[6] Recent developments in posttranslational modification: Intracellular processing

Kivirikko, Kari I.; Myllylä, Raili

doi:10.1016/0076-6879(87)44175-x

Cited by 60 publications

(37 citation statements)

References 49 publications

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“…No differences were found in the maximal velocities obtained with the two types of enzyme tetramer; both gave a velocity of about 12.5-13 mol/sec per mol of enzyme. This value agrees well with that reported for the chick enzyme tetramer, which consists of two differentially glycosylated forms of the a subunit (24,28). The Km values for Fe2+, 2-oxoglutarate, ascorbate, and the peptide substrate were also identical for the two types of recombinant enzyme tetramer and the chick enzyme tetramer (Table 1).…”

Section: Resultssupporting

confidence: 81%

“…4) by an affinity column procedure (24,28). No differences were found in the maximal velocities obtained with the two types of enzyme tetramer; both gave a velocity of about 12.5-13 mol/sec per mol of enzyme.…”

Section: Resultsmentioning

confidence: 89%

“…Prolyl 4-hydroxylase was purified by a procedure consisting of poly(L-proline) affinity chromatography, DEAEcellulose chromatography-, and gel filtration (28).…”

Section: Methodsmentioning

confidence: 99%

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Characterization of the human prolyl 4-hydroxylase tetramer and its multifunctional protein disulfide-isomerase subunit synthesized in a baculovirus expression system.

Vuori

Pihlajaniemi

Marttila

et al. 1992

Proc. Natl. Acad. Sci. U.S.A.

121

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Prolyl 4-hydroxylase (EC 1.14.11.2), an a2132 tetramer, catalyzes the posttranslational formation of 4-hydroxyproline in collagens. The enzyme can easily be dissociated into its subunits, but all attempts to associate a tetramer from the dissociated subunits in vitro have been unsuccessful. Molecular cloning of the catalytically important a subunit has identified two types of cDNA clone due to mutually exclusive alternative splicing. The 13 subunit is a highly unusual multifunctional polypeptide, being identical to the enzyme protein disulfide-isomerase (EC 5.3.4.1). We report here on expression of the a and 13 subunits of prolyl 4-hydroxylase and a fully active enzyme tetramer in Spodopterafrugiperda insect cells by baculovirus vectors. When the 13 subunit was expressed alone, the polypeptide produced was found in a 0.1% Triton X-100 extract of the cell homogenate and was a fully active protein disulfide-isomerase. When either form of the a subunit was expressed alone, only traces of the a subunit could be extracted from the cell homogenate with 0.1% Triton X-100, and 1% SDS was required to obtain efficient solubilization. These a subunits had no prolyl 4-hydroxylase activity. When the cells were coinfected with both a-and 1subunit-producing viruses, an enzyme tetramer was formed, but signifcant amounts of a and 13 subunits remained unassociated. The recombinant tetramer was indistinguishable from that isolated from vertebrate tissue in terms of its specific activity and kinetic constants for cosubstrates and the peptide substrate. The two alternatively spliced forms of the a subunit gave enzyme tetramers with identical catalytic properties. Baculovirus expression seems to be an excellent system for mass production of the enzyme tetramer and for detailed investigation of the mechanisms involved in the association of the monomers.Prolyl 4-hydroxylase (EC 1.14.11.2), an enzyme residing in the lumen of the endoplasmic reticulum, catalyzes the formation of 4-hydroxyproline in collagens and related proteins by the hydroxylation of proline residues in peptide linkages. This cotranslational and posttranslational modification plays a crucial role in collagen synthesis, as the 4-hydroxyproline residues formed are essential for the folding of the newly synthesized procollagen polypeptide chains into triple-helical molecules. The active prolyl 4-hydroxylase is an a2P32 tetramer consisting of two types of inactive monomer with mo-

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Section: Resultssupporting

confidence: 81%

Section: Resultsmentioning

confidence: 89%

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Characterization of the human prolyl 4-hydroxylase tetramer and its multifunctional protein disulfide-isomerase subunit synthesized in a baculovirus expression system.

Vuori

Pihlajaniemi

Marttila

et al. 1992

Proc. Natl. Acad. Sci. U.S.A.

