Additional evidence for a proform to tropoelastin from chick aorta.

Evidence is presented for the presence of precursor to tropoelastin in chick arterial extracts. The precursor is approx. 100 000 daltons in size. It is suggested to be a precursor to tropoelastin (72 000 daltons). This protein may be observed in culture in vitro if appropriate precautions are taken to inhibit proteolysis. Once synthesized, it appears to be converted into tropoelastin within 10--20 min. The protein may also be detected in vivo. When 1-day-old cockerels were fed on a copper-deficient diet (less than 1 p.p.m. to inhibit cross-linking) containing epsilon-aminohexanoic acid (0.2%) to retard proteolysis and then injected wiht [3H]valine, extraction of arterial proteins 12h after injection resulted in detection of two major peaks of [3H]valine-labelled protein with pI values of pH 7.0 and 5.0 respectively. The protein that focused at pH 7.0 was estimated to be about 100 000 daltons in size and could be shown to be converted into a more basic protein with the properties of tropoelastin. It is speculated that the protein with pI 5.0 may be yet another extension peptide. The data appear to be in keeping with similar observations by ourselves and others that a proform of tropoelastin exists, and, in at least one step before conversion into tropoelastin, exists as a 100 000-dalton protein subunit.     

Biosynthesis and secretion of tropoelastin by chick aorta cells

Cells were isolated from the aortae of 17-day old chick embryos by digestion of the vessels with a combination of trypsin and collagenase. When these cells were incubated in suspension culture in Krebs-Ringer media containing pancreatic trypsin inhibitor and radioactive amino acids, they synthesized and secreted labeled proteins into the media. Polyacrylamide gel electrophoresis in sodium dodecyl sulfate of the secreted proteins labeled with [¹⁴C]proline revealed two major components. The larger component with an approximate molecular weight of 125,000 had a [¹⁴C]hydroxyproline content consistent with a form of procollagen. The molecular weight of 70,000 and [¹⁴C]hydroxyproline content of the second component was consistent with that previously reported for tropoelastin extracted from chick aortae. By following the kinetics and secretion of tropoelastin labeled with [³H]valine, we have estimated that 17 minutes are required to synthesize and secrete the molecule under these experimental conditions.     

Partial purification and characterization of collagen galactosyltransferase from chick embryos.

Collagen galactosyltransferase was purified 50-150-fold from chick-embryo extract. The tissue homogenate was prepared in the presence of Triton X-100, since the addition of the detergent doubled the enzyme activity in the homogenate and the extract. Three species of the enzyme activity with different molecular weights were recovered on gel filtration, the mol.wts. being about 450000, 200000 and 50000. Collagen galactosyltransferase activity was strongly inhibited by p-mercuribenzoate, and stimulated by the addition of dithiothreitol to the incubation system. Studies on substrate requirements indicated that denatured citrate-soluble collagen is a more effective substrate than gelatinized insoluble collagen, as judged from their Km values. Experiments on three peptide fractions prepared from citrate-soluble collagen indicated that a fraction with an average mol.wt. of 500-600 contained peptides large enough to meet a minimun requirement for interaction with the enzyme. However, longer peptides were clearly better substrates. When native and heat-denatured citrate-soluble collagens were compared as substrates, practically no synthesis of galactosylhydroxylysine was found with native collagen. This finding suggests that the triple-helical conformation of collagen prevents the galactosylation of hydroxylysine residues.     

Partial purification and characterization of thymidylate kinase from embryonic chick liver.

Thymidylate kinase from the livers of 18-day-old chick embryos was concentrated 423-fold. The purification procedure included acid precipitation, ammonium sulfate fractionation, gel filtration on Sephadex G-100 and G-75 Super Fine, and ion-exchange chromatography on Diethylaminoethyl Sephadex A-50. This enzyme was found to be very labile but could be stabilized for long periods of time by its substrate (thymidine 5′-monophosphate) in the presence of 2-mercaptoethanol. Enzymes responsible for the formation of thymidine 5′-diphosphate and thymidine 5′-triphosphate, respectively, were separated during fractionation procedures. Thymidylate kinase from chick embryo liver was found to be a single protein having a molecular weight of approximately 46,000, Michaelis constant approximately 8 × 10^?5m, and a broad pH optimum between 6.6 and 8.6. A 2–3 mm requirement of Mg²⁺ above the adenosine 5′-triphosphate concentration was shown to be necessary for maximum enzyme activity. The enzyme appears to be competitively inhibited by thymidine, thymidine 5′-diphosphate, and thymidine 5′-triphosphate and noncompetitively inhibited by adenosine 5′-diphosphate.Thymidylate kinase enzymes isolated from two stages of developing embryonic liver and adult chick liver were shown to be identical.     

Tropoelastin production and tropoelastin messenger RNA activity. Relationship to copper and elastin cross-linking in chick aorta.

