Solubilization and characterization of a ouabain-sensitive protein from transverse tubule membranejunctional sarcoplasmic reticulum complexes (TTM-JSR) in cat cardiac muscle

Abstract

A new glycoprotein of 31,500 dalton, which has a high affinity for ouabain, and is independent of (Na+-K+)-ATPase, was solubilized from transverse tubule membrane and junctional sarcoplasmic reticulum complexes (TTM-JSR) of cat cardiac muscle. This protein could be extracted only in small amounts from sarcolemma-plasma membrane (SLM-PL) fragments, suggesting that it indeed originates from the TTM-JSR.

The Inhibition of Fibroblast Growth Factor-2 Export by Cardenolides Implies a Novel Function for the Catalytic Subunit of Na+,K+-ATPase

“…Indeed, Blanco et al (32) have shown that the ␣-subunit itself can organize into stable oligomeric structures that localize on the cell surface but that have no known function. In addition, there have also been reports describing a putative ␥-subunit of Na ϩ ,K ϩ -ATPase (31, 33) and another ouabain receptor protein (29,30) that could be involved in formation of the PMTA mediating FGF-2 protein export. Structural, functional, and/or regulatory similarities may exist between the PMTA mediating protein export and the translocon mediating protein translocation through the ER (34).…”

An Immunohistochemical Study of the Significance of a New 31.5-kD Ouabain Receptor Protein Isolated from Cat Cardiac Muscle

“…Isolation of TTM-JSR complexes and solubilization and purification of NORP Isolation of TTM-JSR complexes and solubilization and purification of NORP, the 31.5-kD protein, were performed according to the previously described methods (15,37) except for the additional use of wheat germ agglutinin (WGA) affinity chromatography to remove all the dihydropyridine (DHP) receptor protein and its subunits. WGA affinity chromatography was done according to Curtis and Catterall (38).…”

“…The 31.5-kD protein was purified as follows: A band with molecular weight of 31.5 kD (a in B-2 of Fig. 1) was cut out of the gel, and this slice was crushed in elution buffer (pH 5.3) comprising 0.1 % SDS, 0.1 mM EDTA, 5 mM dithiothreitol, 150 mM NaCI and buffering reagents (100 mM citric acid and 200 mM Na2HPO4) (15).…”

“…They demonstrated that glycosides bind to a high affinity site on the Na+-K+ ATPase, but the detailed mechanism is still a matter of speculation (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14). However, we recently solubilized a novel 31.5-kD ouabain receptor protein (NORP) from cardiac muscle that is independent of the Na+-K+ ATPase of cardiac muscles (15). This protein is extractable almost selectively from the transverse tubule membrane-junctional sarcoplasmic reticulum (TTM-JSR) system, which is regarded as a regulator of mechanical contraction or force-generation in muscle cells (16)(17)(18)(19); and as shown by 3H-ouabain photolabeling, it is probably responsible for ouabain potentiation (15).…”

“…However, we recently solubilized a novel 31.5-kD ouabain receptor protein (NORP) from cardiac muscle that is independent of the Na+-K+ ATPase of cardiac muscles (15). This protein is extractable almost selectively from the transverse tubule membrane-junctional sarcoplasmic reticulum (TTM-JSR) system, which is regarded as a regulator of mechanical contraction or force-generation in muscle cells (16)(17)(18)(19); and as shown by 3H-ouabain photolabeling, it is probably responsible for ouabain potentiation (15). Although the mechanism of coupling of the electrical events (E) at the TTM with the intracellular Ca-release indispensable for contraction (C) is still not sufficiently understood (17,18,(20)(21)(22)(23)(24)(25), our previous studies (26)(27)(28)(29)(30)(31)(32)(33)(34)(35)(36) have suggested that ouabain potentiation is produced by the action of ouabain on the E-C coupling process in the cardiac muscle.…”