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Biomedical subjects

M L Tanzer

Publications and source records attributed to M L Tanzer.

At least 73 records · Page 4Linked to original sources
PubMed1980

Kinetic studies of the intracellular transport of procollagen and fibronectin in human fibroblasts. Effects of the monovalent ionophore, monensin.

The monovalent ionophore, monensin, has been found to inhibit the secretion of both procollagen and fibronectin from human fibroblasts in cell culture. The kinetics of inhibition, as well as those for the release of inhibition, suggested that both proteins may be cotransported in the cell. In the present study, we have examined the intracellular translocation and release into the culture medium of procollagen and fibronectin, in the presence or absence of monensin. Pulse-chase studies were combined with subcellular fractionation of the fibroblasts to determine the rates of intracellular movement. We found that monensin did not significantly affect either the subcellular fractionation, or the distribution of organelle marker enzymes along the gradient, and that procollagen moved from a region of high buoyant density (primarily endoplasmic reticulum), through a mid-density region (primarily Golgi elements), to a region of low buoyant density before exiting from the cell. Monensin markedly decreased the rate of transit from one region to the other; kinetic analysis of the data showed a greater than 3-fold decrease in the rate constants for intracellular movement into the low buoyant density components and thence to the culture medium. The density gradient distribution of fibronectin was affected by monensin in similar fashion, indicating that it and procollagen probably follow the same intracellular route. Since monensin causes both proteins to accumulate in highest abundance in regions of the density gradient corresponding to Golgi and endoplasmic reticulum, the site of ionophore blockade appears to be within the Golgi apparatus. Thus, procollagen and fibronectin share a common intracellular route prior to entering the Golgi region of the cell.

Biological Transport↗
PubMed1980

Undersulfated proteoglycans are secreted by cultured chondrocytes in the presence of the ionophore monensin.

The monovalent ionophore, monensin, inhibits secretion of many different proteins from a wide variety of cells. The site of blockage is at the golgi complex. We have exposed chick embryo chondrocytes in suspension culture to monensin, at concentrations ranging from 10(-8) to 10(-6) M. At the higher concentrations, between 10(-7) and 10(-6) M, monensin inhibited secretion of type II procollagen, which accumulated in the chondrocytes. At these concentrations of the ionophore, proteoglycan synthesis was inhibited, as measured by radioactive serine incorporation into core proteins and by radioactive glucosamine or SO4 incorporation into glycosaminoglycans. However, at a monensin concentration of 3 x 10(-8) M, the incorporations of serine and glucosamine were close to normal while SO4 incorporation was at 30% of control values. The ratio of glucosamine to serine in pronase-released glycosaminoglycans from culture media was unaffected by 3 x 10(-8) M monensin but the sulfate to serine ratio decreased to 29% of control values. Examination of the glycosaminoglycans by gel filtration showed a progressive increase in Kav values as sulfation decreased. Undersulfation was demonstrated by radiochromatographic analysis of the digestion products following incubation with chondroitinase ABC. The composite results show that monensin interferes with sulfation of newly synthesized proteoglycans.

Animals↗
PubMed1980

Nereis cuticle collagen. Proteolysis by marine vibrial and clostridial collagenases.

Proteolysis of Nereis cuticle collagen by two bacterial collagenases was investigated using viscosimetry, enzyme kinetics, sodium dodecyl sulfate polyacrylamide gel electrophoresis, and ion exchange chromatography of collagenolytic peptides. Collagenase of the marine Vibrio B-30 completely degrades native cuticle collagen at 7 degress C with a turnover number 50 times greater than that of the clostridial collagenase. Although turnover numbers for the two enzymes are comparable when using denatured cuticle collagen as substrate, the vibrial collagenase appears to cleave twice as many peptide bonds per mg of cuticle collagen as does the clostridial enzyme. Sodium dodecyl sulfate gel electrophoresis of collagenase-digested native cuticle collagen reflects the resistance of the collagen to clostridial collagenase; however, the vibrial enzyme completely degrades the cuticle collagen with the formation of one transient intermediate (Mr 400,000). Peptide analysis of fully digested denatured cuticle collagen reveals that the two enzymes have a number of qualitative and quantitative similarities. Despite these, however, only the vibrial collagenase seems capable of extensively degrading native cuticle collagen.

