[10] Posttranslational enzymes in the biosynthesis of collagen: Intracellular enzymes

Kivirikko, Kari I.; Myllylä, Raili

doi:10.1016/0076-6879(82)82067-3

Cited by 372 publications

(300 citation statements)

References 128 publications

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“…5 The first method requires preparation of a radio-labeled substrate ([ 14 C]lysine) and radiochemical determination of the hydroxy[ 14 C]lysine product in a three step process. The second method requires preparation of a radio-labeled substrate (2-oxo-[1-14 C]glutarate) and radiochemical determination of the decarboxylation reaction.…”

Section: Discussionmentioning

confidence: 99%

“…4 The extent of Hyl glycosylation has a direct effect on collagen fibril assembly, in that increased carbohydrate content decreases fibril diameter. 5 Hyl glycoslyation levels may also modulate mineralization of collagen triple-helices 6 and cellular interactions with collagen. 7 Second, the Hyl hydroxyl group participates in collagen cross-link formation, which occurs between an aldehyde derived from a Lys or Hyl residue and the e-amino group of a second Lys or Hyl.…”

Section: Introductionmentioning

confidence: 99%

“…One atom of oxygen is incorporated at the C-5 position of a Lys residue and another one into 2-oxoglutarate, which is decarboxylated to form succinate and CO 2 ( Figure 1). 4,5,9 Ascorbate is needed to regenerate the enzyme after several reaction cycles. 9 LH1 and LH2 gene mutations have been linked to EhlersDanlos syndrome type VI and Bruck syndrome, respectively.…”

Section: Introductionmentioning

confidence: 99%

“…Prior LH assay methods are multi-step, require radio-labeled substrates, and/or measure depletion of 2-oxoglutarate or formation of CO 2 . 5,20 Since the LH reaction with 2-oxoglutarate is uncoupled from Lys hydroxylation, 9,21,22 the most accurate assay of LH activity should monitor the formation of Hyl. Conceptually, we sought to efficiently separate Lys-containing peptide substrates from Hyl-containing peptide products using RP-HPLC.…”

Section: Introductionmentioning

confidence: 99%

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Development of a convenient peptide‐based assay for lysyl hydroxylase

Čudić¹,

Patel²,

Lauer‐Fields³

et al. 2008

Biopolymers

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Hydroxylysine (Hyl) is a posttranslationally modified amino acid found mainly in collagens, the most abundant protein in mammals. Lysyl hydroxylase (LH) catalyzes the hydroxylation of the C‐5 position of a Lys residue, resulting in the production of Hyl. Mechanistically, LH incorporates one oxygen atom into both Lys and 2‐oxoglutarate; the latter is decarboxylated to form succinate and CO2. To develop a convenient, RP‐HPLC based LH assay, we used Fmoc solid‐phase methodology to synthesize three different peptides designed as LH substrates and one peptide corresponding to an LH product. Peptides were characterized by RP‐HPLC, MALDI‐TOF mass spectrometry and CD spectroscopy. Separation of peptides was examined under a variety of RP‐HPLC conditions. The best results were achieved using peptide derivatization (1‐anthroylnitrile for organic phase and dansyl chloride for aqueous phase) prior to RP‐HPLC analysis. The products (di‐ and tetra‐substituted Lys‐ and Hyl‐containing peptides) were well resolved by RP‐HPLC. The resolution of each peak allows for quantification of peak areas, which in turn, when examined as a function of time, can be utilized for studying the kinetics of LH catalyzed reactions. Most significantly, the RP‐HPLC assay directly monitors the Hyl containing product. Prior LH assay methods are multi‐step, require radio‐labeled substrates, and/or measure depletion of 2‐oxoglutarate or formation of CO2. Since the LH reaction with 2‐oxoglutarate is uncoupled from Lys hydroxylation, the most accurate assay of LH activity should monitor the formation of Hyl. © 2007 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 90: 330–338, 2008.This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Development of a convenient peptide‐based assay for lysyl hydroxylase

