Effect of amino acids on collagen biosynthesis

Summary The effect of various amino acids on collagen biosynthesis was studied in organ cultures of chicken embryo tibiae. Competitive interrelationship between selected amino acids influences independently the uptake of proline and lysine, precursors of hydroxyproline and hydroxylysine, respectively, which are the two amino acids characteristics of collagen. On the basis of these influences, the possibility of biosynthesis of anomalous collagens is stressed. Parallel studies of biosynthesis of hydroxyproline and hydroxylysine are necessary.     

Rapid robust separation of hydroxyproline and proline.

The presence of hydroxyproline, and the determination of the ratio of the secondary amino acids proline to hydroxyproline, in amino acid hydrolysates specifically identifies collagen and collagen peptides. o-Phthalaldehyde, and then 9-fluorenylmethyl chloroformate, were used to carry out sequential prederivatization of amino acid hydrolysates in an in-line high-performance liquid chromatography sample loop. After derivatization, reversed-phase high-performance liquid chromatography with a C-18 ODS Hypersil cartridge column was used to resolve the hydroxyproline and proline from all primary amino acids, with resolution and detection of hydroxyproline and proline within 2.0 and 2.8 min, respectively, at concentrations in the range of picomoles per microliter of derivatized amino acid. The assay has a turnaround time of 10.75 min.     

Hydroxylation of peptide-bound proline and lysine before and after chain completion of the polypeptide chains of procollagen

The synthesis of procollagen hydroxyproline and hydroxylysine was examined in matrix-free cells which were isolated from embryonic tendon by controlled enzymic digestion and then incubated in suspension. After the cells were labeled with [¹⁴C]proline for 2 min, or about one-third the synthesis time for a Pro-α chain, [¹⁴C]hydroxyproline was found in short peptides considerably smaller than the Pro-α chains of procollagen. The results, therefore, confirmed previous reports indicating that the hydroxylation of proline can begin on nascent chains. In similar experiments in which the cells were labeled with [¹⁴C]lysine, [¹⁴C]hydroxylysine was found in short, newly synthesized peptides, providing the first evidence that the hydroxylation of lysine can also begin on nascent peptides. However, further experiments demonstrated that the synthesis of hydroxyproline and hydroxylysine continues until some time after assembly of the polypeptide chains is completed.     

The structure of l-CB3, a cyanogen bromide fragment from the central portion of the l chain of rat collagen. The tryptic peptides from skin and dentin collagens

The mixture of peptides released by tryptic hydrolysis of the collagen CNBr peptide, αl-CB3, has been resolved by ion-exchange chromatography. The resultant eleven tryptic peptides ranged in size from 3 to 46 amino acids and accounted for all the amino acids of the parent CNBr peptide. Two of the lysines in αl-CB3 from rat dentin collagen were shown to be hydroxylated to a substantial degree by isolation of the appropriate hydroxylysine-containing tryptic peptides. An analysis of the tryptic peptides indicated that αl-CB3 from dentin collagen is identical in structure to that from skin collagen, if hydroxylysine and hydroxyproline are considered equivalent to lysine and proline, respectively.     

Adducts between nucleophilic amino acids and hexahydrophthalic anhydride,a structure inducing both types I and IV allergy

Haptens causing type I allergy have been shown to predominantly form lysine adducts in the carrier protein, while many haptens giving rise to type IV allergy preferentially form adducts with cysteine residues. Hexahydrophthalic anhydride derivatives are strong sensitizers capable of inducing allergic rhinitis, asthma and urticaria (type I allergy) and allergic contact dermatitis (type IV allergy). The ability of hexahydrophthalic anhydride (HHPA) to form adducts with nucleophilic amino acids and a model peptide in vitro is presented. Adduct formation was monitored by high-performance liquid chromatography with ultraviolet light/vis detection (LC-UV/vis) and high-performance liquid chromatography with mass spectrometric detection (LC/MS). The characterization was obtained by nuclear magnetic resonance spectroscopy (NMR) and mass spectrometry (MS and MS/MS). It was found that HHPA formed adducts with N^α-acetylated lysine and cysteine and the non-acetylated α-amino group of proline and, to some extent, also with other nucleophilic amino acids. The adducts with lysine and proline were chemically stable. Addition of one HHPA to a model carrier peptide with all important nucleophilic amino acid residues showed N-terminal proline to be the major site of reaction. The addition of a second hapten gave a lysine adduct, but a minor cysteine adduct was also found. The cysteine–HHPA adducts were shown to be chemically unstable and participated in further reactions with lysine forming lysine–HHPA adducts. The results will be useful for understanding the formation of HHPA–protein adducts with the capability of being markers of exposure, and also to a deeper understanding of the chemical structures causing types I and IV allergy.     

