TY - JOUR
T1 - Purification and immunochemical characterization of a human-specific urine and tissue esterase
AU - Munjal, D.
AU - Rose, N. R.
N1 - Funding Information:
The authors are grateful to Dr W. B. Elliott of the Department of Biochemistry for allowing us to use his laboratory facilities. This investigation was supported by U.S.P.H.S. Research Grant CA-o52o 3 from the National Cancer Institute.
PY - 1972/12/28
Y1 - 1972/12/28
N2 - An esterase (carboxylic-ester hydrolase, EC 3.1.1.1) has been isolated from human urine, kidney and cell cultures by a combination of ammonium sulfate precipitation, gel permeation chromatography, preparative column electrophoresis and ultra-filtration. It has been characterized by disc gel electrophoresis, immunoelectrophoresis, and the Ouchterlony technique. Studies indicated immunological identity of esterases in urine, kidney and cell cultures using rabbit antiserum prepared against esterase from urine. The enzyme from human urine and cell cultures could hydrolyze substrates having up to a six carbon chain, while esterase from kidney hydrolyzed substrates with a four carbon chain only. Eserine, eserine sulfate, phenazine methosulfate and sodium amytal did not show any inhibitory effect on the hydrolyzing capacity of the urine, kidney and cell culture esterase. Further, urine and cell culture enzyme was found to be strongly inhibited by phenyl methyl sulfonyl fluoride, while NaF and sodium azide showed partial inhibition of the substrate hydrolysis. Experimental slides from urine and cell cultures treated with melittin, EDTA, heparin and sodium deoxycholate, showed strong esterase activity, when later treated with substrates. Esterase from kidney, on the other hand, showed marked inhibition by EDTA and slight inhibition by heparin and sodium deoxycholate. NaF and sodium azide did not inhibit the hydrolyzing capacity of the kidney esterase. Carbohydrate analysis of eluted fractions showed that the cathodal subfraction of urinary enzyme contains about 1.5 to 2.0% of carbohydrate, which may be responsible for some of the heterogeneity of the enzyme and its action on substrates, as compared to kidney and cell culture esterase.
AB - An esterase (carboxylic-ester hydrolase, EC 3.1.1.1) has been isolated from human urine, kidney and cell cultures by a combination of ammonium sulfate precipitation, gel permeation chromatography, preparative column electrophoresis and ultra-filtration. It has been characterized by disc gel electrophoresis, immunoelectrophoresis, and the Ouchterlony technique. Studies indicated immunological identity of esterases in urine, kidney and cell cultures using rabbit antiserum prepared against esterase from urine. The enzyme from human urine and cell cultures could hydrolyze substrates having up to a six carbon chain, while esterase from kidney hydrolyzed substrates with a four carbon chain only. Eserine, eserine sulfate, phenazine methosulfate and sodium amytal did not show any inhibitory effect on the hydrolyzing capacity of the urine, kidney and cell culture esterase. Further, urine and cell culture enzyme was found to be strongly inhibited by phenyl methyl sulfonyl fluoride, while NaF and sodium azide showed partial inhibition of the substrate hydrolysis. Experimental slides from urine and cell cultures treated with melittin, EDTA, heparin and sodium deoxycholate, showed strong esterase activity, when later treated with substrates. Esterase from kidney, on the other hand, showed marked inhibition by EDTA and slight inhibition by heparin and sodium deoxycholate. NaF and sodium azide did not inhibit the hydrolyzing capacity of the kidney esterase. Carbohydrate analysis of eluted fractions showed that the cathodal subfraction of urinary enzyme contains about 1.5 to 2.0% of carbohydrate, which may be responsible for some of the heterogeneity of the enzyme and its action on substrates, as compared to kidney and cell culture esterase.
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U2 - 10.1016/0005-2795(72)90320-0
DO - 10.1016/0005-2795(72)90320-0
M3 - Article
C2 - 4633642
AN - SCOPUS:0015527801
SN - 0005-2795
VL - 285
SP - 352
EP - 363
JO - BBA - Protein Structure
JF - BBA - Protein Structure
IS - 2
ER -