Abstract:Because it is well known that excess branchedchain amino acids (BCAAs) have a profound influence on neurological function, studies were conducted to determine the impact of BCAAs on neuronal and astrocytic metabolism and on trafficking between neurons and astrocytes. The first step in the metabolism of BCAAs is transamination with a-ketoglutarate to form the branched-chain a-keto acids (BCKAs). The brain is unique in that it expresses two separate branched-chain aminotransferase (BOAT) isoenzymes. One is the common peripheral form [mitochondrial (BCATm)], and the other [cytosolic(BCATc)] is unique to cerebral tissue, placenta, and ovaries. Therefore, attempts were made to define the isoenzymes' spatial distribution and whether they might play separate metabolic roles. Studies were conducted on primary rat brain cell cultures enriched in either astroglia or neurons. The data show that over time BCATm becomes the predominant isoenzyme in astrocyte cultures and that BCATc is prominent in early neuronal cultures. The data also show that gabapentin, a structural analogue of leucine with anticonvulsant properties, is a competitive inhibitor of BCATc but that it does not inhibit BCATm. Metabolic studies indicated that BCAAs promote the efflux of glutamine from astrocytes and that gabapentin can replace leucine as an exchange substrate. Studying astrocyte-enriched cultures in the presence of [U-14C]glutamate we found that BOKAs, but not BCAAs, stimulate glutamate transamination to a-ketoglutarate and thus irreversible decarboxylation of glutamate to pyruvate and lactate, thereby promoting glutamate oxidative breakdown. Oxidation of glutamate appeared to be largely dependent on the presence of an a-keto acid acceptor for transamination in astrocyte cultures and independent of astrocytic glutamate dehydrogenase activity. The data are discussed in terms of a putative BCAAJBCKA shuttle, where BOATs and BCAAs provide the amino group for glutamate synthesis from a-ketoglutarate via BCATm in astrocytes and thereby promote glutamine transfer to neurons, whereas BCATc reaminates the amino acids in neurons for another cycle.

We have developed overexpression systems for the human branched-chain aminotransferase isoenzymes. The enzymes function as dimers and have substrate specificity comparable with the rat enzymes. The human cytosolic enzyme appears to turn over 2-5 times faster than the mitochondrial enzyme, and there may be anion and cation effects on the kinetics of both enzymes. The two proteins demonstrate similar absorption profiles, and the far UV circular dichroism spectra show that no global structural changes occur when the proteins are converted from the pyridoxal to pyridoxamine form. On the other hand, the near UV circular dichroism spectra suggest differences in the local environment surrounding tyrosines within these proteins. Both enzymes require a reducing environment for maximal activity, but the mitochondrial enzyme can be inhibited by nickel ions in the presence of reducing agents, while the cytosolic enzyme is unaffected. Chemical denaturation profiles of the proteins show that there are differences in structural stability. Titration of -SH groups with 5,5-dithiobis(2-nitrobenzoic acid) suggests that no disulfide bonds are present in the mitochondrial enzyme and that at least two disulfide bonds are present in the cytosolic enzyme. Two -SH groups are titrated in the native form of the mitochondrial enzyme, leading to complete inhibition of activity, while only one -SH group is titrated in the cytosolic enzyme with no effect on activity. Although these proteins share 58% identity in primary amino acid sequence, the local environment surrounding the active site appears unique for each isoenzyme.

The Escherichia coli RecA protein performs the DNA strand-exchange reaction utilized in both genetic recombination and DNA repair. The binding of nucleotides triggers conformational changes throughout the protein resulting in the RecA-ATP (high DNA affinity) and RecA-ADP (low DNA affinity) structures. Difference infrared spectroscopy has allowed us to study protein structural changes in RecA that occur after binding ADP or ATP. Experiments were performed on control and uniformly (15)N-labeled RecA in an effort to assign vibrational changes to protein structures and study the molecular changes associated with the allosteric regulation of RecA. Comparison of RecA-ATP and RecA-ADP data indicates that the protein adopts unique secondary structures in each form and altered N-H stretching vibrations in the RecA-ADP structure not observed in the RecA-ATP data. Numerous vibrations throughout the 1700-1300 cm(-)(1) region are influenced by isotopic substitution and imply that many nitrogen-containing side chains are altered after ADP binds to RecA. The RecA-ATP data contain unique vibrations that are not observed in the RecA-ADP data and may be associated with Gln, Lys, Arg, or Asn. Model compound studies on control and (15)N-labeled glutamine and lysine provide additional evidence that supports the tentative assignments of vibrations observed in our difference spectra. In addition, we provide evidence that nitrogen-containing amino acids are important in locking in the low-DNA affinity, more compact conformation of the protein and that some of these interactions may not be present in a more extended, flexible RecA-ATP conformation.

The goal of this paper was to determine the contribution of the mitochondrial branched chain aminotransferase (BCATm) to branched chain alpha-keto acid transport within rat heart mitochondria. Isolated heart mitochondria were treated with sulfhydryl reagents of varying permeability, and the data suggest that essential cysteine residues in BCATm are accessible from the cytosolic face of the inner membrane. Treatment with 15 nmol/mg N-ethylmaleimide (NEM) inhibited initial rates of alpha-ketoisocaproate (KIC) uptake in reconstituted mitochondrial detergent extracts by 70% and in the intact organelle by 50%. KIC protected against inhibition suggesting that NEM labeled a cysteine residue that is inaccessible when substrate is bound to the enzyme. Additionally, the apparent mitochondrial equilibrium KIC concentration was decreased 50-60% after NEM labeling, and this difference could not be attributed to effects of NEM on matrix pH or KIC oxidation. In fact, NEM was a better inhibitor of KIC oxidation than rotenone. Measuring matrix aspartate and glutamate levels revealed that the effects of NEM on the steady-state KIC concentration resulted from inhibition of BCATm catalyzed transamination of KIC with matrix glutamate to form leucine. Furthermore, circular dichroism spectra of recombinant human BCATm with liposomes showed that the commercial lipids used in the reconstituted transport assay contain BCAT amino acid substrates. Thus BCATm is distinct from the branched chain alpha-keto acid carrier but may interact with the inner mitochondrial membrane, and it is necessary to inhibit or remove transaminase activity in both intact and reconstituted systems prior to quantifying transport of alpha-keto acids which are transaminase substrates.

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