In vivo turnover of the basement membrane and other heparan sulfate proteoglycans of rat glomerulus.
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.
Beavan
L A
LA
Department of Biochemistry, Charing Cross and Westminster Medical School, London, United Kingdom.
Davies
M
M
Couchman
J R
JR
Williams
M A
MA
Mason
R M
RM
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
D011508:Chondroitin Sulfate Proteoglycans; D006023:Glycoproteins; D006025:Glycosaminoglycans; D019812:Heparan Sulfate Proteoglycans; D006497:Heparan Sulfate; D011509:Proteoglycans; D013462:Sulfur Radioisotopes