4-Aminobutyrate aminotransferase : physical studies of pig liver enzyme, molecular cloning and expression of the human brain cDNA in bacterial cells

Author

Yaa Difie Osei

Date of Award

12-1995

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Major

Biochemistry and Cellular and Molecular Biology

Major Professor

Jorge E. Churchich

Committee Members

Wesley D. Wicks, Solon Georghiou, Cynthia Peterson

Abstract

4-Aminobutyrate aminotransferase (GABA-T) was purified from pig liver using ammonium sulfate precipitation, ion-exchange chromatography and gel filtration techniques. The behavior of the enzyme was studied at acid pH, using analytical gel filtration, circular dichroism (CD) and stopped-flow spectroscopy. The results of gel filtration by FPLC in 50 mM potassium phosphate buffer indicated that the monomeric species predominates, and the quaternary structure was lost at acid pH. The monomers showed no enzyme activity, but activity was restored upon dialysis or dilution at pH 7.0 in potassium phosphate buffer. CD measurements revealed that the secondary structure of the protein remained invariant both pH 5.0 and 70. The kinetics of the dissociation of the dimeric structure at pH 5.0 were characterized by a relaxation time of 18 me when monitored by stopped-flow measurements of ANS-labeled enzyme, however, the rate of association of the monomeric species at pH 7.0 was too fast to be detected. Based on these results it is postulated that there is an increase in the number of positively charged amino acid residues at acid pël, particularly those arising from the protonation of histidine residues. If such residues happen to be at the interface between the subunits, electrostatic repulsion might contribute to destabilization of the quaternary structure

Fluorescence studies of the ANS-labeled enzyme the presence of varied concentrations of α-ketoglutarate, one of the substrates for the enzyme, showed that the fluorescence quenched by the substrate. The removal of at least 7 residues from the COOH-terminal end of the GABA-T by carboxypeptidase Y does not affect its activity.

The cDNA for human brain GABA-T was identified from a human brain cDNA library using a 670 bp Sma 1/Kpe 1 fragment of the pig brain cDNA for GABA-T. The CDNA was isolated and sequenced, and its tissue distribution was determined. Using the GCG program, an open reading frame consisting of 1503bp encoding a protein of 500 amino acids predicted, with the first 27 amino acids being assigned to the presequence. Searches of the Genbank revealed a high sequence identity (95.4%) with the rat brain, pig brain and liver enzymes, 48%, for the A. nidulans enzyme, 44% for S cerevisiae and 28% for that from E. coli

Hybridization using a 1.46 kbp fragment of the human GABA-T CONA probe for multiple human tissue Northern blots revealed that the detected levels of GABA-T mRNAs were follows (in decreasing order): liver pancreas brain kidney heart placenta, with transcript being absent in the lung and skeletal muscle, while multiple transcripts were observed for the liver and pancreas. The size of the major transcript in all the tissues was about 5.7 kb.

The cDNA corresponding to the open reading frame successfully cloned and expressed as a fusion protein using the pET vector expression system (Novagen). The expressed protein was detected by SDS-polyacrylamide gel electrophoresis followed by Coomassie blue staining or Westem blots of whole cell lysates and the protein found to be insoluble A combination of low temperature incubation for the growth of cells after IPTG induction, solubilization of the protein purification by affinity chromatography and subsequent stepwise dialysis in decreasing concentrations of urca, the presence of pyridoxal-5-phosphate and high concentrations of reducing agent, was used to obtain a soluble protein. The protein preparation showed no enzyme activity. Expression of the protein in the presence of the co-chaperones, GroEL/ES, likewise, did not result in the production of a soluble protein. It is envisaged that a soluble and active enzyme could be obtained for characterization by manipulating the solubilization parameters or expressing the gene in a mammalian expression system.

Recommended Citation

Osei, Yaa Difie, "4-Aminobutyrate aminotransferase : physical studies of pig liver enzyme, molecular cloning and expression of the human brain cDNA in bacterial cells. " PhD diss., University of Tennessee, 1995.
https://trace.tennessee.edu/utk_graddiss/10057