Purification and characterization of the cytoplasmic histone acetyltransferase B of maize embryos.
From a soluble cellular fraction of maize embryos we purified to apparent homogeneity a cytoplasmic histone acetyltransferase, which matches all criteria for a B-type enzyme. Using 8 chromatographic steps, we achieved a 6700-fold purification of an enzymatically active protein with a molecular weight of approximately 90 kDa. Under denaturing conditions the protein split into 2 components which migrated at 45 and 50 kDa in SDS-PAGE, suggesting that the native enzyme is a heterodimer. The purified enzyme was characterized in terms of physicochemical and kinetic properties, and substrate specificity. It was specific for histone H4, leading to acetylation of non-acetylated H4 subspecies into the di-acetylated state in vitro. Its activity was coincident with the intensity of DNA replication in meristematic cells during embryo germination. We established an electrophoretic system under non-denaturing conditions for detection of enzyme activity within the gel matrix; in combination with second dimension SDS-PAGE the procedure allowed the unambiguous identification of histone acetyltransferase, even in crude enzyme preparations.
Eberharter
A
A
Department of Microbiology, University of Innsbruck, Medical School, Austria.
Lechner
T
T
Goralik-Schramel
M
M
Loidl
P
P
D000107:Acetylation; D000123:Acetyltransferases / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D005591:Chemical Fractionation; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D002845:Chromatography / Q000379:methods; D003593:Cytoplasm / Q000201:enzymology; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D051548:Histone Acetyltransferases; D006657:Histones / Q000378:metabolism; D007700:Kinetics; D011489:Protein Denaturation; D029701:Saccharomyces cerevisiae Proteins; D012639:Seeds / Q000201:enzymology; D012995:Solubility; D013379:Substrate Specificity; D003313:Zea mays / Q000196:embryology / Q000201:enzymology*
D006657:Histones; D029701:Saccharomyces cerevisiae Proteins; D000123:Acetyltransferases; D051548:Histone Acetyltransferases