Development of the biochemical and morphological changes induced by administration of a beta-xyloside to chick embryos.

4-Methylumbelliferyl beta-D-xyloside was administered to 9-day-old chick embryos, and the morphological and chemical changes in the embryo were followed daily. Increases in wet weight, Na and Cl content, and visible edema were detectable at 10 days and fully apparent at 11 days. Dry weight increased to the same extent in control and treated embryos for four days, but then diverged. The degree of sulfation of chondroitin sulfate was slightly less in treated than control embryos at 10 days, and reached a steady low value at 11 days. Analysis of glycosaminoglycans in skin, muscle, and aorta showed an increase in chondroitin and its sulfates in the two former tissues but not the latter. In muscle and aorta, the degree of sulfation of chondroitin sulfate was markedly reduced; but in skin the results suggested a more complex picture in which the normal metabolism of glycosaminoglycans was altered. A possible physiological role is suggested for chondroitin sulfate in embryonic soft tissues.

Fluid and glycosaminoglycan excretion by chick embryos treated with a beta-D-xyloside.

Wet weights and glycosaminoglycan content were determined for embryo, amnion, and allantois of control chick embryos and embryos injected with 4-methylumbelliferyl beta-D-xyloside at nine days of age. There was an immediate increase in total uronic acid content, but not in uronic acid concentration, in the embryo. No difference could be detected either in fluid volume, nor in content or type of glycosaminoglycan, in the amnion of the two groups. The fluid content of the allantois fo control eggs increased steadily between nine and 14 days, but in treated embryos the fluid content of the allantois remained low for at least a week. Less than 2 mg of uronic acid was present in allantoic fluid of control 16-day-old embryos, while treated embryos had accumulated more than 8 mg. More than 95% of the latter uronic acid was accounted for as chondroitin sulfate linked to methylumbelliferone and with a degree of sulfation of 50-60%. Thus beta-xyloside-treated embryos excrete large amounts of chondroitin sulfate and very little fluid.

Changes in chemical composition of chick embryos treated with a beta-xyloside and a lathyrogen.

Nine-day chick embryos were treated with 4-methylumbelliferyl beta-D-xyloside or beta-aminopropionitrile fumarate, and their gross chemical composition was examined one week later. Total DNA was 10--20% less in embryos treated with either drug than it was in control embryos. Xyloside-treated embryos showed marked increases in percent wet weight and in sodium/DNA and chloride/DNA ratios, and small decreases in protein/DNA, hydroxyproline/DNA and sulfate/DNA. None of these parameters was affected in embryos treated with beta-aminopropionitrile. Approximately 85% of the uronic acid of control embryos was present as chondroitin sulfate, with a degree of sulfation of 80% and charge density of 1.8; all of this chondroitin sulfate was covalently linked to peptide and had a number-average molecular weight of 29,300. In embryos treated with beta-xyloside, 90% of the uronic acid was present as chondroitin sulfate, with a degree of sulfation of 40% and charge density ranging from 1 to 2; 27% of this chondroitin sulfate, with an average molecular weight of 25,400, was peptide linked, while 73% was linked to 4-methylumbelliferone and had an average molecular weight of 22,900. The chemical differences between embryos treated with the xyloside and embryos treated with the lathyrogen reinforce the conclusion on morphological grounds that these are distinct syndromes involving different aspects of the extracellular matrix.

The mode of action of 4-methylumbelliferyl beta-D-xyloside on the synthesis of chondroitin sulphate in embryonic-chicken sternum.

1. Embryonic-chicken sterna, incubated in medium containing 0.1mm-4-methylumbelliferyl beta-d-xyloside (4-methylcoumarin 7-beta-d-xyloside), synthesize proteochondroitin sulphate that is significantly undersulphated and shorter than usual [Gibson, Segen & Audhya (1977) Biochem. J.162, 217-233]. 2. Neither the beta-d-galactoside nor the beta-d-glucuronide of 4-methylumbelliferone, nor 4-methylumbelliferone itself, produced the effects. The only metabolites of 4-methylumbelliferone that were detected in cartilages exposed to 4-methylumbelliferyl beta-d-xyloside were unchanged xyloside and chondroitin sulphate covalently attached to 4-methylumbelliferone. 3. Gel filtration of salt extracts of sterna incubated in medium containing the xyloside showed that there were two pools of chondroitin sulphate in the tissue. One pool was identified, on the basis of its elution pattern and the linear kinetics of incorporation of sulphate into it, as proteochondroitin sulphate. Incorporation into the other pool, whose properties suggested that it was methylumbelliferyl-chondroitin sulphate, indicated that it underwent partial turnover. The molecular weight of this chondroitin sulphate was about 19000, and it appeared to be about 70% sulphated. 4. When sterna were incubated in medium containing the xyloside, there was a very large incorporation of sulphate and glucose into glycosaminoglycans that were released into the incubation medium. This contrasts with incubations of sterna in the absence of the xyloside, in which less than 5% of the sulphate incorporated could be recovered from the medium. The glycosaminoglycan released into the medium was 4-methylumbelliferyl-chondroitin sulphate, whose average molecular weight was 7000-8000 and degree of sulphation more than 95%. 5. Incorporation of sulphate into proteochondroitin sulphate was stimulated more than 3-fold by addition of 20% (v/v) human serum and 10nm-l-3,3',5-tri-iodothyronine. Incorporation into methylumbelliferyl-chondroitin sulphate, in either the tissue or the medium, was not significantly altered. 6. The decrease in chain length and degree of sulphation of proteochondroitin sulphate is explained in terms of competition between peptide-linked primers and methylumbelliferone-containing primers at the intracellular sites of polysaccharidechain elongation and sulphation. The implications of the results for the mechanism of stimulation of proteoglycan synthesis by serum factors are discussed.

