Preparation and properties of leucine aminopeptidase

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Publication Type dissertation
School or College School of Medicine
Department Biochemistry
Author Spackman, Darrel H.
Title Preparation and properties of leucine aminopeptidase
Date 1954-06
Description Swine kidney has been used as a source for leucine aminopeptidase. A purification procedure has been worked out to produce highly purified preparations of this enzyme in good yield. The 8 steps involve conversion to an acetone powder, fractional precipitation with inorganic salts and with organic solvents, heating, aging, and finally separation by zone electrophoresis. Preparations have been obtained with a C1 = 88 for L-leucinamide representing a 1600 fold purification over the level found in crude kidney extracts. Such preparations appear to be homogeneous in the ultracentrifuge, and upon paper electrophoresis and very nearly homogeneous in the Tiselius electrophoresis. A tentative molecular weight of 300,000 has been determined for this enzyme. The aminopeptidase has been further studied by determining its absorption spectrum in the ultraviolet and by determining its amino acid content. Over 97 per cent of the protein has been accounted for as the naturally occurring amino acids and this evidence together with the previous evidence indicate that leucine aminopeptidase is a large, simple protein and there is no evidence for any prosthetic group, The activation and stability of this enzyme in the presence of its two specific metal activators, Mn++ and Mg++, have been studied in some detail and a comparison of the effects of the two metal activators made. Activation times for both metals have decreased from several hours to a few minutes with purification and in the highly purified state, rapid inactivation occurs upon removal of the metal components. A substrate-activation effect has been noted at certain metal concentrations and the implications of this have been discussed. The metal dissociation constants for both metals have been determined and are found to be 1.2 X 10(4) molar-1 for Mg++ and 3.3 1 10(4) molar-1 for Mn++. A relationship between the enzyme and each of the metals has been found which suggests that one metal ion combines for each active site of enzyme. The variation of activity with pH has been studied for 2 substrates and with both metals and the pH optimum for the enzyme appears to be 9.1 to 9.3. Several new amino acid amides have been synthesized making available nearly all of the naturally occurring amino acids in amide form. A total of more than 50 substrates have been subjected to the action of the highly purified leucine aminopeptidase and it has been found that a general activity is shown. L-alanyl-L-leucinamide is hydrolyzed with a C0 of 26,000 corresponding to a turnover number of 208,000 moles hydrolyzed per minute per 100,000 gm. of enzyme. Evidence is reviewed for the elimination of the presence of other enzymes and various aspects of specificity are discussed including a comparison with other well characterized proteolytic enzymes.
Type Text
Publisher University of Utah
Subject Metabolism
Subject MESH Leucine; Aminopeptidases; Proteins
Dissertation Institution University of Utah
Dissertation Name PhD
Language eng
Relation is Version of Digital reproduction of "Preparation and properties of leucine aminopeptidase". Spencer S. Eccles Health Sciences Library.
Rights Management © Darrel H. Spackman.
Format Medium application/pdf
Format Extent 4,963,217 bytes
Identifier undthes,3791
Source Original: University of Utah Spencer S. Eccles Health Sciences Library (no longer available)
Funding/Fellowship United States Public Health Service Predoctoral Fellowship.
Master File Extent 4,963,252 bytes
ARK ark:/87278/s6tb18sd
Setname ir_etd
Date Created 2012-04-24
Date Modified 2012-04-24
ID 192017
Reference URL