Abstract
A range of structurally related zwitterionic detergents, Zwittergents 3-06, 3-08, 3-10, and 3-12, and a derivative of cholic acid (Chaps) were examined for their ability to enhance the extraction of newly synthesized, intracellular proteoglycans and for their effect on the functional properties of cartilage proteoglycan. Although none of the detergents could extract greater than 4% of the intracellular proteoglycans when used alone, Zwittergents 3-10, 3-12, and Chaps proved equally as effective when used in combination with 4 M guanidine HCl extracting greater than 90% of newly synthesized proteoglycans. Rate zonal centrifugation of aggregates containing either 3H-link protein or 3H-monomer, which had been incubated with 2% (w/v) detergent indicated that none of the test detergents caused a disassembly of intact aggregates. However, both Zwittergents 3-10 and 3-12 prevented the reaggregation of components dissociated with 4 M guanidine HCl. Similar to the finding with aggregate, none of the detergents caused a disassembly of monomer-link protein complexes prepared from purified 3H-link protein and proteoglycan monomer, while Zwittergents 3-10 and 3-12 prevented their assembly from free link protein and monomer. However, monomer-link protein complexes once formed were able to associate with hyaluronic acid to form link-stable ternary complexes in the presence of all detergents tested including Zwittergents 3-10 and 3-12.
Highlights
A range of structurally related zwitterionic deter- have been secreted from the cell [8,9,10,11]
Extraction of Intracellular Proteoglycans Using Zwitterionic Detergents-Primary cultures of rat chondrosarcoma chondrocytes were labeled on day 2 of tissue culturewith 35s04for 5 min or with [3H]leucinefor 30 min to label predominantly intracellular proteoglycans and cell-associated proteins
None of the detergents tested were capable of solubilizing more than 4% of the newly synthesized, intracellular proteoglycans when usedalone (Table I)
Summary
$ Recipient of an Arthritis Foundation Postdoctoral Fellowship. J To whom reprint requests should be addressed. The culture medium was subjected to associative CsCl density gradient centrifugationfollowedby dissociative centrifugation [20].The A1D5 fraction containing 3H-link protein was dialyzed first against 0.1 M Tris, 0.1 M sodium acetate, pH 7.2, a t 4 "C and exhaustively against H 2 0 before lyophilization. Stability of the Proteoglycan Aggregate and the Monomer-Link Complex in the Presence of Detergent-Carrier aAl from the Swarm rat chondrosarcoma was added to the3H-link protein-labeled or 3Hmonomer-labeled aggregates prepared as described above to give a final concentration of 6-8 mg/ml. The percentage of proteoglycans still existing in an aggregate or monomer-link complex form was determined by rate zonal centrifugation on 16.5-ml Cs2S04gradients containing 0.5% of the appropriate detergent.500-pl fractions were collected and assayed for radioactivity and uronic acid. The molar ratio of core protein:link protein was calculated assuming that the core protein represented 10% of the monomer weight and that the molecular weight of the core protein in monomer and the link protein were 200,000 and 40,000, respectively [3, 10]
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