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J H Fessler

Publications and source records attributed to J H Fessler.

At least 55 records · Page 3Linked to original sources
PubMed1983

Procollagen IV. Association to tetramers.

Procollagen IV was isolated from culture media of the mouse endodermal cell line PF-HR9. Some of the triple helical procollagen IV molecules were associated at their NH2 ends to tetramers which were identified by electron microscopy, velocity sedimentation, and electrophoresis. The formation of these tetramers in cell cultures and from isolated procollagen IV molecules was investigated. After an initial noncovalent association, which is reversible, disulfide bonds form between molecules. Even alkylated molecules form disulfide-linked tetramers when exposed to a mixture of reduced and oxidized glutathione. This reaction requires an adequate concentration of procollagen and is not facilitated by added laminin, Ca2+, or Mg2+ ions. Cystine, as a normal constituent of cell culture media, interferes in tetramer assembly, presumably by forming mixed disulfides. Tetramers formed normally and under the influence of glutathione are similar, but probably not identical, and resemble those isolated from fragmented basement membranes. We conclude that the NH2 ends of procollagen IV molecules can associate into tetramers without the help of other molecules and that disulfide bridges subsequently stabilize the association in various ways.

Animals↗
PubMed1982

Structural implications from an electronmicroscopic comparison of procollagen V with procollagen I, pC-collagen I, procollagen IV, and a Drosophila procollagen.

Chick embryo procollagen V, procollagen I, and pC-collagen I were sprayed, rotary shadowed, and compared electronmicroscopically with mouse procollagen IV and Drosophila procollagen produced by cell cultures and prepared in the same way. All the molecules appeared as threads and had a prominent knob protrusion at one end. For procollagen I and pC-collagen I this must correspond to the carboxyl propeptides, and most likely corresponds to the similarly sized carboxyl propeptides of procollagen IV. Procollagen V appeared as a thread of nearly the same length as procollagen I but with substantial knobs at both ends, corresponding to its known propeptides. The length of procollagen V and the known, successive processing of its propeptides make procollagen V more similar to the interstitial collagens than to the basement membrane procollagen IV, which is 1.4 times as long and has so far been found not to be processed. Drosophila procollagen resembles procollagen IV in length and appearance.

Animals↗
PubMed1982

Identification of the carboxyl peptides of mouse procollagen IV and its implications for the assembly and structure of basement membrane procollagen.

Clusters of mouse PF-HR9 endoderm cells derived from teratocarcinoma PCC4-F cells were incubated with [3H]proline and [35S]methionine. The synthesis of pro alpha 1 IV and pro alpha 2 IV chains and their association into triple helically folded disulfide-linked molecules were followed. Short incubations and incubations with pactamycin showed that approximately 30,000 molecular weight collagenase-resistant peptides, which are destroyed by pepsin, form the carboxyl end of the pro alpha IV chains. While disulfide links bridge parts of individual peptides, the carboxyl peptides of the three chains of a molecule are not disulfide linked to each other. We propose that these peptides form the knob protrusion seen in electron micrographs of rotary shadowed procollagen IV molecules. The implications of these findings, especially for the relatively slow assembly of procollagen IV, are discussed.

Animals↗
PubMed1982

Mouse procollagen IV. Characterization and supramolecular association.

The endodermal cell line PF-HR9, derived from the murine teratocarcinoma cell line PCC4-F, was grown as monolayers and as cell clusters called embryoid bodies. Procollagen IV and laminin were isolated from both kinds of culture media. Antibodies specific to collagen IV and to laminin demonstrated these materials in association with the cells and in the culture media. The procollagen IV consisted of pro alpha 1 IV and pro alpha 2 IV chains and gave a circular dichroic spectrum characteristic for collagen helices, with thermal transitions at 40, 44, and 51 degrees C. The molecules were visualized electron microscopically after rotary shadowing. Laminin showed the characteristic beaded cross-appearance, and procollagen IV was a 434 +/- 12-nm long linear thread containing a 17-nm carboxyl-terminal knob. The 7% of collagen helix with Tm = 51 degrees C corresponds to about a 30-nm length of the molecule and is probably that section of the amino end through which several procollagen IV molecules form a junctional complex. Several noncovalent associations of procollagen IV molecules were demonstrated by velocity sedimentation and electron microscopy of concentrated culture media, specifically associations of two and four procollagen IV molecules through their amino ends and dimers linked at their carboxyl ends. The results show that procollagen IV molecules associate noncovalently into the components which others have isolated from basement membranes and strongly support a network model of these supramolecular assemblies.

Animals↗
PubMed1981

Chain assembly intermediate in the biosynthesis of type III procollagen in chick embryo blood vessels.

