Effects of glutathione depletion on the synthesis of proteoglycan and collagen in cultured chondrocytes.
We studied the effect of the depletion of glutathione on the synthesis of proteoglycan and collagen in cultured chick chondrocytes. When the cultured chondrocytes were incubated with 1 mM buthionine sulfoximine (BSO), a specific inhibitor of gamma-glutamyl-cysteine synthetase, the intracellular glutathione level markedly dropped within 12 h with no loss of cell viability. Incorporation of 35SO2-4 into proteoglycan was lowered in the presence of BSO. When the 35S-labeled proteoglycans were separated into two fractions by glycerol density gradient centrifugation, the inhibitory effect of BSO on the synthesis of proteoglycan was greater in the fast-sedimenting proteoglycan fraction, which consisted mainly of cartilage specific large proteoglycan (PG-H), than in the slowly sedimenting proteoglycan fraction. The inhibition by BSO of the synthesis of core protein-free glycosaminoglycan chains primed by p-nitrophenyl-beta-D-xyloside was smaller than the inhibition of the synthesis of proteoglycan. Analysis of glycosaminoglycans labeled with [3H]glucosamine indicated that the treatment of chondrocytes with BSO resulted in a small increase in the proportion of synthesis of hyaluronic acid to the synthesis of total glycosaminoglycan. The incorporation of [3H]proline into collagen was also inhibited by BSO. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of the 3H-labeled collagen showed that, in the presence of BSO, processing of Type II collagen appeared to slow down and the proportion of Type X collagen synthesis was reduced.
Habuchi
O
O
Department of Chemistry, Aichi Kyoiku University, Kariya, Japan.
Miyachi
T
T
Kaigawa
S
S
Nakashima
S
S
Fujiwara
C
C
Hisada
M
M
D000818:Animals; D000963:Antimetabolites; D019328:Buthionine Sulfoximine; D002478:Cells, Cultured; D002642:Chick Embryo; D003094:Collagen / Q000096:biosynthesis*; D004591:Electrophoresis, Polyacrylamide Gel; D005944:Glucosamine / Q000378:metabolism; D005978:Glutathione / Q000502:physiology*; D006025:Glycosaminoglycans / Q000378:metabolism; D006027:Glycosides / Q000494:pharmacology; D006132:Growth Plate / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D007700:Kinetics; D008717:Methionine Sulfoximine / Q000031:analogs & derivatives / Q000494:pharmacology; D009596:Nitrophenols / Q000494:pharmacology; D014176:Protein Biosynthesis; D011499:Protein Processing, Post-Translational; D011509:Proteoglycans / Q000096:biosynthesis*; D013461:Sulfur Oxides / Q000378:metabolism
D000963:Antimetabolites; D006025:Glycosaminoglycans; D006027:Glycosides; D009596:Nitrophenols; D011509:Proteoglycans; D013461:Sulfur Oxides; D008717:Methionine Sulfoximine; C008646:4-nitrophenyl beta-D-xyloside; D019328:Buthionine Sulfoximine; D003094:Collagen; D005978:Glutathione; D005944:Glucosamine