Comprehensive analysis of collagen metabolism in vitro using (4(/sup 3/H))/(/sup 14/C)proline dual-labeling and polyacrylamide gel electrophoresis
A method to simultaneously quantify the production, secretion, and prolyl hydroxylation of individual types of collagen in cell culture samples has been developed. Collagens were biosynthetically labeled with a mixture of (/sup 14/C)proline and (4-/sup 3/H)proline. The labeled collagens were isolated and their component alpha-chains were resolved by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. Migration of the collagen alpha-chains was determined by fluorography, and radioactivity in excised bands was quantified by scintillation counting. (/sup 14/C)Proline labeling of collagen chains was used to determine the production and secretion of the different types of collagen. The ratios of the component alpha 1(I) and alpha 2(I) chains of type I collagen were also determined in this way. Prolyl hydroxylation of collagen alpha-chains was readily determined by measurement of their /sup 3/H:/sup 14/C ratios. Following 4-hydroxylation, /sup 3/H was lost from the (4-3H)proline with alteration of this ratio. This dual-labeling method is suitable for the comprehensive analysis of collagen metabolism in multiple samples.
- Research Organization:
- Univ. of Melbourne, Victoria (Australia)
- OSTI ID:
- 7154308
- Journal Information:
- Anal. Biochem.; (United States), Vol. 168:1
- Country of Publication:
- United States
- Language:
- English
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Related Subjects
CARBON 14 COMPOUNDS
ISOTOPE RATIO
COLLAGEN
METABOLISM
TRITIUM COMPOUNDS
CELL CULTURES
DOUBLE LABELLING
ELECTROPHORESIS
FIBROBLASTS
HYDROXYLATION
IN VITRO
METHYLATION
PROLINE
SCINTILLATION COUNTING
TRACER TECHNIQUES
AMINES
AMINO ACIDS
ANIMAL CELLS
AZOLES
CARBOXYLIC ACIDS
CHEMICAL REACTIONS
CONNECTIVE TISSUE CELLS
COUNTING TECHNIQUES
HETEROCYCLIC ACIDS
HETEROCYCLIC COMPOUNDS
ISOTOPE APPLICATIONS
LABELLED COMPOUNDS
LABELLING
ORGANIC ACIDS
ORGANIC COMPOUNDS
ORGANIC NITROGEN COMPOUNDS
PROTEINS
PYRROLES
PYRROLIDINES
SCLEROPROTEINS
SOMATIC CELLS
550501* - Metabolism- Tracer Techniques