BIOCHEMICAL AND MOLECULAR STUDIES OF NORMAL AND MUTANT HUMAN HEME BIOSYNTHETIC ENZYMES.
Item
-
Title
-
BIOCHEMICAL AND MOLECULAR STUDIES OF NORMAL AND MUTANT HUMAN HEME BIOSYNTHETIC ENZYMES.
-
Identifier
-
AAI8629723
-
identifier
-
8629723
-
Creator
-
OSTASIEWICZ, LUDMILA TERESA.
-
Contributor
-
Robert J. Desnick
-
Date
-
1986
-
Language
-
English
-
Publisher
-
City University of New York.
-
Subject
-
Chemistry, Biochemistry
-
Abstract
-
PBG-deaminase was studied in erythrocyte lysates from 165 AIP heterozygotes from 92 unrelated families. Immunologic, physical, and kinetic characterization revealed four classes of PBG-deaminase mutations. In the majority of families, the amount of immunoreactive enzyme protein corresponded to enzymatic activity (CRIM-negative Type 1). In three families, patients had normal levels of erythrocyte PBG-deaminase activity (CRIM-negative Type 2). Two types of CRIM-positive mutations were identified: the Type 1 mutation had a CRIM/activity ratio of (TURNEQ)1.7 and a crossed-immunoelectrophoretic profile in which all the enzyme intermediates were increased with the B intermediate predominant (B > A >> C (TURNEQ) D > E). The mutation altered both the kinetics and stability of the noncatalytic immunoreactive enzyme protein. The second CRIM-positive mutation, Type 2, had a CRIM/activity ratio of (TURNEQ)5.7. Crossed-immunoelectrophoresis revealed markedly increased amounts of substrate bound intermediates (B > C > D > E >> A) with increased resistance to intraerythrocyte proteolysis.;Three monoclonal antibodies were produced to human ALA-D and used to develop a rapid immunoaffinity purification procedure for the enzyme. Crude enzyme preparations were applied to the column and the enzyme eluted with 3 M KSCN resulting in a 19,500-fold increase in purity, an overall recovery of activity of 26% and enyzme which was greater than 95% pure. Three human ALA-D isozymes 1-1, 1-2, and 2-2 were affinity purified and characterized. The isozymes exhibited similar pH optima, apparent k(,m) values, stabilities, and effects of activators and inhibitors. They were distinguishable by starch gel electrophoresis, isoelectric focusing (pI 1-1 = 5.31; pI 1-2 = 5.17-5.31; pI 2-2 = 5.17), and DEAE-cellulose chromatography. In the presence of 0.05 mM zinc, the 1-1, 1-2, and 2-2 isozymes eluted from DEAE-cellulose at 0.180, 0.205, and 0.230 M KCl, respectively.;Human erythrocyte ALA-D was purified to homogeneity and the N-terminal amino acid sequence determined. Mixed oligonucleotide probes corresponding to this sequence, as well as to bovine sequences, were synthesized and used, along with monospecific polyclonal rabbit anti-human ALA-D antibodies in the isolation of an 827 base pair clone for ALA-D from a pEX human liver cDNA expression library. The authenticity of this cDNA was demonstrated by colinearity of the predicted amino acid sequence with peptide sequences from the purified enzyme.
-
Type
-
dissertation
-
Source
-
PQT Legacy CUNY.xlsx
-
degree
-
Ph.D.
-
Program
-
Biomedical Sciences
