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Biomedical subjects

M L Tanzer

Publications and source records attributed to M L Tanzer.

At least 55 records · Page 3Linked to original sources
PubMed1985

Identification and quantitation of O-phosphoserine in human plasma fibronectin.

O-Phosphoserine was positively identified as the phosphorylated moiety in human plasma fibronectin by 31P-NMR of intact peptides. These data correlated completely with chemical analyses which demonstrated the presence of O-phosphoserine at a concentration of 2 residues/molecule. Neither O-phosphothreonine nor O-phosphotyrosine was detected in partial acid and partial alkaline hydrolysates, respectively.

Fibronectins↗
PubMed1985

Partial characteristics of an analog of pyridinoline isolated from human skin.

The most abundant amine in acid hydrolysates of human skin, eluting in the crosslink region of a reversed-phase HPLC chromatogram, has the same retention time as pyridinoline standard. This amine is not pyridinoline, since it is a weak fluorophore and its U/V spectrum does not agree with that of pyridinoline. The unknown amine was isolated and characterized by fast atom bombardment mass spectrometry and its structure is consistent with a deoxy-analogue of pyridinoline. It may be a crosslink component of some biological importance, since it is not detectable in skin from a patient with Marfan's Syndrome.

Amines↗
PubMed1984

High-performance liquid chromatographic separation of glycopeptides from Nereis cuticle collagen.

To facilitate the structural studies of invertebrate collagens, a sensitive and effective method was developed, using reverse-phase high-performance liquid chromatography for preparative isolation of the collagen subunits and their clostridial collagenase-derived peptides; the methods have been applied to Nereis cuticle collagen. The two subunits of denatured Nereis cuticle collagen, termed A and B, were initially separated by high-performance liquid chromatography. These polypeptides, with Mr of about 0.5 million, were each exhaustively digested with clostridial collagenase. The digest of the A subunit, which contains all of the uronic acid, was enriched for the uronic acid-containing glycopeptides by means of gel filtration. These glycopeptides were resolved into 23 major peaks, using reverse-phase HPLC, over a 5-h elution time, with an acetonitrile gradient (0-20%) containing 0.1% TFA. The amino acid composition data suggests that the peptides are of variable length, from 5 to 17 residues, while beta-elimination studies show that the uronic acid-containing moieties are all O-glycosidically linked to threonine residues, in the peptides examined. The amino acid sequence of one of the major glycopeptides was determined and found to be Gly-Hyp-Ala-Gly-Gly-Ile-Gly-Glu-Thr-Gly-Ala-Val-Gly-Leu-Hyp. The amino acid compositions of glycosylated and nonglycosylated peptides which had eluted, numbering about 100, showed a correspondence between hydrophobicity or hydrophilicity and emergence time from the column. We also found that the peptides most enriched in 4-hydroxyproline emerged earliest. These studies provide a foundation for elucidating the detailed structures of the large, unusual subunits of a well-characterized cuticle collagen.

Amino Acid Sequence↗
PubMed1984

Reversibility of monensin inhibition of oligosaccharide processing of human fibronectin.

Monensin impairs oligosaccharide processing in fibronectin primarily by inhibiting the conversion of oligosaccharides from the high mannose type to the complex type. The separate effects of monensin and cations on alpha-mannosidase activity in fibroblasts were examined using an in vitro assay system. The results indicated that monensin did not directly inhibit alpha-mannosidase activity in vitro, although prior treatment of fibroblasts with monensin caused an irreversible suppression of enzyme activity. The reversibility of monensin action on oligosaccharide processing was also examined. Analyses using concanavalin A (ConA) Sepharose affinity chromatography showed that the inhibitory action of monensin on oligosaccharide processing was biologically reversible. A progressive return to complex type oligosaccharides began about 11 h after the removal of the monensin. These composite results indicate that the reversibility of monensin action on oligosaccharide processing in fibronectin may be attributed to the restoration of enzyme activity, although the mechanism by which restoration occurs remains to be deciphered.

Calcium↗
PubMed1983

Swainsonine inhibits macrophage receptor-mediated uptake and degradation of a mannosyl-oligosaccharide.

Rat pulmonary macrophages were incubated in the presence of a radiolabeled mannosyl-oligosaccharide obtained from ovalbumin. Receptor-mediated endocytosis and degradation of this ligand by the cells was followed in the presence or absence of swainsonine, an inhibitor of alpha-mannosidases. The results indicated that at higher concentrations (greater than 1 microgram/ml) of swainsonine, both the internalization and degradation of the radiolabeled ligand were inhibited. At a concentration of 0.1 microgram/ml of swainsonine, only the degradation was inhibited while the uptake was unaltered. The degradation of the oligosaccharide was blocked due to the inhibition of lysosomal alpha-mannosidase. However, the inhibition of lysosomal alpha-mannosidase was reversible upon withdrawal of swainsonine.

