Activation and inhibition of phosphorylase kinase by monospecific antibodies against preparatively isolated alpha, beta and gamma subunits
Open Access
- 28 June 2008
- journal article
- research article
- Published by Wiley in European Journal of Biochemistry
- Vol. 147 (3) , 619-630
- https://doi.org/10.1111/j.0014-2956.1985.00619.x
Abstract
Homogenous .alpha. and .beta. subunits were isolated for the first time in preparative amounts in the presence of sodium dodecyl sulfate. Analysis by analytical polyacrylamide electrophoresis, sedimentation velocity and immunoprecipitation with monospecific antibodies indicated homogeneity. The apparent molecular masses of the purified subunits as determined electrophoretically in the presence of dodecyl sulfate are: .alpha. = 140.2 .+-. 2.1 kDa [kilodaltons] and .beta. = 123 .+-. 1.8 kDa. Amino acid analyses show that per 100 mol amino acid the .alpha.-subunit has a higher serine content (Ser.alpha./Ser.beta. = 1.32, Ser.alpha./Ser.gamma. = 1.42) and a lower aspartic acid/asparagine (Asx) content (Asx.alpha./Asx.beta. = 0.76, Asx.alpha./Asx.gamma. = 0.90) than the .beta. and .gamma. subunits. Monospecific antibodies against the purified .alpha., .beta. and .gamma. subunits were produced in sheep and their action on the catalytic activity of non-activated phosphorylase kinase assayed. Certain antibody fractions of anti-.alpha., anti-.beta. and anti-.gamma. inhibit the Ca2+-dependent and Ca2+-independent activity at pH 6.8 as well as at pH 8.2. Other antibody fractions against the .beta. and .gamma. subunits, however, activate the Ca2+-dependent activity at pH 6.8 3-fold to 4-fold, although they inhibit the activity at pH 8.2. These antibodies lead to an .apprx. 5-fold increase in the pH 6.8/8.2 activity ration. Activating anti-.beta. can even overcome the inhibitory action of anti-.alpha. at pH 6.8. A kinetic analysis shows that inhibition is the result of a mixed type mechanism whereas activation is due to a 5-fold to 10-fold increase in Vmax for phosphorylase b. The results illustrate the importance of possibly large, concerted conformational changes of phosphorylase kinase. It appears that activation or inhibition can be triggered by the antibody binding to conformational determinants of a single subunit type leading to a structural alteration of the holoenzyme.This publication has 50 references indexed in Scilit:
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