A cystatin-based affinity procedure for the isolation and analysis of papain-like cysteine proteinases from tissue extracts.
Cysteine-proteinases (CP) of the papain family can be affinity-adsorbed by egg white cystatin C coupled to Sepharose 4B, thus allowing their selective isolation from either tissue or cultured cell extracts as well as biolological fluids and culture media. CP complexed by immobilized cystatin are further analyzed by means of SDS-PAGE and Western blot followed by serial or parallel immunological detection. The single-step affinity adsorption of papain-like enzymes has the advantage, over immunoprecipitation techniques, of yielding the simultaneous and comprehensive picture of most CP, as both precursor and mature forms, in a given sample. Moreover, cell extraction in the presence of immobilized cystatin ensures a fast complexation of CP, avoiding artifacts, due to conversion, degradation, and, eventually, subtraction of constitutive enzymes from the sample because of their interactions with endogenous inhibitors. This will provide a pattern that might reflect more closely the real CP levels in intact cells. The method may be useful in the field of biochemistry, cell biology, and, possibly, clinical chemistry to perform rapid analyses of papain-like enzymes and to monitor changes in both cellular and extracellular CP profiles along with different physiopathological conditions.
Tombaccini
D
D
Department of Experimental Pathology and Oncology, Faculty of Medicine and Surgery, Viale G. B. Morgagni 50, 50134 Florence, Italy.
Mocali
A
A
Weber
E
E
Paoletti
F
F
D000327:Adsorption; D000818:Animals; D001671:Biochemistry / Q000379:methods; D015153:Blotting, Western; D002401:Cathepsin B / Q000378:metabolism; D056657:Cathepsin K; D056668:Cathepsin L; D002403:Cathepsins / Q000378:metabolism; D002478:Cells, Cultured; D002645:Chickens; D015891:Cystatins / Q000378:metabolism*; D003546:Cysteine Endopeptidases / Q000737:chemistry* / Q000302:isolation & purification*; D004305:Dose-Response Relationship, Drug; D004529:Egg White; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases; D005347:Fibroblasts / Q000378:metabolism; D006801:Humans; D008099:Liver / Q000378:metabolism; D010206:Papain / Q000737:chemistry*; D011233:Precipitin Tests; D051381:Rats; D013997:Time Factors
D015891:Cystatins; D002403:Cathepsins; D010450:Endopeptidases; D003546:Cysteine Endopeptidases; D002401:Cathepsin B; C534281:CTSL protein, human; D056668:Cathepsin L; C534280:Ctsl protein, rat; D010206:Papain; C534130:CTSK protein, human; D056657:Cathepsin K; C534133:Ctsk protein, rat