomethane to form the carboxyl methyl esters of the glucuronic acid residues, their inhibitory activity is abolished. The binding capacity can be fully restored by saponification. The amide derivative, which is formed by condensation of the oligosaccharide carboxyl groups with glycine methyl ester, is also ineffective in blocking the proteoglycan-hyaluronic acid interaction. In this case, binding activity is not restored when the amidated oligomers are subjected to saponification to yield the free carboxylate groups on the glycine residues. Thus the displacement of the carboxylate groups on the polysaccharide chain by the interposition of a glycine residue blocks the interaction between the proteoglycans and the hyaluronic acid oligomers. When the oligosaccharide methyl ester is reduced with NaBH4, the resultant glucose-containing oligomers exhibit decreased binding to proteoglycans. Thus it appears that the hyaluronic acid carboxylate anion in a specific spatial orientation is required for hyaluronic acid-proteoglycan interaction.     

Synthesis and biological activity of some new 3,6-dinitro-1:8-naphthaloyl- and 3,6-diamino-1:8-naphthaloylamino acids and dipeptide derivatives

Synthesis of a series of 3,6-dinitro-1:8-naphthaloylamino acids (II-IX) and some of their corresponding methyl esters (X-XVI) and 3,6-diamino-1:8-naphthaloylamino acid derivatives (XXIX-XXXVI) is described. Coupling of 3,6-dinitro-1:8-naphthaloylamino acids with amino acid methyl ester hydrochlorides in dioxane-DMF-Et3N medium using DCC method furnishes the desired 3,6-dinitro-1:8-naphthaloyldipeptide methyl esters (XVII-XXVIII). Most of the synthesized 3,6-dinitro-1:8-naphthaloylamino acids, esters and dipeptide derivatives (compounds III-VI, XI-XV, XVII, XIX-XXI, XXIII and XXV) and 3,6-diamino-1:8-naphthaloylamino acid derivatives (XXIX-XXXV) were found to be active against a number of microorganisms.     

Selective carboxyl methylation of structurally altered calmodulins in Xenopus oocytes

The eucaryotic protein carboxyl methyltransferase specifically modifies atypical D-aspartyl and L-isoaspartyl residues which are generated spontaneously as proteins age. The selectivity of the enzyme for altered proteins in intact cells was explored by co-injecting Xenopus laevis oocytes with S-adenosyl-L-[methyl-3H]methionine and structurally altered calmodulins generated during a 14-day preincubation in vitro. Control experiments indicated that the oocyte protein carboxyl methyltransferase was not saturated with endogenous substrates, since protein carboxyl methylation rates could be stimulated up to 8-fold by increasing concentrations of injected calmodulin. The oocyte protein carboxyl methyltransferase showed strong selectivities for bovine brain and bacterially synthesized calmodulins which had been preincubated in the presence of 1 mM EDTA relative to calmodulins which had been preincubated with 1 mM CaCl2. Radioactive methyl groups were incorporated into base-stable linkages with recombinant calmodulin as well as into carboxyl methyl esters following its microinjection into oocytes. This base-stable radioactivity most likely represents the trimethylation of lysine 115, a highly conserved post-translational modification which is present in bovine and Xenopus but not in bacterially synthesized calmodulin. Endogenous oocyte calmodulin incorporates radioactivity into both carboxyl methyl esters and into base-stable linkages following microinjection of oocytes with S-adenosyl-[methyl-3H]methionine alone. The rate of oocyte calmodulin carboxyl methylation in injected oocytes is calculated to be similar to that of lysine 115 trimethylation, suggesting that the rate of calmodulin carboxyl methylation is similar to that of calmodulin synthesis. At steady state, oocyte calmodulin contains approximately 0.0002 esters/mol of protein, which turn over rapidly. The results suggest the quantitative significance of carboxyl methylation in the metabolism of oocyte calmodulin.     

Chiral calix[4]arenes bearing amino alcohol functionality as membrane carriers for transport of chiral amino acid methylesters and mandelic acid

Novel chiral calix[4]arene derivatives bearing amino alcohol moieties at the lower rim have been synthesized from the reaction of p-tert-butylcalix[4]arene diester with various amino alcohols. The transport of amino acid esters (phenylglycine, phenylalanine, and tryptophan methyl esters hydrochloride) and mandelic acid were studied through chloroform bulk liquid membrane system using chiral calix[4]arenes 15-20. All these receptors have been found to act as carriers for transport of aromatic amino acid methylesters and mandelic acid from the aqueous source phase to the aqueous receiving phase. The influence of calixarene and guest structures upon transport through liquid membrane is discussed.