caa a22 4500 477119794 CHVBK 20180405111633.0 cr unu---uuuuu 170330e19960301xx s 000 0 eng 10.1007/BF00714329 doi (NATIONALLICENCE)springer-10.1007/BF00714329 Purification and characterization of the carboxyl-domain of human hexokinase type III expressed as fusion protein [Elektronische Daten] [Francesco Palma, Deborah Agostini, Philip Mason, Marina Dachá, Giovanni Piccoli, Beatrice Biagiarelli, Mara Fiorani, Vilberto Stocchi] In mammalian tissues hexokinase (ATP:D-hexose 6-phosphotransferase, EC 2.7.1.1) exists as four isoenzymes encoded by distinct genes. These proteins are homologous and are organized in two homologous domains, with the exception of hexokinase type IV which has only one. This organization is believed to be the result of a duplication and tandem fusion event involving the gene encoding for the ancestral hexokinase. In this study we cloned the carboxyl-domain of human hexokinase type III and expressed it in Escherichia coli as a glutathione S-transferase fusion protein, using the pGEX-2T expression vector. The recombinant protein showed catalytic activity. A comparative study of the kinetic properties of the expressed carboxyl-domain and the enzyme partially purified from human lymphocytes is also shown. The results now allow a better understanding of the role of the carboxyl-domain in determining the catalytic properties of the enzyme. KluwerAcademic Publishers, 1996 hexokinase type III nationallicence pGEX-2T vector nationallicence fusion protein nationallicence glutathione S-transferase nationallicence D-glucose nationallicence SSPE : 0.15 mol/l NaCl/ 10 mmol/l Na-phosphate/ 1 mmol/l EDTA pH 7.4 nationallicence Denhardt's : 0.02 g/l BSA Fractions V/ 0.02 g/l Ficoll 400/ 0.02 g/l polyvinylpyrollidone nationallicence SSC : 0.15 mol/l NaCl/ 15 mmol/l Na3-citrate pH 7.0 nationallicence IPTG : isopropyl β-D-thiogalactopyranoside nationallicence PMSF : phenylmethylsulfonyl fluoride nationallicence Glc-6-P : glucose 6-phosphate nationallicence PAGE : polyacrylamide gel electrophoresis nationallicence Palma Francesco Istituto di Chimica Biologica ‘Giorgio Fornaini', Università di Urbino, Via Saffi, 2, 61029, Urbino, Italy aut Agostini Deborah Istituto di Chimica Biologica ‘Giorgio Fornaini', Università di Urbino, Via Saffi, 2, 61029, Urbino, Italy aut Mason Philip Haematology Department, Hammersmith Hospital, Du Cane Road, W12 ONN, London, UK aut Dachá Marina Istituto di Chimica Biologica ‘Giorgio Fornaini', Università di Urbino, Via Saffi, 2, 61029, Urbino, Italy aut Piccoli Giovanni Istituto di Chimica Biologica ‘Giorgio Fornaini', Università di Urbino, Via Saffi, 2, 61029, Urbino, Italy aut Biagiarelli Beatrice Istituto di Chimica Biologica ‘Giorgio Fornaini', Università di Urbino, Via Saffi, 2, 61029, Urbino, Italy aut Fiorani Mara Istituto di Chimica Biologica ‘Giorgio Fornaini', Università di Urbino, Via Saffi, 2, 61029, Urbino, Italy aut Stocchi Vilberto Istituto di Chimica Biologica ‘Giorgio Fornaini', Università di Urbino, Via Saffi, 2, 61029, Urbino, Italy aut Molecular and Cellular Biochemistry Kluwer Academic Publishers 155/1(1996-03-01), 23-29 0300-8177 155:1<23 1996 155 11010 https://doi.org/10.1007/BF00714329 text/html Onlinezugriff via DOI 1 research-article jats NATIONALLICENCE 856 40 https://doi.org/10.1007/BF00714329 text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Palma Francesco Istituto di Chimica Biologica ‘Giorgio Fornaini', Università di Urbino, Via Saffi, 2, 61029, Urbino, Italy aut NATIONALLICENCE 700 1- Agostini Deborah Istituto di Chimica Biologica ‘Giorgio Fornaini', Università di Urbino, Via Saffi, 2, 61029, Urbino, Italy aut NATIONALLICENCE 700 1- Mason Philip Haematology Department, Hammersmith Hospital, Du Cane Road, W12 ONN, London, UK aut NATIONALLICENCE 700 1- Dachá Marina Istituto di Chimica Biologica ‘Giorgio Fornaini', Università di Urbino, Via Saffi, 2, 61029, Urbino, Italy aut NATIONALLICENCE 700 1- Piccoli Giovanni Istituto di Chimica Biologica ‘Giorgio Fornaini', Università di Urbino, Via Saffi, 2, 61029, Urbino, Italy aut NATIONALLICENCE 700 1- Biagiarelli Beatrice Istituto di Chimica Biologica ‘Giorgio Fornaini', Università di Urbino, Via Saffi, 2, 61029, Urbino, Italy aut NATIONALLICENCE 700 1- Fiorani Mara Istituto di Chimica Biologica ‘Giorgio Fornaini', Università di Urbino, Via Saffi, 2, 61029, Urbino, Italy aut NATIONALLICENCE 700 1- Stocchi Vilberto Istituto di Chimica Biologica ‘Giorgio Fornaini', Università di Urbino, Via Saffi, 2, 61029, Urbino, Italy aut NATIONALLICENCE 773 0- Molecular and Cellular Biochemistry Kluwer Academic Publishers 155/1(1996-03-01), 23-29 0300-8177 155:1<23 1996 155 11010 Metadata rights reserved Springer special CC-BY-NC licence nationallicence BK010053 XK010053 XK010000 NATIONALLICENCE NATIONALLICENCE NL-springer