The elastin content of the chick thoracic aorta increases 2--3-fold during the first 3 weeks post-hatching. The deposition of elastin requires the covalent cross-linking of tropoelastin by means of lysine-derived cross-links. This process is sensitive to dietary copper intake, since copper serves as cofactor for lysyl oxidase, the enzyme that catalyses the oxidative deamination of the lysine residues involved in cross-link formation. Disruption of cross-linking alters tissue concentrations of both elastin and tropoelastin and results in a net decrease in aortic elastin content. Autoregulation of tropoelastin synthesis by changes in the pool sizes of elastin or tropoelastin has been suggested as a possible mechanism for the diminished aortic elastin content. Consequently, dietary copper deficiency was induced to study the effect of impaired elastin cross-link formation on tropoelastin synthesis. Elastin in aortae from copper-deficient chicks was only two-thirds to one-half the amount measured in copper-supplemented chicks, whereas copper-deficient concentrations of tropoelastin in aorta were at least 5-fold higher than normal. In spite of these changes, however, increased amounts of tropoelastin, copper deficiency and decreased amounts of elastin did not influence the amounts of functional elastin mRNA in aorta. Likewise, the production of tropoelastin in aorta explants was the same whether the explants were taken from copper-sufficient or -deficient birds. The lower accumulation of elastin in aorta from copper-deficient chicks appeared to be due to extracellular proteolysis, rather than to a decrease in the rate of synthesis. Electrophoresis of aorta extracts, followed by immunological detection of tropoelastin-derived products, indicated degradation products in aortae from copper-deficient birds. In extracts of aortae from copper-sufficient chicks, tropoelastin was not degraded and appeared to be incorporated into elastin without further proteolytic processing.     

Partial purification and characterization of the first hydrogenase isolated from a thermophilic sulfate-reducing bacterium.

A soluble [NiFe] hydrogenase has been partially purified from the obligate thermophilic sulfate-reducing bacterium Thermodesulfobacterium mobile. A 17% purification yield was obtained after four chromatographic steps and the hydrogenase presents a purity index (A398 nm/A277 nm) equal to 0.21. This protein appears to be 75% pure on SDS-gel electrophoresis showing two major bands of molecular mass around 55 and 15 kDa. This hydrogenase contains 0.6-0.7 nickel atom and 7-8 iron atoms per mole of enzyme and has a specific activity of 783 in the hydrogen uptake reaction, of 231 in the hydrogen production assay and of 84 in the deuterium-proton exchange reaction. The H2/HD ratio is lower than one in the D2-H+ exchange reaction. The enzyme is very sensitive to NO, relatively little inhibited by CO but unaffected by NO2-. The EPR spectrum of the native hydrogenase shows the presence of a [3Fe-4S] oxidized cluster and of a Ni(III) species.     

Carbohydrate content of insoluble elastins prepared from adult bovine and calf ligamentum nuchae and tropoelastin isolated from copper-deficient procine aorta

1. Insoluble elastin has been prepared by several different methods from adult bovine and calf ligamentum nuchae. Highly purified tropoelastin has been prepared from copper-deficient porcine aorta. 2. Amino acid analyses indicated that all preparations, except that obtained from calf ligamentum nuchae by using an EDTA extraction followed by collagenase digestion (preparation E6), were typical of pure elastin having high concentrations of hydrophobic and low concentrations of hydrophilic amino acids. Preparation E6 was found to contain approx. 40% collagen. 3. The determination and composition of the carbohydrates associated with these preparations is reported. With the exception of preparation E6, the insoluble elastins contained only trace amounts of neutral sugars (0.13-0.35%, w/w) and amino sugars (0.01-0.06%, w/w). The porcine tropoelastin contained virtually no carbohydrate. 4. The results suggest that carbohydrate analyses can yield valuable information about the purity of elastin preparations.     

Sialoglycopeptides isolated from bovine aorta.

Intima-media of bovine aorta was digested with pronase, after preliminary extraction of saline (1%)-soluble substances and fat. Crude glycopeptide fraction was then obtained from the resulting complex carbohydrate fraction by fractionation with CPC (cetylpyridinium chloride). Complete separation of sialoglycopeptides was achieved by chromatography on a DEAE-cellulose column at pH 7.2 followed by repeated chromatography on a DEAE-Sephadex A-25 column at pH 5.2. Nine sialoglycopeptides (SGP 1-SGP 9) thus obtained were homogeneous on high-voltage paper electrophoresis at pH 3.5 and pH 5.2. The analytical data showed great heterogeneity of the carbohydrate chains of these preparations, although they consisted of the same monosaccharides (galactose, glucose, mannose, glucosamine, galactosamine, fucose, and sialic acid), except that SGP 1 lacked galactosamine. Heterogeneity was also observed in their peptide chains. It was noticed, however, that the contents of hexose, hexosamine, and aspartic acid of the fractions (SGP 3, SGP 4, and SGP 5) which eluted from the DEAE-Sephadex A-25 column at lower molarity of the eluting salt were higher than those of the fractions (SGP 7, SGP 8, and SGP 9) which eluted at higher molarity, while the contents of sialic acid and hydroxyamino acids were in an opposite relationship. Representative fractions (SGP 7 and SGP 9) of the latter contained many more alkali-sensitive linkages than those (SGP 3 and SGP 5) of the former, indicating the presence of many more O-glycosidic linkages between hydroxyamino acid(s) and sugar(s) in the latter than in the former. The sialoglycopeptides contained significant amounts of sialic acid, ranging from 10% (sgp 1) to 32.4% (SGP 8). The highest contents were in SGP 8 and SGP 9, which contained equimolar amounts of sialic acid and hexosamine. Furthermore, infrared spectra indicated the presence of sulfate groups in most of the sialoglycopeptides.     

Partial characterization of a low molecular weight proteoglycan isolated from bovine parietal pericardium

Knowledge of the nature of pericardial connective tissue components is incomplete. To gain a better understanding of the composition of this tissue, bovine parietal pericardium was extracted with 4 M guanidine hydrochloride yielding a proteoglycan-containing protein mixture. This was fractionated by a three-step chromatographic procedure with the resultant purification of a 75-110 Kd proteoglycan. The purified proteoglycan was susceptible to chondroitinase ABC digestion but resistant to chondroitinase AC and nitrous acid degradation suggesting the presence of dermatan sulfate glycosaminoglycan(s). This is the first reported isolation of a proteoglycan from parietal pericardium.