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Biomedical subjects

W T Butler

Publications and source records attributed to W T Butler.

At least 109 records · Page 6Linked to original sources
PubMed1983

Metabolism of rat bone proteoglycans in vivo.

Former evaluations of the role of proteoglycans in mineralization have neglected to address the possibility that the metabolism of proteoglycans may be of significance in this regard. This problem was studied by using radiolabeling in vivo of rat calvaria with [35Sulphate for 2-72 h and a sequential extraction procedure to yield two pools of newly synthesized proteoglycans: one obtained from non-mineralized tissue by extraction with guanidinium chloride (GdmCl) and another obtained only after demineralization with EDTA. Total radioactivity in calvaria was maximal after 12 h of incorporation, but by 36 h had declined to a level that was about 55-65% of maximum. Radioactivity in the GdmCl extract declined steadily after 12 h, whereas that in the EDTA extract remained constant until 36 h, when it began to increase. Each extract contained a minor proteoglycan that eluted at the void volume (Vo) of a Sepharose CL-6B column. Unlike in the EDTA extract, this proteoglycan gradually disappeared from the GdmCl extract. Each extract also contained a major, smaller proteoglycan, with a Kav. of 0.24 and 0.36 in the GdmCl and EDTA extracts respectively. Papain digestion of each extract yielded glycosaminoglycan chains with Kav. values of 0.32 and 0.50 on CL-6B in the GdmCl and EDTA extracts respectively. Digestion of each extract with chondroitinase ABC and chondroitinase AC showed that the glycosaminoglycans were of similar disaccharide composition, with about 85% being 4-sulphated and the remainder 6-sulphated and/or iduronic acid-containing. These data suggest that about 45% of the newly synthesized proteoglycans are removed from the tissue during the course of mineralization.

Animals↗
PubMed1983

The biosynthesis of dentine gamma-carboxyglutamic acid-containing proteins by rat incisor odontoblasts in organ culture.

Rat odontoblasts were shown to synthesize and secrete gamma-carboxyglutamic acid(Gla)-containing proteins into dentine after organ culture in the presence of radiolabelled amino acid precursors. Purified dentine Gla-containing protein from rat incisors was used as antigen to prepare rabbit antisera as a probe of dentine Gla-containing-protein biosynthesis in organ cultures of dentine (rat incisor) and bone (rat calvaria). Use of the antiserum also pointed out the cross-reactivity of a high-M, glycoprotein present within the dentine matrix. The present results are significant in identifying dentine gla-containing protein as endogenous to mineralizing dentine and may relate to the commonality between calcifying connective tissues in general.

1-Carboxyglutamic Acid↗
PubMed1983

Amelogenins. Sequence homologies in enamel-matrix proteins from three mammalian species.

Partial amino acid sequences for selected amelogenin polypeptides isolated from the developing enamel of cow, pig and human foetuses are reported. It was found that there was an identity of sequence for the initial 28 residues of the polypeptides analysed, irrespective of their origin or size. A tyrosine-rich polypeptide was shown to be the N-terminal fragment of the principal higher-molecular-weight amelogenins, although a leucine-rich polypeptide of similar size was not identified in any other amelogenin structure. The findings demonstrate a striking degree of sequence conservation for the amelogenin proteins of the extracellular enamel matrix and support the concept of a discrete fragmentation of an initial 30 000 Da amelogenin molecule during the mineralization of the enamel.

Amelogenin↗
PubMed1983

Multiple forms of rat dentin phosphoproteins.

