scholarly journals The sulphation of chondroitin sulphate in embryonic chicken cartilage

1969 ◽  
Vol 113 (3) ◽  
pp. 543-549 ◽  
Author(s):  
H. C. Robinson
Keyword(s):  
Ionic Strength ◽  
Incubation Time ◽  
Chondroitin Sulphate ◽  
Soluble Enzyme ◽  
Enzyme Preparations ◽  
Embryonic Chicken ◽  
Inorganic Sulphate ◽  
Effects Of Ph ◽  
Embryonic Cartilage

1. Whole tissue preparations and subcellular fractions from embryonic chicken cartilage were used to measure the rate of incorporation of inorganic sulphate into chondroitin sulphate in vitro. 2. In cartilage from 14-day-old embryos, [35S]sulphate is incorporated to an equal extent into chondroitin 4-sulphate and chondroitin 6-sulphate at a rate of 1·5nmoles of sulphate/hr./mg. dry wt. of cartilage. 3. Microsomal and soluble enzyme preparations from embryonic cartilage catalyse the transfer of sulphate from adenosine 3′-phosphate 5′-sulphatophosphate into both chondroitin 4-sulphate and chondroitin 6-sulphate. 4. The effects of pH, ionic strength, adenosine 3′-phosphate 5′-sulphatophosphate concentration and acceptor chondroitin sulphate concentration on the soluble sulphotransferase activity were examined. These factors all influence the activity of the sulphotransferase, and pH and incubation time also influence the percentage of chondroitin 4-sulphate formed.

scholarly journals Control of chondroitin sulphate biosynthesis. β-d-Xylopyranosides as substrates for UDP-galactose: d-xylose transferase from embryonic-chicken cartilage

1981 ◽  
Vol 194 (3) ◽  
pp. 839-846 ◽  
Author(s):  
J A Robinson ◽  
H C Robinson
Keyword(s):  
Transfer Reaction ◽  
Chondroitin Sulphate ◽  
Acetate Incorporation ◽  
Microsomal Preparation ◽  
Embryonic Chicken ◽  
A Cell ◽  
Embryonic Cartilage ◽  
Chondroitin Sulphate Chain ◽  
Core Production

Embryonic-chicken epiphyseal cartilage was incubated in vitro with a variety of beta-xylosides and the amount of [3H]acetate incorporation into chondroitin sulphate was determined under conditions when normal protein core production was inhibited by cycloheximide. The ability of the different beta-xylosides to relieve thea cycloheximide-mediated inhibition of chondroitin sulphate synthesis was influenced by the nature of the aglycan group of te xyloside. beta-Xylosides with apolar and uncharged aglycan groups were most effective and produced a severalfold stimulation of chondroitin sulphate biosynthesis. beta-Xylosides with charged aglycan groups were less effective initiators of chondroitin sulphate synthesis. The rate of galactose transfer from UDP-galactose to each of the beta-xylosides, catalysed by a cell-free microsomal preparation from embryonic cartilage, was measured. This study showed that the nature of the aglycan group of the beta-xyloside was a factor determining the capacity of the xyloside to act as an acceptor for galactosyltransferase I, the enzyme that catalyses the first galactose transfer reaction of chondroitin sulphate synthesis. The aglycan group of the xyloside also appeared to influence other steps leading to chondroitin sulphate chain initiation in vitro.

Conversion of Flavanone to Flavone, Dihydroflavonol and Flavonol with an Enzyme System from Cell Cultures of Parsley

1981 ◽  
Vol 36 (9-10) ◽  
pp. 742-750 ◽  
Author(s):  
L. Britsch ◽  
W. Heller ◽  
H. Grisebach
Keyword(s):  
Microsomal Fraction ◽  
Specific Activity ◽  
Enzyme System ◽  
High Specific Activity ◽  
Cell Suspension Cultures ◽  
Soluble Enzyme ◽  
Enzyme Preparations ◽  
Malonyl Coa ◽  
In Vitro Biosynthesis

Abstract Soluble enzyme preparations from irradiated cell suspension cultures of parsley (Petroselinum hortense Hoffm.) catalyse the conversion of flavanone to flavone, dihydroflavonol and flavonol. These reactions require 2-oxoglutarate, Fe2+ and ascorbate as cofactors. In the presence of these cofactors conversion of dihydroflavonol to flavonol was also observed. With this system in vitro biosynthesis of radioactive flavone, dihydroflavonol and flavonol from [2-14C]malonyl-CoA and 4-coumaroyl-CoA in good yield and with high specific activity is possible.We postulate that synthesis of flavone and flavonol from flavanone proceeds via 2-hydroxy-and 2,3-dihydroxyflavanone, respectively, with subsequent dehydration.The microsomal fraction of the parsley cells contains an NADPH-dependent flavanone 3'-hydroxylase.

