Renal glomerular proteoglycans. An investigation of their synthesis in vivo using a technique for fixation in situ.
Newly synthesized rat glomerular [35S]proteoglycans were labelled in vivo after injecting Na2[35S]SO4 intraperitoneally. At the end of the labelling period (7 h) the kidneys were perfused in situ with 0.01% (w/v) cetylpyridinium chloride. This fixed proteoglycans in the tissue and increased their recovery 2-3-fold during subsequent isolation of glomeruli from the renal cortex. The glomeruli were fractionated by a modified osmotic lysis and detergent extraction procedure [Meezan, Brendel, Hjelle & Carlson (1978) in The Biology and Chemistry of Basement Membranes (Kefalides, N.A., ed.), Academic Press, New York; Kanwar & Farquhar (1979) Proc. Natl. Acad. Sci. U.S.A. 76, 4493-4497] to obtain a basement membrane preparation. The proteoglycans released at each stage of the procedure were characterized using DEAE-Sephacel ion-exchange chromatography, chondroitinase ABC and HNO2 digestion and Sepharose CL-4B gel-permeation chromatography. About 85% of the [35S]proteoglycans synthesized were of the heparan sulphate variety, the remainder being chondroitin sulphate proteoglycans. Three sizes of heparan sulphate proteoglycans were identified. The largest (HS1, Kav. 0.47) accounts for 44% of the total extractable heparan sulphates. About one third of HS1 were extracted from the glomerular basement-membrane fraction with 8 M-urea and 4 M-guanidine hydrochloride but the remainder were released from the glomerulus during preparation of the fraction. The two smaller molecules (HS2, Kav. 0.56 and HS3, Kav. 0.68) accounted for 27% and 28% of the extractable heparan sulphate respectively and were not associated with the basement membrane fraction. HS1, HS2 and HS3 were also isolated from non-fixed glomeruli labelled in vivo but with much lower recovery. In glomeruli labelled in vitro, heparan sulphate accounted for only 35% of the proteoglycans, the remainder being of the chondroitin sulphate type. Proteoglycans similar to HS1, HS2 and HS3 were present in glomeruli labelled in vitro but, in addition, a large, highly charged heparan sulphate (HS1a) was extracted from the glomerular basement-membrane fraction of these glomeruli. It accounted for 6% of the total heparan sulphate.
Beavan
L A
LA
Department of Biochemistry, Charing Cross and Westminster Medical School, University of London, U.K.
Davies
M
M
Mason
R M
RM
D000818:Animals; D001485:Basement Membrane / Q000378:metabolism; D002594:Cetylpyridinium / Q000494:pharmacology; D011508:Chondroitin Sulfate Proteoglycans / Q000096:biosynthesis; D002853:Chromatography, Liquid; D003851:Deoxyribonucleases / Q000494:pharmacology; D005260:Female; D019812:Heparan Sulfate Proteoglycans; D006497:Heparan Sulfate / Q000096:biosynthesis; D007678:Kidney Glomerulus / Q000187:drug effects / Q000378:metabolism*; D046911:Macromolecular Substances; D008722:Methods; D010477:Perfusion; D011509:Proteoglycans / Q000096:biosynthesis*; D051381:Rats; D011919:Rats, Inbred Strains
D002594:Cetylpyridinium; D011508:Chondroitin Sulfate Proteoglycans; D003851:Deoxyribonucleases; D019812:Heparan Sulfate Proteoglycans; D006497:Heparan Sulfate; D046911:Macromolecular Substances; D011509:Proteoglycans