Bosmann H B
J Cell Biol. 1973 Dec;59(3):601-14. doi: 10.1083/jcb.59.3.601.
The molecules occurring as terminal residues on the external surfaces of nuclei prepared from rat liver by either sucrose-CaCl(2) or citric acid methods and nucleoli derived from the sucrose-CaCl(2) nuclei were studied chemically and electrokinetically. In 0.0145 M NaCl, 4.5% sorbitol, and 0.6 mM NaHCO(3) with pH 7.2 +/- 0.1 at 25 degrees C, the sucrose-CaCl(2) nuclei had an electrophoretic mobility of -1.92 microm/s/V/cm, the citric acid nuclei, -1.63 microm/s/V/cm, and the nucleoli, -2.53 microm/s/V/cm. The citric acid nuclei and the nucleoli contained no measurable sialic acid. The sucrose-CaCl(2) nuclei contained 0.7 nmol of sialic acid/mg nuclear protein; this was essentially located in the nuclear envelope. Treatment of these nuclei with 50 microg neuraminidase/mg protein resulted in release of 0.63 nmol of sialic acid/mg nuclear protein; treatment with 1 % trypsin caused release of 0.39 nmol of the sialic acid/mg nuclear protein. The pH-mobility curves for the particles indicated the sucrose-CaCl(2) nuclei surface had an acid-dissociable group of pK. approximately 2.7 while the pK for the nucleoli was considerably lower. Nucleoli treated with 50 microg neuraminidase/mg particle protein had a mobility of -2.53 microm/s/V/cm while sucrose-CaCl(2) nuclei similarly treated had a mobility of -1.41 microm/s/V/cm. Hyaluronidase at 50 microg/mg protein had no effect on nucleoli mobility but decreased the sucrose-CaCl(2) nuclei mobility to -1.79 microm/s/V/cm. Trypsin at 1 % elevated the electrophoretic mobility of the sucrose-CaCl(2) nuclei slightly but decreased the mobility of the nucleoli to -2.09 microm/s/V/cm. DNase at 50 microg/mg protein had no effect on the mobility of the isolated sucrose-CaCl(2) nuclei but decreased the electrophoretic mobility of the nucleoli to -1.21 microm/s/V/cm. RNase at 50 microg/mg protein also had no effect on the electrophoretic mobility of the sucrose-CaCl(2) nuclei but decreased the nucleoli mobility to -2.10 microm/s/V/cm. Concanavalin A at 50 microg/mg protein did not alter the nucleoli electrophoretic mobility but decreased the sucrose-CaCl(2) nuclei electrophoretic mobility to -1.64 microm/s/V/cm. The results are interpreted to mean that the sucrose-CaCl(2) nuclear external surface contains terminal sialic acid residues in trypsin-sensitive glycoproteins, contains small amounts of hyaluronic acid, is completely devoid of nucleic acids, and binds concanavalin A. The nucleolus surface is interpreted to contain a complex made up of protein, RNA, and primarily DNA, to be devoid of sialic acid and hyaluronic acid, and not to bind concanavalin A.
采用蔗糖 - 氯化钙法或柠檬酸法从大鼠肝脏制备的细胞核以及从蔗糖 - 氯化钙法制备的细胞核中分离得到的核仁，对其外表面作为末端残基存在的分子进行了化学和电动学研究。在25℃下，于0.0145 M氯化钠、4.5%山梨醇和0.6 mM碳酸氢钠（pH 7.2±0.1）中，蔗糖 - 氯化钙法制备的细胞核的电泳迁移率为 -1.92微米/秒/伏/厘米，柠檬酸法制备的细胞核为 -1.63微米/秒/伏/厘米，核仁为 -2.53微米/秒/伏/厘米。柠檬酸法制备的细胞核和核仁中未检测到可测量的唾液酸。蔗糖 - 氯化钙法制备的细胞核每毫克核蛋白含有0.7纳摩尔唾液酸；这些唾液酸主要位于核膜中。用50微克神经氨酸酶/毫克蛋白处理这些细胞核，导致每毫克核蛋白释放0.63纳摩尔唾液酸；用1%胰蛋白酶处理导致每毫克核蛋白释放0.39纳摩尔唾液酸。这些颗粒的pH - 迁移率曲线表明，蔗糖 - 氯化钙法制备的细胞核表面有一个pK约为2.7的酸可解离基团，而核仁的pK则低得多。用50微克神经氨酸酶/毫克颗粒蛋白处理的核仁迁移率为 -2.53微米/秒/伏/厘米，而同样处理的蔗糖 - 氯化钙法制备的细胞核迁移率为 -1.41微米/秒/伏/厘米。50微克/毫克蛋白的透明质酸酶对核仁迁移率无影响，但使蔗糖 - 氯化钙法制备的细胞核迁移率降至 -1.79微米/秒/伏/厘米。1%的胰蛋白酶使蔗糖 - 氯化钙法制备的细胞核的电泳迁移率略有升高，但使核仁迁移率降至 -2.09微米/秒/伏/厘米。50微克/毫克蛋白的脱氧核糖核酸酶对分离的蔗糖 - 氯化钙法制备的细胞核的迁移率无影响，但使核仁的电泳迁移率降至 -1.21微米/秒/伏/厘米。50微克/毫克蛋白的核糖核酸酶对蔗糖 - 氯化钙法制备的细胞核的电泳迁移率也无影响，但使核仁迁移率降至 -2.10微米/秒/伏/厘米。50微克/毫克蛋白的伴刀豆球蛋白A未改变核仁的电泳迁移率，但使蔗糖 - 氯化钙法制备的细胞核的电泳迁移率降至 -1.64微米/秒/伏/厘米。结果表明，蔗糖 - 氯化钙法制备的细胞核外表面在对胰蛋白酶敏感的糖蛋白中含有末端唾液酸残基，含有少量透明质酸，完全不含核酸，并能结合伴刀豆球蛋白A。核仁表面被认为含有由蛋白质、RNA和主要是DNA组成的复合物，不含唾液酸和透明质酸，且不结合伴刀豆球蛋白A。