Alkaloids↗
PubMed1983

Abnormal glycosylation of human cellular fibronectin in the presence of swainsonine.

The relationship between post-translational modifications of macromolecules and their intracellular routing is of fundamental importance. The availability of the indolizidine alkaloid, swainsonine, which interferes with glycoprotein processing, provides a new probe for studying relationships between glycosylation of proteins and their cellular routing. Using fibronectin as a model glycoprotein, we have explored the effect of swainsonine upon oligosaccharide structure, glycoprotein synthesis, and secretion. Confluent human fibroblasts were labeled with radioactive mannose or glucosamine in the presence or absence of swainsonine. Fibronectin was secreted into the medium of swainsonine-treated cultures and found to contain endo-beta-N-acetylglucosaminidase H-sensitive oligosaccharides, instead of the normal complex endo-beta-N-acetylglucosaminidase H-resistant oligosaccharides. Pronase-released glycopeptides and hydrazine-released oligosaccharides of isolated fibronectin from the culture media were analyzed using endo-beta-N-acetylglucosaminidase H and specific exoglycosidase digestions in conjunction with calibrated gel filtration chromatography. The structure of the swainsonine-modified endo-beta-N-acetylglucosaminidase H-sensitive oligosaccharide was found to be a hybrid type, Gal beta leads to GlcNAc beta leads to Man alpha leads to [Man alpha leads to (Man alpha leads to) Man alpha leads to]Man beta leads to GlcNAc beta leads to (+/- Fuc alpha leads to)-GlcNAc. Other experiments showed that the synthesis and secretion of fibronectin were not affected by its change in glycosylation. The results also infer that swainsonine inhibits Golgi mannosidase II in intact cells, and that removal of two mannose residues by that mannosidase is not a prerequisite for addition of galactose to the existing peripheral nonreducing GlcNAc or for the addition of fucose to the innermost reducing GlcNAc. The composite results indicate that significant changes in oligosaccharide structure had little effect upon the routing and cellular release of a typical N-asparagine-linked glycoprotein.

Alkaloids↗
PubMed1983

Abnormal collagen synthesis in skeletal muscle of dystrophic chicken.

Specific molecular properties of skeletal muscle collagens from normal and dystrophic chickens have been compared. When dystrophy develops in skeletal muscle tissue there was an increase in the amount of total collagen and an increased proportion of Type III collagen in the tissue. The results from the cross-link study as well as the analysis of the solubility of collagen showed that skeletal muscle of dystrophic chicken produces more immature collagen fibers compared to normal chicken. These findings strongly indicate an important role of collagen in the pathogenesis of the extensive connective tissue prolipheration characteristic of muscular dystrophies.

Animals↗
PubMed1983

Abnormal glycosylation of human fibronectin secreted in the presence of monensin.

Detailed studies of the effects of the ionophore monensin upon the glycosylation of secreted fibronectin have been carried out. Human fibroblasts in culture were incubated in 1 microM monensin for several hours, following which radiolabeled glucosamine or mannose was added to the cultures. Parallel incubation and labeling of control cultures were done. Labeled fibronectin was isolated from the culture media by gelatin-Sepharose chromatography, from cell surfaces by urea extraction, and from intracellular locations by cell lysis followed by immunoprecipitation. Detailed comparison of the glycopeptides released from fibronectin by pronase and of the oligosaccharides liberated by hydrazinolysis was carried out, particularly focusing on the secreted fibronectin, using gel filtration, high performance liquid chromatography, and concanavalin A chromatography, in conjunction with the use of endoglycosidase H and specific exoglycosidases. We demonstrate that fibronectin in the medium of monensin-treated cultures differs in its glycosylation pattern from the control fibronectin. High mannose oligosaccharides are abundant in the monensin-derived fibronectin, whereas the control protein contains primarily complex oligosaccharides. Monensin apparently does not alter the initial glycosylation of fibronectin since the high mannose oligosaccharides are present on both control and monensin-treated intracellular fibronectin. We suggest, therefore, that monensin, by impairing intracellular translocation through the Golgi region, allows incompletely processed forms of fibronectin to reach the cell surface and to be released into the culture medium.

Adult↗
PubMed1983

Characterization of a large fragment from annelid cuticle collagen and its relationship to the intact molecule.

