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L I Fessler

Publications and source records attributed to L I Fessler.

52 records · Page 3Linked to original sources
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

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

Synthesis of types I, III and AB2 collagen by chick tendon fibroblasts in vitro.

Tendons from 14--17-day-old chick embryos contain predominantly type I collagen and about 5% AB2 collagen; type III collagen is not detectable by biochemical methods, such as sodium dodecyl sulfate/polyacrylamide gel electrophoresis or cyanogen bromide pattern, but can be visualized by immunofluorescence staining with collagen-type-specific antibodies. Similarly, freshly dissociated tendon cells secrete only type I collagen into the culture medium but no significant amounts of type III collagen [Uitto, J., Lichtenstein, J. R., and Bauer, E. A. (1976) Biochemistry, 15, 4935--4942]. Transfer of tendon cells from chick embryos to monolayer conditions, however, initiated synthesis of type III collagen in about 10% of the cells within three days, as visualized by immunofluorescence staining. Secretion of type III collagen into the culture medium can also be detected by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. With increasing number of passages the number of cells producing type III collagen reached levels of about 80% after the third passage, while 90% of all cells stained positively for type I collagen. This is reflected by an increase of production of type III collagen as determined by CM-cellulose chromatography. Using velocity sedimentation, the secretion of type III procollagen and of pN-collagen (carrying the amino-terminal extension only), into the culture medium of a second-passage tendon cell culture was detected. This study provides new evidence that the phenotype of cells may alter during transfer from the environment in vivo to conditions in vitro and that additional changes may occur with time in culture.

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

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↗
PubMed1973

Collagen synthesis: a disulfide-linked collagen precursor in chick bone.

Chick calvaria labeled in vitro contain a triple-stranded collagen precursor that contains two pro alpha1 and one pro alpha2 chains. All chains contain cystine and are linked by S-S bonds. Ultracentrifugal analysis was used to measure the relative size of the disulfide-linked units. It is proposed that the S-S links help to determine the correct alignment of the triple-stranded structure.

Amides↗