Molecular insights into prolyl and lysyl hydroxylation of fibrillar collagens in health and disease

Collagen is a macromolecule that has versatile roles in physiology, ranging from structural support to mediating cell signaling. Formation of mature collagen fibrils out of procollagen α-chains requires a variety of enzymes and chaperones in a complex process spanning both intracellular and extracellular post-translational modifications. These processes include modifications of amino acids, folding of procollagen α-chains into a triple-helical configuration and subsequent stabilization, facilitation of transportation out of the cell, cleavage of propeptides, aggregation, cross-link formation, and finally the formation of mature fibrils. Disruption of any of the proteins involved in these biosynthesis steps potentially result in a variety of connective tissue diseases because of a destabilized extracellular matrix. In this review, we give a revised overview of the enzymes and chaperones currently known to be relevant to the conversion of lysine and proline into hydroxyproline and hydroxylysine, respectively, and the O-glycosylation of hydroxylysine and give insights into the consequences when these steps are disrupted.     

The synthesis of connective tissue protein in smooth muscle cells.

The synthesis of elastin by smooth muscle cells was clearly demonstrated by amino acid analyses and the presence of lysine-derived crosslinks. The values obtained were compatible with those found in amorphous elastin isolated from rabbit aortic tissue. Collagen synthesis by these same cells was monitored by the appearance of [14C] hydroxyproline when the cells were grown in the presence of [14C] proline. When the cells were pulsed with [14C] lysine, one could detect [14C] hydroxylysine and [14C] glucosylgalactosylhydroxylysine. Further evidence for the synthesis of elastin and collagen was the finding of radiolabelled epsilon-hydroxynorleucine and the reduced aldol condensate of two residues of allysine after reduction of [14C] lysine pulsed cells with NaBH4.     

Studies on the rat glomerular basement membrane: Age-related changes in composition

To examine the effect of age on the glomerular basement membrane, compositional analyses were performed on membranes isolated in highly purified form from rats at different stages of their growth (35 to 200 days old). Substantial age-related changes were observed in the amino acid composition of the basement membranes. A significant correlation with age (P < 0.01) was evident in the contents of 3- and 4-hydroxyproline, threonine, serine, alanine, valine, half-cystine, hydroxylysine, and lysine. Of these amino acids, hydroxylysine and both isomers of hydroxyproline demonstrated a progressive increase with age, while the others were found to decline. The direct relationship of hydroxylysine content with age (P < 0.001) was associated with an inverse correlation of lysine with age (P < 0.001) so that the ratio of hydroxylysine to lysine increased in a highly significant manner from 0.92 at 35 days to 1.33 at 200 days. This elevation in the hydroxylysine content was accompanied by an augmentation in the number of saccharide units linked to it so that the percentage glycosylation of this amino acid was not significantly affected by age. The relative differences in the hydroxylysine and lysine levels between young and older rats were maintained in sodium dodecyl sulfateextracted membranes. These results suggest that the compositional changes observed during the aging process reflect an alteration in the subunit makeup of the basement membrane, possibly due to an increased synthesis or decreased degradation of the more collagen-like polypeptide components.     

Neutrophils as a source of branched-chain,aromatic and positively charged free amino acids

Neutrophils release branched-chain (valine, isoleucine, leucine), aromatic (tyrosine, phenylalanine) and positively charged free amino acids (arginine, ornithine, lysine, hydroxylysine, histidine) when adhere and spread onto fibronectin. In the presence of agents that impair cell spreading or adhesion (cytochalasin D, fMLP, nonadhesive substrate), neutrophils release the same amino acids, except for a sharp decrease in hydroxylysine and an increase in phenylalanine, indicating their special connection with cell adhesion. Plasma of patients with diabetes is characterized by an increased content of branched-chain and aromatic amino acids and a reduced ratio of arginine/ornithine compared to healthy human plasma. Our data showed that the secretion of neutrophils, regardless of their adhesion state, can contribute to this shift in the amino acid content.

