Purification of Urokinase by a Beta-Naphthamidine Affinity Column

Åstedt, Birger; Holmberg, Lars; Wagner, G.; Richter, P. Konstantin; Ploug, Jørgen

doi:10.1055/s-0038-1666941

Cited by 10 publications

(4 citation statements)

References 2 publications

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“…4-Aminobenzamidine itself has been used in the purification of a number of trypsin-like enzymes including trypsin (Hixson and Nishikawa, 1973), plasminogen (Ito et al, 1985) and urokinase (Holmberg et al, 1976). A number of derivatives of this compound have also been used (Hixson and Nishikawa, 1973;Astedt et al, 1979;Clonis et al, 1987) in purification schemes. Despite its low affinity for porcine pancreatic kallikrein, 4-aminobenzamidine (K,=240 PM; Geratz, 1969) has been used in its purification but as one step in a multistep protocol (Honda et al, 1986).…”

Section: Introductionmentioning

confidence: 99%

Design of novel affinity adsorbents for the purification of trypsin‐like proteases

Burton

Lowe

1992

J of Molecular Recognition

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A number of ligands for the selective purification by affinity chromatography of the trypsin-like protease, porcine pancreatic kallikrein, were designed de novo by computer-aided molecular design. The ligands were designed to mimic the side-chains of a number of arginyl dipeptides and included a benzamidine moiety substituted on a triazine ring. The ligands displayed inhibitory activities against pancreatic kallikrein which mirrored the specificity constants of the dipeptides they were designed to mimic. The ligand with the highest affinity for the enzyme, an analogue of a Phe-Arg dipeptide, when immobilized to Sepharose CL-4B via a hexamethylene spacer arm, purified pancreatic kallikrein 110-fold in one step from a crude pancreatic acetone extract.

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Section: Introductionmentioning

confidence: 99%

Design of novel affinity adsorbents for the purification of trypsin‐like proteases

Burton

Lowe

1992

J of Molecular Recognition

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“…Two types of plasminogen-activating enzymes can be distinguished, as based on mol. wt., immunological reactivity, and data on the amino acid sequences of the proteins and the nucleotide sequences of the corresponding cDNA (Aoki and Kaulla, 1971;Unkeless et al, 1974;Christman et al, 1975;Dan0 and Reich, 1978;Granelli-Piperno and Astedt, 1979;Vetterlein et al, 1979;Dan0 et al, 1980b;Rijken et al, 1980;Roblin and Young, 1980;Wilson et al, 1980;Rijken and Collen, 1981;Gunzler et al, 1982;Kaltoft et al, 1982;Schaller et al, 1982;Steffens et al, 1982; Ic IRL Press Limited, Oxford, England. Edlund et al 1983;Nielsen et al, 1983;Pennica et al, 1983).…”

Section: Introductionmentioning

confidence: 99%

Inactive proenzyme to tissue-type plasminogen activator from human melanoma cells, identified after affinity purification with a monoclonal antibody.

Andreasen¹,

Nielsen²,

Grøndahl-Hansen³

et al. 1984

The EMBO Journal

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The human 66 000 mol. wt. plasminogen activator (HPA66; tissue‐type plasminogen activator) has been purified from melanoma cells by a one‐step affinity method with a monoclonal antibody. HPA66 purified in this way consists mainly of a one‐polypeptide chain form with small amounts (15%) of a form containing two polypeptide chains held together by one or more disulphide bridges. The one‐chain form was converted to the two‐chain form by catalytic amounts of plasmin. During the conversion, the enzyme activity of HPA66, as measured by an [125I]plasminogen conversion assay and with a chromogenic substrate, increased linearly with the percentage of the two‐chain form. A linear regression analysis showed that all enzyme activity could be accounted for by the two‐chain form, while the one‐chain form had no measurable enzyme activity (detection limit approximately 5% of the activity of the two‐chain form). Together with previous findings of inactive proenzymes to murine and human approximately 50 000 mol. wt. (urokinase‐type) plasminogen activators, these findings indicate that plasminogen activators are generally formed from inactive one‐chain proenzymes which are converted to active two‐chain enzymes by limited proteolysis, thus demonstrating a third step in a cascade reaction leading to extracellular proteolysis.

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“…At present, mammalian plasminogen-activating enzymes seem to fall into two distinct types differing in mol. wt., immunological reactivity and possibly in biological function (Aoki and Kaulla, 1971;Unkeless et al, 1974b;Rifkin et al, 1974;Christman et al, 1975;Granelli-Piperno and Reich, 1978;Dan0 and Reich, 1978;Astedt, 1979;Vetterlein et al, 1979;Dano et al, 1980b; Roblin and Young, 1980;Rijken et al, 1980;Wilson et al, 1980;Matsuo et al, 1981;Rijken and Collen, 1981; *To whom reprint requests should be sent.…”

Section: Introductionmentioning

confidence: 99%

Monoclonal antibody to human 66,000 molecular weight plasminogen activator from melanoma cells. Specific enzyme inhibition and one-step affinity purification.

et al. 1983

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Hybridomas producing a monoclonal IgG, antibody to a human plasminogen-activating enzyme with an apparent mol. wt. of 66 000 (66 K, HPA66) from human melanoma cells were obtained by fusion of NSI-Ag 4/1 mouse myeloma cells with spleen cells from a mouse immunized with a partially purified preparation of the enzyme. Screening for clones of hybridomas producing antibodies to HPA66 was performed with the impure enzyme preparation. A preliminary screening included enzyme-linked immunosorbent assay and SDS-polyacrylamide gel electrophoresis (SDS-PAGE) followed by immunoblotting; the final identification was based on inhibition of the enzymatic activity of HPA66 which was complete at high antibody concentrations. No inhibition of three other human and murine plasminogen activators or of plasmin was observed. Employing a one-step affinity procedure with the antibody coupled to Sepharose, HPA66 was purified -200-fold from conditioned medium from the melanoma cells with a yield of 79Wo. The purified HPA66 was homogeneous as evaluated by SDS-PAGE. Electrophoresis under reducing conditions indicated that it consisted of one polypeptide chain. The binding constant between the antibody and 1251-labelled HPA66 was 2.5 x 109 l/mol. The antibody did not bind to a variety of other plasminogen activators, including 52-K and 36-K human enzymes and 48-K and 75-K murine enzymes. Previously, a monoclonal antibody against another enzyme was derived by the sole use of enzyme inhibition for screening. The present study represents a modification of this procedure that can be used when antibody-unrelated inhibitors of the enzyme are present in hybridoma culture fluid.

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Purification of Urokinase by a Beta-Naphthamidine Affinity Column

Cited by 10 publications

References 2 publications

Design of novel affinity adsorbents for the purification of trypsin‐like proteases

Design of novel affinity adsorbents for the purification of trypsin‐like proteases

Inactive proenzyme to tissue-type plasminogen activator from human melanoma cells, identified after affinity purification with a monoclonal antibody.

Monoclonal antibody to human 66,000 molecular weight plasminogen activator from melanoma cells. Specific enzyme inhibition and one-step affinity purification.

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