Abstract
Abstract A proenzyme (hereafter propeptidase), with a high activity on N-acetyl-l-tyrosine ethyl ester, was found associated with proelastase through several steps of purification. Propeptidase is distinct from chymotrypsinogen A as shown by its electrophoretic mobility and its high resistance, in the activated form, to inhibition by l-1-tosylamido-2-phenyl-ethyl chloromethyl ketone. Evidence indicates that propeptidase is probably the proenzyme form of elastomucase. Activation of proelastase and propeptidase with trypsin changed their electrophoretic mobilities. The activated proelastase migrated at the same rate as authentic, pure elastase. The proenzymes that were originally highly insoluble could be solubilized by treatment with alumina Cγ gel. The solubilizing effect of alumina Cγ gel was reversible. The solubilized proenzymes could be reprecipitated by recombination with the eluate of alumina Cγ gel. Activation of the proenzymes had no effect on their precipitation with Cγ eluate. To a lesser degree, trypsin and chymotrypsinogen A also precipitated upon mixing them with Cγ eluate. Proelastase and propeptidase each were obtained in an electrophoretically pure state by column chromatography on hydroxylapatite.
Highlights
Tauber [13] achieveda 40-fold purification, by euglobulin precipitation, but their preliminary attempts to purify it further on a carboxymethyl cellulosecolumn resulted in almost complete loss of activity
The chromatographicisolationof native proelastaseon hydroxylapatite columnwasreported by mein 1967in a changed their electrophoretic mobilities
The activated proelastase migrated at the same rate as authentic, pure elastase
Summary
Enzyme Assay--Elastin, for the measurementof proelastase, waspreparedaccordingto the method of Partridge, Davis, and. Adair [16], as modified by Donovan [17]. The dry elastin powder was dyed with Congo Red as describedby Naughton and Sanger [3]. Proelastasewas activated in 0.1 M Tris-HCl, pH 8.9, at roomtemperaturewith 25pg of trypsin per ml. After 20 min, activation was stoppedwith the addition of a 2-fold excessof STI.. Control sampleswere treated except that the trypsin andST1 wereaddedin reverseorder. Aliquots of activated and control preparations, each at two different concentrations,wereassayedin 0.1 M Tris-HCl, pH 8.9, containing 1.40mg of CongoRed-dyed elastin per ml, in 2.0 ml final
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