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M Bhown

Publications and source records attributed to M Bhown.

16 recordsLinked to original sources
PubMed1992

Isolation, characterization and immunolocalization of a 53-kDal dentin sialoprotein (DSP).

We isolated a sialic-rich protein from rat dentin extracts and have named it dentin sialoprotein, DSP (formerly called 95K glycoprotein). DSP is rich in aspartic acid, glutamic acid, glycine and serine, but contains no cysteine or phosphate. The 30% carbohydrate content includes about 9% sialic acid and indicates that several N-glycosides and O-glycosides are present. Sedimentation equilibrium analysis gave a M(r) of 52,570. Based on this molecular weight we calculated that DSP contains about 350-amino acids and 75 monosaccharides. With automated Edman degradation the sequence of the first 8-amino acids was shown to be: Ile-Pro-Val-Pro-Gln-Leu-Val-Pro. The initial 3 residues of this sequence are identical to the first 3 in human osteopontin (OPN) and are closely similar to the Leu-Pro-Val sequences of OPN from other species, as well as at the beginning of bone acidic glycoprotein-75 (BAG-75). On Western immunoblots, purified polyclonal antibodies reacted only with DSP in dentin extracts and with none of the proteins from bone. Similarly, immunolocalization experiments showed the presence of DSP in dentin but not in enamel or alveolar bone. Along with immunohistochemical localization data reported elsewhere, these observations suggest that DSP may be an important marker for cells in the odontoblast lineage.

Amino Acid Sequence↗
PubMed1987

Isolation, characterization, and biosynthesis of a phosphorylated glycoprotein from rat bone.

A phosphorylated glycoprotein was purified from the mixture of proteins extracted by demineralization of rat bone with 0.5 M EDTA in 4 M guanidinium chloride. A high level of purity for the preparation was indicated by a single band on sodium dodecyl sulfate (SDS)-gradient gel electrophoresis, sedimentation equilibrium ultracentrifugal data, and by automated Edman degradation results. The molecular weight of the phosphoprotein was shown to be about 44,000 by sedimentation equilibrium analyses in 4 M guanidinium chloride, even though an Mr of 75,000 was obtained by 5-15% SDS-polyacrylamide gel electrophoresis. Subsequent analysis by 15% SDS-polyacrylamide gel electrophoresis gave an Mr of 45,000. Analytical data showed that the protein contained 16.6% carbohydrate, possibly including 1 N-linked oligosaccharide and 5-6 O-linked oligosaccharides. The aspartic acid- and glutamic acid-rich protein contained about 300 amino acid residues including 1 phosphothreonine and 12 phosphoserine residues. Alkaline beta-elimination/NaBH4 reduction data showed that the phosphate obtained by complete acid hydrolysis prior to amino acid analysis was equivalent to the phosphate subject to alkaline beta-elimination. In this experiment, the losses of serine plus threonine exceeded the amount of phosphate liberated by 5-6 residues/protein. These serine and threonine residues probably represent O-linked oligosaccharides, since the protein contained about this number of N-acetyl-galactosamine residues. That the phosphoprotein is synthesized and secreted by osteoblast-like cells was shown with cultures of clonal rat osteosarcoma cells. After pulsing with 32PO4 the proteins secreted into the medium were precipitated with trichloroacetic acid and the radiolabeled proteins were immunoadsorbed. A protein migrating in the same position, on 5-15% SDS-polyacrylamide gel electrophoresis (i.e. with an Mr = 75,000) and on 15% gels (Mr = 45,000), as the phosphoprotein obtained from bone could be specifically immunoprecipitated.

Amino Acids↗
PubMed1987

A comparative immunocytochemical study on the subcellular distributions of 44 kDa bone phosphoprotein and bone gamma-carboxyglutamic acid (Gla)-containing protein in osteoblasts.

