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1.
The effects of lead upon collagen synthesis and proline hydroxylation were examined in the Swiss mouse 3T6 fibroblast. The results indicate that lead reduces proline hydroxylation in stationary phase cultures of 3T6 cells, resulting in increased cellular retention of unhydroxylated procollagen. Inhibition of proline hydroxylation by lead was prevented by increasing the extracellular Fe2+Pb2+ molar ratio. Interference by lead in the hydroxylation of proline in logarithmic phase cultures of 3T6 cells resulted in increases in the 0.5 n HClO4 soluble/insoluble hydroxyproline ratio. This was attributed to an increase in the rate of breakdown of lead-induced unhydroxylated procollagen. Kinetic analysis of the lead-iron interaction with proline hydroxylase suggests that the mechanism is competitive.  相似文献   

2.
The synthesis of procollagen hydroxyproline and hydroxylysine was examined in matrix-free cells which were isolated from embryonic tendon by controlled enzymic digestion and then incubated in suspension. After the cells were labeled with [14C]proline for 2 min, or about one-third the synthesis time for a Pro-α chain, [14C]hydroxyproline was found in short peptides considerably smaller than the Pro-α chains of procollagen. The results, therefore, confirmed previous reports indicating that the hydroxylation of proline can begin on nascent chains. In similar experiments in which the cells were labeled with [14C]lysine, [14C]hydroxylysine was found in short, newly synthesized peptides, providing the first evidence that the hydroxylation of lysine can also begin on nascent peptides. However, further experiments demonstrated that the synthesis of hydroxyproline and hydroxylysine continues until some time after assembly of the polypeptide chains is completed.  相似文献   

3.
Matrix-free cells from embryonic tendons were incubated under conditions in which they synthesized and accumulated protocollagen, the unhydroxylated form of procollagen, which is non-helical at 37°. Limited digestion with pepsin demonstrated that when the accumulated protocollagen was hydroxylated intracellularly to procollagen, or when the cells were cooled below the Tm of protocollagen, the protein became triple-helical in about 5 min, or in a fraction of the time required for isolated α chains to become helical. When disulfide bonds in the NH2-terminal extensions of protocollagen were reduced by treating the cells with dithiothreitol, the rate of helix formation was markedly decreased. The results demonstrated that the NH2-terminal extensions found in protocollagen and procollagen play an important role in formation of the triple-helix during biosynthesis.  相似文献   

4.
THE hydroxyproline and hydroxylysine in collagen are synthesized by hydroxylation of proline and lysine after these amino-acids have been incorporated into peptide linkages (for review see ref. 1). Experiments with embryonic cartilage in vitro in which the hydroxylases were intermittently inhibited demonstrated that the hydroxylations can occur after the proline-rich and lysine-rich polypeptide precursor protocollagen is released from ribosomal complexes1,2. There has been controversy, however, over the question of whether in uninhibited systems the hydroxylation of the appropriate prolyl and lysyl residues occurs while nascent polypeptide chains are still being assembled on ribosomes1,3,4.  相似文献   

5.
Partial characterization of protocollagen from embryonic cartilage   总被引:4,自引:1,他引:3       下载免费PDF全文
1. Attempts were made to isolate and characterize the protocollagen that accumulates in connective tissue when the hydroxylation of proline and lysine is inhibited. The term protocollagen has been used to describe the proline-rich and lysine-rich polypeptide or polypeptides that serve as substrates for the formation of hydroxyproline and hydroxylysine during the synthesis of collagen. 2. Both protocollagen and newly synthesized collagen from embryonic cartilage were isolated as complex aggregates, which contained sulphated mucopolysaccharides and other proteins or polypeptides from the same tissue. The complexes containing protocollagen were similar to those containing newly synthesized collagen when examined with several different techniques. 3. After the complexes were denatured and disaggregated, zone centrifugation and gel filtration indicated that the denatured protocollagen was similar to the denatured newly synthesized collagen obtained from cartilage in which the hydroxylation was not inhibited, and it was also similar to purified alpha-collagen. The results suggest that, when the hydroxylation is inhibited, most of the protocollagen polypeptides that accumulate are as large as complete alpha-chains of collagen. 4. Significant purification of the protocollagen polypeptides was obtained with a new technique for DEAE-Sephadex chromatography in which urea was used to prevent aggregation of the samples and the column was eluted with guanidine thiocyanate. 5. Protocollagen polypeptides were completely hydrolysed to diffusible peptides by a specific collagenase. 6. It is not entirely clear whether the hydroxylation normally begins while relatively short protocollagen molecules are still attached to polysomes, or whether protocollagen molecules of the size of alpha-collagen are synthesized even when the hydroxylation is not inhibited. 7. Results obtained with puromycin suggest that some hydroxylation occurs with smaller polypeptides, but polypeptide chains approaching the size of alpha-collagen are required to obtain complete hydroxylation of the appropriate amino acid residues of protocollagen.  相似文献   

