Abstract
A mammary-uterine-pituitary tumor cell growth factor has been purified from lyophilized powders of pregnant sheep uteri by a five-step procedure. Uterine-derived growth factor (UDGF) was extracted from the powders with 0.1 M acetic acid, heated at 95 degrees C, and further purified by sulfopropyl-Sephadex C-25, Sephadex G-50, and carboxymethyl-Sephadex C-25 chromatography. From 500 g of uterine powder, 40 to 50 mg of UDGF can be isolated at an overall yield of 33%. The degree of homogeneity of the final preparations was estimated by 8 M urea, 0.1% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (PAGE), and by PAGE under nondissociating conditions at either pH 8.5 or 4.5. In all PAGE experiments, the purified UDGF preparation showed a single Coomassie blue-stained band that directly corresponded to the only area of elution of UDGF activity from duplicate unstained gels. Molecular sieve high performance liquid chromatography HPLC, reverse phase HPLC on an octylsilyl (C8) column, and hydrophobic chromatography on octyl-Sepharose CL-4B all confirm a similar degree (i.e. greater than 90%) of homogeneity. The Mr of UDGF estimated by urea/sodium dodecyl sulfate-PAGE was 4200 +/- 500 and, by molecular sieve HPLC, 6200 +/- 1000. The isoelectric point of UDGF was estimated as pI = 7.3. The UDGF isolated showed marked cell-type specificity for established cell lines that were derived from estrogen-responsive tumors; purified sheep UDGF was mitogenic for MTW9/PL rat mammary tumor cells (at 10(-10) to 10(-9) M concentrations) while showing no mitogenic activity toward normal rat diploid fibroblasts. UDGF also promoted growth of uterine-derived tumor cells and the GH3/C14 rat pituitary line. Measuring growth as an increase in cell number, UDGF supported the logarithmic growth of the MTW9/PL rat mammary tumor cells over 6 days; other known hormones and growth factors were not able to substitute for the UDGF mitogenic action on MTW9/PL cells. It is concluded that a rapid, high-yield method of purification of a new uterine-derived growth factor activity has been developed.
Highlights
The UOGF laolated by the 5 step ll1ethod descd.bed in ene n'l4in tt"lIt was stUdied to establish plo~rt1eB pt"rtlllent to its identification as a unique factor
Since we are interested in identifying polypeptide growth factors that may be involved in estrogen responsive mammary, pituitary, and uterine tumor cell growth in vivo, and since we have reported evidence suggesting that newly identified estrogen-inducible growth factors from uterus [42, 43], kidney [42], and pituitary [44] may be involved in mammary tumor growth in vivo, we describe here the continuation of our studies aimed at purifying to homogeneity a mammary-uterine-pituitary tumor growth factor from lyophilized powders of early pregnant sheep uteri
Before initiating a large scale growth factor purification from species other than rat, the new source was evaluated from hormone responsiveness ofthe Uterinederived growth factor (UDGF) activity
Summary
The UOGF laolated by the 5 step ll1ethod descd.bed in ene n'l4in tt"lIt was stUdied to establish plo~rt1eB pt"rtlllent to its identification as a unique factor. The activity was assayed to deter.tne the \;.ineticB of inductiol'l of O»J\. Aynthe!ll.& in MTW9!PL cells in serum-free culture. The uocr activity willa assayed for the ability to promote cell 9(0",th lUI measured by increase in cell nullber. Ttl1rd, the UOGP cell type specificity w.,& dete>rllined. Several other hormones and grQwth rect.cr e were /lyed for the ability to either replace or supple-
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