Čudić¹,

Patel²,

Lauer‐Fields³

et al. 2008

Biopolymers

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“…This assay is based on mild oxidation of the product, i.e. the hypusine residue [19] and subsequent selective extraction of the generated aldehydes with organic solvent [20]. Using this assay, we were able to analyze the mode of inhibition of the metal chelators and the specificity of the metal ion dependency of the human deoxyhypusine hydroxylase from HeLa cells in vitro.…”

Section: Introductionmentioning

confidence: 99%

Evaluation of the metal ion requirement of the human deoxyhypusine hydroxylase from HeLa cells using a novel enzyme assay

Csonga¹,

Ettmayer²,

Auer³

et al. 1996

FEBS Letters

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Hypusine synthesis in the eukaryotic initiation factor 5A is a unique two-step posttranslational modification. After deoxyhypusine is generated by the deoxyhypusine synthase, the deoxyhypusine hydroxylase (EC 1.14.99.29) catalyzes the formation of mature hypusine. A rapid assay for monitoring the deoxyhypusine hydroxylase activity was established, employing the oxidative cleavage of the hypusyl residue and subsequent extraction of the generated aldehydes. As metal ion chelators have been reported to inhibit the deoxyhypusine hydroxylase, the mechanism of this inhibition and the effect of transition metal ions on the enzyme activity were investigated. A ferric ion appears to be essential for enzymatic activity, the inhibition of which is entirely attributed to the metal ion binding capacity of the chelators.

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Differential effects of ascorbate depletion and α,α′-dipyridyl treatment on the stability, but not on the secretion, of type IV collagen in differentiated F9 cells

Kim

Peterkofsky

1997

J. Cell. Biochem.

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Ascorbic acid stimulates secretion of type I collagen because of its role in 4-hydroxyproline synthesis, but there is some controversy as to whether secretion of type IV collagen is similarly affected. This question was examined in differentiated F9 cells, which produce only type IV collagen, by labeling proteins with [14C]proline and measuring collagen synthesis and secretion. Hydroxylation of proline residues in collagen was inhibited to a greater extent in cells treated with the iron chelator alpha,alpha'-dipyridyl (97.7%) than in cells incubated without ascorbate (63.1%), but both conditions completely inhibited the rate of collagen secretion after 2-4 h, respectively. Neither treatment affected laminin secretion. Collagen synthesis was not stimulated by ascorbate even after treatment for 2 days. On SDS polyacrylamide gels, collagen produced by alpha,alpha'-dipyridyl-treated cells consisted mainly of a single band that migrated faster than either fully (+ ascorbate) or partially (- ascorbate) hydroxylated alpha 1(IV) or alpha 2(IV) chains. It did not contain interchain disulfide bonds or asn-linked glycosyl groups, and was completely digested by pepsin at 15 degrees C. These results suggested that it was a degraded product lacking the 7 S domain and that it could not form a triple helical structure. In contrast, the partially hydroxylated molecule contained interchain disulfide bonds and it was cleaved by pepsin to collagenous fragments similar in size to those obtained from the fully hydroxylated molecule, but at a faster rate. Kinetic experiments and monensin treatment suggested that completely unhydroxylated type IV collagen was degraded intracellularly in the endoplasmic reticulum or cis Golgi. These studies indicate that partial hydroxylation of type IV collagen confers sufficient helical structure to allow interchain disulfide bond formation and resistance to pepsin and intracellular degradation, but not sufficient for optimal secretion.

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[10] Posttranslational enzymes in the biosynthesis of collagen: Intracellular enzymes

Cited by 372 publications

References 128 publications

Development of a convenient peptide‐based assay for lysyl hydroxylase

Development of a convenient peptide‐based assay for lysyl hydroxylase

Evaluation of the metal ion requirement of the human deoxyhypusine hydroxylase from HeLa cells using a novel enzyme assay

Differential effects of ascorbate depletion and α,α′-dipyridyl treatment on the stability, but not on the secretion, of type IV collagen in differentiated F9 cells

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