121

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“…Samples were electrophoresed in an 8% polyacrylamide gel containing 0.5 M urea and 0.1% NaDodSO4 (20). The fractionated proteins were electroblotted onto nitrocellulose filters (BA85, Schleicher & Schuell) as described (19). The filters were stained with heparin/toluidine, cut into strips, and destained (19).…”

Section: Methodsmentioning

confidence: 99%

“…Both peptide regions were selected on the basis of National Biomedical Research Foundation and Swiss-Prot data base searches for uniqueness to the PF19 collagen sequence-i.e., not represented in other protein sequences and, in particular, other collagen types. Approximately 2 mg ofeach peptide was conjugated to 10 mg of keyhole limpet hemocyanin using glutaraldehyde (19) and each peptide solution was divided into eight aliquots. For primary immunization, two aliquots ofeach peptide were emulsified in Freund's complete adjuvant and injected intradermally into two rabbits.…”

Section: Methodsmentioning

confidence: 99%

Identification of a previously unknown human collagen chain, alpha 1(XV), characterized by extensive interruptions in the triple-helical region.

Myers

Kivirikko

Gordon

et al. 1992

Proc. Natl. Acad. Sci. U.S.A.

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A previously unknown collagen cDNA clone, PF19, was isolated from a human placenta library. The 2.1-kilobase Insert has a complete open reading frame of709 amIno adds that includes 12 amino acids ofthe NHrterminal domain, a principally collagenous region of 577 residues, and 120 residues of the noncollagenous COOH terminus. The cIanous part of the sequence encoded by PF19 Is characterized by 13 interruptions ranging in size from 2 to 45 amino aids. Within four interruptions are consensus sequences for attachment of serine-linked glycosamlnoglycans and araginelinked oligosaccharides suggeting that this collagen may be extensively glycosylated. A synthetic decapeptide-reprting a sequence at the b nng of the COOH-terminal ncollagenous domain was used to prepare an antibody in rabbits. This antiserum deteced a 125-kDa bacteria lagenase-senstive protein in Western blots ofHeLa cell lysate. Consistent with the size of the collagen chin, Northern blot hybridization revealed a major transcript of 5.3 kilobases and two minor ones of 4.7 and 4.4 kilobases that are present in cultured human fibroblasts but absent from umbilcal vein endothelial celis. We propose that the previously unidentified polypeptide descibed in this report be designated the al chain of type XV collagen.

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Opposing effects on mitochondrial membrane potential by malonate and levamisole, whose effect on cell-mediated mineralization is antagonistic

et al. 1996

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The act of chondrocyte preparation for primary, enchondral, mineralization is associated with a decline in mitochondrial respiration toward the end of the proliferative zone and the hypertrophic zone in the growth plate. Dexamethasone (Dex)-stimulated cultures of rat marrow stroma constitute a differentiation model simulating, in its energy metabolism, chondrocyte mineralization. In this model, early inhibition of succinate dehydrogenase (SDH) enriches the culture with mineralizing cells, whereas levamisole inhibits mineralization. Dex also increases mitochondrial membrane potential in stromal cells, especially on days 7-8 of stimulation. In the present study, suicide inhibition of SDH, by nitropropionic acid (NPA), in Dex-stimulated cells showed a dose-dependent increase in day 21 mineralization; the maximal effect was induced on days 2-4 of stimulation. Mineralization under 2-day-long exposure to NPA showed a similar trend to the previously studied effect of continuous exposure to malonate applied between days 3-11. Unlike malonate, the effect of NPA required its presence in the cultures for only 2 days and resulted in higher mineralization than that seen under 8 days of malonate. NPA delineated a period, days 2/4 to 7/9, in which inhibition of succinate oxidation is necessary to augment mineralization. During this period, NPA also exhibited OPC selection capacity. Early application of levamisole, under conditions previously shown to decrease day 21 mineralization, maintained mitochondrial membrane potential at the beginning of Dex stimulation but decreased or had little effect on it during days 5-10. By contrast, malonate previously found to increase day 21 mineralization decreased the membrane potential at the beginning of Dex stimulation but increased it later on day 7, or during days 5-10. These results indicate that during osteoprogenitor differentiation, before the mineralization stage, a surge in mitochondrial inner membrane potential during late matrix maturation may be a marker that heralds the extracellular matrix mineralization.

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[6] Recent developments in posttranslational modification: Intracellular processing

Cited by 60 publications

References 49 publications

Characterization of the human prolyl 4-hydroxylase tetramer and its multifunctional protein disulfide-isomerase subunit synthesized in a baculovirus expression system.

Characterization of the human prolyl 4-hydroxylase tetramer and its multifunctional protein disulfide-isomerase subunit synthesized in a baculovirus expression system.

Identification of a previously unknown human collagen chain, alpha 1(XV), characterized by extensive interruptions in the triple-helical region.

Opposing effects on mitochondrial membrane potential by malonate and levamisole, whose effect on cell-mediated mineralization is antagonistic

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