A general mechanism for the assembly of procollagens is proposed from a biosynthetic study of procollagen III. This was shown to proceed by a stepwise process punctuated by disulfide bond formation and an assembly intermediate was recovered. The biosynthesis of type III procollagen in excised chick embryo blood vessels was studied by radioactive labeling for 30 min. Velocity sedimentation under denaturing conditions and purified antibodies specific against bovine amino propeptide III were used to identify and characterize monomeric pro alpha 1 III chains and a type III procollagen intermediate which is interchain disulfide-linked only at the carboxyl end but not at the amino end. The monomeric chains presumably have intrachain disulfide bonds within the propeptides. The monomeric pro alpha 1 III chains were also found when alpha, alpha'-dipyridyl was present during incubation. Pulse-chase experiments show that the monomeric chains and the intermediate are biosynthetic precursors of type III procollagen. Furthermore, it is shown that monomeric pro alpha 1 chains are not triple helical when extracted under nondenaturing conditions. The results indicate that the assembly of pro alpha 1 III chains into type III procollagen starts with the association of the folded carboxyl propeptides and is followed by formation of disulfide bonds between carboxyl propeptides, folding of the triple helix, and formation of disulfide bonds between amino propeptides. All procollagens may follow a similar assembly sequence.

Animals↗
PubMed1981

Assembly and processing of procollagen V (AB) in chick blood vessels and other tissues.

The biosynthesis and processing of type V procollagens was investigated in chick embryo blood vessels labeled with radioactive amino acids. Monomeric, pepsin-sensitive pro alpha 1 V and pro alpha 2 V chains are slowly assembled into triple helically folded molecules. A small proportion of these procollagen V molecules contain interchain disulfide bridges, and the disulfide-linked heterodimer and heterotrimer (pro alpha 1 V)2pro alpha 2 V were found. A relatively fast conversion of procollagen V to p-collagen V is followed by slow change to collagen V. This time course is similar to the processing of procollagen III and is much slower than the rate of appearance of type I collagen in blood vessels. A combination of sedimentation and electrophoretic analyses was used to measure the relative size of the type V chains and to demonstrate attachment of noncollagenous peptides (Mr = 33,000) to p alpha 2 V by disulfide linkage. Similar quantitative pulse-chase studies were made with calvaria and crop. As the same unusual features of assembly and processing of type V chains were seen in muscle, bone, and blood vessels, we conclude that these are characteristic of type V collagen biosynthesis.

Animals↗
PubMed1981

Propeptides of procollagen V (A,B) in chick embryo crop.

The biosynthesis of type V (A,B) collagens was recently found to proceed through the sequential forms pro alpha (A,B), p alpha (A,B), and f alpha (A,B). All these chains are larger than the A,B chains extracted from tissues after pepsin digestion. This report shows that all forms contain substantial peptides which are resistant to bacterial collagenase and concludes that type V collagens differ from the intestitial collagens (types I, II, III) in retaining large noncollagenous peptides in tissues. A peptide becomes transiently attached to the processing intermediate p alpha A by a reducible linkage. The conversion of procollagen V to p-collagen V was inhibited by colchicine and arginine. Previously, the disulfide-linked heterotrimer [(pro alpha B)2 (pro alpha A)] was found and additional procollagens containing only B type molecules were inferred. Further investigations reported here agree with these conclusions and also indicate that some trimeric molecules containing more than one chain related to A may exist.

Amino Acids↗
PubMed1981

Assembly and processing of procollagen type III in chick embryo blood vessels.

The processing of [3H]proline-labeled procollagen III in excised chick embryo blood vessels was found to differ significantly from that of procollagen I in the same tissue. While first the amino propeptides and then the carboxyl propeptides were fairly rapidly cleaved from procollagen I, only the carboxyl propeptides were split off procollagen III, leaving pN-collagen III. This intermediate, which is only slowly converted to collagen III by loss of amino propeptides, was characterized by its sedimentation properties, isolation of the amino propeptide, and reaction with purified antibodies that are specific against bovine amino propeptide III. It is interchain disulfide-linked, both through the amino propeptide and the carboxyl ends of the collagen chains. The conversion of procollagen III to pN-collagen III either in blood vessels, or after isolation by a carboxyl procollagen peptidase obtained from chick tendon fibroblast cultures, is inhibited by 50 mM arginine. Underhydroxylated procollagen III was isolated from blood vessels treated with alpha, alpha'-dipyridyl. Its amino propeptides reacted with the above antibodies but were not linked to each other. In contrast, its carboxyl propeptides were interchain disulfide-bridged, supporting previous suggestions that the carboxyl propeptides play a role in the assembly of procollagen trimer.

Animals↗
PubMed1980

Biosynthesis of A,B procollagen.

