Abstract
A 53,000-dalton intrinsic glycoprotein of the sarcoplasmic reticulum was separated from the Ca2+ + Mg2+-ATPase by dissolution with low concentrations of deoxycholate in the presence of 1 M KCl and purified in two successive gel filtration steps. It was aggregated and eluted at the void volume when subjected to gel filtration in the presence or absence of deoxycholate. When subsequently chromatographed in the presence of sodium dodecyl sulfate, the glycoprotein eluted in pure form as a monomer. The glycoprotein contained 48% nonpolar amino acids. It also contained 4 mol of glucosamine and 18 mol of mannose per mol of protein, suggesting that it contained two chains of (GlcNAc)2, (Man)9. The 53,000-dalton glycoprotein was completely removed from deoxycholate extracts of sarcoplasmic reticulum by affinity chromatography on concanavalin A Sepharose. Elution of glycoproteins with alpha-methyl-D-mannoside and deoxycholate resulted in co-purification of the 53,000-dalton glycoprotein and 160,000-dalton glycoprotein previously observed in sarcoplasmic reticulum. The apparent molecular weight of the glycoprotein was reduced from 53,000 to 49,000 after digestion with endo-beta-N-acetylglucosaminidase H (Endo H) and its reactivity with concanavalin A (Con A) was lost. There was no change in molecular weight of the glycoprotein and no diminution of its reactivity with Con A when sealed vesicles of sarcoplasmic reticulum were treated with Endo H. Endo H reduced the molecular weight and the Con A reactivity of the protein when the vesicles were made permeable by detergents. These observations, together with our previous demonstration that the glycoprotein reacts with a cycloheptaamylose-fluorescamine complex in sealed vesicles (Michalak, M., Campbell, K. P., and MacLennan, D. H. (1980) J. Biol. Chem. 255, 1317-1326), show that the glycoprotein is a transmembrane protein. A protein of approximately 53,000 daltons was labeled when the sarcoplasmic reticulum was reacted with the photoaffinity label [32P]8-N3-cAMP. The labeled protein was neither the glycoprotein nor the high affinity calcium-binding protein since it was not sensitive to Endo H and was sensitive to trypsin digestion.
Highlights
ATPase by dissolution with low concentrations of de- tubular system of skeletal muscle (1).We have found four oxycholate in the presence of l M KC1 and purified in glycoproteins of M, 160,000, 63,000, 60,OOO, and 53,000 in the two successive gel filtration steps
It was aggregated sarcoplasmic reticulum, and we showed that a 190,000-dalton and eluted at the void volume when subjected to gel glycoprotein was concentrated in fractions enriched in the filtration in the presence or absence of deoxycholate
There exposed on the cytoplasmic surface of the sarcoplasmic reticwas no change in molecular weight of the glycoprotein ulum (I),but thefact that thesarcoplasmicreticulum has the and no diminution of its reactivity with concanavalin A (Con A) when same orientation as therough endoplasmic reticulum implies sealed vesicles of sarcoplasmic reticulum were treated that thecarbohydrate moieties should lie in the lumen of the with Endo H
Summary
Gel Electrophoresis-SDS-polyacrylamide slab gels were run acenter the 25% sucrose by centrifugation at 78,000 X g for 2 h and cording to themethod of Laemmli (16) or of Weber and Osborn (17). FKst peak were concentrated by ultrafdtration using an XM 50 mem- Amino Acid Analysis-Amino acid compositions were determined brane to approximately 5 ml and dialyzed overnight against 4 liters of after hydrolysis of the proteins in 6 N HCI for 24,48, and 72 h in. Purification of Glycoprotein using Concanavalin A Affinity Col- mined by amino acid analysis followinghydrolysis in 6 N HCI at llO°C umns-Con A Sepharose 4B was either washed with 0.5 M n-methyl- for 24 h. The deoxycholate extract of sarcoplasmic reticulum vesicles or the void peak from the Bio-Gel A-5m column was applied directly (without concentration) to a column (1.5 x 5 cm) of Con A Sepharose 4B analysis revealed that 4 N HC1 hydrolysates of the trichloroacetic acid-washed preparations of glycoprotein were free of glucose but contained normal amounts of mannose. The protein eluted with a-methyl-D-mannoside was concentrated using an XM 50 membrane andthen dialyzed
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