Date of Award
1-1-1986
Document Type
Dissertation
Degree Name
Doctor of Philosophy (PhD)
Department
Biochemistry and Molecular Biology
Abstract
The purpose of this study was to purify rabbit hepatic mitochondrial phosphoenolpyruvate carboxykinase (PEPCK) and characterize certain physical properties of the purified cytoplasmic and mitochondrial forms of PEPCK in rabbit liver.Purification to >95 percent homogeneity of rabbit hepatic mitochondrial PEPCK was accomplished with the use of QAE Sephadex chromatography followed by Bio-Gel HTP (hydroxyapatite) chromatography and Reactive Blue 2-Agarose chromatography as the final step. A 134-fold purification of the enzyme was achieved with an overall yield of 14 percent and a final specific activity of 13.6 (mu)moles/min/mg protein. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of the enzyme indicated a molecular weight on the order of 64,000 for the mitochondrial enzyme.Intramitochondrial localization of PEPCK by mitochondrial swelling followed by discontinuous sucrose gradient centrifugation and Lubrol PX treatment indicated that mitochondrial enzymatic activity is located entirely within the matrix of the mitochondria.Polyacrylamide gel isoelectric focusing of rabbit hepatic PEPCKs indicated that the cytoplasmic enzyme has a pI of 5.30 while the mitochondrial form of the enzyme has a pI of 6.20.Using reverse phase high performance liquid chromatography, peptide maps were developed of trypsin digestion and cyanogen bromide cleavages of purified rabbit hepatic cytoplasmic and mitochondrial PEPCKs. The results indicated substantial differences in primary amino acid structure between the two enzymes.Polyclonal IgG antibodies were developed in pygmy goats against purified rabbit hepatic cytoplasmic and mitochondrial PEPCKs. Partial purification of the antibodies by 45 percent ammonium sulfate fractionation followed by DEAE cellulose chromatography led to titers greater than 1/2048. Antibody titers were determined using a Dot Enzyme-Linked Immunosorbent Assay modified for PEPCK antigen-antibody reactions. Inhibition of enzymatic activity by the antibodies developed against their respective antigens was investigated. The results indicated that 22.5 mg/ml cytoplasmic PEPCK antibody totally inhibited the activity by 10 (mu)g purified cytoplasmic PEPCK while the same concentration of antibody inhibit 10 (mu)g purified mitochondrial PEPCK enzyme 25 percent. Mitochondrial PEPCK antibody, 10 mg/ml, totally inhibited the activity of 10 mg purified mitochondrial PEPCK while the same concentration of antibody inhibit 10 (mu)g purified cytoplasmic enzyme 10 percent.
Recommended Citation
Gallwitz, Wolfgang Ernst, "Characterization Of Certain Physical Properties Of The Purified Cytoplasmic And Mitochondrial Forms Of Phosphoenolpyruvate Carboxykinase In Rabbit Liver." (1986). Theses and Dissertations. 8641.
https://commons.und.edu/theses/8641