Rats were implanted with mini osmotic pumps delivering sodium [35S]sulphate and their newly synthesized proteoglycans were labelled over a 146 h period (steady-state labelling). Proteoglycan turnover was measured in vivo using a chase protocol. Glomerular proteoglycans were recovered quantitatively and the perlecan present was isolated by immunoprecipitation. The procedure allows newly synthesized proteoglycans to be quantified in mass units (pmol of glycosaminoglycan sulphate) after labelling and during the chase. Ultrastructural-immunogold experiments identified the location of perlecan as the glomerular basement membrane and mesangial matrix. Perlecan in the basement membrane was quantified using the ultrastructural-immunogold technique. Perlecan comprises about 10% of the total glomerular proteoglycans, which are otherwise associated with glomerular cells and the mesangium. Both the total glomerular heparan sulphate proteoglycans and perlecan turn over rapidly (t½ ∼ 3–4 h and < 3 h respectively). In contrast, turnover of proteoglycans in other tissues was slow, except in the liver where the heparan sulphate and chondroitin sulphate t½ values were 16 h and 9 h respectively. Microalbuminuria was induced with a low-dose regimen of puromycin aminonucleoside. At the onset of microalbuminuria (5 days) there was no change in the level of newly synthesized perlecan, or in perlecan in the glomerular basement membrane detected by immunogold labelling. Newly synthesized perlecan had undergone a minimal change in turnover rate by day 5 in puromycin aminonucleoside-treated rats. In contrast, the total glomerular proteoglycan population showed a dramatic decrease in turnover by day 5. Since there was no evidence of accumulation of glomerular proteoglycans on either day 5 or day 6, it is likely that decreased turnover of cell-associated proteoglycans is accompanied by an equivalent decrease in their synthesis.
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November 1996
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Research Article|
November 15 1996
A steady-state labelling approach to the measurement of proteoglycan turnover in vivo and its application to glomerular proteoglycans Available to Purchase
Emma L AKUFFO;
Emma L AKUFFO
*Department of Biochemistry, Charing Cross and Westminster Medical School, Fulham Palace Road, London W6 8RF
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Jayne R HUNT;
Jayne R HUNT
*Department of Biochemistry, Charing Cross and Westminster Medical School, Fulham Palace Road, London W6 8RF
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Jill MOSS;
Jill MOSS
†Department of Histopathology, Charing Cross and Westminster Medical School, Fulham Palace Road, London W6 8RF
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David WOODROW;
David WOODROW
†Department of Histopathology, Charing Cross and Westminster Medical School, Fulham Palace Road, London W6 8RF
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Malcolm DAVIES;
Malcolm DAVIES
‡Institute of Nephrology, University of Wales College of Medicine, Cardiff Royal Infirmary, Newport Road, Cardiff CF2 1SZ, Wales, U.K.
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Roger M. MASON
Roger M. MASON
§
*Department of Biochemistry, Charing Cross and Westminster Medical School, Fulham Palace Road, London W6 8RF
§To whom correspondence should be addressed.
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Publisher: Portland Press Ltd
Received:
January 16 1996
Revision Received:
July 23 1996
Accepted:
July 30 1996
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London © 1996
1996
Biochem J (1996) 320 (1): 301–308.
Article history
Received:
January 16 1996
Revision Received:
July 23 1996
Accepted:
July 30 1996
Citation
Emma L AKUFFO, Jayne R HUNT, Jill MOSS, David WOODROW, Malcolm DAVIES, Roger M. MASON; A steady-state labelling approach to the measurement of proteoglycan turnover in vivo and its application to glomerular proteoglycans. Biochem J 15 November 1996; 320 (1): 301–308. doi: https://doi.org/10.1042/bj3200301
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