Principles of nuclear organization.
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Cell Cycle↗
Biomedical subjects
L de Jong
Publications and source records attributed to L de Jong.
Three-dimensional distribution of DNase I-sensitive chromatin regions in interphase nuclei of embryonal carcinoma cells.
In situ nick-translation allows the visualization of nuclease-sensitive chromatin regions in interphase nuclei. We have analyzed the three-dimensional (3-D) distribution of DNase I-sensitive regions of chromatin in nuclei from mouse P19 embryonal carcinoma cells by making optical sections using confocal scanning laser microscopy. In undifferentiated as well as embryonal carcinoma cells differentiated in vitro, DNase I-sensitive regions of chromatin are observed as discrete spots in the nucleus. These spots represent clusters of DNase I-sensitive sites. By optical sectioning, we show that these spots are preferentially, but not exclusively, localized at the nuclear periphery. No differences were observed in the spatial distribution of DNase I-sensitive sites in P19 EC cells or the differentiated P19 END-2 cells. Furthermore, we did not observe differences in the distribution of DNase I-sensitive chromatin regions during the cell cycle. These findings indicate, at least for P19 mouse embryonal carcinoma cells and their differentiated derivative END-2, that the compartmentalization of DNase I-sensitive chromatin regions is a general characteristic of the nucleus, independent of cell cycle stage or differentiation state. Since evidence has been presented that DNase I-sensitive sites are associated with actively transcribed chromatin, our results indicate that active transcribing chromatin is compartmentalized, preferentially in the periphery of the nucleus.
Animals↗
Processive action of the two peptide binding sites of prolyl 4-hydroxylase in the hydroxylation of procollagen.
The number of peptide binding sites of prolyl 4-hydroxylase was manipulated with the peptide photoaffinity label N-(4-azido-2-nitrophenyl)glycyl-(Pro-Pro-Gly)5, and the effect on hydroxylation of the relatively short peptide substrate (Pro-Pro-Gly)5 and of the long natural substrate procollagen was studied. With (Pro-Pro-Gly)5 as a substrate, a linear relation was found between enzyme activity and the amount of covalently bound photoaffinity label, approximately 50% inactivation being reached at 1 mol of label/mol of enzyme. No difference in Km value for (Pro-Pro-Gly)5 was detected between unlabeled and partially labeled enzyme preparations. These results indicate that enzyme molecules with only one free active site hydroxylated the synthetic substrate (Pro-Pro-Gly)5 with the same Km and at half the rate of native enzyme. In contrast, with procollagen as a substrate a 5-10-fold increase in Km was found with the fraction of enzyme containing only one free active site, as compared to the Km for procollagen with nonlabeled enzyme. This finding is explained by an enzyme-kinetic model based on a processive action of the two peptide substrate binding sites of prolyl 4-hydroxylase, preventing dissociation of the enzyme-substrate complex between successive hydroxylations of a long peptide with multiple substrate sites. Such a mechanism leads to a low Km for a long peptide by overcoming the diffusional constraints on the rate of association between the enzyme and the individual substrate sites.
Affinity Labels↗
Photoaffinity labelling of the 2-oxoglutarate binding site of prolyl 4-hydroxylase with 5-azidopyridine-2-carboxylic acid.
The synthesis of the photoaffinity label 5-azidopyridine-2-carboxylic acid is described. The 2-oxoglutarate analogue photoaffinity label is a competitive inhibitor with respect to 2-oxoglutarate with a Ki value of 9 X 10(-3) M. Upon ultraviolet irradiation, 5-azidopyridine-2-carboxylic acid inactivated prolyl 4-hydroxylase irreversibly by up to 50%. The extent of inactivation depended on the 5-azidopyridine-2-carboxylic acid concentration and the irradiation time. Inactivation was prevented in the presence of an excess of 2-oxoglutarate. It is concluded that the 5-azidopyridine-2-carboxylic acid became covalently bound to the alpha subunit of prolyl 4-hydroxylase, as the alpha subunit of the photoaffinity labelled enzyme had a decreased electrophoretic mobility in polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate.
Affinity Labels↗
Collagen synthesis in cultured myoblasts and myotubes from patients with Duchenne muscular dystrophy.
Collagen synthesis was studied in cultured myoblasts and myotubes from 4 patients with Duchenne muscular dystrophy (DMD) and 4 control persons. Incorporation of [3H]proline into collagen of DMD cells and control cells was not significantly different. The same types of collagen, i.e., type I and type III were synthesized by myogenic cells from DMD patients and controls. In contrast to others, we could not obtain evidence for an increase in the degree of prolyl hydroxylation in collagen of the DMD muscle cells.
Biopsy↗
Environmental and biological monitoring of workers occupationally exposed to dimethylacetamide.
