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IN VITRO SYNTHESIS OF PROTEOGLYCANS OF MEDULLARY BONE IN ESTROGEN-STIMULATED MALE QUAIL

Author
HUNTER, SUSAN JULIA
Physical Description
151 pages
Additional Creators
Pennsylvania State University
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Summary
A short-term incubation system was developed for the study of the early stages of medullary bone formation in estrogen-treated male Japanese quail. Initial experiments with ('3)H-thymidine uptake were inconclusive, however; the work showed that the synthesis of glycosamino-glycans (GAGs) and proteoglycans associated with medullary bone occurred.
Quail were given estradiol-17(beta) (5 mg/kg) or sesame oil carrier and killed 17, 22, 27, and 34 hours later. Excised femoral shafts were reamed with a stainless-steel probe to remove some of the marrow and then incubated in Joklik-modified Minimal Essential Medium containing 0.1% bovine serum albumin to remove more marrow. Shaft fragments were then incubated in BGJ(,b) with 0.1% bovine serum albumin supplemented with 10% fetal calf serum. Two hours after incubation began, H(,2)('35)SO(,4) (60 (mu)Ci/ml) or Na(,2)('35)SO(,4) (60 (mu)Ci/ml) was added to each vial. Glycosamino-glycans were extracted by a 24-hour papain digestion and chromatographed initially on a DEAE BioGel A column eluted with a 400-ml linear 0.05 M Tris-HCl-NaCl gradient (0-1 M NaCl). Fractions were monitored for radioactivity.
Elution profiles of material from birds treated with estrogen for 34 hours followed by incubation for 3 hours showed two distinct peaks. Material from carrier-injected birds showed only a single peak corresponding to the second peak in the estrogenized sample. The onset of synthesis of GAG peak I was determined by a time-course experiment. GAG peak I was present after 20 hours of estrogen treatment and increased dramatically between 25 and 30 hours after estrogenization.
The material eluted from the DEAE BioGel A column was characterized by treatment with various agents and chromatographed on a Sepharose CL-6B column using a buffer of 4 M guanidine-HCl, 0.05 M sodium acetate and 0.02% sodium azide, pH 7.0. GAG peak I was identified as keratan sulfate (M(,r) (TURNEQ) 7800) by virtue of its degradation by keratan sulfate (beta)-endogalactosidase and its resistance to chondroitinase ABC and nitrous acid. GAG peak II was identified as chondroitin sulfate (M(,r) (TURNEQ) 27,000) primarily. Identification was based on its susceptibility to chondroitinase ABC and resistance to keratinase. GAG peak II may also contain some heparin or heparan sulfate since 12% of the total cpm was degraded by nitrous acid and 14% of the total cpm was resistant to chondroitinase ABC.
Intact proteoglycans were extracted from the bone fragments and incubation medium with 4 M guanidine-HCl containing protease inhibitors and chromatographed on a DEAE BioGel A column. The elution profile of material from birds treated with estrogen showed two peaks while that from control samples had only one peak corresponding to the second peak in the estrogenized sample.
Proteoglycan peaks 1 and 2 from bone samples of estrogenized birds were eluted from the DEAE BioGel A column and chromatographed on Sepharose CL-4B using a buffer of 0.5 M guanidine-HCl, 0.5 M sodium acetate and 0.02% sodium azide, pH 7.0. Peak 1 had a M(,r) (TURNEQ) 40,000 - 70,000, and peak 2 had a M(,r) > 150,000.
Proteoglycans from the incubation medium of estrogenized samples were characterized by treatment with various agents and chromatography on Sepharose CL-4B using the buffer mentioned above. The molecular weight of this material was found to be less than that of the bone proteoglycans. This may be due to protease activity in the field calf serum added to the medium. Peak 1 proteoglycan was found to consist of keratan sulfate glycosaminoglycan, and peak 2 consisted predominantly of chondroitin sulfate. Peak 1 proteoglycan had a buoyant density of 1.5 g/ml.
Other Subject(s)
Dissertation Note
Ph.D. The Pennsylvania State University 1980.
Note
Source: Dissertation Abstracts International, Volume: 41-10, Section: B, page: 3658.
Part Of
Dissertation Abstracts International
41-10B
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