Abbreviations: BCAAs: branched-chain amino acids; Е2: 17β-estradiol; LPS: lipopolysaccharide from Salmonella enterica serovar Typhimurium; fMLP: N-formylmethionyl-leucyl-phenylalanine.     

Reversed-phase high-performance liquid chromatographic analysis of hydroxyproline and proline from collagen by derivatization with dabsyl chloride

A high-performance liquid chromatographic method for the analysis of hydroxyproline and proline has been developed. The method is based on the derivatization of the secondary amino group with dabsyl-chloride after blocking of the primary amino group with o-phthalaldehyde. Dabsyl-hydroxyproline and dabsyl-proline were separated from other amino acids by high-performance liquid chromatography in the gradient elution mode, and eluted at 10.27 and 16.02 min, respectively. The correlations between the peak areas of dabsyl-hydroxyproline and dabsyl-proline were linear in the range from 20–200 pmol, with equations y = 1.10x − 0.80 (r = 0.999) and y = 1.12x − 0.52 (r = 0.999), respectively. The method was applied to the analysis of rat tail collagen, and the contents of hydroxyproline and proline were 1.55 ± 0.04 and 2.03 ± 0.04 nmol/μg, respectively.     

Preparation and Enantioselectivity Binding Studies of a New Chiral Cobalt(II)porphyrin‐Tröger's Base Conjugate

A new bis[cobalt(II)porphyrin]‐Tröger's base conjugate was studied as a potential receptor for methyl esters of several amino acids. The conjugate was prepared as racemate, and then resolved via preparative high‐performance liquid chromatography (HPLC) on a chiral column. The high affinity to lysine, histidine, and proline methyl esters was found by complexation studies followed by UV‐Vis spectroscopy. The studies of pure enantiomers, followed by UV‐Vis and electronic circular dichroism spectroscopy, revealed the highest enantioselectivity for lysine methyl ester. Chirality 26:361–367, 2014. © 2014 Wiley Periodicals, Inc.     

The kinetics of inhibition of Vigna catjang cotyledon and buffalo liver arginase by L-proline and branched-chain amino acids

The effect of proline, isoleucine, leucine, valine, lysine and ornithine under standard physiological conditions, on purified Vigna catjang cotyledon and buffalo liver arginases was studied. The results showed that V. catjang cotyledon arginase is inhibited by proline at a lower concentration than buffalo liver arginase and the inhibition was found to be linear competitive for both enzymes. Buffalo liver arginase was more sensitive to inhibition by branched-chain amino acids than V. catjang cotyledon. Leucine, lysine, ornithine and valine are competitive inhibitors while isoleucine is a mixed type of inhibitor of liver arginase. We have also studied the effect of manganese concentration which acts as a cofactor and leads to activation of arginase. The optimum Mn^{2 +} concentration for Vigna catjang cotyledon arginase is 0.6 mM and liver arginase is 2.0 mM. The preincubation period required for liver arginase is 20 min at 55°C, the preincubation period and temperature required for activation of cotyledon arginase was found to be 8 min at 35°C. The function of cotyledon arginase in polyamine biosynthesis and a possible role of branched chain amino acids in hydrolysis of arginine in liver are discussed.     

OH-PRED: prediction of protein hydroxylation sites by incorporating adapted normal distribution bi-profile Bayes feature extraction and physicochemical properties of amino acids

Hydroxylation of proline or lysine residues in proteins is a common post-translational modification event, and such modifications are found in many physiological and pathological processes. Nonetheless, the exact molecular mechanism of hydroxylation remains under investigation. Because experimental identification of hydroxylation is time-consuming and expensive, bioinformatics tools with high accuracy represent desirable alternatives for large-scale rapid identification of protein hydroxylation sites. In view of this, we developed a supporter vector machine-based tool, OH-PRED, for the prediction of protein hydroxylation sites using the adapted normal distribution bi-profile Bayes feature extraction in combination with the physicochemical property indexes of the amino acids. In a jackknife cross validation, OH-PRED yields an accuracy of 91.88% and a Matthew’s correlation coefficient (MCC) of 0.838 for the prediction of hydroxyproline sites, and yields an accuracy of 97.42% and a MCC of 0.949 for the prediction of hydroxylysine sites. These results demonstrate that OH-PRED increased significantly the prediction accuracy of hydroxyproline and hydroxylysine sites by 7.37 and 14.09%, respectively, when compared with the latest predictor PredHydroxy. In independent tests, OH-PRED also outperforms previously published methods.     