Exposure to dimethylacetamide was measured in a plant where a prefabricated synthetic product was handled and mechanically processed. Stationary monitoring, personal ambient monitoring, and biological monitoring were employed to evaluate exposure. Personal exposure in the breathing zone varied considerably in comparison with a relatively constant level observed with stationary monitoring. No correlation between personal airborne exposure and excretion of mono-methylacetamide in urine was detected during a full workshift (5 days). Most (n = 6) workers studied (n = 8) excreted about 13% of the calculated inhaled dose as metabolite in urine. For two workers this parameter was about 30%. It is concluded that for a substance like dimethylacetamide, which is easily absorbed through the skin, biological monitoring is superior to airborne concentration monitoring in determining total uptake and (possible) health risk.
Acetamides↗
Photoaffinity labeling of peptide binding sites of prolyl 4-hydroxylase with N-(4-azido-2-nitrophenyl)glycyl-(Pro-Pro-Gly)5.
The synthesis is described of the photoaffinity label N-(4-azido-2-nitrophenyl)glycyl-(Pro-Pro-Gly)5 for the peptide binding site of prolyl 4-hydroxylase. The photoaffinity label is a good substrate and is capable of light-induced inactivation of prolyl 4-hydroxylase activity. Inactivation depends on the concentration of photoaffinity label and is prevented by competition with excess (Pro-Pro-Gly)5. Two moles of photoaffinity label per mole of enzyme is needed for 100% inactivation of enzymic activity. Oxidative decarboxylation of 2-oxoglutarate measured in the absence of added peptide substrate is not affected by labeling. We conclude that the covalently bound nitreno derivative of N-(4-azido-2-nitrophenyl)glycyl-(Pro-Pro-Gly)5 acts by preventing the binding of peptide substrate to the catalytic site without interfering with the binding of the other substrates and cofactors 2-oxoglutarate, O2, Fe2+, and ascorbate. Labeling is specific for the alpha subunit of the tetrameric alpha 2 beta 2 enzyme. In addition to two catalytic binding sites that are blocked by the photoaffinity label, the enzyme contains binding subsites for peptide substrates, as judged from the capability of photoinactivated enzyme to bind to a poly(L-proline) affinity column. These binding subsites may account for the rapidly increasing affinity for peptide substrates with increasing chain length.
Affinity Labels↗
Plasma prolidase in the rat: no index of liver fibrosis.
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Animals↗
The influence of 2-oxoglutarate on the activity of prolyl 4-hydroxylase.
1. Treatment of prolyl 4-hydroxylase (prolyl-glycyl-peptide, 2-oxoglutarate:oxygen oxidoreductase (4-hydroxylating), EC 1.14.11.2) with 2-oxoglutarate in the absence of added Fe2+ for 10 s causes partial inactivation of the enzyme which is not reversed by subsequent addition of Fe2+. It appears that 2-oxoglutarate prevents loss of enzyme-bound iron and prevents access of added iron to its binding site. 2. For optimal enzyme activity the enzyme should be preincubated for 15 s with Fe2+ (5 microM). 3. Under turnover conditions prolyl 4-hydroxylase does not release iron. 4. The inactivation brought about by pre-incubation with 2-oxoglutarate and O2 in the absence of ascorbate is partly reversed by removal of 2-oxoglutarate. 5. It is proposed that dead-end complex formation with 2-oxoglutarate is responsible for the inactivation of the enzyme by 2-oxoglutarate in the absence of either ascorbate or Fe2+. 6. Optimal enzyme activity is obtained if the reactants are added to the reaction medium in the following order: enzyme, iron and ascorbate in any order, then after at least 15 s 2-oxoglutarate and finally (Pro-Pro-Gly)5 to start the reaction.
Animals↗
Prolyl 4-hydroxylase activity in relation to the oxidation state of enzyme-bound iron. The role of ascorbate in peptidyl proline hydroxylation.
In agreement with others (Myllylä, R., Kuutti-Savolainen, E.-R. and Kivirikko, K.I. (1978) Biochem. Biophys. Res. Commun. 83, 441-448), it was found that, in the absence of ascorbate, prolyl 4-hydroxylase (prolyl-glycyl-peptide, 2-oxoglutarate:oxygen oxidoreductase (4-hydroxylating), EC 1.14.11.2) catalyses the hydroxylation of peptidyl proline, stoicheiometrically coupled to the oxidative decarboxylation of 2-oxoglutarate, at a high initial rate. Under these conditions the enzyme becomes inactivated by at least 90% within 1 min in the presence of 400 microM 2-oxoglutarate, in the presence or absence of the peptide substrate (Pro-Pro-Gly)10. The enzyme can be partly reactivated by ascorbate, but not by Fe2+. Addition of a stoicheiometric amount of iron to the enzyme gives rise to a small EPR signal at g = 4.3, which is typical of a high-spin d 5 ion in a rhombic environment. After subsequent incubation for 30 s at 37 degrees C in the presence of 2-oxoglutarate, the amplitude of the EPR signal at g = 4.3 increases 3-4-fold and corresponds to virtually all of the iron added. In addition, an EPR signal at g = 2.0 is formed under these conditions. The signal at g = 4.3 decreases after subsequent addition of ascorbate. It is concluded that in the presence of 2-oxoglutarate enzyme-bound Fe2+ is rapidly converted to Fe3+, leading to inactivation of the enzyme. Enzyme-bound Fe3+ can be reduced again by ascorbate, thus reactivating the enzyme, or, in the absence of 2-oxoglutarate, by Fe2+.
