The release of glycoconjugates by rat parathyroid glands in tissue culture. II. Studies on uptake and incorporation of isotopically labeled glucosamine and characterization of the secreted material.

Abstract 1. 1. The uptake and incorporation of [3H] glucosamine into rat parathyroid glands were examined in organ culture. Both uptake and incorporation were stimulated by a low Ca2+ concentration (0.75 mM) and inhibited by a high Ca2+ concentration (2.25 mM). 2. 2. Uptake of [3H]glucosamine into the trichloroacetic acid-soluble pool was rapid, and the majority of the radioactivity was present as glucosamine 6-phosphate, N-acetylglucosamine or UDP-N-acetylglucosamine. Addition of actinomycin D and cycloheximide did not inhibit this uptake process. 3. 3. There was incresing incorporation of labeled glucosamine into trichloroacetic acid-insoluble material with time. Only small amounts of label were lipid soluble. When the trichloroacetic acid-precipitate of the gland was solubilized with dodecysulphate, it eluted with the void volume of Sephadex G-100, but after treatment with trypsin, it was degraded to material which eluted just before the salt volume. Brief treatment of intact parathyroid glands which had been incubated with [3H]glucosamine with trypsin released a peak migrating on Sephadex G-100 similar to that of the smallest peak secreted by the glands during culture. 4. 4. The medium from cultures of parathyroid glands incubated with [3H] glucosamine showed labeled macromolecular material in three peaks. The largest, Peak 1, was released continuously throughout 10 days of culture. The two smaller peaks, Peak 2, which migrated in the region of ovalbumin, and Peak 3, which migrated in the region of parathyroid hormone, were released in increasing amounts for 4 days in a low-Ca2+ medium. High Ca2+ inhibited their release. Bioassay showed that the maximum parathyroid hormone activity was secreted at 4 days in these cultures. 5. 5. Rat liver, thyroid or salivary gland culture in the presence of [3H]glucosamine released labeled macromolecular material which migrated in the void volume and with serum albumin, but no Peak 3 material. 6. 6. Further incubation of Peaks 1 or 2 with serum or culture medium and dual-labeling studies failed to demonstrate any precursor product relation between Peak 3 and Peaks 1 or 2. 7. 7. Peaks 1, 2, and 3 were all nearly insoluble in lipid solvents. Peaks 1 and 2 were easily digested by trypsin and pronase, and Peak 3 was partially digested by papain and contained labeled serine as well as glucosamine. 8. 8. On gel filtration, Peak 1 was heterogeneous having a molecular weight range of 100 000 to 150 000 while Peak 2 was more homogeneous, with a molecular weight of 45 000 to 46 000 daltons. On DEAE-cellulose or CM-cellulose Peaks 1 and 2 were heterogeneous consisting of basic and acidic as well as neutral moieties. 9. 9. Peak 3 was homogeneous on Sephadex G-100 and P-30 gel chromatography and by scurose gradient ultracentrifugation. Its calculated molecular weight was 6200, while labeled rat parathyroid hormone had an average weight of 7500. 10. 10. Peak 3 was eluted from DEAE-cellulose with 0.02 M phosphate before hyaluronate or chondroitin sulfate, Peak 3 was not digested by testicular hyaluronidase or bacterial chondroitinase nor precipitated by cetyl trimethyl ammonium bromide. It was not labeled when parathyroid glands were cultured with 3 5SO42− or [14C]glucuronate. 11. 11. Isotopically labeled fucose, mannose, and galactosamine, as well as glucosamine, were incorporated into Peak 3, and analysis of the hydrolysis products revealed multiple labeled sugars.