Affinity labelling of smooth-muscle myosin light-chain kinase with 5'-[p-(fluorosulphonyl)benzoyl]adenosine.
5'-(p-(Fluorosulphonyl)[14C]benzoyl)adenosine (FSBA) was synthesized and used as a probe to study the ATP-binding site of smooth-muscle myosin light-chain kinase (MLCK). FSBA modified both free MLCK and calmodulin/MLCK complex, resulting in inactivation of the kinase activity. Nearly complete protection of the calmodulin/MLCK complex against FSBA modification was obtained by addition of excess ATP whereas MLCK activity alone was lost in a dose-dependent manner even in the presence of excess ATP. These results suggest that FSBA modified ATP-binding sites and ATP-independent sites, and the latter sites are protected by calmodulin binding. The results also suggest that the ATP-binding site is accessible to the nucleotide substrate regardless of calmodulin binding. The FSBA-labelled MLCK was completely proteolysed by alpha-chymotrypsin, and the 14C-labelled peptides were isolated and sequenced. The sequence of the labelled peptide was Ala-Gly-X-Phe, where X is the labelled residue. The sequence was compared with the known MLCK sequence, and the labelled residue was identified as lysine-548, which is located downstream of the GXGXXG motif conserved among ATP-utilizing enzymes.
Komatsu
H
H
Department of Physiology and Biophysics, Case Western Reserve University, School of Medicine, Cleveland, OH 44106.
Ikebe
M
M
D000241:Adenosine / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000378:metabolism; D000345:Affinity Labels; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002848:Chromatography, DEAE-Cellulose; D002851:Chromatography, High Pressure Liquid; D002918:Chymotrypsin; D016384:Consensus Sequence; D005895:Gizzard, Avian / Q000201:enzymology; D007700:Kinetics; D008239:Lysine; D008969:Molecular Sequence Data; D009130:Muscle, Smooth / Q000201:enzymology*; D009219:Myosin-Light-Chain Kinase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D010446:Peptide Fragments / Q000302:isolation & purification; D011494:Protein Kinases / Q000737:chemistry; D012265:Ribonucleotides / Q000378:metabolism / Q000494:pharmacology; D017386:Sequence Homology, Amino Acid; D014422:Turkeys
D000345:Affinity Labels; D010446:Peptide Fragments; D012265:Ribonucleotides; C010959:5'-(4-fluorosulfonylbenzoyl)adenosine; D000255:Adenosine Triphosphate; D011494:Protein Kinases; D009219:Myosin-Light-Chain Kinase; D002918:Chymotrypsin; D008239:Lysine; D000241:Adenosine