Animals↗
The biogenesis of rat mitochondrial ATPase. Two subunits are synthesized inside the mitochondria.
Isolated mitochondria from regenerating rat liver synthesize at least five different polypeptides with molecular weights ranging from 19 000 to 43 000. Among these, two polypeptides with molecular weights of 22 000 and 25 ooo could be identified as ATPase subunits. It has previously been shown that these subunits, designated 6 and 7, are lacking in the ATPase complex that is formed in vivo when mitochondrial protein synthesis is blocked by thiamphenicol treatment. The 22 000 Mr protein is enriched in the fraction containing the fully assembled ATPase complex, whereas the 25 000 Mr protein is not.
Adenosine Triphosphatases↗
The biogenesis of rat-liver mitochondrial ATPase. Evidence that the N, N'-dicyclohexyl carbodiimide-binding protein is synthesized outside the mitochondria.
1. Radioactive N,N'-dicyclohexyl carbodiimide (DCCD) is bound as effectively to the N, N'-dicyclohexyl carbodiimide- and oligomycin-sensitive ATPase complex in submitochondrial particles of normal rat liver as to the similar but partially N,N'-dicyclohexyl carbodiimide- and oligomycin-insensitive complex of thiamphenicol-treated rats. The latter complex is deficient in 3 subunits (subunit 6, 7 and 10). 2. Radioactive N,N'-dicyclohexyl carbodiimide is exclusively bound to the subunits present in the bands 8 and 11 of SDS-PAA gels of the purified ATPase complex. These subunits, most likely the dimer and monomer of the N,N'-dicyclohexyl carbodiimide-binding protein, are products of the cytoplasmic protein synthesis. 3. The results together indicate that the N,N'-dicyclohexyl carbodiimide-insensitivity of the ATPase complex formed during in vitro inhibition of mitochondrial protein synthesis, is not caused by a lack of inhibitor binding protein. The same holds for the oligomycin-insensitivity.
Adenosine Triphosphatases↗
The biogenesis of rat liver mitochondrial ATPase. Subunit composition of the normal ATPase complex and of the deficient complex formed when mitochondrial protein synthesis is blocked.
1. An ATPase complex containing 12 subunits was isoalted from rat liver mitochondria. 2. In vivo inhibition of mitochondrial protein synthesis by the chloramphenicol analogue thiamphenicol leads to the formation of an oligomycin-insensitive membrane-bound ATPase complex in mitochondria of regenerating rat liver. 3. This oligomycin-insensitive, membrane-bound ATPase was isolated by the same procedure as the ATPase complex from regenerating livers of untreated animals. 4. SDS-polyacrylamide gel electrophoresis of in vivo labelled ATPase complexes from control and from thiamphenicol-treated rats reveals that three subunits out of the 12 are not synthesized or assembled when the mitochondrial translation activity is blocked. 5. From the subunits synthesized and assembled when mitochondrial pror (Fo) of the ATPase complex (subunit 5). 6. The oligomycin sensitivity-conferring protein seems absent in the ATPase complex formed in the presence of thiamphenicol.
Adenosine Triphosphatases↗
The use of antibiotics to study mitochondrial protein synthesis.
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Animals↗
Relation between reduced nicotinamide adenine dinucleotide oxidation and amino acid transport in membrane vesicles from Bacillus subtilis.
The rate of reduced nicotinamide adenine dinucleotide (NADH) oxidation by membrane vesicles from Bacillus subtilis W23 increases three- to fourfold during logarithmic growth, reaching maximal levels in early stationary phase. Initial rates of L-proline, L-alanine, and L-glutamate transport energized by NADH closely parallel the increase in NADH oxidation. In vesicles prepared at different stages of growth, a constant number of NADH molecules varying from 150 to 260 have to be oxidized to transport one molecule of amino acid. Membrane vesicles from B. subtilis aroD (strain RB163), a mutant defective in menaquinone synthesis, do not transport amino acids in the presence of NADH. Ascorbate plus phenazine methosulfate, however, energizes amino acid transport equally well as in vesicles of B. subtilis W23. NADH oxidation and NADH-driven amino acid transport can be restored instantaneously by the addition of menadione (vitamin K3).
Alanine↗