Gamma-subunit of skeletal muscle phosphorylase kinase contains two noncontiguous domains that act in concert to bind calmodulin.

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Publication Type Journal Article
School or College College of Pharmacy; School of Medicine
Department Biomedical Informatics; Biochemistry; Pharmacology & Toxicology
Creator Blumenthal, Donald K.
Other Author Dasgupta, Maitrayee; Honeycutt, Tereas
Title Gamma-subunit of skeletal muscle phosphorylase kinase contains two noncontiguous domains that act in concert to bind calmodulin.
Date 1989-10-15
Description Phosphorylase kinase is a Ca2+-regulated, multisubunit enzyme that contains calmodulin as an integral subunit (termed the delta-subunit). Ca2+-dependent activity of the enzyme is thought to be regulated by direct interaction of the delta-subunit with the catalytic subunit (the gamma-subunit) in the holoenzyme complex. In order to systematically search for putative calmodulin (delta-subunit)-binding domain(s) in the gamma-subunit of phosphorylase kinase, a series of 18 overlapping peptides corresponding to the C terminus of the gamma-subunit was chemically synthesized using a tea bag method. The calmodulin-binding activity of each peptide was tested for its ability to inhibit Ca2+/calmodulin-dependent activation of myosin light chain kinase. Data were obtained indicating that two distinct regions in the gamma-subunit, one spanning residues 287-331 (termed domain-N) and the other residues 332-371 (domain-C), are capable of binding calmodulin with nanomolar affinity. Peptides from both of these two domains also inhibited calmodulin-dependent reactivation of denatured gamma-subunit. The interactions of peptides from both domain-N and domain-C with calmodulin were found to be Ca2+-dependent. Dixon plots obtained using mixtures of peptides from domain-N and domain-C indicate that these two domains can bind simultaneously to a single molecule of calmodulin. Multiple contacts between the gamma-subunit and calmodulin (delta-subunit), as indicated by our data, may help to explain why strongly denaturing conditions are required to dissociate these two subunits, whereas complexes of calmodulin with most other target enzymes can be readily dissociated by merely lowering Ca2+ to submicromolar concentrations. Comparison of the sequences of the two calmodulin-binding domains in the gamma-subunit of phosphorylase kinase with corresponding regions in troponin I indicates similarities that may have functional and evolutionary significance.
Type Text
Publisher American Society for Biochemistry and Molecular Biology (ASBMB)
Volume 264
Issue 29
First Page 17156
Last Page 17163
Subject Pharmacology; Metabolism; Enzymology
Subject MESH Binding Sites; Calcium; Calmodulin; Egtazic Acid; Enzyme Activation; Kinetics; Macromolecular Substances; Molecular Sequence Data; Muscles; Myosin-Light-Chain Kinase; Peptide Fragments; Phosphorylase Kinase; Protein Denaturation
Language eng
Bibliographic Citation Dasgupta M, Honeycutt T, Blumenthal DK. The gamma-subunit of skeletal muscle phosphorylase kinase contains two noncontiguous domains that act in concert to bind calmodulin. J Biol Chem. 1989 Oct 15;264(29):17156-63. Retrieved August 31, 2006 from http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=2507540&query_hl=21&itool=pubmed_docsum
Rights Management Copyright © American Society for Biochemistry and Molecular Biology, Copyright © American Society for Biochemistry and Molecular Biology, Copyright © American Society for Biochemistry and Molecular Biology, J Biol Chem., 264,17156-17163,1989.
Format Medium application/pdf
Identifier ir-main,365
ARK ark:/87278/s6xs6d02
Setname ir_uspace
Date Created 2012-06-13
Date Modified 2012-06-13
ID 706734
Reference URL https://collections.lib.utah.edu/ark:/87278/s6xs6d02
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