Digestion of the cuticle collagen from the annelid Nereis virens with Clostridium histolyticum collagenase yields a native, collagenase-resistant fragment (CCRF) of the molecule with an Mr of about 900,000 (Kimura and Tanzer, J. Biol. Chem. 252: 8018, 1977). We have produced 940 nm long, SLS-crystallites from the collagenase-resistant fragment; the SLS pattern matches a region at one end of the 2,400 nm SLS obtained from intact cuticle collagen. Upon denaturation, the fragment yields two subunits, CCRFA and CCRFB, which can be separated by SDS polyacrylamide gel electrophoresis or by chromatography on DEAE-cellulose; the subunits are in about a 2:1 ratio. The subunits have amino acid compositions which are similar to those of the original A and B chains, although the fragments have a higher content of acidic residues and a lower content of hydroxyl residues. Previous studies of the intact B chain have shown that there is about one methionine in the chain, and that it is located near the COOH terminus. The CCRFB subunit also contains about one methionine, indicating that CCRFB is probably derived from the COOH end of the intact molecule. Based on these composite data, we have provisionally defined the amino and carboxyl ends of the cuticle collagen SLS and provide additional evidence that the molecule is a heteropolymer with the formula A(B)2.

Animals↗
PubMed1982

Effects of the ionophore monensin on type II collagen and proteoglycan synthesis and secretion by cultured chondrocytes.

Detailed studies of the effects of the ionophore monensin upon avian chondrocyte ultrastructure, macromolecular synthesis, and macromolecular secretion have been carried out. Embryonic avian chondrocytes in suspension culture were incubated in concentrations of monensin ranging from 1 X 10(-7) to 1 X 10(-6) M for durations up to 8 h. Electron microscopy revealed that the treated chondrocytes developed abnormal Golgi structures and a markedly distended rough endoplasmic reticulum. Biochemical and immunoassay studies showed that while total protein synthesis was only slightly impaired by monensin, the ionophore had pronounced effects upon the secretion of both type II collagen and proteoglycans. These two macromolecules responded to monensin inhibition in a similar fashion and accumulated within the affected chondrocytes. The kinetics of response over the monensin concentration range used was virtually identical for type II collagen and proteoglycan. Undersulfation of proteoglycan, caused by monensin, was examined by ion exchange chromatography and analysis of the products of chondroitinase ABC digestion. The results indicated that undersulfation affected all glycosaminoglycan chains in a general fashion rather than affecting a specific population of chains.

Animals↗
PubMed1982

Proteoglycan core protein is accumulated in cultured chondrocytes in the presence of the ionophore monensin.

The nature of the proteoglycan antigen which accumulates in chick embryo chondrocytes that had been incubated in monensin was examined. Both the culture media and cell lysates were immunoprecipitated using antibody which primarily is directed against core protein. Gel filtration and electrophoresis of the immunoprecipitates showed that molecules corresponding to completed proteoglycan, core protein, and link protein were present in the immunoprecipitates. Monensin caused the cellular accumulation of core protein which was both underglycosylated and undersulfated. These results suggest that monensin affects early events of proteoglycan biosynthesis and that it may be useful for elucidating those events.

Animals↗
PubMed1982

The phosphate content of human fibronectin.

The nature and distribution of the covalently bound phosphate of human fibronectin have been investigated. Phosphate was detected only as phosphoserine and is not present on asparagine-linked oligosaccharides. Fibroblast fibronectin labeled with 3H-amino acids and [32P]orthophosphate was subjected to limited tryptic digestion. Initial cleavages which reduced the molecule to nondisulfide-bonded polypeptides of Mr approximately 210,000-220,000 did not entail the loss of phosphate. Subsequent cleavage products included intermediate-sized 32P-phosphopeptides (43,000 and 37,000), but all 32P label became increasingly associated with lower molecular weight material (13,000 and 9,000) eventually terminating in a phosphopeptide (or peptides) of Mr approximately 6,000. The phosphorylated region(s) of human fibronectin are apparently not located at the extreme COOH-terminal region containing the interchain disulfide bond, but seem to be within a 40,000-50,000 segment near one end of the molecule. Cyanogen bromide cleavage of 32P-labeled fibronectin isolated from fibroblast culture medium produced one major phosphopeptide (Mr approximately 5,000) which co-purified through two chromatographic procedures with a CNBr phosphopeptide derived from plasma fibronectin. The phosphorylation of fibronectin is evidently restricted to a small region of the protein molecule, implying a very site-specific event. The phosphorylated region is present on human fibronectins obtained from plasma, fibroblast surfaces, and fibroblast culture medium, indicating that the role of the phosphate may be important to all forms of fibronectin.

Cell Line↗