Jerome M. Seyer

Glycosaminoglycan synthesis by the early embryonic chick heart.

Abstract Glycosaminoglycans of embryonic chick hearts labeled in situ were characterized by means of labeled precursor incorporation, electrophoretic mobility, sensitivity to testicular hyaluronidase, elution characteristics from CPC-cellulose columns, and hexosamine content. During the initial period of overt cardiac muscle differentiation (approximately stage 10) chondroitin sulfates are not detectable but an undersulfated component is present. Chondroitin sulfate synthesis appears shortly after overt muscle differentiation. Hyaluronate is present both during and after overt myocardial differentiation. Although epimerization of 3H-glucosamine-derived labeled UDP-N-acetyl- d -glucosamine occurs (determined by recovery of incorporated labeled galactosamine), label does not appear in chondroitin sulfate. 3H-Glucosamine is thus a relatively specific precursor for unsulfated glycosaminoglycans, a fact that we exploited in demonstrating their distribution radioautographically. Glycosaminoglycan synthesis was also examined in hearts labeled (a) in isolated organ culture, (b) in situ but exposed directly to the medium by removal of the splanchnopleure. In both cases fully sulfated chondroitin sulfate and chondroitin are not synthesized. Hearts make only hyaluronate and undersulfated chondroitin sulfate.

The biochemical and ultrastructural demonstration of collagen during early heart development

Abstract A correlative ultrastructural and biochemical study was made of cardiac collagen in the chick embryo, spanning stages 9- to 11 (6 to 13 somites). Analysis (carboxymethyl cellulose chromatography, SDS acrylamide gel electrophoresis and levels of proline hydroxylation) of collagen synthesized in situ permitted classification of this collagen as type I-like (α1:α2 = 2:1). Correlative electron microscopy of hearts fixed in situ showed the appearance of striated collagen fibrils in the cardiac jelly, thus complementing the biochemical findings. Although the electron microscope showed the presence of developing basal laminae and laminalike material, synthesis of the type of collagen reported as unique to basal laminae was not detected, and we propose that these basal laminae may lack type IV collagen. Embryonic stages 9- to 11 are the earliest stages in which collagen synthesis has been demonstrated, and this is the first report of the occurrence of collagen in cardiac jelly of early hearts.

The isolation of phosphorylated polypeptide components of the organic matrix of embryonic bovine enamel

Abstract 1. (1)The organic matrix of developing embryonic enamel was obtained from bovine fetuses of various ages. This material was extracted sequentially using three neutral and two acid pH extractions at 4° and the amount of protein in each extract measured. 2. (2)Fractionation of the proteins which were extracted at neutral pH was achieved by three procedures: (a) Free flow, horizontal electrophoresis in 6 M urea at pH 8.3; (b) Preparative polyacrylamide gel electrophoresis at pH 8.8 in 6 M urea; and (c) gel filtration using columns of Bio-gel P-10 and P-20 in 6 M urea at pH 8.3. 3. (3)Four highly purified organic phosphorus containing polypeptides were obtained by preparative polyacrylamide gel electrophoresis. The four polypeptides (E 1 , E 2 , E 3 , E 4 ) contained 0.39; 0.45; 1.60 and 2.10% phosphorus, respectively. Two of the fractions (E 1 , E 2 ) constitute only approx. 0.2% of the enamel proteins which are soluble at neutral pH, while the other two fractions (E 3 , E 4 ) represent 15–20% of the neutral pH-soluble proteins. 4. (4)The E 3 fraction was characterized by its high content of leucine while the E 4 fraction contained relatively large amounts of tyrosine. The results of molecular sieving indicate that these two components had similar mol. wt. of approx. 6000.

The content and nature of the carbohydrate components of the organic matrix of embryonic bovine enamel

Abstract 1. 1. The carbohydrate content of the organic matrix of developing, embryonic bovine enamel has been investigated. The carbohydrates accounted for slightly more than 1% of the total weight of the organic matrix and of the fractions soluble at neutral pH. Hexose and hexosamine were present in approximately equal amounts. Galactose and galactosamine were the principal sugars identified, although mannose and trace amounts of glucose were consistently found. The neutral-insoluble, but acid-soluble fraction of the organic matrix contained slightly greater amounts of carbohydrates. These were identified as galactose and galactosamine in equimolar quantities. 2. 2. Acid mucopolysaccharides and glycopeptides were isolated after papain digestion of the neutral-soluble fraction. From uronic acid vaues of the fraction of the enamel matrix soluble at neutral pH, the acid mucopolysaccharide content of the organic matrix was estimated to be 0.20%. Glycopeptides containing as much as 20% carbohydrate were isolated by preparative polyacrylamide-gel electrophoresis. From the carbohydrate analyses of these glycopeptides, it was suggested that the major oligosaccharide moiety of the glycopeptides contains galactosamine, galactose, and mannose in a ratio of 4:3:1. 3. 3. Aldehydes and hydroxylamine-sensitive, ester-like bonds were not identified in any significant quantity in the organic matrix of embryonic bovine enamel.

