Relevance of feedback inhibition applied to the biosynthesis of hexosamines.
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Biomedical subjects
Publications and source records attributed to C F Phelps.
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The acid glycosaminoglycans were extracted from the skins of young rats less than 1 day post partum. The isolated products were fractionated by a cetylpyridinium chloride-cellulose column technique and identified by chemical analysis, electrophoretic mobility and susceptibility to testicular hyaluronidase digestion. Hyaluronic acid (56%) dermatan sulphate (15.6%) and chondroitin 6-sulphate (9.1%) were the major components, but chondroitin 4-sulphate, heparan sulphate and heparin were also present, together with two further fractions tentatively suggested to be a heparan sulphate-like fraction and a dermatan sulphate fraction, both of short chain length or low degree of sulphation.
1. The incorporation of [(35)S]sulphate in vivo into the acid-soluble intermediates extracted from young rat skin showed three sulphated hexosamine-containing components. 2. The rates of synthesis of these components were determined in vivo by measuring the incorporation of radioactivity from [U-(14)C]glucose into their isolated hexosamine moieties. 3. The incorporation of radioactivity from [U-(14)C]glucose into the isolated hexosamine and uronic acid moieties of the acid glycosaminoglycans was also measured. These results, combined with those obtained on the intermediary pathways of hexosamine and uronic acid biosynthesis previously determined in this tissue, indicated that the acid-soluble sulphated hexosamine-containing components were not precursors of the sulphated hexosamine found in the acid glycosaminoglycans. 4. The rates of synthesis of the acid glycosaminoglycan fractions were calculated from the incorporation of radioactivity from [U-(14)C]glucose into the hexosamine moiety. The sulphated components containing principally dermatan sulphate, chondroitin 6-sulphate and in smaller amounts, chondroitin 4-sulphate, heparan sulphate and heparin appeared to be turning over about twice as rapidly as hyaluronic acid and about four times as rapidly as the small keratan sulphate fraction. The relative rates of synthesis of the sulphated glycosaminoglycans were calculated from the incorporation of [(35)S]sulphate and were in agreement with those from (14)C-labelling studies.
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1. The tissue contents of hexose monophosphate, N-acetylglucosamine 6-phosphate, UDP-glucose, UDP-galactose, UDP-N-acetylglucosamine, UDP-N-acetylgalactosamine and UDP-glucuronic acid were determined in the skin of young rats less than 1 day post partum. Tissue-space determinations were used to calculate their average cellular concentrations. 2. The incorporation of [U-(14)C]-glucose into the intermediates was recorded with time and their rates of turnover were calculated. The results demonstrated product-precursor relationships along the pathway of hexosamine synthesis and that of hexuronic acid synthesis. The rates of synthesis of UDP-N-acetylhexosamine and UDP-glucuronic acid were 1.5+/-0.3 and 0.24+/-0.03mmumoles/min./g. of tissue respectively. These results indicated the average turnover time of the total tissue glycosaminoglycans to be about 5 days.
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1. Protein-polysaccharide complexes were prepared in three different ways and the gross stoicheiometry of the complexes compared. 2. The neutral sugar content was ascertained and the possibility of a glycoprotein occurring with chondroitin sulphate and keratosulphate is discussed. 3. Physical data support a molecule of molecular weight 3.2x10(6)-5.8x10(6) with a roughly spherical domain and an average radius of gyration of 1390A. Such a particle is highly solvated. The complex is heavily charged with the sulphate groups on the outside. 4. These findings are discussed in the light of the physiological role of protein-polysaccharide light fraction (PPL) in cartilage.
1. Fragments from enzymic degradation of protein-polysaccharide light fraction (PPL) have been analysed. 2. The time-course of action of some proteolytic enzymes and of hyaluronidase on PPL has been followed by viscometric techniques. 3. It is suggested that papain acts to produce single polysaccharide chains, whereas other proteolytic enzymes tried give evidence of twin-chain residues. 4. The molecular weight of the fragments derived from complete enzyme action on PPL supports this postulate. 5. A structure of the PPL complex is suggested.
1. A non-recirculatory perfusion system has been developed suitable for the perfusion of the hind limbs of small experimental animals.2. By means of it a solution of T. 1824-labelled serum albumin has been introduced into the vascular compartment of the hind limbs of female rats under isogravimetric conditions. Excision and analysis of certain muscles has been used to provide information concerning the percentage distribution of the labelled albumin within these muscles.3. Experiments have been carried out in vivo employing [(131)I]labelled serum albumin and [(51)Cr]labelled erythrocytes in order to compare the vascular volumes determined under in vivo conditions and in perfusions, and to estimate the capillary haematocrit in vivo.4. The physiological validity of the methods used and the results obtained has been discussed.
1. The sucrose, raffinose and inulin spaces within the gastrocnemius of the female rat have been estimated at 12.0 +/- 2.2%, 10.7 +/- 0.6%, and 10.0 +/- 0.3% respectively, expressed as a percentage of the total muscle volume.2. The mucopolysaccharide and hexosamine contents of the gastrocnemius have been found to be inversely dependent upon the weight of the muscle.3. The possibility that a relation exists between the saccharide spaces and the muscle musopolysaccharide content has been discussed in terms of saccharide molecular radii.