Three distinct molecular species of proteoglycan synthesized by the rat limb bud at the prechondrogenic stage.
To characterize proteoglycans in the prechondrogenic limb bud, proteoglycans were extracted with 4 M guanidine HCl containing a detergent and protease inhibitors from Day 13 fetal rat limb buds which had been labeled with [35S]sulfate for 3 h in vitro. About 90% of 35S-labeled proteoglycans was solubilized under the conditions used. The proteoglycan preparation was separated by DEAE-Sephacel column chromatography into three peaks; peak I eluted at 0.45 M NaCl concentration, peak II at 0.52 M, and peak III at 1.4 M. Peaks I and III were identified as proteoglycans bearing heparan sulfate side chains. The heparan sulfate proteoglycan in peak III was larger in hydrodynamic size than the proteoglycan in peak I. The heparan sulfate side chains of peak III proteoglycan were smaller in the size and more abundant in N-sulfated glucosamine than those of peak I proteoglycan. Peak II contained a chondroitin sulfate proteoglycan with a core protein of a doublet of Mr 550,000 and 500,000. The chondroitin sulfate proteoglycan was easily solubilized with a physiological salt solution and the heparan sulfate proteoglycan in peak I was partially solubilized with the physiological salt solution. The remainder of the proteoglycan in peak I and the heparan sulfate proteoglycan in peak III could be solubilized effectively only with a solution containing a detergent, such as nonanoyl-N-methylglucamide. This observation indicates the difference in the localization among these three proteoglycans in the developing rat limb bud.
Matsui
F
F
Department of Embryology, Central Hospital, Aichi, Japan.
Oohira
A
A
Shoji
R
R
Nogami
H
H
D000596:Amino Acids / Q000378:metabolism; D000818:Animals; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D015180:Electrophoresis, Gel, Two-Dimensional; D005121:Extremities / Q000196:embryology; D008970:Molecular Weight; D010012:Osteogenesis; D011509:Proteoglycans / Q000096:biosynthesis* / Q000302:isolation & purification; D051381:Rats; D011919:Rats, Inbred Strains; D013431:Sulfates / Q000378:metabolism; D013462:Sulfur Radioisotopes
D000596:Amino Acids; D011509:Proteoglycans; D013431:Sulfates; D013462:Sulfur Radioisotopes