6.
In permanent blastulae of the sea urchin, which were obtained by culture in SO2?4-free artificial seawater from the time of fertilization, ascorbate and α-ketoglutarate, activators of protocollagen proline hydroxylase, induced the formation of archenteron. By adding either ascorbate or α-ketoglutarate to the SO2?4-free culture at 12 hr of fertilization, spherical embryos with archenteron were obtained by successive 12 hr cultures at 20°C. The embryos thus obtained did not develop to plutei. Archenteron formation induced by these compounds in SO2?4-free-cultured embryos, as well as in the normal embryos, was inhibited by α,α′-dipyridyl, an inhibitor of protocollagen proline hydroxylase. Glutamate, malate, citrate, and fumarate did not stimulate archenteron formation in SO2?4-free cultured embryos. In the SO2?4-free-cultured embryos exposed to [14C]proline, considerable radioactivity was found in hot trichloroacetic acid-extractable proteins but the radioactivity of [14C]hydroxyproline residue, produced by hydroxylation of proline residue of protocollagen, was markedly lower than that in normal embryos. In the presence of ascorbate and α-ketoglutarate, the radioactivity of [14C]hydroxyproline residue became high and was lowered by α,α′-dipyridyl. Archenteron formation induced by ascorbate and α-ketoglutarate in the embryos kept in SO2?4-free artificial seawater probably results from the stimulated protocollagen hydroxylation.  相似文献   

7.
The synthesis of collagen can be interrupted, after the assembly of proline-rich and lysine-rich polypeptide chains called protocollagen, by incubating connective tissues anaerobically. Under these conditions the proline and lysine residues in protocollagen are not hydroxylated to hydroxyproline and hydroxylysine, and protocollagen molecules accumulate intracellularly. Chemical data and radioautographs at the level of the light and electron microscopes indicated that in tissues labeled with proline-3,4-3H under nitrogen, there appeared to be an accumulation of radioactivity over the ground cytoplasm. When the inhibition of protocollagen hydroxylase was reversed by exposing the tissue to oxygen, the accumulated protocollagen-3H was converted to collagen-3H and there was a rapid transfer of label from the ground cytoplasm to the extracellular matrix. There was no significant change in distribution of label over either the Golgi vacuoles or the cisternae of the endoplasmic reticulum. The failure to find a significant change in distribution of label over the Golgi vacuoles or the cisternae does not completely exclude the possibility that these two compartments are involved in the extrusion, but the data are consistent with the simpler notion that the completed collagen molecules pass directly from the ground cytoplasm to the extracellular matrix.  相似文献   

8.
Procollagen mRNA was purified from collagen synthesizing polysomes obtained from an experimental guinea pig granuloma, and iodinated in vitro. The procollagen 125I-labelled mRNA was hibridized with granuloma and liver guinea pig DNA in vast DNA excess conditions. A Cot 12 800–900 mol · s · l?1 for both tissues was obtained from the hybridization curves. With these results, we could suggest the existence of 11–13 procollagen genes per haploid genome. By the analysis of the hybridization data it was possible to infer that there is no genomic amplification in tissues highly specialized in the synthesis of collagen such as granuloma.  相似文献   

9.
Matrix-free chick embryo tendon cells were incubated with [14C]proline for 60 minutes and protein synthesis was stopped by the addition of cycloheximide. Newly synthesized collagen precursors recovered in the incubation medium were mostly intact procollagen molecules which contain both amino-terminal and carboxy-terminal extensions. If the cells were further incubated for 2 hours in the presence of cycloheximide, most of the procollagen was converted to precursor molecules which were devoid of amino-terminal extensions. Removal of the carboxy-terminal extensions from procollagen was not observed. Similar experiments with intact tendons demonstrated that procollagen synthesized by the intact tissues invitro was readily converted to an intermediate form devoid of amino-terminal extensions and then to collagen. The results suggest that the removal of the amino-terminal and carboxy-terminal extensions from procollagen is catalyzed by two separate enzymic activities.  相似文献   

