Da. Malencik et Sr. Anderson, DITYROSINE FORMATION IN CALMODULIN - CROSS-LINKING AND POLYMERIZATIONCATALYZED BY ARTHROMYCES PEROXIDASE, Biochemistry, 35(14), 1996, pp. 4375-4386
We employ bovine brain calmodulin, a protein that is subject to photoa
ctivated dityrosine formation [Malencik, D. A., & Anderson, S, R. (198
7) Biochemistry 26, 695; (1994) Biochemistry 33, 13363], as a model fo
r the development of an efficient enzyme-catalyzed protein cross-linki
ng technique. Key steps in the elaboration of the procedure are (1) id
entification of a peroxidase, from Arthromyces ramosus, that catalyzes
dityrosine production in proteins that are not acted on by other comm
on peroxidases, (2) monitoring of the intrinsic fluorescence of dityro
sine to determine optimum reaction conditions, achieved with calmoduli
n in solutions containing boric acid-sodium berate (concentration grea
ter than or equal to 0.2 M), similar to pH 8.3, similar to 40 degrees
C, and (3) quenching of the reaction with reduced glutathione. Arthrom
yces,ces peroxidase is the only common peroxidase able to catalyze sig
nificant dityrosine production in calmodulin, through a reaction that
is largely intermolecular. Gel filtration yields fractions (accounting
for similar to 40% of the initial calmodulin) that represent differin
g mobility ranges in NaDodSO(4) polyacrylamide gel electrophoresis and
contain close to the maximum possible amounts of dityrosine. The vari
ous fractions undergo Ca2+-dependent conformational changes detected i
n sedimentation velocity and/or fluorescence anisotropy measurements.
Most of the samples stimulate the Ca2+-dependent activity of smooth mu
scle myosin light chain kinase. In catalytic assays utilizing the synt
hetic phosphate acceptor peptide, the average activities range from 50
to 100% of that determined for native calmodulin. However, only the l
east polymerized fraction and the photogenerated calmodulin dimers sig
nificantly enhance the p-nitrophenylphosphatase activity of calcineuri
n. The ability to prepare soluble calmodulin polymers that retain a su
bstantial degree of biological activity and exhibit the intense visibl
e fluorescence of dityrosine illustrates the potential usefulness of A
rthromyces peroxidase in the zero-length cross-linking of proteins.