Biochimica et Biophysica Acta (BBA) - Protein Structure
Amidated carboxyl groups in elastin
Abstract
Dicarboxylic amino acids constitute the most numerous residues of insoluble elastin which are potentially ionizable in the physiological range of pH. These residues are essential in facilitating productive electrostatic interaction between elastase and elastin. The present study has investigated the possibility that the glutamic and aspartic acid residues of elastin are amidated. Acid-labile amide-bound ammonia of elastin was quantitated after hydrolysis of the insoluble protein with 2 M HCl by incubating aliquots of microdistilled hydrolysates with glutamate dehydrogenase, excess α-ketoglutarate, and reduced nicotinamide adenine dinucleotide and measuring the resultant decrease in A340 due to oxidation of the dinucleotide cofactor. It was found that ligament elastin purified by repeated autoclaving contains approximately 2.29 μmol of acid-labile amide nitrogen per 10 mg of protein, a value equivalent to approximately 70% of the total number of dicarboxylic amino acid residues. Independent analysis of the amide content was obtained by amino acid analysis of an esterified and reduced elastin sample in which the free dicarboxylic amino acid residues had been converted to the corresponding alcohol derivatives. This analysis indicated that autoclaved ligament elastin contains approximately 18 glutamine, 3 asparagine, 4 glutamic acid and 5 aspartic acid residues per 1000 residues, in good agreement with the analysis of total acid-labile ammonia. The esterified and reduced elastin derivative was nearly inert as an elastase substrate, consistent with a lack of free dicarboxylic amino acid residues. However, addition of sodium dodecyl sulfate to this elastin derivative restores enzyme-substrate charge complementarity, and elastin-ligand complex was readily hydrolyzed by elastase at the fully stimulated rate, emphasizing the control such ligands can exert in elastolysis. The amide bonds of elastin were found to be significantly more resistant to hydrolysis by 0.1 M NaOH at 98 °C than were those of lysozyme or free amidated amino acids. The finding that most of dicarboxylic amino acid residues of elastin exist as neutral amides further emphasizes the apolar character of elastin and has bearing upon the metabolic susceptibility, ligandbinding ability and structural aspects of this connective tissue protein.
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Interaction of human leukocyte elastase with soluble and insoluble protein substrates. A practical kinetic approach
1990, Biochimica et Biophysica Acta (BBA)/Protein Structure and MolecularA progress-curve kinetic method was developed to investigate the interaction between human leukocyte elastase and macromolecular substrates, such as insoluble elastin and soluble plasma proteins. A fluorogenic, synthetic peptide (reporter substrate) was incubated in the presence of finely powdered elastin and enzyme under continuous stirring. The progress curves, which corresponded to the release of product from the reporter substrate, were very sensitive to the presence of various amounts of the macromolecular substrate. The kinetic parameters for the interaction between elastase and elastin were calculated using a pre-steady-state approach characteristic of slow-binding inhibitors. The interaction of elastase with the soluble protein substrates was studied with similar techniques, but formally treating the substrates as classical, fully competitive inhibitors. The adsorption of elastase on insoluble elastin was a time-dependent process cosisting of at least three observable phases: The first step was a rapid formation of an encounter complex followed by a very slow step lasting several minutes, and the third step consisted of a steady-state release of products. On the contrary, elastase very rapidly formed productive complexes with bovine serum albumin and a human monoclonal immunoglobulin G. The progress-curve method was also suitable for analyzing the behavior of inhibitors in the presence of protein substrates. The kinetic parameters which characterize the interaction between elastase and protein substrates represent a practical tool to formulate hypotheses on the efficiency of inhibitors in vivo.
Proteolysis of Insoluble Elastin
1982, Methods in EnzymologyA number of sensitive assay methods now exist that can be relied upon to establish and quantify the ability of a protease to degrade elastin. This chapter describes assay procedures found useful for quantifying elastolytic activity. The identification of an enzyme as having elastase activity must include the demonstrated ability of such an enzyme to digest an insoluble elastin substrate. The chapter describes that method, utilizing a tritiated reduced derivative of insoluble elastin as a substrate; it is believed this to be a reliable and extremely sensitive method for demonstrating and quantifying elastolytic activity. Underivatized insoluble elastin is also useful for solubilization assays because the intrinsic protein nature of the enzyme-solubilized material can be quantified by spectrofluorometric, spectrophotometric, conductimetric, or chemical means. Visual measurement of the area of clearing around sample wells in a suspension of finely divided elastin in agar is also widely used.
The microfibrillar components of porcine lung elastic fiber
1981, BBA - Protein StructureLung elastic fiber was isolated from anatomically defined porcine parenchymal tissue. Constituent microfibrillar acidic structural glycoproteins (external and meso) were obtained by mild dissociative quantitative procedures, leaving the elastin-rich fraction as the insoluble residue. The presumptive cross-link lysylnorleucine was identified in both of the highly aggregated acidic structural glycoprotein fractions. The external-acidic structural glycoproteins require dithiothreitol but not sodium dodecyl sulfate for solubility. Both sodium dodecyl sulfate and dithiothreitol at 55°C are required to release meso-acidic structural glycoproteins from the elastic fiber. On the basis of physical characteristics, I propose a new fraction from elastic fiber, the meso-acidic structural glycoprotein. The meso-acidic structural glycoproteins may have been in longest and closest contact with the elastin core of the elastic fiber. Alkali-soluble peptides from the elastin-rich fraction contained desmosine and high concentrations of hydroxyproline, polar amino acids and lysylnorleucine. The cross-link, lysylnorleucine, may be evidence of covalent bonding of glycoprotein to elastin, explaining my inability to obtain the biopolymer elastin with the composition of tropoelastin corrected for the desmosine-lysine content.
Characterization of insoluble elastin from copper-deficient pigs. Its usefulness in elastin sequence studies
1979, BBA - Protein StructureInsoluble elastin from copper-deficient animals has an amino acid composition intermediate between mature elastin and salt-soluble elastin (a higher lysine content and correspondingly low number of cross-links relative to the normal protein) and is solubilized by successive treatment with trypsin and chymotrypsin at 4 and 37°C.
Small amounts of B3H4 (11 mg–2 g of elastin) reduced allysine, allysine aldol, dehydronorleucine, and dehydromerodesmosine in insoluble elastin from copper-deficient pig aorta. In contrast, desmosine and isodesmosine were reduced only when a large excess of reductant (400 mg borohydride) was included in the reaction mixture. Reduction studies indicated that lysinonorleucine and merodesmosine were present in their dehydro forms to a greater extent in copper-deficient pig elastin than in normal elastin. After reduction with borohydride approximately 35% of the reduced form of the insoluble elastin remained insoluble after digestion with trypsin and chymotrypsin.
A peptide containing the aldehyde oxidation product of lysine (allysine) and demonstrating an enrichment in glutamic acid was purified from the reduced form of copper-deficient pig elastin and partially sequenced. Its sequence (Gly-Ala-Glu-allysine-(Glu)…) and amino acid composition suggest: (1) clustering of glutamic acid residues in the elastin molecule, and (2) that allysine residues are not restricted to the alanine-enriched sites described for other elastin crosslinks. Insoluble elastin from copper-deficient animals promises to be a useful tool for elastin sequence studies.
Isolation of elastin from bovine auricular cartilage
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