Biomedical subjects

K Majamaa

Publications and source records attributed to K Majamaa.

64 records · Page 4Linked to original sources

PubMed1984

Ascorbate is consumed stoichiometrically in the uncoupled reactions catalyzed by prolyl 4-hydroxylase and lysyl hydroxylase.

The hydroxylation of proline and lysine residues by the collagen hydroxylases is coupled with a stoichiometric decarboxylation of 2-oxoglutarate. Ascorbate is virtually a specific requirement for these enzymes, but previous studies have demonstrated that it is not consumed during most catalytic cycles. Prolyl 4-hydroxylase and lysyl hydroxylase are known also to catalyze an uncoupled decarboxylation of 2-oxoglutarate in the absence of the peptide substrate. It is shown here that, unlike the complete hydroxylation reaction, the uncoupled decarboxylation reaction involves stoichiometric ascorbate consumption. This stoichiometric ascorbate consumption was also seen when the rate of the uncoupled prolyl 4-hydroxylase reaction was enhanced by the addition of poly(L-proline). Since collagen hydroxylases may catalyze occasional uncoupled reaction cycles even in the presence of the peptide substrates, the main function of ascorbate in these reactions in vivo is suggested to be that of reactivating the enzymes after such uncoupled cycles.

Animals↗

PubMed1984

The 2-oxoglutarate binding site of prolyl 4-hydroxylase. Identification of distinct subsites and evidence for 2-oxoglutarate decarboxylation in a ligand reaction at the enzyme-bound ferrous ion.

The structure and function of the 2-oxoglutarate binding site of prolyl 4-hydroxylase was studied by assaying the inhibitory potential of 24 selected aliphatic or aromatic compounds. All except one of them inhibited the enzyme competitively with respect to 2-oxoglutarate and noncompetitively with respect to Fe2+, the Ki values ranging from 0.8 microM to over 15 mM. The Ki values for the two most effective inhibitors, pyridine 2,5-dicarboxylate and 2,4-dicarboxylate, were about 0.8 microM and 2 microM, these compounds being the most potent inhibitors of prolyl 4-hydroxylase with respect to 2-oxoglutarate known so far. Only one of the compounds tested, 2-oxoadipinate, was able to support hydroxylation by replacing 2-oxoglutarate as a cosubstrate. The data suggest that the 2-oxoglutarate binding site can be divided into three distinct subsites. Subsite I is probably a positively charged side chain of the enzyme that ionically binds the C5 carboxyl group of the 2-oxoglutarate, subsite II consists of two cis-positioned equatorial coordination sites of the enzyme-bound ferrous ion and is chelated by the C1-C2 moiety, while subsite III involves a hydrophobic binding site in the C3-C4 region of the cosubstrate. The sp3 rehybridization of C2 within the chelating moiety of the cosubstrate appears to be a crucial event during decarboxylation that proceeds in the form of a ligand reaction inside the Fe2+ coordination sphere.

Animals↗

PubMed1982

Regulation of proline 3-hydroxylation and prolyl 3-hydroxylase and 4-hydroxylase activities in transformed cells.

Prolyl 3-hydroxylase activity and the extent of collagen proline 3-hydroxylation were studied in six transformed and three control human cell lines. In the transformed cell lines, the enzyme activity was markedly high in two, similar to that in control cells in two and significantly low in two. The extent of proline 3-hydroxylation was markedly high in cell lines with high enzyme activity, but it was also significantly high in some transformed cell lines with enzyme activities similar to those in the controls. The results thus suggest that, in addition to the amount of enzyme activity present, the rate of collagen synthesis also affects the extent of proline 3-hydroxylation in the newly synthesized collagen. The effect of acute cell transformation on prolyl 3-hydroxylase and 4-hydroxylase activities was studied by infecting chick-embryo fibroblasts with Rous sarcoma virus mutant NY68, temperature-sensitive for transformation. At the permissive temperature prolyl 3-hydroxylase activity showed a more rapid increase and decrease than did prolyl 4-hydroxylase activity, the maximal activity for both enzymes being about 2.5 times that in the control chick fibroblasts. When the transformed cells were shifted to the non-permissive temperature the decays in the elevated enzyme activities were similar, suggesting identical half-lives.

