title: In vivo turnover of the basement membrane and other heparan sulfate proteoglycans of rat glomerulus.
abstract: The metabolic turnover of rat glomerular proteoglycans in vivo was investigated. Newly synthesized proteoglycans were labeled during a 7-h period after injecting sodium [35S]sulfate intraperitoneally. At the end of the labeling period a chase dose of sodium sulfate was given. Subsequently at defined times (0-163 h) the kidneys were perfused in situ with 0.01% cetylpyridinium chloride in phosphate-buffered saline to maximize the recovery of 35S-proteoglycans. Glomeruli were isolated from the renal cortex and analyzed for 35S-proteoglycans by autoradiographic, biochemical, and immunochemical methods. Grain counting of autoradiographs revealed a complex turnover pattern of 35S-labeled macromolecules, commencing with a rapid phase followed by a slower phase. Biochemical analysis confirmed the biphasic pattern and showed that the total population of [35S]heparan sulfate proteoglycans had a metabolic half-life (t1/2) of 20 and 60 h in the early and late phases, respectively. Heparan sulfate proteoglycans accounted for 80% of total 35S-proteoglycans, the remainder being chondroitin/dermatan sulfate proteoglycans. Whole glomeruli were extracted with 4% 3-[(cholamidopropyl)dimethy-lammonio]-1-propanesulfonate-4 M guanidine hydrochloride, a procedure which solubilized greater than 95% of the 35S-labeled macromolecules. Of these 11-13% was immunoprecipitated by an antiserum against heparan sulfate proteoglycan which, in immunolocalization experiments, showed specificity for staining the basement membrane of rat glomeruli. Autoradiographic analysis showed that 18% of total radioactivity present at the end of the labeling period was associated with the glomerular basement membrane. The glomerular basement membrane [35S]heparan sulfate proteoglycans, identified by immunoprecipitation, have a very rapid turnover with an initial phase, t1/2 = 5 h, and a later phase t1/2 = 20 h.
authors.affiliation: Department of Biochemistry, Charing Cross and Westminster Medical School, London, United Kingdom.
authors.forename: L A
authors.initials: LA
authors.lastname: Beavan
authors.forename: M
authors.initials: M
authors.lastname: Davies
authors.forename: J R
authors.initials: JR
authors.lastname: Couchman
authors.forename: M A
authors.initials: MA
authors.lastname: Williams
authors.forename: R M
authors.initials: RM
authors.lastname: Mason
mesh_terms: D000818:Animals; D001345:Autoradiography; D001485:Basement Membrane / Q000378:metabolism*; D011508:Chondroitin Sulfate Proteoglycans / Q000378:metabolism*; D005260:Female; D006023:Glycoproteins / Q000302:isolation & purification; D006025:Glycosaminoglycans / Q000378:metabolism*; D019812:Heparan Sulfate Proteoglycans; D006497:Heparan Sulfate / Q000378:metabolism*; D007678:Kidney Glomerulus / Q000378:metabolism*; D007700:Kinetics; D011509:Proteoglycans / Q000302:isolation & purification / Q000378:metabolism*; D051381:Rats; D011919:Rats, Inbred Strains; D013462:Sulfur Radioisotopes
chemical_list: D011508:Chondroitin Sulfate Proteoglycans; D006023:Glycoproteins; D006025:Glycosaminoglycans; D019812:Heparan Sulfate Proteoglycans; D006497:Heparan Sulfate; D011509:Proteoglycans; D013462:Sulfur Radioisotopes