Evidence for the presence of numerous protein components in immature bovine dental enamel.

SummaryThe proteins and peptides of immature enamel were extracted from freshly slaughtered bovine embryos in solutions containing protease inhibitors. No detectable differences were noted in the number of components, their overall amino acid composition, or molecular weights from the proteins and peptides extracted 12–16 h postmortem in solutions which contained no protease inhibitors. These data indicate that the large number of components found in developing bovine enamel is not due to proteolysis occurring during their isolation. Significant amounts of protein components having molecular weights greater than ∼15,000 were not detected. Therefore, if the ameloblasts initially synthesize only a few high molecular weight protein species, the present data imply that in vivo degradation of the high molecular weight enamel proteins occurs very rapidly after their synthesis and precedes the massive loss of protein which accompanies the final stages of enamel mineralization and maturation.

Isolation, characterization and partial amino acid sequence of a phosphorylated polypeptide (E4) from bovine embryonic dental enamel.

A phosphorylated polypeptide (E4) of molecular weight 5000-6000, has been isolated from bovine embryonic enamel by Bio-Gel P-10 gel filtration and DE-52 ion-exchange chromatography. The peptide contains three serine residues all of which are phosphorylated. All three O-phosphoserine residues are in glutamic acid-O-phosphoserine-tyrosine sequences that are distributed relatively evenly along the polypeptide chain. Although it was not possible to sequence the entire polypeptide chain directly by automatic peptide sequencing, a partial sequence and peptide map was constructed on the basis of the sequence and composition of peptides derived by cyanogen bromide, trypsin and chymotrypsin digestion. The presence of glutamic acid, tyrosine and leucine adjacent to and near the O-phosphoserine residues may be important in calcium binding and in mineralization.

The comparative biochemistry of the organic matrix proteins of developing enamel—I: Amino acid composition

Abstract The amino acid composition of the proteins of developing enamel from eleven different species was determined. All of them were characterized by their relatively high concentrations of proline, glutamic acid, leucine and histidine. Combined with previous studies of four other species, the data clearly indicate that the enamel proteins represent a separate class of structural proteins with a characteristic amino acid composition.

Synthesis of type III collagen by embryonic chick skin

Analysis of cyanogen bromide peptides from the α1 chains synthesized by lathyritic embryonic chick skin during short-term tissue culture revealed significant levels of both Types I and III collagen prior to thirteen days of development, but mainly Type I collagen thereafter. The elevated ratio of α1 to α2 polypeptide chain synthesis during the period of maximal Type III production supports the proposed [α1(III)]3 molecular structure for Type III collagen. Maximal synthesis of Type III collagen occurs during a period when collagen production is apparently necessary for normal dermal and hence normal ectodermal development.

The comparative biochemistry of the organic matrix of developing enamel--II. Ultracentrifugal and electrophoretic characterization of proteins soluble at neutral pH.

Abstract At a pH of 7.7 the neutral soluble proteins of both embryonic ovine and equine enamel have a concentration-dependent trinodal sedimentation boundary very similar to that previously found for the embryonic bovine enamel proteins. In the latter case, the 1 S node was primarily due to a number of chemically unique subunits, the 2 S node to intermediate(s), and the 12 S node to various high molecular weight aggregates, some of which are labile and in a state of (partial) equilibrium with the subunits during centrifugation, and others that are stable to dilution. When dissolved in 6 M urea, the sedimentation velocity patterns of the ovine and equine proteins are similar to that of the bovine enamel proteins: they show but one peak with an apparent sedimentation coefficient of about 1 S. The polyacrylamide gel electrophoresis patterns for these three proteins in 6 M urea are likewise similar: each shows approximately twenty bands, which, with a few exceptions, have a band-to-band correspondence. It was concluded that the physico-chemical data add further evidence that the neutral soluble proteins of developing enamel constitute a distinct class of structural proteins.

The amino acid sequence of two O-phosphoserine containing tripeptides isolated from the organic matrix of embryonic bovine enamel.

Abstract 1. 1.|Two tripeptides containing O -phosphoserine were isolated from an acid hydrolyzate of embryonic, bovine enamel proteins soluble at neutral pH. The tripeptides were purified by ion-exchange chromatography. 2. 2.|One of the tripeptides (Peptide I) contained equimolar amounts of glutamic acid, serine, leucine and phosphorus; the other tripeptide (Peptide II) contained glutamic acid, serine, tyrosine and phosphorus in equimolar amounts. 3. 3.|Sequential analysis by phenylisothiocyanate degradation and dansylation as well as by enzymatic hydrolysis with leucine aminopeptidase and carboxypeptidase A revealed that Peptide I had the sequence of glutamic acid- O -phosphoserine-leucine, while the amino acid sequence of Peptide II was glutamic acid- O -phosphoserine-tyrosine. Enzymatic hydrolysis of both tripeptides with a phosphomonoesterase indicated that phosphorus was covalently bound to the hydroxyl group of serine as a monoester.