Purification, characterization and antitumor activity of L-asparaginase isolated from Pseudomonas stutzeri MB-405.
An L-asparaginase produced by Pseudomonas stutzeri MB-405 was isolated and characterized. After initial ammonium sulfate fractionation, the enzyme was purified by consecutive column chromatography on Sephadex G-100, Ca-hydroxylapatite, and DEAE-Sephadex A-50. The 665.5-fold purified enzyme thus obtained has the specific activity of 732.3 units mg protein-1 with an overall recovery of 27.2%. The apparent M(r) of the enzyme under nondenaturing and denaturing conditions was 34 kDa and 33 kDa respectively, and the isoelectric point was 6.38 +/- 0.02. It displayed optimum activity at pH 9.0 and 37 degrees C. The enzyme was very specific for L-asparagine and did not hydrolyze L-glutaminate. The Km of the L-asparaginase was found to be 1.45 x 10(-4) M towards L-asparagine and was competitively inhibited by 5-diazo-4-oxo-L- norvaline (DONV) with a Ki of 0.03 mM. Metal ions such as Mn2+, Zn2+, Hg2+, Fe3+, Ni2+, and Cd2+ potentially inhibited the enzyme activity. The activity was enhanced in the presence of thiol-protecting reagents such as DTT, 2-ME, and glutathione (reduced), but inhibited by PCMB and iodoacetamide. The tumor inhibition study with Dalton's lymphoma tumor cells in vivo indicated that this enzyme possesses antitumor properties.
Manna
S
S
Department of Microbiology, Bose Institute, Calcutta, India.
Sinha
A
A
Sadhukhan
R
R
Chakrabarty
S L
SL
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D000970:Antineoplastic Agents / Q000302:isolation & purification* / Q000378:metabolism / Q000494:pharmacology; D001215:Asparaginase / Q000302:isolation & purification* / Q000378:metabolism / Q000494:pharmacology; D001216:Asparagine; D007526:Isoelectric Point; D007700:Kinetics; D008297:Male; D008670:Metals / Q000494:pharmacology; D051379:Mice; D008970:Molecular Weight; D009374:Neoplasms, Experimental / Q000188:drug therapy; D011549:Pseudomonas / Q000201:enzymology*; D013379:Substrate Specificity
D000596:Amino Acids; D000970:Antineoplastic Agents; D008670:Metals; D001216:Asparagine; D001215:Asparaginase