The (αF357C)3(βR372C)3γ Subcomplex of the F1-ATPase from the ThermophilicBacillusPS3 Has Altered ATPase Activity after Cross-Linking α and β Subunits at Noncatalytic Site Interfaces†

The Mg(2+)-ATPase present in rabbit skeletal-muscle transverse tubules is an integral membrane enzyme which has been solubilized and purified previously in this laboratory [Kirley (1988) J. Biol. Chem. 263, 12682-12689]. The present study indicates that, in addition to the approx. 100 kDa protein (distinct from the sarcoplasmic-reticulum Ca(2+)-ATPase) seen previously to co-purify with the Mg(2+)-ATPase activity, there are also proteins having molecular masses of 160, 70 and 43 kDa. The 70 and 43 kDa glycosylated proteins (50 and 31 kDa after deglycosylation) are difficult to detect by SDS/PAGE before deglycosylation, owing to the broadness of the bands. Additional purification procedures, cross-linking studies and chemical and enzymic deglycosylation studies were undertaken to determine the structure and relationship of these proteins. Both the 97 and 160 kDa proteins were demonstrated to be N-glycosylated at multiple sites, the 97 kDa protein being reduced to a peptide core of 84 kDa and the 160 kDa protein to a peptide core of 131 kDa after deglycosylation. Although the Mg(2+)-ATPase activity is resistant to a number of chemical modification reagents, cross-linking inactivates the enzyme at low concentrations. This inactivation is accompanied by cross-linking of two 97 kDa molecules to one another, suggesting that the 97 kDa protein is involved in ATP hydrolysis. The existence of several proteins along with the inhibition of ATPase activity by cross-linking is consistent with the interpretation of the susceptibility of this enzyme to inactivation by most detergents as being due to the disruption of a protein complex of associated subunits by the inactivating detergents. The 160 kDa glycoprotein can be partially resolved from the Mg(2+)-ATPase activity, and is identified by its N-terminal amino acid sequence as angiotensin-converting enzyme.

Download Full-text

Structure of beef heart mitochondrial F1-ATPase Arrangement of subunits as disclosed by cross-linking reagents and selective labeling by radioactive ligands

Biochimica et Biophysica Acta (BBA) - Protein Structure ◽

10.1016/0005-2795(76)90255-5 ◽

1976 ◽

Vol 453 (1) ◽

pp. 111-120 ◽

Cited By ~ 26

Author(s):

M. Satre ◽

G. Klein ◽

P.V. Vignais

Keyword(s):

Cross Linking ◽

Beef Heart ◽

Selective Labeling ◽

F1 Atpase

Download Full-text

Dissociation of clathrin coats coupled to the hydrolysis of ATP: role of an uncoating ATPase.

The Journal of Cell Biology ◽

10.1083/jcb.99.2.734 ◽

1984 ◽

Vol 99 (2) ◽

pp. 734-741 ◽

Cited By ~ 104

Author(s):

W A Braell ◽

D M Schlossman ◽

S L Schmid ◽

J E Rothman

Keyword(s):

Atpase Activity ◽

Atp Hydrolysis ◽

Low Ph ◽

Coated Vesicles ◽

Magnesium Concentration ◽

Cross Linking ◽

Dependent Atpase ◽

Event Triggering ◽

Hydrolysis Of

ATP hydrolysis was used to power the enzymatic release of clathrin from coated vesicles. The 70,000-mol-wt protein, purified on the basis of its ATP-dependent ability to disassemble clathrin cages, was found to possess a clathrin-dependent ATPase activity. Hydrolysis was specific for ATP; neither dATP nor other ribonucleotide triphosphates would either substitute for ATP or inhibit the hydrolysis of ATP in the presence of clathrin cages. The ATPase activity is elicited by clathrin in the form of assembled cages, but not by clathrin trimers, the product of cage disassembly. The 70,000-mol-wt polypeptide, but not clathrin, was labeled by ATP in photochemical cross-linking, indicating that the hydrolytic site for ATP resides on the uncoating protein. Conditions of low pH or high magnesium concentration uncouple ATP hydrolysis from clathrin release, as ATP is hydrolyzed although essentially no clathrin is released. This suggests that the recognition event triggering clathrin-dependent ATP hydrolysis occurs in the absence of clathrin release, and presumably precedes such release.

Download Full-text