Effect of monoclonal antibodies raised against Ca2+,Mg2+-ATPase from rabbit skeletal muscle sarcoplasmic reticulum on ATPase activity and its correlation with epitope location

1988 ◽  
Vol 16 (5) ◽  
pp. 771-772
Author(s):  
ANA M. MATA ◽  
JOHN COLYER ◽  
RICHARD E. A. TUNWELL ◽  
ANTHONY G. LEE ◽  
J. MALCOLM EAST
Keyword(s):  
Skeletal Muscle ◽  
Monoclonal Antibodies ◽  
Sarcoplasmic Reticulum ◽  
Atpase Activity ◽  
Rabbit Skeletal Muscle

scholarly journals Effects on ATPase activity of monoclonal antibodies raised against (Ca2+ + Mg2+)-ATPase from rabbit skeletal muscle sarcoplasmic reticulum and their correlation with epitope location

1989 ◽  
Vol 262 (2) ◽  
pp. 439-447 ◽  
Author(s):  
J Colyer ◽  
A M Mata ◽  
A G Lee ◽  
J M East
Keyword(s):  
Skeletal Muscle ◽  
Monoclonal Antibodies ◽  
Sarcoplasmic Reticulum ◽  
Epitope Mapping ◽  
Structural Models ◽  
Rabbit Skeletal Muscle ◽  
Protein Fragments ◽  
Binding Domains ◽  
Inhibitory Antibodies ◽  
Phosphorylation Domain

A total of 28 monoclonal antibodies have been raised against the (Ca2+ + Mg2+)-ATPase of rabbit skeletal muscle sarcoplasmic reticulum. Epitope mapping, using protein fragments generated by proteolysis, indicates that these antibodies include examples binding to at least four distinct epitopes on the A1 and B tryptic fragments of the ATPase. Competition data also show that the 28 antibodies are directed against at least five spatially distinct regions. Altogether, nine inhibitory antibodies were produced: six of these inhibitory antibodies mapped to the same spatial region, although they appear to bind to two distinct epitopes located within the hinge region and the nucleotide-binding domains of current structural models; one antibody bound to an epitope located within the phosphorylation domain and the stalk-transmembranous region designated M4S4 by Brandl, Green, Korczak & MacLennan [(1986) Cell 44, 597-607]. Two of the inhibitory antibodies recognized assembled epitopes exclusively and could not be mapped. Binding to four of the five identified spatial regions was without effect on activity. These data show that the inhibition of catalytic activity by monoclonal antibodies is achieved only by binding to defined regions of the ATPase and they may therefore provide useful probes of structure-function relationships.

scholarly journals The Mg2+-ATPase of rabbit skeletal-muscle transverse tubule is a highly glycosylated multiple-subunit enzyme

1991 ◽  
Vol 278 (2) ◽  
pp. 375-380 ◽  
Author(s):  
T L Kirley
Keyword(s):  
Skeletal Muscle ◽  
Atpase Activity ◽  
Atp Hydrolysis ◽  
Rabbit Skeletal Muscle ◽  
Cross Linking ◽  
Low Concentrations ◽  
Sds Page ◽  
Molecular Masses ◽  
Relationship Of ◽  
Peptide Core

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.

scholarly journals A model for the uptake and release of Ca2+ by sarcoplasmic reticulum

1987 ◽  
Vol 245 (3) ◽  
pp. 739-749 ◽  
Author(s):  
G W Gould ◽  
J M McWhirter ◽  
J M East ◽  
A G Lee
Keyword(s):  
Skeletal Muscle ◽  
Sarcoplasmic Reticulum ◽  
Atpase Activity ◽  
External Medium ◽  
Spontaneous Release ◽  
Calculated Concentration ◽  
Effects Of Ph ◽  
Hydrolysis Of ◽  
Uptake And Release

On addition of ATP to vesicles derived from the sarcoplasmic reticulum (SR) of skeletal muscle, Ca2+ is accumulated from the external medium. Following uptake, spontaneous release of Ca2+ occurs in the presence or in the absence of ATP. These processes of Ca2+ uptake and release were simulated by using the models derived for ATPase activity [Gould, East, Froud, McWhirter, Stefanova & Lee (1986) Biochem. J. 237, 217-227; Stefanova, Napier, East & Lee (1987) Biochem. J. 245, 723-730] and for Ca2+ release from passively loaded vesicles [McWhirter, Gould, East & Lee (1987) Biochem. J. 245, 713-722]. The simulations are consistent with measurements of the effects of pH, K+, Ca2+ and Mg2+ on uptake and release of Ca2+. The increase in maximal Ca2+ accumulation observed in the presence of maleate is explained in terms of complexing of Ca2+ and maleate within the SR. The calculated concentration of ADP generated by hydrolysis of ATP has a large effect on the simulations. The effects of an ATP-regenerating system on the measured Ca2+ uptake is explained in terms of both removal of ADP and precipitation of Ca3(PO4)2 within the vesicles. It is concluded that both the process of Ca2+ uptake and the process of Ca2+ release seen with SR vesicles can be interpreted quantitatively in terms solely of the properties of the Ca2+ + Mg2+-activated ATPase.

Sign in / Sign up

Export Citation Format

Share Document