Eur. J. Biochem. 54, 531-540 (1975)
The Biosynthesis of Basement-Membrane Collagen by Isolated Rat Glomeruli Michael E. GRANT, Richard HARWOOD, and Isabel F. WILLIAMS Department of Medical Biochemistry, University of Manchester (Received January 9/February 27, 1975)
A technique is described for the rapid isolation of highly purified preparations of viable glomeruli from rat kidney cortex. The synthesis of protein as judged by the incorporation of ['4C]proline into non-diffusible material was shown to be linear for up to 6 h. The synthesis of collagen, measured as non-diffusible 4-hydro~y['~CIproline, was also linear over this period but represented only a small proportion of total protein synthesis. Similar studies conducted in vivo confirmed that collagen synthesis accounted for less than 5 % of total protein synthesis in glomeruli. When isolated glomeruli were incubated with [14C]proline,it was found that approximately 16 % of the hydroxyproline present in the collagenous component occurred as the 3-isomer. When glosynthesised was glycomeruli were incubated with [14C]lysine over 90 % of the hydr~xy['~C]lysine sylated and most of the glycosylated hydr~xy['~C]lysine was present as glucosyl-galactosyl-hydroxy['4C]lysine. The size of the basement membrane collagen synthesised by the isolated glomeruli was estimated by treating the I4C-labelled protein with mercaptoethanol and sodium dodecyl sulphate and then chromatographing the 14C-labelledprotein on an agarose column equilibrated and eluted with buffer containing 0.1 % (w/v) sodium dodecyl sulphate. The initial form of ['4C]collagen synthesised was found to consist of polypeptide chains which had molecular weights of approximately 140000 and which were shown to be distinctly larger than the polypeptide chains from embryonic chick tendon procollagen. Also when glomeruli were labelled with ['4C]proline for 2 h and chased with unlabelled proline for 4 h there was a time-dependent conversion of the initially synthesised collagen moiety to collagen polypeptide chains which co-chromatograph with tendon pro-a chains (molecular weight approx. 120000).
Basement membranes are specialised forms of connective tissues which are widely distributed in nature and are believed to be comprised of a collagenous component and one or more non-collagenous glycoproteins (for review, see [2]). The renal glomerular basement membrane, an important example of these extracellular membranes, is of considerable physiological and pathological interest because of its involvement in the initial ultrafiltration process of urine formation and its possible modification in a variety of renal diseases. That the morphology of the glomerular basement membrane alters in diseases such as diabetes mellitus and nephrosis is well documented [3-51 but whether or not the chemical A preliminary report on this work has been presented elsewhere [I]. Eur. J. Biochem. 54 (1975)
structure of the basement membrane is altered is the subject of conflicting reports [6,7]. Indeed, considerable speculation and controversy exists as to the structure of basement membranes and to the interrelationship of the various components which have been extracted by a variety of techniques [2,8,9]. The significance of the heterogeneity of the glomerular basement membrane components isolated by Hudson and Spiro [lo, 111is not at all clear but it seems unlikely that these components represent the initial biosynthetic subunits of the basement membrane [12]. In an attempt to elucidate the biogenesis of the glomerular basement membrane we have undertaken a study of the synthesis of the first-formed subunits using radioactive labelling techniques. We here report procedures employing the sieving of rat cortical tissue
532
and a discontinuous Ficoll gradient to achieve the rapid isolation of pure glomeruli. The isolated glomeruli exhibit a linear incorporation of labelled amino acids into protein and a study of the synthesis of a collagenous molecule having the characteristics of glomerular basement membrane collagen is described. EXPERIMENTAL PROCEDURE Materids Male Sprague Dawley rats (150 f 10 g) were supplied by the animal unit of the University of Manchester Medical School. [2-3H]Glycine (2.0 Ci/ mmol), [U-"C]proline (290 mCi/mmol) and [4,5-3H]lysine (5.3 Ci/mmol) were purchased from the Radiochemical Centre (Amersham, Bucks). Eagle's basal medium with Earle's salts and foetal calf serum were purchased from Flow Laboratories (Irvine, Ayrshire). Cycloheximide was obtained from Sigma Chemical Co. (London SW6) and 2,2'-bipyridyl was obtained from Calbiochem (Los Angeles, Calif.). Ficoll400 was purchased from