scholarly journals The effect of β-d-xylosides on chondroitin sulphate biosynthesis in embryonic chicken cartilage in the absence of protein synthesis inhibitors

1977 ◽  
Vol 162 (2) ◽  
pp. 217-233 ◽  
Author(s):  
K D Gibson ◽  
B J Segen ◽  
T K Audhya
Keyword(s):  
Protein Synthesis ◽  
Incubation Medium ◽  
Chondroitin Sulphate ◽  
Gel Filtration ◽  
Protein Synthesis Inhibitors ◽  
Serum Factors ◽  
Embryonic Chicken ◽  
Low Concentrations ◽  
Normal Human

Incorporation of [35S]]sulphate, [3H]glucose and [3H]serine into glycosaminoglycans and proteoglycans of embryonic-chicken sternum was measured in vitro in incubation medium containing 4-methylumbelliferyl beta-D-xyloside or p-nitrophenyl beta-D-xyloside at low concentrations, and in the absence of inhibitors of protein synthesis. Incorporation of sulphate was decreased by 80% in incubations in which 1mM-4-methylumbelliferyl beta-xyloside or 2.5 mM-p-nitrophenyl beta-xyloside was present; under these conditions, serum factors stimulated incorporation to only a small extent. When the concentration of the xyloside was decreased tenfold, incorporation of sulphate was inhibited by 60-70%, but when normal human serum or L-3,3′,5-tri-iodothyronine or both were also added to the incubation medium, incorporation was markedly stimulated. Experiments in which [35S]sulphate and [3H]glucose were incorporated simultaneously, and enzymic analysis of glycosaminoglycans formed in such experiments, indicated that chondroitin sulphate formed in the presence of 0.1 mM-4-methylumbelliferyl beta-xyloside contained 30-40% less sulphate than did chondrotin sulphate synthesized in the absence of xylosides. Similar experiments, with [3H]serine instead of [3H]glucose, suggested also a 20-30% decrease in chain length of the chondroitin sulphate; this was confirmed by direct gel filtration of labelled glycosaminoglycans on a calibrated column. Incorporation of [3H]glucose or [3H]serine was stimulated by serum and tri-iodothyronine in parallel with incorporation of sulphate. The changes seen in the total chondroitin sulphate were mirrored in the major proteoglycan fraction, purified by isopycnic centrifugation of salt-extracted proteoglycans. The labelling pattern of chondroitin sulphate from this proteoglycan indicated that decreased sulphation of chondroitin sulphate was largely due to the inferior ability of short polysaccharide chains to accept sulphate, with some direct interference with transfer of sulphate to all chains. The results also suggested that the action of serum factors on synthesis of proteochondroitin sulphate is exercised at the level of either protein synthesis or transport to the sites of initiation of polysaccharide synthesis.

scholarly journals The metabolic fate of intravenously injected peptide-bound chondroitin sulphate in the rat

1976 ◽  
Vol 158 (1) ◽  
pp. 39-46 ◽  
Author(s):  
K M Wood ◽  
C G Curtis ◽  
G M Powell ◽  
F S Wusteman
Keyword(s):  
Chondroitin Sulphate ◽  
Rat Kidney ◽  
Average Molecular Weight ◽  
The Body ◽  
Connective Tissues ◽  
Peptide Fraction ◽  
Inorganic Sulphate ◽  
Average Size ◽  
Carbohydrate Components

The degradation of intravenously administered chondroitin sulphate-peptide, obtained by trypsin digestion of rat cartilage preparations labelled in vitro with 35S (and, in some cases, with 3H), was studied in rats. As with free chains of chondroitin sulphate, the major site of accumulation and degradation in the body was the liver, although peptide-linked chains were taken up more rapidly than free chains. In the first 2h after intravenous injection of a chondroitin sulphate-peptide fraction, labelled macromolecular components were excreted in the urine. These were shown to be chondroitin sulphate-peptide of the same degree of sulphation but of smaller average size than the injected material. A similar observation was made when free chains of chondroitin sulphate from the same source were administered intravenously. An isolated perfused rat kidney failed to de-sulphate circulating chondroitin sulphate-peptide, but a component of lower average molecular weight was excreted in the urine. When a chondroitin sulphate-peptide fraction of relatively larger hydrodynamic volume was administered, very little chondroitin sulphate appeared in the urine in the first 2h. It was concluded that, depending on size and/or peptide content, the chondroitin sulphate-peptide released from connective tissues into the circulation would probably be subjected to one of two alternative fates. The smaller fragments are more likely to be excreted in the urine, whereas the larger ones are taken up by the liver and there degraded to inorganic sulphate and undefined carbohydrate components.

