Isolation of two high-molecular-mass proteinases from human erythrocytes.
Two forms of a neutral--alkaline high-molecular-mass proteinase (termed A1 and A2) have been purified from human erythrocytes by a procedure including a DEAE-cellulose batchwise treatment of erythrocyte cytosol, gel filtration and DEAE-cellulose chromatography. Both enzymes show distinctive properties of multicatalytic proteinases. They have an apparent molecular mass of 700 kDa and are composed by eight major subunits (23-32 kDa). Both enzymes show a proteinase activity towards casein and hydrolyze synthetic peptides with tyrosine, arginine or lysine at the P1 position. Among the synthetic peptides tested, the tetrapeptide succinyl-leucyl-leucyl-valyl-tyrosyl-7-amido-4-methylcoumarin and tripeptides with arginine in the P1 position (benzyloxycarbonyl-valyl-leucyl-arginyl-4-methoxy-2-naphthylamide and benzyloxycarbonyl-alanyl-arginyl-arginyl-4-methoxy-2-naphthylamide) are the most effective substrates. The proteinases are devoid of amino and diaminopeptidase activity. Both enzymes are completely inhibited by hemin, chymostatin and thiol-group reagents. However, the enzymes can be distinguished by the isoelectric point, the different effect of nucleotides, glutathione disulphide, sodium dodecyl sulfate and cations on the catalytic activity.
Istituto di Scienze Biochimiche, Facoltà di Medicina, Università G. D'Annunzio, Chieti, Italy.
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D000595:Amino Acid Sequence; D002848:Chromatography, DEAE-Cellulose; D002850:Chromatography, Gel; D003546:Cysteine Endopeptidases / Q000302:isolation & purification; D010450:Endopeptidases / Q000097:blood / Q000302:isolation & purification*; D004912:Erythrocytes / Q000201:enzymology*; D006427:Hemin / Q000494:pharmacology; D006801:Humans; D007526:Isoelectric Point; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000097:blood / Q000302:isolation & purification*; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000302:isolation & purification; D009842:Oligopeptides / Q000494:pharmacology; D010455:Peptides / Q000378:metabolism; D046988:Proteasome Endopeptidase Complex; D013379:Substrate Specificity; D013438:Sulfhydryl Compounds / Q000494:pharmacology
D007527:Isoenzymes; D009097:Multienzyme Complexes; D009842:Oligopeptides; D010455:Peptides; D013438:Sulfhydryl Compounds; D006427:Hemin; C002101:chymostatin; D010450:Endopeptidases; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex