scholarly journals Effect of prevention of procollagen triple-helix formation on proline 3-hydroxylation in freshly isolated chick-embryo tendon cells

1981 ◽  
Vol 196 (1) ◽  
pp. 203-206 ◽  
Author(s):  
K Majamaa
Keyword(s):  
Carboxylic Acid ◽  
Chick Embryo ◽  
Triple Helix ◽  
Hydroxylase Activity ◽  
Intact Cells ◽  
Helix Formation ◽  
Tendon Cells ◽  
Triple Helix Formation

Inhibition of procollagen triple-helix formation by the addition of cis-hydroxyproline or azetidine-2-carboxylic acid increased the synthesis of 3-hydroxy[14C]proline 1.7-1.8-fold in pulse-chase experiments with freshly isolated chick-embryo tendon cells. The amount of 3-hydroxy[14C]proline, expressed as a percentage of the total 14C radioactivity in hydroxyproline, reached 8.4%. Control experiments indicated that the two analogues had no effect on the prolyl 3-hydroxylase activity of these cells. The data suggest that the time available before triple-helix formation in part regulates the extent of the 3-hydroxylation of proline in the biosynthesis of collagen in intact cells.

scholarly journals Effect of L-azetidine-2-carboxylic acid on glycosylations of collagen in chick-embryo tendon cells

1976 ◽  
Vol 160 (3) ◽  
pp. 639-645 ◽  
Author(s):  
A Oikarinen ◽  
H Anttinen ◽  
K I Kivirikko
Keyword(s):  
Carboxylic Acid ◽  
Chick Embryo ◽  
Type I Collagen ◽  
Type I ◽  
Pulse Label ◽  
Chase Period ◽  
Helix Formation ◽  
Collagen Secretion ◽  
Tendon Cells ◽  
Triple Helix Formation

The glycosylations of hydroxylysine during collagen biosynthesis in isolated chick-embryo tendon cells were studied by using pulse-chase labelling experiments with [14C]-lysine. The hydroxylation of lysine and the glycosylations of hydroxylysine continued after a 5 min pulse label for up to about 10 min during the chase period. These data differ from those obtained previously in isolated chick-embryo cartilage cells, in which, after a similar 5 min pulse label, these reactions continued during the chase period for up to about 20 min. The collagen synthesized by the isolated chick-embryo tendon cells differed markedly from the type I collagen of adult tissues in its degree of hydroxylation of lysine residues and glycosylations of hydroxylysine residues. When the isolated tendon cells were incubated in the presence of L-azetidine-2-carboxylic acid, the degree of glycosylations of hydroxylysine during the first 10 min of the chase period was identical with that in cells incubated without thcarboxylic acid for at least 60 min, whereas no additional glycosylations took place in the control cells after the 10 min time-point. As a consequence, the collagen synthesized in the presence of this compound contained more carbohydrate than did the collagen synthesized by the control cells. Additional experiments indicated that azetidine-2-carboxylic acid did not increase the collagen glycosyltransferase activities in the tendon cells or the rate of glycosylation reactions when added directly to the enzyme incubation mixture. Control experiments with colchicine indicated that the delay in the rate of collagen secretion, which was observed in the presence of azetidine-2-carboxylic acid, did not in itself affect the degree of glycosylations of collagen. The results thus suggest that the increased glycosylations were due to inhibition of the collagen triple-helix formation, which is known to occur in the presence of azetidine-2-carboxylic acid.

scholarly journals Further studies on the effect of the collagen triple-helix formation on the hydroxylation of lysine and the glycosylations of hydroxylysine in chick-embryo tendon and cartilage cells

1977 ◽  
Vol 166 (3) ◽  
pp. 357-362 ◽  
Author(s):  
Aarne Oikarinen ◽  
Henrik Anttinen ◽  
Kari I. Kivirikko
Keyword(s):  
Chick Embryo ◽  
Triple Helix ◽  
Collagen Triple Helix ◽  
Helix Formation ◽  
Tendon Cells ◽  
Triple Helix Formation ◽  
Lysine Residues ◽  
Cartilage Cells ◽  
Helical Molecules ◽  
And Cartilage

The hydroxylation of lysine and glycosylations of hydroxylysine were studied in isolated chick-embryo tendon and cartilage cells under conditions in which collagen triple-helix formation was either inhibited or accelerated. The former situation was obtained by incubating the tendon cells with 0.6mm-dithiothreitol, thus decreasing their proline hydroxylase activity by about 99%. After labelling with [14C]proline, the formation of hydroxy[14C]proline was found to have declined by about 95%. Since the hydroxylation of a relatively large number of proline residues is required for triple-helix formation at 37°C, the pro-α-chains synthesized under these conditions apparently cannot form triple-helical molecules. Labelling experiments with [14C]lysine indicated that the degree of hydroxylation of the lysine residues in the collagen synthesized was slightly increased and the degree of the glycosylations of the hydroxylysine residues more than doubled, the largest increase being in the content of glucosylgalactosylhydroxylysine. Recovery of chick-embryo cartilage cells from temporary anoxia was used to obtain accelerated triple-helix formation. A marked decrease was found in the extent of hydroxylation of the lysine residues in the collagen synthesized under these conditions, and an even larger decrease occurred in the glycosylations of the hydroxylysine residues. The results support the previous suggestion that the triple-helix formation of the pro-α-chains prevents further hydroxylation of lysine residues and glycosylations of hydroxylysine residues during collagen biosynthesis.

The detrimental effect of orotic acid substitution in the peptide nucleic acid strand on the stability of PNA2:NA triple helices

2005 ◽  
Vol 83 (10) ◽  
pp. 1731-1740 ◽  
Author(s):  
Robert HE Hudson ◽  
Filip Wojciechowski
Keyword(s):  
Nucleic Acid ◽  
Carboxylic Acid ◽  
Peptide Nucleic Acid ◽  
Triple Helix ◽  
Orotic Acid ◽  
Uv Spectroscopy ◽  
Triplex Formation ◽  
Helix Formation ◽  
Triple Helix Formation ◽  
The Stability

We have investigated the incorporation of C6 derivatives of uracil into polypyrimidine peptide nucleic acid oligomers. Starting with uracil-6-carboxylic acid (orotic acid), a peptide nucleic acid monomer compatible with Fmoc-based synthesis was prepared. This monomer then served as a convertible nucleobase whereupon treatment of the resin-bound methyl orotate containing hexamers with hydroxide or amines cleanly converted the ester to an orotic acid or orotamide-containing peptide nucleic acid. Peptide nucleic acid hexamers containing the C6-modified nucleobase hybridized to both poly(riboadenylic acid) and poly(deoxyriboadenylic acid) via triplex formation. Complexes formed with poly(riboadenylic acid) were more stable than those formed with poly(dexoyriboadenylic acid), as measured by temperature-dependent UV spectroscopy. However, both of these complexes were destabilized relative to the complexes formed by an unmodified peptide nucleic acid oligomers. Internal or doubly substituted hexamers are destabilized more strongly than a terminally substituted one, and the type of substitution (carboxamide, ester, carboxylic acid) affects the overall triplex stability. These results clearly show that incorporation of a C6-substituted uracil into polypyrimidine PNA is detrimental to triplex formation. We have also extended this chemistry to incorporate uracil-5-methylcarboxylate into a peptide nucleic acid hexamer. After on-resin conversion of the C5 ester to the 3-(N,N-dimethylamino)propylamide, significant stabilization of the triplex formed with poly(riboadenylic acid) was observed, which illustrates the compatibility of C5 substitution with peptide nucleic acid directed triple helix formation. Key words: peptide nucleic acid, triple helix, orotic acid, orotamide, PNA.

scholarly journals Type-III procollagen assembly in semi-intact cells: chain association, nucleation and triple-helix folding do not require formation of inter-chain disulphide bonds but triple-helix nucleation does require hydroxylation

1996 ◽  
Vol 317 (1) ◽  
pp. 195-202 ◽  
Author(s):  
Neil J. BULLEID ◽  
Richard WILSON ◽  
Janice F. LEES
Keyword(s):  
Triple Helix ◽  
Intact Cells ◽  
Permeabilized Cells ◽  
Type Iii ◽  
Helical Domain ◽  
Helix Formation ◽  
Disulphide Bonds ◽  
Type Iii Procollagen ◽  
Triple Helix Formation ◽  
Triple Helical Domain

Procollagen assembly is initiated within the endoplasmic reticulum by three α-chains associating via their C-propeptides (C-terminal propeptides). To study the requirements for the association of procollagen monomers at synthesis we have reconstituted the initial stages in the folding, assembly and modification of procollagen using semi-permeabilized cells. By translating a type-III procollagen ‘mini-gene’ which lacks part of the triple-helical domain, we demonstrate that these cells efficiently carry out the assembly of hydroxylated, triple-helical, procollagen trimers and allow the identification of specific disulphide-bonded intermediates in the folding pathway. Mutant chains, which lack the ability to form inter-chain disulphide bonds within the C-propeptide, were still able to assemble within this system. Furthermore, characterization of the trimeric molecules formed suggested that inter-chain disulphide bonds had formed within the C-telopeptide (C-terminal telopeptide). However, when hydroxylation of prolyl and lysyl residues was inhibited no inter-chain disulphide bonds were formed in the C-telopeptide, indicating that hydroxylation is required for the initial nucleation of the triple-helical domain. Mutant chains which lacked the ability to form inter-chain disulphide bonds within the C-propeptide or the C-telopeptide could still assemble to form trimeric triple-helical molecules linked by inter-chain disulphide bonds within the N-propeptide (N-terminal propeptide). These results indicate that inter-chain disulphide bond formation within the C-propeptide or the C-telopeptide is not required for chain association and triple-helix formation.

scholarly journals A physico-chemical study of triple helix formation by an oligodeoxythymidylate with N3'->P5' phosphoramidate linkages

1997 ◽  
Vol 25 (9) ◽  
pp. 1782-1787 ◽  
Author(s):  
B.-w. Zhou-Sun ◽  
J. Liquier ◽  
E. Taillandier ◽  
J.-s. Sun ◽  
T. Garestier ◽  
...  
Keyword(s):  
Triple Helix ◽  
Chemical Study ◽  
Helix Formation ◽  
Physico Chemical ◽  
Triple Helix Formation
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