scholarly journals The effect of chondroitin sulphate–protein on the formation of collagen fibrils in vitro

1968 ◽  
Vol 109 (5) ◽  
pp. 857-866 ◽  
Author(s):  
B. P. Toole ◽  
D. A. Lowther
Keyword(s):  
Ionic Strength ◽  
Chondroitin Sulphate ◽  
Collagen Fibrils ◽  
Lag Phase ◽  
Second Stage ◽  
Sequence Of Events

1. It was found that the precipitation of collagen fibrils at 37° from mixtures of chondroitin sulphate–protein and tropocollagen at physiological ionic strength and pH takes place in two distinct phases. The first occurs immediately on mixing either at 4° or at 37°, and the second occurs only at 37° and after a lag phase whose magnitude depends on the proportions of components. 2. When the second stage of precipitation was inhibited by mixing the reactants at 4°, the initial precipitate was found to contain ‘native-type’ collagen fibrils and chondroitin sulphate–protein. 3. On the basis of kinetic experiments it was concluded that aggregates of chondroitin sulphate–protein and tropocollagen form instantaneously and that these act as sites for the second stage of precipitation of fibrils. 4. The gels that result after continued incubation at 37° are fibrous in appearance if formed in the presence of the initial precipitate of chondroitin sulphate–protein and tropocollagen. 5. On the basis of these experiments in vitro the authors propose a sequence of events for collagen fibrogenesis in vivo.

scholarly journals Iron bound to pectin is utilised by rats

2011 ◽  
Vol 106 (1) ◽  
pp. 73-78 ◽  
Author(s):  
Tomihiro Miyada ◽  
Akira Nakajima ◽  
Kiyoshi Ebihara
Keyword(s):  
Ionic Strength ◽  
Ferrous Iron ◽  
Ferric Iron ◽  
Ph Value ◽  
Effects Of Ph ◽  
Hb Concentration

In the present in vitro study, the effects of pH and ionic strength on the release of iron from pectin and the ability of pectin to reduce ferric iron to ferrous iron were examined. The bioavailability of Fe bound to pectin was evaluated in rats. The amount of Fe released from pectin was at a maximum at pH 2·0 and decreased as the pH value increased. At pH 2·0, the amount of Fe released from pectin increased as the ion length increased; at pH 5·0, ion length had no effect on pectin release. Pectin effectively reduced Fe from the ferric form to the ferrous form. In rats fed a pectin diet, where Fe bound to pectin was the only Fe source, the final Hb concentration using diets containing 4·4–5·7, 7·2 or 11·5 mg Fe/kg diet was equal to the concentration in rats fed diets containing 4·5, 7·6 or 13·5 mg ferrous iron/kg diet, respectively. Hb regeneration efficiencies in rats fed pectin diets were significantly different from rats fed a diet containing 13·5 mg ferrous iron/kg diet. In rats fed a diet with or without pectin, where ferric iron was the only Fe source, pectin increased the final Hb concentration. These results suggest that Fe bound to pectin is utilised by rats.

scholarly journals The degradation of intravenously injected chondroitin 4-sulphate in the rat

1973 ◽  
Vol 134 (4) ◽  
pp. 1009-1013 ◽  
Author(s):  
Keith M. Wood ◽  
Frederick S. Wusteman ◽  
C. Gerald Curtis
Keyword(s):  
Molecular Weight ◽  
Rat Liver ◽  
Degradation Products ◽  
Chondroitin Sulphate ◽  
Whole Body ◽  
Low Molecular Weight ◽  
Inorganic Sulphate ◽  
Liver Lysosomes ◽  
Isolated Liver

