Abstract
The primary amino acid sequence for a highly abundant junctional sarcoplasmic reticulum glycoprotein (triadin) has been deduced from the cDNA sequence. Based on both biochemical analysis and the predicted amino acid sequence we suggest that this protein is an intrinsic membrane glycoprotein containing a single transmembrane domain that separates the protein into cytoplasmic and luminal domains. The cytoplasmic domain is proposed to contain the amino-terminal 47 amino acids. The remainder of the protein including the carboxyl terminus is proposed to be found within the lumen of the sarcoplasmic reticulum and contains an extremely high concentration of basic residues. Protease analysis of intact triads was consistent with the topological predictions. Western and Northern blots suggest that the protein is specifically expressed in skeletal muscle and not cardiac muscle or brain. The abundance and localization of this protein suggest that it plays an important regulatory or structural role in excitation-contraction coupling in skeletal muscle.
Highlights
The primary amino acid sequence for a highly abupnat-tern of migration on SDS-PAGE’ when run in the absence dant junctional sarcoplasmic reticulum glycoprotein of reducing agents
The tissue distribution of the 94-kDa glycoprotein was assessed using both Western and Northernblots, which showed that theprotein is expressed in skeletal muscle but notcardiac muscle or brain
These results suggest that the94kDa glycoprotein performs an important function in calcium regulation at the triajudnction
Summary
94-kDa Glycoprotein performed using nonfat dry milk as a blocking agent as described phase HPLC using an Applied Biosystems model 130AHPLC system previously (Leung et al, 1987). Chromatography was a-Chymotrypsin Digestion of Rabbit Skeletal Triads-Rabbit skel- performed initially in 0.1% trifluoroacetic acid at 0.05 ml/min, and etal muscle triads were treated with a 1:160 or 1:20 ratio of a- individual peaks were repurified on the same column in 0.1% amchymotrypsin to protein inthe presence or absence of 0.25% CHAPS monium acetate. In both cases, elution was performed with a gradient for 15 min at 37 “C. Methylsulfonyl fluoride and 3% SDS in Laemmli sample buffer and Materials -Isopropyl-1-thio-P-D-galactopyranoside)was from analyzed by 3-12% SDS-PAGE. Used as a probe to isolate two clones (p94kl and p94k3) from an oligo(dT)-primed cDNA expression library constructed inX g t l l from
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