Acid phosphatases from the liver of Labeo rohita: purification and characterization.
Low molecular weight acid phosphatase (LM-ACP) peak 2 (the isoenzyme corresponding to isoform 2, IF-2) from the liver of fish Rahu (Labeo rohita) was purified to homogeneity. 900 times purification resulted with specific activity of 35 U/mg of protein and recovery of 0.2%. The enzyme was found homogeneous on sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). Molecular weight of 18 killo Daltons (kDa) was obtained. The peak 1 isoenzyme corresponding to isoform1 (IF-1) was partially purified about 160 times with specific activity of 7 U/mg of protein. Major protein band corresponding to 18 kDa was seen along with other protein faint bands. High molecular weight acid phosphatase (HM-ACP) was also partially purified. The molecular weight was estimated to be a 100 kDa by gel filtration on Sephadex G-100. LM-ACP isoenzymes and HM-ACP enzyme were studied for their substrate specificity, sensitivity to inhibitors or activators and other kinetic parameters. LM-ACP isoenzymes were not inhibited by tartrate and fluoride but were inhibited by sulfhydryl reagent whereas high molecular weight enzyme was strongly inhibited by fluoride and tartrate. Phosphate vanadate and molybdate inhibited both types of enzymes competitively, but their action was more pronounced in HM-ACP enzyme. LM-ACP was effectively activated by purine compounds whereas HM-ACP was not. LM-ACP showed strict substrate specificity while HM-ACP showed broad substrate specificity. The two types of acid phosphatases also differed in their rate of hydrolysis of alpha-naphthyl phosphate and beta-glyerophosphate.
Siddiqua
Aisha
A
Department of Chemistry, Gomal University, Dera Ismail Khan 29050, Pakistan.
Rehmat
Mamoona
M
Saeed
Asma
A
Amin
Shazia
S
Naz
Rubina
R
Sherazi
Mehrin
M
Khan
Gul Majeed
GM
Saeed
Ahmad
A
D000135:Acid Phosphatase / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D000818:Animals; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D005399:Fishes / Q000378:metabolism*; D007527:Isoenzymes / Q000737:chemistry / Q000302:isolation & purification; D008099:Liver / Q000201:enzymology*; D008970:Molecular Weight; D011687:Purines / Q000737:chemistry; D013379:Substrate Specificity
D007527:Isoenzymes; D011687:Purines; D000135:Acid Phosphatase