Previous studies have shown that the phosphoprotein from rat dentin is heterogenous and can be partially separated into two fractions by ion-exchange chromatography. These proteins were further characterized by polyacrylamide gel electrophoresis, gel chromatography, and amino acid and phosphate analysis, after chromatographic separations on ion-exchange columns. On 5-15% gradient gels, the phosphoproteins extracted from rat dentin and precipitated by CaCl2 gave three Alcian blue-staining bands with apparent molecular weights in the 90-95,000 range. The two slower-moving bands corresponded to highly phosphorylated proteins (HP) that had phosphoserine contents of greater than 400 residues per thousand and contained little or no valine, leucine, phenylalanine, or arginine. The faster-moving band corresponded to a moderately phosphorylated protein that contained about 250 residues per thousand of phosphoserine and greater quantities of glutamic acid, proline, and several other amino acids than HP. The nature of the phosphoproteins in HP was further studied after total removal of the phosphate with an insoluble form of bovine intestinal alkaline phosphatase. The dephosphorylated product (dP-HP) gave a single major band on gel electrophoresis but showed evidence for two closely related NH2-terminal sequences, Asp-Asp-Asp-Asn and Asp-Asp-Pro-Asn. The dephosphorylated material was separated into two components (dP-HP1 and dP-HP2) by chromatography on QAE-Sephadex A-25. The amino acid compositions of the two components showed that they differed in their primary structures. This conclusion was verified by the finding of the proline-containing sequence in dP-HP2. In addition to these two groups of phosphoproteins, a third class, LP, contains low levels of phosphoserine and high amounts of glutamic acid (W.T. Butler, M. Bhown, M.T. DiMuzio, and A. Linde, (1981) Coll. Res. 1, 187-199).

Amino Acids↗
PubMed1982

Evidence for several gamma-carboxyglutamic acid-containing proteins in dentin.

With anion-exchange chromatography, the gamma-carboxyglutamic acid (Gla)-containing proteins of rat dentin were separated into four closely related fractions (gamma 1-gamma 4). Edman degradation of gamma 2 gave two NH2-terminal sequences with a minor sequence beginning five residues shorter than the major one. Gel electrophoresis of gamma 2 yielded one major and one minor protein band. Fraction gamma 3 gave one band on gel electrophoresis and a single NH2-terminal sequence. The composition of gamma 4 suggested that, compared to gamma 2 and gamma 3, a portion of the COOH-terminal was missing. Thus some of the heterogeneity of rat dentin Gla-containing proteins can be explained by shortened ends.

1-Carboxyglutamic Acid↗
PubMed1981

Alignment of biologically active domains in the fibronectin molecule.

Gelatin-binding material was isolated from a human plasma cryoprecipitate by affinity chromatography on gelatin-Sepharose. Individual fragments of fibronectin with Mr = 170,000, 100,000, and 80,000 and a mixture of fragments with Mr = 205,000 and 190,000 (200K fraction) were isolated from this material. These fragments reacted with antifibronectin and with antibodies to a gelatin-binding Mr = 70,000 tryptic fragment of fibronectin. They all shared the same NH2-terminal amino acid sequence. The 205K and 190K fragments bound also to heparin-Sepharose, whereas the smaller fragments did not. The 200K fraction and the 170K fragment mediated cell attachment when used to coat plastic, whereas the 100K and 80K fragments were inactive in this assay. Further digestion of the 205K and 190K fragments with chymotrypsin yielded separate sets of smaller fragments that bound to either gelatin-Sepharose or heparin-Sepharose, as well as fragments that did not show either of these binding activities but mediated cell attachment. Since the NH2-terminal ends of the 205K, 190K, 100K, and 80K fragments are the same, the results define the order of the active sites in the fibronectin molecule as gelatin-binding site, cell attachment site, and heparin-binding site.

Amino Acid Sequence↗
PubMed1981

Non-collagenous proteins of rat dentin. Evidence that phosphoprotein is not covalently bound to collagen.

The non-collagenous proteins of rat dentin that remain firmly bound to the matrix after demineralization were studied in order to ascertain if they are covalently linked to insoluble dentin collagen. After solubilization with CNBr or with bacterial collagenase, unusually small amounts of dentin phosphoprotein were detected in the matrix. The phosphoprotein obtained by CNBr digestion of the matrix was separated from collagen peptides using two chromatographic steps. Thus even this small quantity of phosphoprotein found in decalcified rat dentin matrix was not covalently bound to collagen.

Amino Acids↗
PubMed1981

Dental enamel matrix: sequences of two amelogenin polypeptides.

