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.
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Istituto di Scienze Biochimiche, Facoltà di Medicina, Università G. D'Annunzio, Chieti, Italy.
Santarone
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Battista
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Di Cola
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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