Phenothiazine-binding and attachment sites of CAPP sub 1 -calmodulin

• Published in: Biochemistry (Easton) 1989-05, Vol.28:9
• Main Authors: Newton, D.L., Klee, C.B.
• Format: Article
• Language: English
• Subjects: 550601 - Medicine- Unsealed Radionuclides in Diagnostics; 560120 - Radiation Effects on Biochemicals, Cells, & Tissue Culture; ACYLATION; AMINO ACID SEQUENCE; AMINO ACIDS; AZINES; BIOLOGICAL EFFECTS; BIOLOGICAL RADIATION EFFECTS; CALMODULIN; CARBON 14 COMPOUNDS; CARBOXYLIC ACIDS; CHEMICAL REACTIONS; CROSS-LINKING; ELECTROMAGNETIC RADIATION; HETEROCYCLIC COMPOUNDS; HYDROGEN COMPOUNDS; LABELLED COMPOUNDS; LYSINE; MOLECULAR STRUCTURE; ORGANIC ACIDS; ORGANIC COMPOUNDS; ORGANIC NITROGEN COMPOUNDS; ORGANIC SULFUR COMPOUNDS; PHENOTHIAZINES; POLYMERIZATION; PROTEINS; RADIATION EFFECTS; RADIATION, THERMAL, AND OTHER ENVIRON. POLLUTANT EFFECTS ON LIVING ORGS. AND BIOL. MAT; RADIATIONS; RADIOLOGY AND NUCLEAR MEDICINE; TRITIUM COMPOUNDS; ULTRAVIOLET RADIATION
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi00435a019

In the presence of Ca{sup 2+} norchlorpromazine isothiocyanate forms a monocovalent complex with calmodulin: CAPP{sub 1}-calmodulin. Trypsin digestion of ({sup 3}H)CAPP{sub 1}-calmodulin yields as the major radioactive peptide N{sup {epsilon}}-CAPP-Lys-Met-Lys, corresponding to residues 75-77 of calmodulin. Stoichiometric amounts of all other expected tryptic peptides are also found, indicating that norchlorpromazine isothiocyanate selectively acylates Lys 75. A second molecule of CAPP-NCS can react, albeit slowly, with calmodulin to form CAPP{sub 2}-calmodulin. Fragments 38-74 and 127-148 are completely missing from the trypsin digests of CAPP{sub 2}-calmodulin without deliberate exposure to UV irradiation. Possibly the lengthy preparation of CAPP{sub 2}-calmodulin favors photolysis, caused by room lights, of the putative CAPP-binding domains located in these two peptides. Lys 148, the sole lysyl residue in fragment 127-148, is a probable site of attachment of the second molecule of CAPP. UV irradiation of CAPP{sub 1}-calmodulin, followed by digestion with trypsin, results in the selective loss of 50% each of peptides containing residues 38-74 and 127-148, suggesting that these peptides contain the hydrophobic amino acids that form the phenothiazine-binding sites. Computer modeling of CAPP{sub 1}-calmodulin with the X-ray coordinates of calmodulin indicates that CAPP attached to Lys 75 cannot interact with the carboxyl-terminal phenothiazine-binding site. Thus, in solution, the structure of CAPP{sub 1}-calmodulin may be different from that of the crystal, and flexibility of the central helix may allow CAPP linked to Lys 75 to bind at either one of the two drug-binding sites with equal probability.