Bone gamma-carboxyglutamic acid (Gla)-containing protein (BGP or osteocalcin) and 44 kDa bone phosphoprotein (44K BPP, also called Sialoprotein I or osteopontin) have been localized at the ultrastructural level in osteoblasts from woven bones of newborn rats. Frozen, undecalcified sections of periodate-lysine-paraformaldehyde fixed specimens were incubated with affinity purified, monospecific antibodies against BGP or 44K BPP. The sites of the antigen-antibody reaction were demonstrated by the avidin-biotin-peroxidase complex method using the Hanker-Yates reagent as a peroxidase substrate. In some cases immunostaining could only be achieved after detergent treatment. The immunostained sections were then flat-embedded in Epon 812 and processed for electron microscopy. Strong specific intracellular labeling was obtained with both antibodies, but the patterns of staining differed significantly: BGP antigenicity was mainly located in the endoplasmic reticulum (ER), whereas 44K BPP behaved as a Golgi-specific antigen. In both cases, however, we found no evidence for immunostained secretory vesicles. There was no correlation between the expression of BGP by osteoblasts and the morphological aspect of these cells, their apparent degree of polarization with respect to the bone matrix, or their relation with the mineralized phase.

Animals↗
PubMed1985

The biosynthesis of dentin phosphophoryns by rat incisor odontoblasts in organ culture.

An in vitro system consisting of rat incisor fragments was used to study the process of dentinogenesis. In order to establish the usefulness of the organ culture, the biosynthesis and deposition of the major noncollagenous components of dentin, the phosphophoryns, were followed for specific lengths of time in culture. Three criteria were satisfied: (1) the synthesis of proteins which appeared to be chemically identical to the native proteins of dentin, (2) the accumulation of the phosphophoryns within the matrix or time, and (3) the association of the secreted proteins with the mineral phase of dentin. The synthesis of phosphophoryns was determined by using both (3H)-serine and (32P)-inorganic phosphate as precursors for synthesis of protein and post-translational modification of serine to phosphoserine. In vitro synthesized phosphophoryns were characterized by 1) their accumulation and EDTA extractability from within dentin, 2) calcium chloride precipitability, 3) elution on anion-exchange columns (DEAE cellulose and AGMP50), and 4) Mr's on SDS-PAGE and Sepharose CL-6B columns. This novel system of studying dentinogenesis provides a model with which to study the regulation of extracellular matrix protein synthesis and may be useful for revealing the effect of other agents which influence tooth development and mineralized tissue metabolism in general.

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

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

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

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↗
PubMed1977

Effect of acetylsalicylic acid on the amphibian gastric mucosa. I. Electrophysiological and permeability changes.

The effects of acetylsalicylic acid on the in vitro bullfrog gastric mucosa were defined. After mucosal exposure of 10 or 20 mM acetylsalicylic acid, a characteristic series of changes evolved. The potential difference and resistance rose initially. After 15 to 30 min the potential difference declined to zero. Transmucosal resistance remained elevated considerably longer but began to decline toward zero after 30 to 60 min of exposure. Mucosal permeability to mannitol increased as transmucosal resistance declined, but was not markedly elevated until the 2nd hr of exposure to acetylsalicylic acid. The initial rise of potential difference was associated with inhibition of H+ secretion whereas the later decline of potential difference was due to inhibition of Cl- transport. The abolition of short circuit current could not be attributed to mucosal to serosal flux of acetylsalicylate.

Animals↗
PubMed1977

Effect of acetylsalicylic acid on gastric mucosa. II. Mucosal ATP and phosphocreatine content, and salicylate effects on mitochondrial metabolism.

The effects of acetylsalicylic acid on gastric mucosal ATP and phosphocreatine and mucosal O2 consumption were studied using in vitro bullfrog gastric mucosa. Electrophysiological parameters (potential difference and resistance) changed rapidly after exposure to 10 or 20 mM acetylsalicylic acid at pH 3.0, 4.0, or 6.0 in the mucosal solution. ATP and phosphocreatine content decreased rapidly after exposure. Control mucosae contained 11.8 +/- 0.98 nmoles of ATP and 11.5 +/- 0.68 nmoles of phosphocreatine per mg of protein. ATP content decreased 21% and phosphocreatine decreased 45% after 15 to 30 min of exposure. Longer durations of exposure reduced both ATP and phosphocreatine to 10 to 15% of control levels. O2 consumption was increased during the first 15 to 30 min after exposure to acetylsalicylate. Longer durations of exposure reduced O2 consumption to near control levels. Study of isolated mitochondria confirmed that acetylsalicylate itself did not significantly affect mitochondrial respiration, respiratory control ratio, or ADP:O ratio. Salicylate had a biphasic effect which was concentration dependent. Salicylate initially stimulated and later inhibited mitochondrial respiration. Using various substrates the site of inhibition appeared to be located in the segment of the respiratory chain between coenzyme Q and cytochrome c1.

Adenosine Triphosphate↗