Analysis of methylthiohydantoins of amino acids by gas-liquid chromatography of their trimethylsilyl derivatives

A procedure is described for the analysis of methylthiohydantoins of amino acids from the Edman degradation of proteins and peptides by gas-liquid chromatography of their trimethylsilyl derivatives. The procedure is applicable to the methylthiohydantoins of all the amino acids commonly found in proteins with the exception of arginine, which did not yield a volatile derivative, and hydroxyproline and hydroxylysine, which were not investigated. Chromatographic separation is achieved in a single run with only one unresolved pair, which can be separated by a supplementary procedure requiring only 4.5 min.     

High-performance liquid chromatographic quantitation of collagen biosynthesis in explant cultures

The capacity of lung explant cultures to synthesize collagen can be estimated by determining the content of [³H]hydroxyproline in protein following incubation with [³H]proline. The technique requires acid hydrolysis followed by quantitative separation of hydroxyproline from proline for scintillation counting and is often restricted to methods that can accommodate large samples because of relatively low specific radioactivity. A method which is useful for such samples, providing rapid separation of nonderivatized amino acids by ion-exchange HPLC, is described here. The HPLC system employs an HPX-87C cation-exchange column in 10 mm calcium acetate, pH 5.5, at 85°C. Under isocratic conditions hydroxyproline is completely resolved from proline with quantitative recovery of the ³H cpm applied to the column. Large amounts of material, equivalent to at least 150 mg wet wt of lung, can be applied without affecting resolution or recovery, and samples can be injected at intervals as short as 40 min. This method was used to study collagen biosynthesis in a model of pulmonary fibrosis induced in rabbits by the tumor-promoting agent, phorbol myristate acetate (PMA), and provides information concerning total protein synthesis as well as production of collagen. The data show a doubling in the rate of collagen production in lung explants prepared from animals treated with PMA compared with explants from control animals.     

Amino acid synthesis from glucose-U-14C in Glossina morsitans

Amino acid synthesis from glucose-U-¹⁴C was investigated in 2 day post-emergent and pregnant females of Glossina morsitans. This insect can synthesize alanine, aspartic acid, cystine, glutamic acid, glycine, proline, and serine from glucose. Arginine, histidine, hydroxyproline, isoleucine, leucine, lysine, methionine, phenylalanine, taurine, threonine, and valine showed no radioactivity and hence may be classified as nutritionally indispensable amino acids. Although tyrosine and hydroxyproline were not synthesized from glucose, they are at least partially dispensable nutrients for this insect because their synthesis from phenylalanine has been demonstrated. After the labelled glucose injection the highest radioactivity was recovered in the proline fraction. This is probably related to its rôle as an important energy reserve for flight. The radioactive amino acids recovered from females and from their offspring following glucose-U-¹⁴C injection were similar to those recovered from younger females. Radioactivity was also detected in the expired CO₂ and the excreta. The amino acids alanine, arginine, cystine, glycine, histidine, leucine/isoleucine, lysine, methionine, proline, and valine were identified in the excreta, of which arginine and histidine were in the largest amounts. Only excreted alanine, glycine, and proline showed radioactivity.     

Determination of amino acids in human serum by capillary gas chromatography

A simple and rapid method for the determination of serum amino acids by gas chromatography (GC) has been developed. Following deproteinization of serum with perchloric acid, free amino acids in the supernatant were converted into their N(O,S)-isobutoxycarbonyl methyl ester derivatives and measured by GC with flame ionization detection using a DB-17 capillary column. All the derivatives of the 22 protein amino acids were completely resolved as single peaks within 9 min by GC. The calibration curves were linear in the range 0.2–50 μg of each amino acid, and the correlation coefficients were above 0.998. By using this method, serum amino acids could be directly analysed without prior clean-up procedure such as ion-exchange column chromatography except for deproteinization of the samples, and without any interference from coexisting substances. Overall recoveries of amino acids added to serum samples were 88–108%. Analytical results for serum amino acids from normal subjects are presented.     

