This is a proposal to study mitochondrial and cytosolic aspartate transaminase isozymes.
We aim to: a) understand molecular forces responsible for each protein's stability, and the characterization of structural elements associated with specific roles in each of reaction mechanisms (chemistry); b) investigate their susceptibility to endocellular protein processing events (proteases) and the mechanisms of protein passage into organelles such as mitochondria (biology); and c) manipulate chemically and genetically to perturb specific enzymatic functions, structural protein elements or those necessary for recognition for passage through membranes (molecular biology). To this end we propose to: a) utilize differential scanning and batch calorimetry, 31P and 13CNMR, as well as FTIR to analyze structural factors affecting each isozyme's stability induced as a consequence of modification by action of selected proteases or chemical manipulation b) characterize the pyrophosphatase-like activity of the apo form of the isozymes; c) study the effects of protease action or chemical modification of the isozymes on mitochondrial recognition and/or protein transport mechanisms; d) produce semisynthetic enzymes in which the 14-19 residue N-terminal portion is judiciously altered and follow the consequences of such alterations; e) search for a mechanisms of action of factors, such as polyphosphates, controlling endocellular protease processing of the isozymes; and f) isolate and express the mitochondrial isozyme's cDNA in cells to produce precursor forms of this isozyme to study the molecular properties of the precursor, its mechanism of transport into mitochondria, and process of conversion into mature isozyme.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL038412-03
Application #
3354648
Study Section
Biochemistry Study Section (BIO)
Project Start
1986-09-01
Project End
1991-08-31
Budget Start
1988-09-01
Budget End
1989-08-31
Support Year
3
Fiscal Year
1988
Total Cost
Indirect Cost
Name
University of Missouri Kansas City
Department
Type
Overall Medical
DUNS #
800772162
City
Kansas City
State
MO
Country
United States
Zip Code
64110
Mattingly Jr, J R; Yanez, A J; Martinez-Carrion, M (2000) The folding of nascent mitochondrial aspartate aminotransferase synthesized in a cell-free extract can be assisted by GroEL and GroES. Arch Biochem Biophys 382:113-22
Artigues, A; Crawford, D L; Iriarte, A et al. (1998) Divergent Hsc70 binding properties of mitochondrial and cytosolic aspartate aminotransferase. Implications for their segregation to different cellular compartments. J Biol Chem 273:33130-4
Torella, C; Mattingly Jr, J R; Artigues, A et al. (1998) Insight into the conformation of protein folding intermediate(s) trapped by GroEL. J Biol Chem 273:3915-25
Lain, B; Yanez, A; Iriarte, A et al. (1998) Aminotransferase variants as probes for the role of the N-terminal region of a mature protein in mitochondrial precursor import and processing. J Biol Chem 273:4406-15
Donate, F; Artigues, A; Iriarte, A et al. (1998) Opposite behavior of two isozymes when refolding in the presence of non-ionic detergents. Protein Sci 7:1811-20
Artigues, A; Iriarte, A; Martinez-Carrion, M (1997) Refolding intermediates of acid-unfolded mitochondrial aspartate aminotransferase bind to hsp70. J Biol Chem 272:16852-61
Mattingly Jr, J R; Iriarte, A; Martinez-Carrion, M (1995) Homologous proteins with different affinities for groEL. The refolding of the aspartate aminotransferase isozymes at varying temperatures. J Biol Chem 270:1138-48
Lain, B; Iriarte, A; Mattingly Jr, J R et al. (1995) Structural features of the precursor to mitochondrial aspartate aminotransferase responsible for binding to hsp70. J Biol Chem 270:24732-9
Reyes, A M; Iriarte, A; Martinez-Carrion, M (1993) Refolding of the precursor and mature forms of mitochondrial aspartate aminotransferase after guanidine hydrochloride denaturation. J Biol Chem 268:22281-91
Mattingly Jr, J R; Iriarte, A; Martinez-Carrion, M (1993) Structural features which control folding of homologous proteins in cell-free translation systems. The effect of a mitochondrial-targeting presequence on aspartate aminotransferase. J Biol Chem 268:26320-7

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