De novo production of alpha 2-macroglobulin in cultured astroglia from rat brain.
Previous studies from our laboratory demonstrated that alpha 2-macroglobulin (alpha 2M) is one of the neurite-promoting factors in the conditioned medium of astroglia. In the present study, we further examined the de novo production of alpha 2M in cultured astroglia by determining the expression of alpha 2M mRNA, and the biosynthesis of [35S]methionine-labeled alpha 2M protein. We analyzed the mRNA of cultured astroglia by differential hybridization using specific probes to alpha 2M and its homologous protein, alpha 1-inhibitor 3 (alpha 1I3), after amplification of reverse-transcribed cDNA with the polymerase chain reaction. The result clearly showed that only alpha 2M mRNA is expressed in cultured astroglia. Northern blotting analysis revealed that alpha 2M mRNA is expressed mainly in the astroglia and is not detected in neurons, microglia and meningeal fibroblasts. Furthermore, the biosynthesis of alpha 2M protein in the astroglia was confirmed by an immunoprecipitation experiment after labeling of each type of cell with [35S]methionine. It was concluded that alpha 2M is produced in the cultured astroglia which is the major source of alpha 2M production among various types of cells in rat brain.
Saitoh
S
S
Department of Neurochemistry, National Institute of Neuroscience, Tokyo, Japan.
Iijima
N
N
Ikeda
M
M
Nakajima
K
K
Kimura
M
M
Katsuki
M
M
Mori
T
T
Kohsaka
S
S
D000818:Animals; D000831:Animals, Newborn; D001253:Astrocytes / Q000166:cytology / Q000378:metabolism*; D001483:Base Sequence; D015152:Blotting, Northern; D001921:Brain / Q000166:cytology / Q000378:metabolism*; D002478:Cells, Cultured; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism; D008715:Methionine / Q000378:metabolism; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes; D016133:Polymerase Chain Reaction; D012313:RNA / Q000235:genetics / Q000302:isolation & purification; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D051381:Rats; D011919:Rats, Inbred Strains; D012689:Sequence Homology, Nucleic Acid; D013462:Sulfur Radioisotopes; D000511:alpha-Macroglobulins / Q000096:biosynthesis* / Q000235:genetics
D015345:Oligonucleotide Probes; D012333:RNA, Messenger; D013462:Sulfur Radioisotopes; D000511:alpha-Macroglobulins; D012313:RNA; D008715:Methionine