| 1. |
- Lohmander, S., et al.
(författare)
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Secretion of proteoglycans by chondrocytes. Influence of colchicine, cytochalasin B, and β-d-xyloside
- 1979
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Ingår i: Archives of Biochemistry and Biophysics. - : Elsevier BV. - 0003-9861. ; 192:1, s. 148-157
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Tidskriftsartikel (refereegranskat)abstract
- Chondrocytes obtained from epiphyseal cartilage of fetal guinea pigs or ear cartilage of young rabbits were cultured in monolayer. The influence of colchicine, cytochalasin B, and p-nitrophenyl-β-d-xylopyranoside on secretion of proteoglycans was investigated. Radioactive sulfate was used as a precursor. As observed previously in other systems, β-d-xylosides initiated the synthesis of free chondroitin sulfate chains, competing with the endogenous proteoglycan core protein acceptor. The molecular weights of the chondroitin sulfate chains synthesized both on the xyloside and on the core-protein acceptor in maximally stimulated cells were similar and significantly lower than in proteoglycans synthesized in the absence of xyloside. The size of the chondroitin sulfate chains synthesized on the xyloside was inversely related to the concentration of this compound. This finding suggests that the chain length is dependent on the ratio between available acceptor and chain-lengthening enzymes or precursors. Cytochalasin B, a microfilament-modifying agent, inhibited proteoglycan synthesis, without any effect on secretion. Cells treated with cytochalasin B could be stimulated with β-d-xyloside to synthesize free chondroitin sulfate chains to the same relative degree as cells with intact microfilaments. Colchicine, an antimicrotubular agent, partially inhibited synthesis and secretion of proteoglycan. However, cells treated with colchicine could be stimulated with β-d-xyloside to synthesize and secrete free chondroitin sulfate chains to about the same relative degree as cells with intact microtubules. The data suggest that microtubules may have a facilitatory rather than an obligatory role in the secretion of proteoglycans and that at least part of the effect of colchicine is located at or after the site of glycosaminoglycan synthesis.
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| 2. |
- Lohmander, Stefan
(författare)
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Turnover of proteoglycans in guinea pig costal cartilage
- 1977
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Ingår i: Archives of Biochemistry and Biophysics. - : Elsevier BV. - 0003-9861. ; 180:1, s. 93-101
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Tidskriftsartikel (refereegranskat)abstract
- The turnover in vivo of proteoglycans of guinea pig costal cartilage was investigated using Na2 35SO4 as precursor. Proteoglycans were extracted with guanidine · HCl, at both low and high ionic strength, and purified and fractionated by ultracentrifugation in CsCl gradients under associative and dissociative conditions. The results suggest that the sulfate is incorporated into macromolecules of at least two major metabolic pools with half-lives of about 3 days and about 60-70 days, respectively. Molecules with a fast turnover were enriched in the low ionic strength extracts and in fractions containing small, nonaggregated proteoglycans. No substantial evidence was found for a precursor-product relationship between different fractions.
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| 3. |
- Madsen, Kjell, et al.
(författare)
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Production of cartilage-typic proteoglycans in cultures of chondrocytes from elastic cartilage
- 1979
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Ingår i: Archives of Biochemistry and Biophysics. - : Elsevier BV. - 0003-9861. ; 196:1, s. 192-198
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Tidskriftsartikel (refereegranskat)abstract
- Chondrocytes from rabbit ear cartilage were isolated and cultured as monolayers in Ham's F-12 medium. The proteoglycans synthesized by short-term cultures formed a high proportion of aggregates and contained chrondroitin-4- and -6-sulfate in a 2:1 proportion. Dermatan sulfate was not present. The average molecular weight of the chondroitin sulfate was about 20,000. Keratan sulfate with an average molecular weight of about 6000 could be isolated from the proteoglycan monomers. Rabbit ear chondrocytes in culture thus produced proteoglycans comparable to those isolated from hyaline cartilage. Culture for longer periods and plating at lower density caused a decrease in the proportion of aggregated proteoglycans. Primary cultures continued to synthesize aggregated proteoglycans for at least 2 weeks, while subdivision of the cultures caused a shift toward the production of small-sized "ubiquitous proteoglycans." The synthesis of proteoglycan aggregates could, however, be partly restored by transfer of the monolayer cells to a suspension culture.
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| 4. |
- Brodelius, Peter, et al.
(författare)
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Studies of Bovine Liver Glutamate Dehydrogenase by Analytical Affinity Chromatography on Immobilized AMP Analogs
- 1979
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Ingår i: Archives of Biochemistry and Biophysics. - : Elsevier BV. - 0003-9861. ; 194:2, s. 449-456
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Tidskriftsartikel (refereegranskat)abstract
- Bovine liver glutamate dehydrogenase has been studied by analytical affinity chromatography on two immobilized AMP analogs, i.e., N6-(6-aminohexyl)-AMP and 8-(6-aminohexyl)-amino-AMP. The existence of various enzyme-coenzyme and enzyme-effector complexes has been verified. Also the cooperative formation of two ternary complexes, i.e., glutamic dehydrogenase (GHD)-NADP-glutamate and GDH-ADP-leucine, has been shown. The results of this study have been rationalized by the “ligand exclusion theory.” which has been proposed for the regulation of the glutamic dehydrogenase. It has been shown that the active site and the ADP-binding effector site are oriented close to each other on the enzyme. Furthermore, the data suggest that the adenylic site is not identical to the nonactive coenzyme binding site. A mechanism based on electrostatic interactions is suggested for the cooperative binding of oxidized coenzyme and substrate. Dissociation constants for complexes between the enzyme and two coenzyme fragments (P-ADPR and 2′,5′-ADP) have been estimated.
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| 5. |
- Brodelius, Peter, et al.
(författare)
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The Synthesis of 8-(6-Aminohexyl)-Amino-GMP and Its Applications as a General Ligand in Affinity Chromatography
- 1978
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Ingår i: Archives of Biochemistry and Biophysics. - : Elsevier BV. - 0003-9861. ; 188:1, s. 228-231
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Tidskriftsartikel (refereegranskat)abstract
- The synthesis of a new general ligand, i.e., 8-(6-aminohexyl)-amino-GMP, has been achieved by bromination of GMP and subsequent substitution of the bromine for hexamethylene diamine. The overall yield of the synthesis has been 60 to 70%. This new general ligand was immobilized on BrN-activated Sepharose and used as an affinity adsorbent for inosinic acid dehydrogenase (E.C. 1.2.1.14) from Aerobacter aerogenes. Various elution methods were investigated in order to increase the specific activity of the purified enzyme.
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