Animals↗
PubMed1980

Separation of cyanogen bromide peptides of collagen by means of high-performance liquid chromatography.

Cyanogen bromide peptides of bovine collagen Types I, II nad III were analyzed using high-performance liquid chromatography (HPLC). Elution patterns of each collagen type were unique and reproducible. Elution patterns of the CNBr peptides of the alpha 1 and alpha 2 chains of Type I collagen were also unique and together accounted for the major components of Type I collagen. Analysis of the eluted peptides from HPLC of each collagen type by sodium dodecyl sulphate-polyacrylamide gel electrophoresis showed specific patterns for each collagen. Thus, unique and reproducible HPLC chromatograms were obtained, providing a new analytical method that is simple, sensitive and rapid.

Animals↗
PubMed1980

Immunocytochemical localization of procollagen and fibronectin in human fibroblasts: effects of the monovalent ionophore, monensin.

The monovalent ionophore monensin inhibits the secretion of both procollagen and fibronectin from human fibroblasts in culture. The distribution of these proteins in control and inhibited (5 x 10(-7) M monensin) cells has been studied by immunofluorescence microscopy. In control cells, both antigens are present throughout the cytoplasm and in specific deposits in a region adjacent to the nucleus, which we identify as a Golgi zone by electron microscopy. Treatment of cells with monensin causes intracellular accumulation of procollagen and fibronectin, initially in the juxta-nuclear region and also subsequently in peripheral regions. Electron microscope studies reveal that in such cells the juxta-nuclear Golgi zone becomes filled with a new population of smooth-membraned vacuoles and that normal Golgi complexes are not found. Immunocytochemically detected procollagen and fibronectin are localized in the region of these vacuoles, whereas more peripheral deposits correspond to the dilated cisternae of rough endoplasmic reticulum, which are also caused by monensin. Procollagen and fibronectin are often codistributed in these peripheral deposits. Accumulation of exportable proteins in Golgi-related vacuoles is consistent with previous analyses of the monensin effect. The subsequent development of dilated rough endoplasmic reticulum also containing accumulated proteins may indicate that there is an additional blockade at the exit from the endoplasmic reticulum, or that the synthesized proteins exceed the capacity of the Golgi compartment and that their accumulation extends into the endoplasmic reticulum.

Cell Line↗
PubMed1979

Isolation and characterization of a double chain intermolecular cross-linked peptide from insoluble calf bone collagen.

A double chain peptide containing the sodium borohydride-reduced intermolecular cross-link, hydroxylysinohydroxynorleucine, was isolated following sequential cyanogen bromide digestion and limited alkaline hydrolysis of insoluble calf bone collagen. Amino acid composition and NH2-terminal sequence analysis indicated that the peptide was highly acidic and consisted of 19 amino acid residues including the cross-link. Amino acid composition and automated sequence analysis of this peptide before and after cleavage of the cross-link, using periodic acid, provided the data from which the following structure was deduced. (formula: see text). The sequence of the larger peptide is identical with that of residues 8c to 19c in the COOH-terminal nonhelical region of the homologous skin collagen alpha1 chain. The hydroxylysine residue located at position 17c in the alpha chain of type I collagen appears to be a predominant site for intermolecular cross-link formation. Assignment of the smaller peptide component within the known primary structure of the collagen molecule currently cannot be made.

Amino Acids↗
PubMed1979

Monovalent ionophores inhibit secretion of procollagen and fibronectin from cultured human fibroblasts.

Procollagen and fibronectin are major products of confluent fibroblasts in culture and both are released from the cells. Procollagen is secreted by known pathways, while the mechanism of fibronectin release is controversial. We find that the secretion of both these proteins can be reduced to 20% by low concentrations (0.1-1 muM) of ionophores that have affinity for monovalent cations. In contrast, little effect upon secretion was found for similar concentrations of an ionophore that binds divalent cations. Electron microscopy showed that the inhibition of secretion is accompanied by accumulation of membranous vacuoles. We believe that the ionophores impede secretion by acting on the secretory structures rather than on the proteins themselves. Biochemical studies supported this interpretation because no changes were detected in hydroxylation or glycosylation of procollagen or glycosylation of fibronectin, nor were significant changes in cellular amino acid incorporation observed. Pulse-chase studies indicated that the rates of secretion were impaired by the ionophore without enhancing intracellular degradation. The decreased secretory rates accounted for the lower levels of procollagen and fibronectin in the culture medium; no evidence for increased catabolism of the secreted proteins was found. Secretion could be readily restored by removing the ionophore from the culture medium. The results indicate that procollagen and fibronectin may be simultaneously secreted, possibly utilizing a common pathway for secretion; the ionophores effectively interfere with cellular secretory pathways without impairing protein synthesis or protein glycosylation or altering protein catabolism.