Animals↗

PubMed1982

Labelling of prolyl hydroxylase tetrameric subunits in freshly isolated chick-embryo tendon cells and in certain chick-embryo tissues in vivo.

The labelling of the subunits of prolyl 4-hydroxylase tetramers was studied in freshly isolated chick-embryo tendon cells and in chick-embryo tissues. In the former both the alpha- and beta-subunits of the tetramer were labelled during a 4 h labelling and 2 h chase period, although the radioactivity in the beta-subunit was much lower than in the alpha-subunit. The corresponding subunits of the enzyme from 12-day chick-embryo cartilaginous bone and heart were labelled in 7 h, again the beta-subunit much less than the alpha-subunit, the ratio of radioactivity in the beta-subunit to that in the alpha-subunit (beta/alpha-radioactivity) being 0.20 and 0.32 respectively. The beta/alpha-radioactivity then increased almost linearily with time between 7 and 24 h, by 9.5-fold in the cartilaginous bone and 3-fold in the heart, and beta/alpha-radioactivity values above 1.0 were reached. The free beta-subunit-size protein (the beta'-protein), which is also present in cells, had been labelled quite heavily by 7 h. The beta/alpha-radioactivity at 7h, determined in four tissues with different ratios of prolyl hydroxylase tetramers to total immunoreactive protein (tetramer percentage), was low in tissues with a high tetramer percentage. It is thus proposed that only a minor fraction of the beta'-protein must be processed to the tetrameric beta-subunit and utilized in the synthesis of the prolyl 4-hydroxylase tetramers.

Animals↗

PubMed1981

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

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.

Animals↗

PubMed1979

Partial purification and characterization of chick-embryo prolyl 3-hydroxylase.

Prolyl 3-hydroxylase was purified up to about 5000-fold from an (NH4)2SO4 fraction of chick-embryo extract by a procedure consisting of affinity chromatography on denatured collagen linked to agarose, elution with ethylene glycol and gel filtration. The molecular weight of the purified enzyme is about 160000 by gel filtration The enzyme is probably a glycoprotein, since (a) its activity is inhibited by concanavalin A, and (b) the enzyme is bound to columns of this lectin coupled to agarose and can be eluted with a buffer containing methyl alpha-D-mannoside. The Km values for Fe2+, 2-oxoglutarate, O2 and ascorbate in the prolyl 3-hydroxylase reaction were found to be very similar to those previously reported for these co-substrates in the prolyl 4-hydroxylase and lysyl hydroxylase reactions.

Animals↗

PubMed1979

Turnover of prolyl hydroxylase tetramers and the monomer-size protein in chick-embryo cartilaginous bone and lung in vivo.

The turnover of prolyl hydroxylase and an immunoreactive protein that corresponds in size to the smaller subunit of the enzyme was studied in vivo after injection of [(3)H]leucine into 11-day chick embryos. The specific radioactivity and total radioactivity of the monomer-size protein were much higher than those of the enzyme tetramers in the cartilaginous bone at 3h and 12h after the radioisotope injection, indicating that the monomer-size protein represents precursors rather than degradation products of the enzyme tetramers. Between 24 and 144h after the injection the specific radioactivity and total radioactivity of the two forms of the enzyme protein showed essentially identical decay rates, the observed specific radioactivity of the monomer-size protein being about 120-130% and total radioactivity about 80% of that of the enzyme tetramers. The true half-life, when corrected for dilution caused by tissue growth and re-utilization of the [(3)H]leucine, was 37.9h for the monomer-size protein and 39.0h for the tetramers. The results obtained in the lung were less reliable owing to high blank radioactivity values in the immunoprecipitation, but even so some definite differences were found between this tissue and the cartilaginous bone. The specific radioactivity of both forms of the enzyme protein at 24h was only about 20-25% of that in the cartilaginous bone. The total radioactivity of the monomer-size protein in the lung remained about 5 times that of the enzyme tetramers, whereas it was only about 0.8 times that of the tetramers in the cartilaginous bone. As in the cartilaginous bone, the decay rates of both forms of the enzyme protein were essentially identical in the lung, with a true half-life of about 46h. The results suggest that the rate of prolyl hydroxylase synthesis is slower in the lung than in the cartilaginous bone, whereas the degradation rates are fairly similar in these two tissues. The data further suggest that, in the lung at least, a large part of the monomer-size protein became degraded without being converted into enzyme tetramers.