10.
The relationship between progesterone (P4) synthesis in vitro by luteal tissue and prostaglandin F (PGF) synthesis in vitro by endometrium and luteal tissue from two stages of the cycle, Days 7 to 8 and 15 to 16, was determined. Luteal and endometrial tissues were collected from pigs in three experimental groups at two stages of the cycle: (A) 6 pigs on Days 7 to 8 with spontaneous, 5 to 6 day old corpora lutea (CL); (B) 5 pigs on Days 15 to 16 with spontaneous, 13 to 14 day old CL; and (C) 6 pigs on Days 15 to 16 with spontaneous, 13 to 14 day old CL and 5 to 6 day old CL induced by pregnant mares serum gonadotropin (PMSG) and human chorionic gonadotropin (HCG) injections. Pigs with spontaneous, 13 to 14 day old CL of the cycle and PMSG-HCG induced accessory, 5 to 6 day old CL were used so that P4 and PGF synthesis in tissue from old and new CL could be compared in the same pig on Day 15 to 16 of the cycle. Tissues (100 mg minces) were incubated in 5 ml of Krebs Ringer solution in an atmosphere of 95% 02:5% CO2 for 2 hours at 0° C, 37° C, or 37° C with 1.3 x 10?4M indomethacin (IND). An aliquot of the incubation medium and an aliquot of the supernatant after homogenization of the tissue in the remaining medium of each flask was quantified for P4 and PGF by radioimmunoassay. P4 and PGF release into the medium and total accumulation of P4 and PGF in the flasks indicated that de novo synthesis had occured at 37° C. Compared to tissue from 13 to 14 day old CL, tissue from 5 to 6 day old CL synthesized more P4 per flask (53.9 vs 25.0 ng/mg tissue, P<.001) and released more P4 into the medium (20.8 vs 8.8 ng/mg, P<.001). P4 synthesis by luteal tissue from 5 to 6 day old and 13 to 14 day old CL from pigs in group C was similar to P4 synthesis by luteal tissue from pigs in group A and group B, respectively. Luteul PGF synthesis was not affected significantly by either the age of the CL or by PMSG-HCG treatment. For endometrial samples, the synthesis of PGF was not significantly different among pigs in groups A, B and C. If uterine PGF is involved in luteal regression in the pig, the sensitivity of the CL to PGF may be more important than an increase in PGF secretion during the late luteal phase of the estrous cycle.  相似文献   

11.
A tridecapeptide containing tritium-labelled lysine and corresponding closely to residues 98 to 110 of the α chain of type I collagen was synthesized by the solid-phase method. Gly-Leu-Hyp-Gly-Nle-[4,5-3H]Lys-Gly-His-Arg-Gly-Phe-Ser-Gly was used as a substrate of human protocollagen lysyl hydroxylase (peptidyllysine, 2-oxoglutarate: oxygen 5-oxidoreductase, EC 1.14.11.4) obtained from dermal fibroblasts. L-[4,5-3H]Lysine was converted to Nα-t-butyloxycarbonyl-N?-o-chlorobenzyloxycarbonyl[3H]lysine which was incorporated during stepwise synthesis of the peptide. The chemical and radiochemical purities and specific activity of the completed peptide were characterized. A non-radiolabelled analogue of the peptide inhibited the hydroxylation of [3H]lysine-containing protocollagen by human lysyl hydroxylase, indicating that the synthetic peptide interacted with the enzyme. The peptide containing [3H]lysine was a substrate for lysyl hydroxylase and permitted direct measurement of enzyme activity in relatively crude cell extracts by a tritium-release assay. Extracts of cultured fibroblasts from a patient with an autosomal recessive pattern of inheritance for Ehlers-Danlos syndrome type VI had activities for tritium release from either the radiolabelled synthetic peptide or from [3H]lysine-containing protocollagen that were only 30% of those from control cells. These data indicate that a stable, well-defined synthetic peptide containing [3H]lysine is a useful substrate for studies of genetically variant lysyl hydroxylase from cultured human cells.  相似文献   

12.
Rabbit antibodies to bovine basement membrane collagen were used to compare the antigenic determinants of rat parietal yolk sac basement membrane [14C]procollagen with [14C]protocollagen. Basement membrane [14C]protocollagen was found to be less antigenic than basement membrane [14C]procollagen. Hydroxylation of basement membrane [14C]protocollagen, either intracellularly or in vitro with protocollagen prolyl hydroxylase, resulted in restoration of antigenicity. The difference in antigenicity observed between basement membrane [14C]procollagen and basement membrane [14C]protocollagen appeared to depend primarily upon the presence of hydroxyproline in the collagen molecule. Glucosylgalactosylhydroxylysine was found to be unimportant for antigenicity.  相似文献   