Carbohydrate Effects on Amino Acid Transport by Trypanosoma equiperdum*

SYNOPSIS. Uptake of ¹⁴C-labeled alanine, glutamate, lysine, methionine, proline, and phenylalanine by Trypanosoma equiperdum during 2-minute incubations occurred by diffusion and membrane-mediated processes. Amino acid metabolism was not detected by paper chromatography of trypanosome extracts. Most of 18 carbohydrates tested for ability to alter amino acid transport neither changed nor significantly inhibited transport. Glucose, however, stimulated glutamate, lysine and proline transport; fructose stimulated lysine uptake and 2-deoxy-D-glucose increased phenylalanine and methionine absorption. No evidence was found that the carbohydrates acted by binding to amino acid transport “sites.” Glucose inhibition of alanine, phenylalanine, and methionine uptake was linked to glycolysis. The rapid formation of alanine from glucose stimulated alanine release and, when glycolysis was blocked, glucose no longer inhibited alanine transport. Methionine and phenylalanine release was also stimulated by glucose. Glucose changed the ability of lysine, glutamate, and proline to inhibit each others’uptake, indicating that certain amino acids are preferentially absorbed by respiring cells. Analysis of free pool amino acid levels suggested that some amino acid transport systems in T. equiperdum are linked in such a way to glycolysis as to control the cell concentrations of these amino acids.     

Rapid quantitation of free fatty acids in human plasma by high-performance liquid chromatography

We report a rapid and sensitive method for separation and quantitation of free fatty acids (FFAs) in human plasma using high-performance liquid chromatography (HPLC). Two established techniques of lipid extraction were investigated and modified to achieve maximal FFA recovery in a reasonably short time period. A modified Dole extraction method exhibited greater recovery (90%) and short processing times (30 min) compared to the method of Miles et al. Reversed-phase HPLC using UV detection was used for plasma FFA separation and quantitation. Two phenacyl ester derivatives, phenacyl bromide and p-bromophenacyl bromide, were investigated in order to achieve optimal separation of individual plasma FFAs (saturated and unsaturated) with desirable detection limits. Different chromatographic parameters including column temperature, column type and elution profiles (isocratic and gradient) were tested to achieve optimal separation and recovery of fatty acids. Phenacyl bromide esters of plasma fatty acids were best resolved using an octadecylsilyl column with endcapped silanol groups. An isocratic elution method using acetonitrile–water (83:17) at 2 ml/min with UV detection at 242 nm and a column temperature of 45°C was found to optimally resolve the six major free fatty acids present in human plasma (myristic [14:0], palmitic [16:0], palmitoleic [16:1], stearic [18:0], oleic [18:1] and linoleic [18:2]), with a run time of less than 35 min and detection limits in the nmol range. The entire process including plasma extraction, pre-column derivatization, and HPLC quantitation can be completed in 90 min with plasma samples as small as 50 μl. Over a wide physiological range, plasma FFA concentrations determined using our HPLC method agree closely with measurements using established TLC–GC methods (r²≥0.95). In addition, by measuring [¹⁴C] or [³H] radioactivity in eluent fractions following HPLC separation of plasma FFA, this method can also quantitate rates of FFA turnover in vivo in human metabolic studies employing isotopic tracers of one or more fatty acids.     

Automated fluorometric amino acid analysis: the determination of proline and hydroxyproline.

A fluorometric method for the automated determination of the imino acids proline and hydroxyproline has been developed. The assay is based on the postcolumn reaction of the imino acids with alkaline sodium hypochlorite, which yields oxidation products amenable to detection with fluorogenic amine reagents. The method is simple and can be adapted readily to high-sensitivity amino acid analyzers which use o-phthalaldehyde for detection. As little as 10 pmol proline and 20 pmol hydroxyproline can be determined accurately. Thus the full array of natural imino and amino acids can now be determined on a high-sensitivity amino acid analyzer using o-phthalaldehyde.