Animals↗

PubMed1979

Prolyl 3-hydroxylase and 4-hydroxylase activities in certain rat and chick-embryo tissues and age-related changes in their activities in the rat.

Prolyl 3-hydroxylase activity, expressed per unit of extract protein, was much higher in rat kidney cortex than in the lung, liver or skin. A marked decrease in activity was found in the kidney cortex, liver and skin beyond 10 days of age. The ratio of prolyl 3-hydroxylase to 4-hydroxylase activity in the kidney cortex was 13--17 times that in the skin, that in the liver 6--8 times, and that in the lung about twice the value for the skin, there being no changes in this ratio with age. In 16-day chick embryos the highest ratios of prolyl 3-hydroxylase to 4-hydroxylase activity were found in the liver, heart, lens, aorta and kidney, and the lowest ratios in tendon, cartilage, cartilaginous and membranous bone and skin. The results suggest that the differences in the extent of prolyl 3-hydroxylation between various collagens can in part be explained by differences in the amount of prolyl e-hydroxylase activity among different cells.

Aging↗

PubMedDate not supplied

Modifier locus for mitochondrial DNA disease: linkage and linkage disequilibrium mapping of a nuclear modifier gene for maternally inherited deafness.

PURPOSE: To examine the role of the nuclear genome in affecting the phenotypic expression of the simplest model of a mitochondrial DNA disease, maternally transmitted deafness. METHODS: Linkage analysis in families with maternally inherited deafness associated with the homoplasmic A1555G mutation. RESULTS: Significant linkage and linkage disequilibrium on chromosome 8 was identified. CONCLUSIONS: This finding represents the first identification of a modifier locus for a human mitochondrial DNA disease and supports the concept of mitochondrial DNA diseases having complex genetic inheritance. The eventual identification of this modifier gene will provide insights into the pathophysiological pathways determining the clinical expression of mitochondrial DNA diseases, an important step toward diagnostic and therapeutic interventions.

Cell Nucleus↗

PubMedDate not supplied

Connective tissue metabolites in serum as markers of disease activity in patients with rheumatoid arthritis.

The serum levels of aminoterminal type III procollagen peptide (S-PIIINP), immunoreactive prolyl 4-hydroxylase protein (S-IRPH), 7S domain of collagen type IV (S-Col IV, 7S), and fragment P1 of laminin (S-Lam), which are associated with the metabolism of extracellular interstitial collagens and basement membranes, were measured sequentially for two years in 14 rheumatoid arthritis (RA) patients undergoing disease modifying antirheumatic drug treatment. Elevated S-PIIINP, S-IRPH, and S-Col IV, 7S levels were demonstrated in active RA. In active disease the metabolites showed some correlation with clinical and serological signs of disease activity. A high average synovial fluid/serum concentration ratio of PIIINP and of Col IV, 7S supports the concept that the increased serum levels of PIIINP and Col IV, 7S originated from the diseased joints. After 2 years of treatment a decline was observed in S-PIIINP and S-Col IV, 7S in treatment responders. However, the median levels of S-PIIINP and S-IRPH were still above the upper limit of normal, suggesting smouldering, subclinical inflammatory processes. S-Lam remained within the normal range in active and inactive disease.

Adult↗