The degradation of chondroitin 4-[35S]sulphate isolated from chick-embryo cartilage was studied in the rat by experiments on free-range animals, on wholly anaesthetized animals with ureter cannulae, by perfusion of isolated liver, by whole-body radioautography and by isolation of liver lysosomes. After injection into rats 68% of the radioactivity was recovered in the urine after 24h, approximately one-half of this being in the form of low-molecular-weight material, chiefly inorganic sulphate. Cannulation experiments demonstrated that the proportion of low-molecular-weight components excreted in the urine increased with time until, after 12h, virtually all was inorganic sulphate. Whole-body radioautography identified the liver as the major site of radioisotope accumulation after injection of labelled polysaccharide. Perfusion through isolated liver indicated that this organ has the ability to metabolize the polymer with the release of low-molecular-weight products, principally inorganic sulphate. Incubation of a lysosomal fraction prepared from rat liver after injection of chondroitin 4-[35S]sulphate gave rise to degradation products of low molecular weight, and experiments in vitro with rat liver lysosomes confirmed that these organelles are capable of the entire degradative process from chondroitin sulphate to free inorganic sulphate.

scholarly journals The effect of D-xylose, β-D-xylosides and β-D-galactosides on chondroitin sulphate biosynthesis in embryonic chicken cartilage

1975 ◽  
Vol 148 (1) ◽  
pp. 25-34 ◽  
Author(s):  
H C Robinson ◽  
M J Brett ◽  
P J Tralaggan ◽  
D A Lowther ◽  
M Okayama
Keyword(s):  
Incubation Medium ◽  
Chondroitin Sulphate ◽  
Polysaccharide Chain ◽  
Embryonic Chicken ◽  
Chain Synthesis

The incorporation of [3H]acetate into chondroitin sulphate was used as a measure of the rate of synthesis of this polysaccharide in whole tibias and femurs of embryonic chicken cartilage in vitro. The incorporation is inhibited by puromycin and by cycloheximide, but the inhibition is relieved by the addition of D-xylose, xβ-D-xylosides and β-D-galactosides to the incubation medium. β-D-Xylosides can stimulate the incorporation to 300% of that of controls incubated in the absence of cycloheximide or puromycin, D-Xylose, β-D-xylosides and β-D-galactosides appear to act as artificial initiators of chondroitin sulphate synthesis and enable polysaccharide-chain synthesis to be studied as an event separate from the synthesis of intact proteoglycan.

Effect of pH and inhibitors on beta-amyloid fibril assembly

1996 ◽  
Vol 54 ◽  
pp. 752-753
Author(s):  
Beverly E. Maleeff ◽  
Timothy K. Hart ◽  
Stephen J. Wood ◽  
Ronald Wetzel
Keyword(s):  
Amyloid Fibril ◽  
Peptide Fragment ◽  
Hydrophobic Peptides ◽  
Amyloid Fibril Formation ◽  
Effects Of Ph ◽  
Severity Of The Disease ◽  
Kinetics Of ◽  
The Brain

Alzheimer's disease is characterized post-mortem in part by abnormal extracellular neuritic plaques found in brain tissue. There appears to be a correlation between the severity of Alzheimer's dementia in vivo and the number of plaques found in particular areas of the brain. These plaques are known to be the deposition sites of fibrils of the protein β-amyloid. It is thought that if the assembly of these plaques could be inhibited, the severity of the disease would be decreased. The peptide fragment Aβ, a precursor of the p-amyloid protein, has a 40 amino acid sequence, and has been shown to be toxic to neuronal cells in culture after an aging process of several days. This toxicity corresponds to the kinetics of in vitro amyloid fibril formation. In this study, we report the biochemical and ultrastructural effects of pH and the inhibitory agent hexadecyl-N-methylpiperidinium (HMP) bromide, one of a class of ionic micellar detergents known to be capable of solubilizing hydrophobic peptides, on the in vitro assembly of the peptide fragment Aβ.

IN VITRO UPTAKE OF RADIOACTIVE 131I LABELLED L-TRIIODOTHYRONINE BY HUMAN ERYTHROCYTES AS AN INDEX OF THYROID FUNCTION

1960 ◽  
Vol XXXIV (II) ◽  
pp. 305-311 ◽  
Author(s):  
M. G. Woldring ◽  
A. Bakker ◽  
H. Doorenbos
Keyword(s):  
Thyroid Function ◽  
Incubation Time ◽  
Clinical Results ◽  
Human Erythrocytes ◽  
Red Cell ◽  
Clinical Value ◽  
Blood Samples ◽  
In Vitro Uptake

ABSTRACT The red cell triiodothyronine uptake technique as used in our hospital is described. Incubation time is of almost no importance. The temperature during incubation should be 37° C. Further improvement of the technique is obtained when all blood samples are brought up to 40 % haematocrit prior to incubation. Clinical results are discussed. It is yet too early to give a definite assessment of its clinical value, but it is definitely superior to the measurement of the BMR.

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