A novel muscle protein located inside the terminal cisternae of the sarcoplasmic reticulum.
An immunofluorescence study of adult rat muscle tissues with a polyclonal antibody against the RGD-directed fibronectin receptor of Friend's erythroleukemia cells (alpha5beta1-integrin) unexpectedly revealed a pattern of intracellular antigen distribution. Western blotting analysis of rat and rabbit membrane fractions indicated that the antibody recognizes a 167-kDa protein expressed both in heart and in skeletal muscle (relative abundance: heart > slow muscle > fast muscle), but not in liver and kidney. The 167-kDa protein did not show altered electrophoretic mobility upon reduction and failed to bind several lectins, including wheat germ agglutinin. A study of its subcellular distribution in rabbit skeletal muscle revealed that the 167-kDa protein is mostly associated with the terminal cisternae of the sarcoplasmic reticulum (SR) and, to a smaller extent, with the sarcolemma, while it is absent in the longitudinal tubules of the SR. The 167-kDa protein is not an integral membrane protein since it can be extracted at pH >/=10. This protein can be proteolytically cleaved only in the presence of detergent, indicating that it resides on the luminal side of the SR. The 167-kDa protein could be resolved from the closely spaced sarcalumenin and histidine-rich protein by column chromatography followed by detergent dialysis and two-dimensional gel electrophoresis. The N terminus and the internal sequences did not match any known sequence in protein and DNA data bases, indicating that the 167-kDa protein is a novel muscle protein selectively localized to the SR. Integrins from rat kidney fibroblasts were not recognized by either (i) a polyclonal antiserum against the purified 167-kDa protein or (ii) the anti-alpha5beta1-integrin antiserum after affinity purification onto the 167-kDa protein. These data indicate that the 167-kDa protein is not immunologically cross-reactive with integrins, despite its reaction with a polyclonal anti-integrin antibody.
Presotto
C
C
Department of Biomedical Sciences, University of Padova Medical School, Padova, Italy.
Agnolucci
L
L
Biral
D
D
Dainese
P
P
Bernardi
P
P
Salviati
G
G
D000818:Animals; D003902:Detergents; D015180:Electrophoresis, Gel, Two-Dimensional; D006863:Hydrogen-Ion Concentration; D016023:Integrins / Q000737:chemistry / Q000302:isolation & purification*; D008297:Male; D008970:Molecular Weight; D009124:Muscle Proteins / Q000302:isolation & purification*; D011817:Rabbits; D051381:Rats; D017208:Rats, Wistar; D012519:Sarcoplasmic Reticulum / Q000737:chemistry*; D014357:Trypsin
D003902:Detergents; D016023:Integrins; D009124:Muscle Proteins; D014357:Trypsin