Pharmacia Ltd (London). Stainless steel sieves (mesh 150,gauge 18 mm) were obtained from N. Greening Ltd (Warrington, Lancs.). Highly purified bacterial collagenase which was free of proteolytic activity towards a [3H]tryptophan-labelled substrate was a generous gift from Mr V. Lee-Own of this Department. Inactin was obtained from Promonta (Hamburg, Federal Republic of Germany) and trypan blue from Bio-Cult Laboratories (Glasgow). Chick tendon procollagen was prepared by incubating I S x 10' cells isolated from embryonic chick tendons in 15 ml of modified Krebs medium with 100 pCi of [3H]glycinefor 3 h [13]. The medium was removed by centrifugation at 3000 x g for 10 min and dialysed against 0.1 N acetic acid. The [3H]procollagen was stored at - 20 C and aliquots containing approx. 10' countsjmin were incorporated as an internal standard into some of the gel-filtration analyses of the ['4C]collagen synthesised by isolated glomeruli. Isolation of Glomeruli Kidneys were removed from rats killed by a blow on the head. The outer 1-2 mm of the cortical tissue was obtained by dissection and kept at 4 "C in modified Krebs medium [14]. This cortical tissue was passed through a 150-mesh stainless steel sieve (105 pm pore size) held taut in a metal holder. In general the tissue from 20 kidneys was gently pressed through an area of approx. 175 cm2 and then 50 ml Krebs medium was washed through the sieve. The material emerging through the sieve was allowed to
Basement-Membrane Biosynthesis
sediment under unit gravity for 10 min in two 35-ml polycarbonate centrifuge tubes. The upper threequarters of the supernatant was removed and the sediment resuspended in 20 ml fresh medium and allowed to settle for a further 10 min. The sediment was resuspended in Krebs medium (10 ml) containing lo"/, (v/v) foetal calf serum and made 127; (wp) in Ficoll, then layered on a discontinuous Ficoll gradient (Fig. 1) comprising 6 ml of each of the following concentrations: 20%, 18% and 15% (w/v). All the above procedures were carried out at 4 "C. After centrifugation at 300 x g for 2.5 min the glomeruli banded at the interface of the 15 18 "/, layers and tubules and large aggregates penetrated to the interface of the 18 %: 20 layers (Fig. 1). The glomerular fraction was diluted with an excess of Krebs medium and centrifuged at 3000 x g for 5 min. In most experiments the glomeruli were resuspended in Eagle's medium and an aliquot (0.02 ml) was taken for examination and counting under the light microscope.
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The Biosynthesis of Basement-Membrane Collagen by Isolated Rat Glomeruli Michael E. GRANT, Richard HARWOOD, and Isabel F. WILLIAMS Department of Medical Biochemistry, University of Manchester (Received January 9/February 27, 1975)
A technique is described for the rapid isolation of highly purified preparations of viable glomeruli from rat kidney cortex. The synthesis of protein as judged by the incorporation of ['4C]proline into non-diffusible material was shown to be linear for up to 6 h. The synthesis of collagen, measured as non-diffusible 4-hydro~y['~CIproline, was also linear over this period but represented only a small proportion of total protein synthesis. Similar studies conducted in vivo confirmed that collagen synthesis accounted for less than 5 % of total protein synthesis in glomeruli. When isolated glomeruli were incubated with [14C]proline,it was found that approximately 16 % of the hydroxyproline present in the collagenous component occurred as the 3-isomer. When glosynthesised was glycomeruli were incubated with [14C]lysine over 90 % of the hydr~xy['~C]lysine sylated and most of the glycosylated hydr~xy['~C]lysine was present as glucosyl-galactosyl-hydroxy['4C]lysine. The size of the basement membrane collagen synthesised by the isolated glomeruli was estimated by treating the I4C-labelled protein with mercaptoethanol and sodium dodecyl sulphate and then chromatographing the 14C-labelledprotein on an agarose column equilibrated and eluted with buffer containing 0.1 % (w/v) sodium dodecyl sulphate. The initial form of ['4C]collagen synthesised was found to consist of polypeptide chains which had molecular weights of approximately 140000 and which were shown to be distinctly larger than the polypeptide chains from embryonic chick tendon procollagen. Also when glomeruli were labelled with ['4C]proline for 2 h and chased with unlabelled proline for 4 h there was a time-dependent conversion of the initially synthesised collagen moiety to collagen polypeptide chains which co-chromatograph with tendon pro-a chains (molecular weight approx. 120000).
