Protein composition of human metaphase chromosomes analyzed by two-dimensional electrophoreses.
A large amount of metaphase chromosomes were isolated from synchronized human cell lines by a polyamine procedure. All the chromosomal proteins extracted by an acetic acid extraction method were fully dissolved into the sample solutions for isoelectric focusing (IEF) or radical free and highly reduced (RFHR) two-dimensional electrophoreses (2-DEs). As a result, well-separated and highly reproducible 2-DE patterns were obtained. This could not be attained by an ordinary acetone precipitation method. The 2-DE patterns visualized using Coomassie Brilliant Blue (CBB) staining indicated that more than one hundred proteins were involved in the isolated metaphase chromosomes, although the most abundant proteins, histones, occupied a greater part of the chromosomal proteins. It was also shown that colcemid treatment for cell cycle synchronization had little effect on the 2-DE pattern compared to that obtained without the treatment. Furthermore, no significant differences were observed in the 2-DE patterns among the chromosomal proteins prepared from two different human cell lines, BALL-1 and K562. However, 2-DE analysis of isolated metaphase chromosomes from HeLa cells apparently showed a smaller number of proteins than the BALL-1 and K562 cell lines at a neutral pI range. The present study paves the way for elucidating protein composition of human metaphase chromosomes.
Department of Biotechnology, Graduate School of Engineering, Osaka University, Suita, Japan.
S
S
Uchiyama
S
S
Kobayashi
H
H
Takata
T
T
Ishihara
T
T
Sone
S
S
Matsunaga
K
K
Fukui
D045744:Cell Line, Tumor; D002877:Chromosomes, Human / Q000737:chemistry* / Q000235:genetics*; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D006367:HeLa Cells / Q000737:chemistry / Q000378:metabolism; D006657:Histones / Q000378:metabolism; D006801:Humans; D007525:Isoelectric Focusing / Q000379:methods; D020014:K562 Cells / Q000737:chemistry / Q000378:metabolism; D008677:Metaphase / Q000235:genetics*; D009698:Nucleoproteins / Q000378:metabolism*; D010449:Peptide Mapping / Q000379:methods; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods
D006657:Histones; D009698:Nucleoproteins