Isolation of Cathepsin Band a-N-Benzoylarginine-B-naphthylamide Hydrolase by Covalent Chromatography on Activated Thiol Sepharose

Cathepsin Band a-N-benzoylarginine-P,-naphthylamide (BANA) hydrolase have been isolated from bovine lymph nodes using a novel procedure that includes besides gel filtration and ion exchange chromatography also covalent chromatography as the essential step. Both enzymes were selectively bound to the activated thiol-Sepharose and afterwards eluted with cysteine. The homogeneity of enzymes was proved by polyacrylamide gel electrophoresis

Purification of Cathepsin D by Affinity Chromatography on Pepstatin Sepharose Column

A method was developed for the isolation of cathepsin D by affinity chromatography on immobilized pepstatin. This inhibitor was coupled to agarose by water soluble carbodiimide. Further purification included gel filtration on Sephadex G-100. The obtained cathepsin D exists in three active forms which were resolved on DEAE cellulose. Electrophoresis in the presence of sodium. dodecyl sulphate revealed that the first form consists of only one polypeptide chain having molecular weight 42 000. The second and the third form contain also polypeptides having molecular weight 27 000 and 14 000

Some Properties of Thymus Cathepsin D

Cathepsin D has been purified from calf thymus using ammonium sulphate precipitation and gel chromatography on Se.phadex G-100. Preparative electrophoresis in polyacrylamide gel, used as the final step in the purification procedure, yielded four active forms of cathepsin D that dissociated further .into several polypepUde bands in the presence of sodium dodecyl sulphate. All four forms were stable over a range of pH from 4-11. They were completely inhibited by pepstatin whereas other metal ions had no appreciable effect upon their activity

Acid Proteinases from Calf Lymph Nodes

Acid proteinases were isolated from calf lymph nodes using acid extraction, ammonium sulphate and acetone precipitation, followed by ion exchange chromatography on CM-cellulose and _ gel chromatography on Sephadex G-100. Cathepsin D (E. C. 3.4.23.5) is present in lymph nodes. It has the molecular weight of 39 000 as determined by gel filtration on Sephadex G-100. Cathepsins Bl and B2 (E. C. 3.4.22.1) were also isolated. Molecular weights of 22 000 and 51 000 were determined for cathepsins Bl and B2, respectively. Calf lymph nodes contain also another proteinase which degrades haemoglobin at an optimum at pH = 3.0. This proteinase has a molecular weight of 14 000 as was determined by gel filtration. Its activity is not inhibited with 0,25 ~tM pepstatin which inhibits 903/o of the cathepsin D activity

Purification and some Properties of Proteinases from Calf Thymus

Calf thymus was used for the investigation of intracellular proteinases. The tissue was homogenized, centrifuged at a low speed and applied on DEAE-cellulose. Active fractions were collected and their proteolytic activity toward different protein substrates was tested. It was found that several proteinases are present in thymus; cathepsin D being the most abundant acid proteinase. The activitiy at neutral pH was ascribed to fibrinogen degrading proteinases

Some Properties of Thymus Cathepsin D

Cathepsin D has been purified from calf thymus using ammonium sulphate precipitation and gel chromatography on Se.phadex G-100. Preparative electrophoresis in polyacrylamide gel, used as the final step in the purification procedure, yielded four active forms of cathepsin D that dissociated further .into several polypepUde bands in the presence of sodium dodecyl sulphate. All four forms were stable over a range of pH from 4-11. They were completely inhibited by pepstatin whereas other metal ions had no appreciable effect upon their activity

Some Characteristics of Cathepsin Band a-N-Benzoylarginine- B-Naphthylamide Hydrolase From Bovine Lymph Nodes

Some properties of cathepsin B and a-N-benzoylarginine-~- naphthylamide (BANA) hydrolase from bovine lymph nodes have been studies. a-N-benzoylarginine-~-naphthylamide was a sensitive substrate for both enzymes. Leucine-2-naphthylamide was cleaved only by BANA hydrolase. Degradation of low molecular weight substrates was optimal at pH = 6.0. At this pH value, the enzymes were most stable. Cathepsin B inactivated aldolase, was inhibited by 1 μM leupeptin and by thiol blocking compounds. BANA hydrolase was not inhibited by 1 μM leupeptin but showed that it required thiol compounds and EDTA for full activation. It was concluded that BANA hydrolase is very similar or identical to cathepsin H from rat liver lysosomes