Basement membranes are specialised forms of connective tissues which are widely distributed in nature and are believed to be comprised of a collagenous component and one or more non-collagenous glycoproteins (for review, see [2]). The renal glomerular basement membrane, an important example of these extracellular membranes, is of considerable physiological and pathological interest because of its involvement in the initial ultrafiltration process of urine formation and its possible modification in a variety of renal diseases. That the morphology of the glomerular basement membrane alters in diseases such as diabetes mellitus and nephrosis is well documented [3-51 but whether or not the chemical A preliminary report on this work has been presented elsewhere [I]. Eur. J. Biochem. 54 (1975)
structure of the basement membrane is altered is the subject of conflicting reports [6,7]. Indeed, considerable speculation and controversy exists as to the structure of basement membranes and to the interrelationship of the various components which have been extracted by a variety of techniques [2,8,9]. The significance of the heterogeneity of the glomerular basement membrane components isolated by Hudson and Spiro [lo, 111is not at all clear but it seems unlikely that these components represent the initial biosynthetic subunits of the basement membrane [12]. In an attempt to elucidate the biogenesis of the glomerular basement membrane we have undertaken a study of the synthesis of the first-formed subunits using radioactive labelling techniques. We here report procedures employing the sieving of rat cortical tissue
532
and a discontinuous Ficoll gradient to achieve the rapid isolation of pure glomeruli. The isolated glomeruli exhibit a linear incorporation of labelled amino acids into protein and a study of the synthesis of a collagenous molecule having the characteristics of glomerular basement membrane collagen is described. EXPERIMENTAL PROCEDURE Materids Male Sprague Dawley rats (150 f 10 g) were supplied by the animal unit of the University of Manchester Medical School. [2-3H]Glycine (2.0 Ci/ mmol), [U-"C]proline (290 mCi/mmol) and [4,5-3H]lysine (5.3 Ci/mmol) were purchased from the Radiochemical Centre (Amersham, Bucks). Eagle's basal medium with Earle's salts and foetal calf serum were purchased from Flow Laboratories (Irvine, Ayrshire). Cycloheximide was obtained from Sigma Chemical Co. (London SW6) and 2,2'-bipyridyl was obtained from Calbiochem (Los Angeles, Calif.). Ficoll400 was purchased from Pharmacia Ltd (London). Stainless steel sieves (mesh 150,gauge 18 mm) were obtained from N. Greening Ltd (Warrington, Lancs.). Highly purified bacterial collagenase which was free of proteolytic activity towards a [3H]tryptophan-labelled substrate was a generous gift from Mr V. Lee-Own of this Department. Inactin was obtained from Promonta (Hamburg, Federal Republic of Germany) and trypan blue from Bio-Cult Laboratories (Glasgow). Chick tendon procollagen was prepared by incubating I S x 10' cells isolated from embryonic chick tendons in 15 ml of modified Krebs medium with 100 pCi of [3H]glycinefor 3 h [13]. The medium was removed by centrifugation at 3000 x g for 10 min and dialysed against 0.1 N acetic acid. The [3H]procollagen was stored at - 20 C and aliquots containing approx. 10' countsjmin were incorporated as an internal standard into some of the gel-filtration analyses of the ['4C]collagen synthesised by isolated glomeruli. Isolation of Glomeruli Kidneys were removed from rats killed by a blow on the head. The outer 1-2 mm of the cortical tissue was obtained by dissection and kept at 4 "C in modified Krebs medium [14]. This cortical tissue was passed through a 150-mesh stainless steel sieve (105 pm pore size) held taut in a metal holder. In general the tissue from 20 kidneys was gently pressed through an area of approx. 175 cm2 and then 50 ml Krebs medium was washed through the sieve. The material emerging through the sieve was allowed to
Basement-Membrane Biosynthesis
sediment under unit gravity for 10 min in two 35-ml polycarbonate centrifuge tubes. The upper threequarters of the supernatant was removed and the sediment resuspended in 20 ml fresh medium and allowed to settle for a further 10 min. The sediment was resuspended in Krebs medium (10 ml) containing lo"/, (v/v) foetal calf serum and made 127; (wp) in Ficoll, then layered on a discontinuous Ficoll gradient (Fig. 1) comprising 6 ml of each of the following concentrations: 20%, 18% and 15% (w/v). All the above procedures were carried out at 4 "C. After centrifugation at 300 x g for 2.5 min the glomeruli banded at the interface of the 15 18 "/, layers and tubules and large aggregates penetrated to the interface of the 18 %: 20 layers (Fig. 1). The glomerular fraction was diluted with an excess of Krebs medium and centrifuged at 3000 x g for 5 min. In most experiments the glomeruli were resuspended in Eagle's medium and an aliquot (0.02 ml) was taken for examination and counting under the light microscope.
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