The amino acid sequences of a leucine-rich amelogenin polypeptide (LRAP) and a tyrosine-rich amelogenin polypeptide (TRAP), isolated from foetal bovine enamel matrix, were determined. Both LRAP and TRAP occurred in two forms; in each case, one of the molecular species appeared to be shortened at the COOH terminus by 2 and 4 residues, respectively. A striking finding was that LRAP and TRAP had identical sequences for the first 33 residues but were almost completely different for the remaining 12 amino acids.

Amelogenin↗
PubMed1981

Nonocollagenous proteins of dentin. Isolation and partial characterization of rat dentin proteins and proteoglycans using a three-step preparative method.

The purpose of this study was to develop a method for fractionation of dentin proteins and proteoglycans into pools. The sequential procedure consisted of: (1) addition of 1.0 M CaCl2 to solutions of EDTA extracts of rat dentin in the presence of protease inhibitors to form a CaCl2 precipitate (Fraction I), (2) dialysis of the resultant supernatant against 0.1 M formic acid to form an acid precipitate (Fraction II), and (3) passage of the 0.1 M formic acid supernatant over a Sephadex G-50 column to obtain a high molecular weight, excluded peak (Fraction III) and a lower molecular weight, included peak (Fraction IV). Each of the four fractions was characterized by amino acid analysis, slab gel electrophoresis and ion-exchange chromatography on DEAE-cellulose. Fraction I contained almost exclusively phosphoproteins while Fraction II consisted of several acidic proteins, albumin, proteoglycans and a protein with a relatively low level of organic phosphate. A unique glycoprotein with an apparent Mr = 95,000 was found in Fraction III along with smaller amounts of other proteins, including albumin and a phosphoprotein with a relatively low level of organic phosphate. Fraction IV contained several low molecular weight, gamma-carboxyglutamate-containing proteins similar to those found in bone. The data show that the method selectively fractionates the proteins and proteoglycans of rat dentin. Furthermore the method is rapid and allows preparative steps to be performed in the presence of protease inhibitors. This new procedure should be a useful step in the comprehensive isolation of dentin proteins in experiments designed to study their detailed chemical nature and metabolism.

Albumins↗
PubMed1980

Noncollagenous proteins of dentin. A re-examination of proteins from rat incisor dentin utilizing techniques to avoid artifacts.

Noncollagenous proteins (NCPs) were obtained rrom rat dentin using several precautionary measures to prevent artifactual degradation and losses of the proteins. Prior to demineralization, rat incisor dentin was extracted with 4 M guanidine hydrochloride (GdmCl) containing enzyme inhibitors. The only major component extracted with GdmCl was a proteoglycan fraction. Most of the NCPs were extracted when the incisors were decalcified with an EDTA solution containing protease inhibitors. The EDTA extract contained four types of macromolecules: acidic glycoproteins, gamma-carboxyglutamic acid (Gla)-containing proteins, phosphoproteins, and proteoglycans. With two exceptions, the apparent molecular weights of these NCPs were greater than 50,000. Our observations contrast sharply with the results obtained by others for human dentin NCPs and suggest that artifactual degradation and losses of some NCPs occurred in these previous studies. The organic phosphate-containing fraction was biphasic when the EDTA extract was chromatographed on DEAE-cellulose. Rechromatography of this fraction by two different procedures separated the material into a complex glycoprotein-containing fraction and, a single rat incisor phosphoprotein peak (RIP). Thus, the earlier interpretation by others that the biphasic nature of the RIP-containing fraction represents two widely differing phosphoprotein species was premature. Highly purified RIP, prepared by passage through a sulfonated polystyrene column, contained no cysteine, valine, methionine, leucine, phenylalanine, or arginine, and the level of phosphoseryl residues was higher than for any previous report. When this preparation of phosphoprotein was dephosphorylated (dP-RIP) and rechromatographed on DEAE-cellulose, a partial separation into two fractions was observed. Automated Edman degradation of fraction dP-RIP suggested the presence of two NH2-terminal sequences: Asp-Asp-Asp-Asn and Asp-Asp-Pro-Asn. However, this material displayed a single protein band when applied to 7.5% sodium dodecyl sulfate polyacrylamide gel electrophoresis and stained with Coomassie brilliant blue. The apparent molecular weight of dP-RIP, compared to standard globular proteins, was about 72,000. The data suggest that rat dentin contains at least two major molecular species of RIP that are closely related in size and-structure. In addition, preliminary evidence suggested that other minor forms of related phosphoproteins may exist.

