PLANT PHYSIOLOGY , Vol 105, Issue 3 911-919, Copyright © 1994 by American Society of Plant Biologists
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MOLECULAR BIOLOGY AND GENE REGULATION |
Microheterogeneous Cytosolic High-Mobility Group Proteins from Broccoli Co-Purify with and Are Phosphorylated by Casein Kinase II
L. J. Klimczak and A. R. Cashmore
Plant Science Institute, Department of Biology, University of Pennsylvania, Philadelphia, Pennsylvania 19104-6018
A group of low molecular weight protein substrates was found to co-purify
with casein kinase II from broccoli (Brassica oleracea var italica). These
substrates showed very high affinity toward casein kinase II and were
efficiently phosphorylated even in the presence of an excess of exogenous
substrates. The broccoli substrates were purified from cytosolic extracts
as a double band of related proteins migrating at 18.7 and 20 kD. Further
microheterogeneity was revealed by anion-exchange high-performance liquid
chromatography and mass spectroscopy. The actual molecular masses of the
three major components identified by mass spectroscopy were determined to
be 12,691, 13,256, and 14,128 D. The substrates showed characteristic amino
acid composition with a high content of polar amino acids, including about
20% each of acidic and basic amino acids. They were soluble in 2%
trichloroacetic acid. The substrates cross-reacted with an antibody against
wheat high-mobility group protein d (HMGd) but not HMGa. The isolated
broccoli HMGs showed general DNA-binding activity without preference for
AT-rich DNA. The presence of these HMG proteins in the cytosolic fraction
is similar to the distribution characteristics of the animal HMG-1
subgroup. On the basis of amino acid composition and DNA-binding
specificity, the isolated broccoli HMGs resemble other plant HMGs
homologous to the HMG-1 subgroup.
