Affinity of chromatin constituents for isopeptidase.
Isopeptidase is a novel eukaryotic enzyme that cleaves a structural chromatin protein, A24, stoichiometrically into H2A and ubiquitin. To understand the rapid turnover of ubiquitin in mitosis as well as the high specific activity of the enzyme associated with metaphase chromosomes, attempts were made to determine chromatin constituents that show high affinity for this enzyme. Endogenous protease-free isopeptidase was prepared from calf thymus and applied to a Sepharose 4B affinity column on which histones, DNA, NHCP and ubiquitin were respectively immobilized. The enzyme proved to bind only histones. To further determine which of the histone fractions is involved, affinity columns with each histone fraction were also used. The enzyme showed affinity for all histone fractions. However, the strength of affinity varied in the order H2A greater than H3 greater than H2B greater than or equal to H4 much greater than H1, being inversely correlated with the ratio of basic/acidic amino acids in these molecules. These results suggest that the turnover of A24 in mitosis is controlled, at least in part, by the affinity of enzyme for histones, and also that such affinity is caused by a mechanism which cannot be explained simply by the electrostatic interaction between negatively charged enzyme molecules and positively charged histones.
D000818:Animals; D019759:Carbon-Nitrogen Lyases; D002417:Cattle; D002460:Cell Line; D002467:Cell Nucleus / Q000032:analysis; D002843:Chromatin / Q000032:analysis; D002846:Chromatography, Affinity / Q000379:methods; D002868:Chromosomal Proteins, Non-Histone / Q000032:analysis; D006224:Cricetinae; D003412:Cricetulus; D004247:DNA / Q000032:analysis; D006657:Histones / Q000032:analysis; D008168:Lung; D008190:Lyases / Q000302:isolation & purification* / Q000378:metabolism; D013950:Thymus Gland / Q000201:enzymology*
D002843:Chromatin; D002868:Chromosomal Proteins, Non-Histone; D006657:Histones; D004247:DNA; D008190:Lyases; D019759:Carbon-Nitrogen Lyases; C034300:isopeptidase