Amino Acid Sequence↗
PubMed1980

IgA1 proteases from Haemophilus influenzae, Streptococcus pneumoniae, Neisseria meningitidis, and Streptococcus sanguis: comparative immunochemical studies.

IgA1 proteases from H. influenzae, N. meningitidis, S. pneumoniae, and S. sanguis were compared with respect to site of cleavage in the IgA1 molecule and EDTA sensitivity. Proteases from S. sanguis and S. pneumoniae cleaved the Pro (227)-Thr (228) bond within the hinge region of the alpha 1 chain and were inhibited by EDTA. H. influenzae IgA1 protease cleaved the Pro (231)-Ser (232) peptide bond. The activity of IgA1 proteases from H. influenzae and N. meningitidis was unaffected by EDTA. Purified and denatured alpha 1 chain was cleaved only in the hinge region. Other component chains of secretory IgA (secretory component, light and J chains) were not susceptible. In addition to IgA1 protease, S. pneumoniae released exo- and endoglycosidases that removed a considerable portion of carbohydrate side chains of IgA1; this activity was absent from crude IgA1 protease preparations of the other three bacterial species. Association in vitro of polymeric IgA1 with SC did not inhibit the degradation of IgA1 proteases. The considerable resistance of secretory IgA to cleavage by IgA1 proteases may be explained in part by the presence of IgA1 protease-neutralizing antibodies in secretory IgA.

Amino Acid Sequence↗
PubMed1979

Dentin proteins: chemistry, structure and biosynthesis.

One of the most abundant noncollagenous proteins of dentin is a phosphoprotein rich in aspartic acid and phosphoserine. This protein occurs in soluble and inextractable forms, the latter being associated with the insoluble collagenous matrix. This protein is capable of tightly binding a relatively high level of calcium. Biosynthetic and radioautographic data suggest that shortly after its biosynthesis, the phosphoprotein is transported and bound to the collagen at the predentin-dentin junction. This event is probably central to the mineralization process, though other glycoproteins may be involved.

Amino Acids↗
PubMed1978

The covalent structure of cartilage collagen. Amino acid sequence of residues 552-661 of bovine alpha1(II) chains.

The covalent structure of the first 111 residues from the N-terminus of peptide alpha1(II)-CB10 from bovine nasal-cartilage collagen is presented. This region comprises residues 552-661 of the alpha1(II) chain. The sequence was determined by automated Edman degradation of peptide alpha1(II)-CB10 and of peptides produced by cleavage with trypsin and hydroxylamine. Comparison of this region of the alpha1(II) chain with the homologous segment of the alpha1(I) chain indicated a homology level of 85%, slightly higher than that of 81% reported for the N-terminal region of the alpha1(II) chain (Butler, Miller & Finch (1976) Biochemistry15, 3000-3006). The occurrence of two residues of glycosylated hydroxylysine was established at positions 564 and 603, the first present exclusively as galactosylhydroxylysine and the latter as a mixture of galactosylhydroxylysine and glucosylgalactosylhydroxylysine. Also, two residues at positions 648 and 657 were tentatively identified as glycosylated hydroxylysines. The amino acid sequences adjacent to the hydroxylysine residues so far identified in the alpha1(II) chain were compared with the homologous regions of the alpha1(I) and alpha2 chains, but no obvious prerequisite for hydroxylation could be seen. From comparison with the homologous sequence of the alpha1(I) chain, it appears that the alpha1(II)-chain sequence presented here contains three more amino acids than that reported for the alpha1(I) chain. This triplet would be interposed between residues 63 and 64 of the reported sequence of peptide alpha1(I)-CB7 from calf skin collagen. Data on the purification of the subpeptides and their amino acid compositions have been deposited as Supplementary Publication SUP 50087 (7 pages) at the British Library Lending Division, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1978) 169, 5.

Amino Acid Sequence↗