Affinity chromatography for purification of two urokinases from human urine

Takahashi, Ryoki; Akiba, Kiyoshi; Koike, Motofumi; Noguchi, Takayasu; Ezure, Yohji

doi:10.1016/s0378-4347(00)00127-4

Cited by 3 publications

(2 citation statements)

References 27 publications

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“…Also, as can be seen in the SDS-PAGE and zymography results here, the LMW and HMW forms of urokinase have eluted together into the same fraction. It has been suggested previously that it might be impossible to separate completely the HMW urokinase from the LMW form 29 . The LMW and HMW forms of urokinase are known to have different efficiencies as regards their plasminogen activating roles.…”

Section: Discussionmentioning

confidence: 97%

See 1 more Smart Citation

Urokinase Separation from Cell Culture Broth of a Human Kidney Cell Line

Bansal

Roychoudhury²,

Kumar

2007

Int. J. Biol. Sci.

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A single step ion-exchange chromatography on a sulfo-propyl (SP)-Sepharose column was performed to separate both the high molecular weight (HMW)-and low molecular weight (LMW)-forms of enzymatically active urokinase type plasminogen activator from human kidney (HT1080) cell culture media. The level of urokinase secreted by the cell line reached to about 145 Plough units/ml culture broth within 48 h of cultivation. The conditioned cell culture media was applied directly to the column without any prior concentration steps. Polyacrylamide gel electrophoresis of the column eluates in the presence of sodium dodecyl sulphate showed that the cell line secretes three forms of two-chain high molecular weight (HMW) urokinase of molecular weights (M r ) 64,000, 60,900 and 55,000. In addition, two low molecular weight (LMW) forms of M r 22,000 and 20,000; proteolytic cleavage products of HMW, were also found. The HMW and LMW forms had intrinsic plasminogen dependent proteolytic activity as judged by zymographic analysis. The specific activity of the pooled peak fractions increased (approximately 93-fold) to values as high as 1481 Plough units/ mg protein. Both HMW as well as LMW forms were obtained in significantly high yields.

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Section: Discussionmentioning

confidence: 97%

“…It has been suggested previously that it might be impossible to separate completely the HMW urokinase from the LMW form 29. The LMW and HMW forms of urokinase are known to have different efficiencies as regards their plasminogen activating roles.…”

Section: Discussionmentioning

confidence: 99%