The effect of beta-D-xylosides on chondroitin sulphate biosynthesis in embryonic chicken cartilage in the absence of protein synthesis inhibitors.

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.

Nonsuppressible insulin-like activity and thyroid hormones: major pituitary-dependent sulfation factors for chick embryo cartilage.

Serum from hypothyroid hypophysectomized rats did not stimulate sulfation or incorporation of amino acids into chick embryo sterna. When such rats were treated for a short time with growth hormone (somatotropin), their serum stimulated incorporation both of sulfate and of amino acids. The different actions of the two types of sera were not due to changes in thyroid state. The results support the existence in serum of a sulfation factor for chick embryo cartilage that is dependent upon growth hormone. Highly purified preparations of nonsuppressible insulin-like activity from human serum stimulated incorporation of amino acids, and of uridine into RNA, in chick embryo sterna in vitro; chondrocytes prepared from this tissue had specific high-affinity binding sites for this insulin-like activity. However, sulfate incorporation was stimulated very little, unless serum from hypothyroid hypophysectomized rats was also present. When L-3,5,3'-triiodothyronine was added as well, the stimulation was enhanced further. From these and other experiments, we conclude that (i) nonsuppressible insulin-like activity or a closely related peptide is the growth-hormone-dependent growth and sulfation factor for chick embryo cartilage: (ii) a second, unidentified factor must be present for the insulin-like activity to stimulate sulfation; and (iii) stimulation of sulfation by thyroid hormones in vitro is additive to that of nonsuppressible insulin-like activity.

Stimulation of proteoglycan synthesis in chick embryo sternum by serum and L-3,5,3'-triiodothyronine.

Incorporation of sulfate into alcian blue-precipitable glycosaminoglycan of 12-day-old chick embryo sterna is stimulated by addition, separately or together, of normal human serum and physiological concentrations of thyroid hormones (Audhya, T.K., and Gibson, K.D. (1975) Proc. Natl. Acad, Sci. U. S. A. 72, 604--608). We present evidence that this stimulation is due to increased synthesis of at least one proteoglycan, with minor alterations in the size and chemical composition of the glycosaminoglycans. Pulse-chase experiments showed no detectable loss of label during the chase, in control sterna or sterna incubated with serum and L-3,5,3'-triiodothyronine; thus, all incorporation was the result of synthesis of glycosaminoglycans. In double-label experiments, with 35SO4(2-) and [3H]acetate, the molar ratio of 3H and 35S incorporated into glycosaminoglycans was changed little, if at all, by addition of serum or triiodothyronine or both, at concentrations which increased incorporation up to 2-fold. Glycosaminoglycans isolated from these and other incubations gave similar elution patterns from agarose columns, and identical electrophoretic patterns on cellulose acetate. Digestion with chondroitinase ABC (chondroitin ABC lyase; EC 4.2.2.4.) showed that incorporation was into chondroitin sulfate and possibly hyaluronic acid, and that the proportions of non-sulfated, 4-sulfated, and 6-sulfated disaccharide units differed little between stimulated and unstimulated sterna. Incorporation of [3H]serine into glycosaminoglycans from papain digest of sterna paralleled incorporation of 35SO4(2-), and indicated a number average molecular weight between 21,000 and 25,000 for the newly synthesized chondroitin sulfate. This value was confirmed by gel filtration chromatography, which also showed that the average molecular weight of the newly synthesized chondroitin sulfate decreased up to 15% under conditions of 2-fold stimulation. Proteoglycans were extracted from sterna incubated with [3H]serine and 35SO4(2-) and analyzed by isopycinic centrifugation in CsCl and by zone sedimentation in a sucrose gradient. A major proteoglycan fraction could be separated by either method. Incorporation of both isotopes into this proteoglycan fraction, and into glycosaminoglycans isolated after papain digestion, was stimulated in a coordinate manner. Almost identical results were obtained with both separation techniques. The results indicate that the synthesis of the major proteoglycan, and probably also of a minor one, is stimulated by serum and triiodothyronine.

Deficiencies of chromatophore proteins in some mutants of Rhodopseudomonas spheroides with altered carotenoids.

Chromatophore proteins of a wild type and three mutant strains of Rhodopseudomonas spheroides were examined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The mutants consisted of a green and a blue-green one, whose phenotypes were essentially the same as those of known mutants, and a brown one, which may be a double mutant and represents a new phenotype. Wild-type chromatophores contained at least six major and seven minor protein bands, with molecular weights ranging from 10,000 to 65,000. The green mutant contained the same protein bands in the same relative quantities. The brown mutant had one protein completely missing and no other alterations. The blue-green mutant was deficient in a different protein, and had reduced quantities of all proteins with molecular weights less than 25,000. Chromatophores were separated into a fraction containing the reaction centers and a fraction containing the light-harvesting bacteriochlorophyll by treatment with sodium dodecyl sulfate. Eight of the proteins were found only in the reaction center fraction, one was only in the light-harvesting fraction, and the remainder were present in both fractions. The protein missing from the brown mutant was found to be a component of the reaction center fraction, whereas the proteins which were missing from the blue-green mutant were all components of the light-harvesting fraction. Some implications for the structure and biogenesis of chromatophores are discussed.