Anti-Bacterial Agents↗
PubMed1977

Nereis cuticle collagen. Isolation and properties of a large fragment resistant to proteolysis by bacterial collagenase.

Native cuticle collagen, obtained from Nereis virens, was incubated with purified bacterial collagenase (EC 3.4.4.19). The kinetics of proteolysis were monitored by viscometry, in parallel with similar digestions of calf skin collagen. Comparison of the kinetics of digestion of the two collagens, at similar enzyme to substrate ratios (w/w), showed that the native cuticle collagen was relatively refractory to digestion by bacterial collagenase. Characterization of the cuticle collagen digest by sodium dodecyl sulfate-polyacrylamide electrophoresis and agarose gel filtration in CaCl2 showed a large polypeptide, of about 300,000 daltons, to be a major product. The native form of this product, a unique fragment, was isolated from the digest by ethanol precipitation. It was found to have an intrinsic viscosity of 120 dl/g, to have an optical rotary dispersion curve characteristic of collagen, to undergo a typical collagenous thermal transition with a Tm of 23.2 degrees, and to have a calculated molar mass of 900,000 g with molecular dimensions of 9,000 X 13 A. It had an amino acid composition which was similar, but not identical with the native cuticle collagen. Although the original substrate contained two dissimilar chains, A and B, in a molar ratio of 1:2, the collagenase-resistant product appeared to be composed of only one type of polypeptide fragment. Possibly, the original subunits contain similar, if not identical collagenase-resistant regions.

Animals↗
PubMed1977

Inhibitory effects of tunicamycin on procollagen biosynthesis and secretion.

Chick embryo cells were briefly exposed to the antibiotic, tunicamycin. Pre-exposed cells, compared to control cultures, showed a severe, progressive inhibition of the incorporation of glucosamine and mannose into total cellular macromolecules. Inhibition of the incorporation of glycine, leucine and proline was also progressive but not as marked as for the carbohydrates. Cellular secretion of all macromolecules was severely impaired. while comparison of the procollagens showed no difference in their subunit size or in their degree of glycosylation; the intracellular content of procollagen polypeptides was similar for both types of cells. In vitro studies showed that tunicamycin selectively inhibited glucosamine, but not mannose, incorporation into macromolecules. The composite results indicate that tunicamycin effectively inhibits protein synthesis, protein glycosylation and protein secretion in chick embryo cells.

Animals↗
PubMed1977

Osteogenesis imperfecta: biochemical studies of bone collagen.

The biochemical properties of tibial bone collagen, obtained from patients with osteogenesis imperfecta, were studied by investigating amino acid composition, subunit composition and crosslink formation. Direct comparison of this bone was made with normal bone, age and sex matched, which had been removed from the tibiae of individuals within 48 hours after accidental death. The amino acid compositions of OI and normal bone collagen were almost identical and the pepsin solubilized collagen fraction as well as the CNBr peptides of insoluble bone collagen were very similar, indicating that no differences in collagen genetic type occurred in OI compared to normal. The crosslink contents and the specific radioactivities of the insoluble collagens were determined following NaB3H4 reduction. The specific radioactivity values of OI bone collagen were found to average 50 per cent higher than normal collagen. Analyses of OI collagen showed abundant formation of the major reducible aldehydes and crosslinks. Compared to the controls there were much higher proportions of the reduced aldehyde, dihydroxynorleucine and the reduced crosslink, dihydroxylysinonorleucine. These results may indicate delayed maturation of crosslinking in OI bone collagen and may reflect diminished stability of such collagen during bone development.

Adolescent↗