Mesoderm-inducing factor from bovine amniotic fluid: purification and N-terminal amino acid sequence determination.
Bovine amniotic fluid contains a protein capable of inducing mesoderm formation from animal cap cells isolated from Xenopus laevis embryos at the late blastula stage. The procedure for purification of this protein is described and includes the following steps: CM-cellulose cation-exchange chromatography, chromatography on hydroxylapatite, reverse-phase hplc, size exclusion hplc, and a second reverse-phase hplc. A partially purified preparation of a mesoderm-inducing factor with high mesoderm-inducing activity was thus obtained. SDS polyacrylamide gel electrophoresis with subsequent transfer of proteins onto Immobilon membrane was used to obtain the protein in pure form. The membrane was cut, the proteins eluted, and each fraction tested for mesoderm-inducing activity. The mesoderm-inducing activity corresponded to a protein of molecular mass 25 kDa (in nonreducing conditions) consisting of two identical polypeptides connected by disulphide bonds. The purified factor, at concentrations of 0.3-1 pmol ml-1, induced development of notochord and muscle cells and, at lower concentrations, mesenchyme and blood cells. The twenty N-terminal amino acid residues of the protein were sequenced and found to be identical to those of the N-terminus of the beta A-chain of inhibin and activin, known regulators of FSH secretion by pituitary cells. The beta A-chain is homologous with the transforming growth factor-beta family of proteins. On the basis of the estimated molecular mass of the mesoderm-inducing factor and its N-terminal sequence it is suggested that the protein is activin or a related protein.
Chertov OYu
Shemyakin Institute of Bioorganic Chemistry, Academy of Sciences of the USSR, Moscow.
Krasnosel'skii
A L
AL
Bogdanov
M E
ME
Hoperskaya
O A
OA
D028341:Activins; D000595:Amino Acid Sequence; D000653:Amniotic Fluid / Q000737:chemistry*; D000818:Animals; D002417:Cattle; D004627:Embryonic Induction / Q000187:drug effects; D005260:Female; D006133:Growth Substances / Q000737:chemistry / Q000302:isolation & purification* / Q000494:pharmacology; D007265:Inhibins / Q000737:chemistry; D008648:Mesoderm / Q000187:drug effects*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011247:Pregnancy; D011506:Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000494:pharmacology; D012689:Sequence Homology, Nucleic Acid; D014982:Xenopus laevis
D006133:Growth Substances; D011506:Proteins; D028341:Activins; D007265:Inhibins