Characterization of nucleoside-diphosphate kinase-associated guanine nucleotide-binding proteins from HeLa S3 cells

• Published in: Biochimica et Biophysica Acta (BBA) - General Subjects Vol. 882; no. 3; pp. 322 - 330
• Main Authors: Ohtsuki, Kenzo, Ikeuchi, Tohru, Yokoyama, Minehiko
• Format: Journal Article
• Language: English
• Published: Amsterdam Elsevier B.V 16.07.1986; Elsevier BV; Elsevier; North-Holland
• Subjects: Analytical, structural and metabolic biochemistry; Binding and carrier proteins; Biological and medical sciences; Calcium; Calcium - metabolism; Centrifugation, Density Gradient; Chlorides; Chlorides - metabolism; Chromatography, DEAE-Cellulose; Chromatography, Gel; Edetic Acid; Edetic Acid - metabolism; Fundamental and applied biological sciences. Psychology; GTP Phosphohydrolases; GTP Phosphohydrolases - metabolism; GTP-Binding Proteins; GTP-Binding Proteins - metabolism; GTPase activity; Guanine nucleotide-binding protein; Guanosine Triphosphate; Guanosine Triphosphate - metabolism; HeLa Cells; HeLa Cells - analysis; HeLa S3 cell; Hot Temperature; Humans; Lithium; Lithium - metabolism; Lithium Chloride; Macromolecular Substances; Magnesium; Magnesium - metabolism; Molecular Weight; Nucleoside-Diphosphate Kinase; Nucleoside-Diphosphate Kinase - metabolism; p21 ras Oncogene product; Phosphotransferases; Phosphotransferases - metabolism; Proteins; NDP-kinase; SDS; HeLa S3 cell; pp[CH 2]pG; [γS]pppG; PMSF; p[NH]ppG; Guanine nucleotide-binding protein; p21 ras Oncogene product; Nucleoside-diphosphate kinase; GTPase activity; Binding protein; Purification; Enzyme; Gene product; Guanine nucleotide; Onc gene; Nucleoside diphosphate kinase
• ISSN: 0304-4165; 0006-3002; 1872-8006
• DOI: 10.1016/0304-4165(86)90254-0

Nucleoside-diphosphate (NDP) kinase-associated [ α- 32P]GTP-incorporating proteins from HeLa S3 cells have been biochemically characterized. Two distinct NDP-kinases (F-I and F-II) had been partially purified from HeLa S3 cells by Sephacryl S-300 gel filtration and DEAE-cellulose column chromatography. The [ α- 32P]GTP-incorporating proteins (approx. M r 20 000) could be separated from NDP-kinases (approx. M r 80 000) by 5–25% glycerol density-gradient centrifugation analysis after treatment with 7 M urea in the presence of 1 mM EDTA. [ α- 32P]GPT incorporation into these two proteins (G1 and G2) from NDP-kinases required 5 mM Mg 2+ and was highly inhibited by either GDP or GTP analogues, such as guanylyl imidodiphosphate and guanylyl methylenediphosphate. [ 3H]GDP, but not other nucleoside 5′-diphosphates, was also bound to these two proteins in the presence of Mg 2+ (5 mM). Moreover, incubation of [ α- 32P]GPT with either G1 or G2 in the presence of Mg 2+ (5 mM) resulted in the formation of [ 32P]GDP and P i. The data presented here indicated that (a) the guanine nucleotide-binding activity, (b) the GTPase activity, and (c) the molecular weight (approx. M r 20 000) of NDP kinase-associated proteins from HeLa S3 cells are similar to those reported for ras oncogene products (p21 proteins).