13.
Type I procollagen secreted by matrix-free chick embryo tendon cells was labeled with L-[3,3'-3H] cystine and purified by DEAE-cellulose chromatography. After bacterial collagenase digestion, the NH2- and COOH-terminal propeptides were partially characterized by ion exchange chromatography and gel filtration. Similar experiments were then conducted after labeling with either D-[6-3H] glucosamine, D-[2-3H] mannose, or D-[U-14C] glucose. On the basis of these studies and subsequent carbohydrate analysis, it was concluded that the COOH-terminal peptide contained greater than 90% of the radioactive carbohydrate which consisted predominantly of glucosamine and mannose with traces of galactosamine and galactose. Only radioactive glucosamine could be detected in the NH2-terminal propeptide. Under conditions which inhibit hydroxylation of lysine and glycosylation of hydroxylysine, unhydroxylated procollagen (protocollagen) could still be labeled with [3H] glucosamine and [3H] mannose. This suggested that glycosylation of the propeptides is at least initiated at the level of the rough endoplasmic reticulum.  相似文献   

14.
15.
16.
Antibody against NADPH-cytochrome c reductase inhibited the NADPH-dependent omega and penultimate hydroxylation of lauric acid by microsomes from kidney cortex and liver of rats, but did not inhibit the NADH-dependent hydroxylation of lauric acid. By contrast, an antibody against cytochrome b5 inhibited both the NADH and the NADPH-dependent hydroxylation of lauric acid by these microsomal preparations. Although the antibody against cytochrome b5 did not inhibit NADPH-oxidation, this lack of inhibition could not be attributed to the presence of an endogenous substrate or an uncoupling inhibitor in the antibody preparation. These findings suggest that NADPH-cytochrome c reductase mediates the NADPH-dependent hydroxylation of lauric acid but not its NADH-dependent hydroxylation, whereas cytochrome b5 plays a role in both the NADPH and the NADH-dependent hydroxylation of the fatty acid.  相似文献   

17.
The fatty acid oxygenase of sheep vesicular glands was solubilized with Tween-40 and purified 60-fold using ammonium sulfate precipitation and DEAE-cellulose chromatography. Glycerol (50%) stabilized the activity at all stages of purification and allowed long-term storage at −60°. The partially purified enzyme contained less than 0.7 nmoles of iron per mg of protein and less than 0.1 nmole of copper per mg of protein. Although the KI values for aspirin, BL-2338, flurbiprofen and ibuprofen remained relatively unchanged during purification, the apparent KI value for inhibition by indomethacin decreased from 120 to 2.7 μM.  相似文献   

18.
A highly purified cytochrome b?c1 complex which is free of QPs (see BBRC 78, 259 and 79, 939) yields 20–30% of semiubiquinone (based on the total Q content in the system) in the presence of catalytic amounts of QPs and succinate dehydrogenase (at or lower than 2 × 10?9 M. The radical shows typical g = 2.00 signals with line widths of 8 and 9 gauss, respectively, at about 22°C and 77°K. The appearance of the radicals approximately parallels that of b reduction but not its disappearance. However, addition of theonytrifluoroacetone or antimycin A immediately abolishes both radical formation and b reduction. These and other observations indicate that the true carrier property of Q is through its binding with proper proteins but not the protei-free form.  相似文献   

19.
During incubation with physiological buffers at 37°, as well as during in vivo maturation, native collagen fibers display a progressive increase in tensile strength and insolubility. This is paralleled by a progressive loss of reducible, intermolecular crosslinks. The experiments described in this paper indicate that nucleophilic addition of lysine and/or hydroxylysine residues to the electrophilic double bond of the reducible crosslinks transforms them into more stable, non-reducible crosslinks. Indeed, modification of lysine/hydroxylysine residues completely blocks this transformation, while modification of his, arg, glu and asp is without effect. On the basis of these and other experiments, tentative structures are proposed for the stable crosslinks.  相似文献   

20.
Dihydroorotic acid is converted to orotic acid in Crithidia by hydroxylation and subsequent dehydration. The hydroxylase is soluble, stable to acid (pH 4.0), destroyed by alkali (pH 11) and by heat (55° for 3 min.). Activity is rapidly lost upon standing at 4° and upon freezing. Its activity is optimum at pH 7.4. Its isoelectric point is 6.2. It has an absolute dependence on O2 and a reduced pteridine. Pteridine reductases are present in cell extracts which, in the presence of NADH, permits the efficient use of biopterin (the oxidized form of the naturally occurring pteridine in this organism) as a cofactor for the hydroxylase.  相似文献   

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