Interest in cell-associated collagens led others to isolate A and B collagen chains, also known as type V collagen, from many tissues, but only after pepsin cleavage. Soluble precursors of these chains are synthesized in vitro by crop, a chicken embryo muscle tissue, and are converted by at least two processing steps from procollagens via intermediates to final forms which are large than the pepsin-derived A and B chains. Heterotrimeric, disulfide-bridged procollagen molecules corresponding to B2A exist. Components were separated by ion exchange chromatography, velocity sedimentation, and electrophoresis, and the relationships between them were established by sequential radioactive labeling and comparison of peptides generated by protease cleavage.

Animals↗
PubMed1979

Separate amino and carboxyl procollagen peptidases in chick embryo tendon.

Procollagen synthesized by freshly excised chick enbryo leg tendons is efficiently processed by proteolytic removal of first the amino propeptides and then the carboxyl propeptides. The same processes proceed in confluent short term cell cultures derived from such tendon explants; in sparse cultures cleavage of the amino propeptides predominates. Separate amino and carboxyl procollagen peptidase activities were demonstrated by specific assays in enzymes obtained from cell culture media by ammonium sulfate precipitation, ion exchange chromatography, and velocity sedimentation. Both enzymes are inhibited by EDTA and 1:10 phenanthroline but not by inhibitors of serine proteases. Evidence is provided that the proteolytic scissions are specific and similar to the physiologically occurring processes. The collagen telopeptides left after cutting by the enzymes can participate in lysyl oxidase-induced cross-linking. The enzymes can remove propeptides from cross-linked procollagens without destroying these links which occur through telopeptides. The enzymes act on the separated amino and carboxyl portions of procollagen fragmented by vertebrate collagenase and can act on procollagens which have been associated as well as on molecules in solution.

Aminopeptidases↗
PubMed1975

Procollagen assembly and secretion in embryonic chick bone.

Chick embryo skull bones incorporated radioactive proline and cystein into procollagen in short term organ culture. Pulse-chase experiments showed that individual precursor chains (pro-alpha1 and pro-alpha2) were formed first and that these were subsequently linked by disulfide bounds into trimers. Radioautography showed that labeled material was secreted 30 min after adding label to the cells, and electrophoretic analyses showed that after this time completed labeled collagen molecules appeared. Conversion from disulfide-linked procollagen to collagen proceeded in more than one step. An intermediate form consisting of shorter chains, which were still trimerically linked, was found.

Animals↗
PubMed1975

Interactions between collagen chains and fiber formation.

The temperature-dependent dissociation of neutral salt-soluble collagen into its component chains was measured in 0.6-1.6 M urea solutions at pH 7.3. The temperature-dependent association of the same radioactively labeled collagen into fibers was measured in 0-0.4 M urea solutions, pH 7.3. The effect of urea on the temperature, Tm(G), for half dissociation into chains was small, and the value extrapolated to zero urea concentration was 39 degrees C. In contrast, the effect of urea on the temperature, Tm(F), for half association into fibers was large, and the value at zero urea concentration was 30 degrees C. We conclude that while body temperature provides excellent conditions for the matching of collagen chains to form molecules, the conditions are not optimal for the formation of highly ordered fibers. The large effects of 0.1 M urea suggest that other factors in vivo may help to destabilize mismatched molecular association during fiber growth. Alternately this might be facilitated by parts of the extension peptides of procollagen.

Animals↗
PubMed1975

Procollagen: biological scission of amino and carboxyl extension peptides.

Procollagen, the triple-stranded precursor of chick embryo skull bone collagen, contains two pro alpha1 and one pro alpha2 chains. We find that each of these is a collagen chain with both an NH2-terminal and a COOH-terminal extension peptide. The NH2-peptide of pro alpha1 contains cysteine and differs from the NH2-peptide of pro alpha2. The three NH2-peptides are cut off, giving a disulfide-linked intermediate, named altered procollagen; then the disulfide-linked COOH-peptides, which contain cysteine and tryptophan, are cut off, leaving collagen. Procollagen, altered procollagen, and COOH-peptide were isolated. Collagenase digestion of procollagen gave both NH2- and COOH-peptides, while altered procollagen gave only COOH-peptides. The following results of sequential, in vitro labeling at 37 degrees and of specific cleavage of procollagen proved the structure: [(NH2-peptide)-collagen-(COOH-peptide)]3 with interstrand S-S links between only the COOH-peptides. (i) The COOH-peptides of pro alpha chains were labeled with [3H]proline before the remainders of the chains; (ii) [35S]cysteine appeared in the COOH-peptides of completed covalent molecules 5 min earlier than in the NH2-peptides; (iii) tadpole tail collagenase, which cuts native collagen into triple-stranded 3/4 pieces containing the NH2 termini and 1/4 pieces containing the COOH ends, cuts procollagen into 3/4 pieces with NH2-peptides attached and 1/4 pieces attached to the disulfide-linked COOH-peptides. The COOH-peptides of pro alpha 1 and pro alpha2 were labeled in a 2:1 ratio at 4 min, indicating simultaneous translation of pro alpha1 and pro alpha2.

Animals↗