. Branched-chain amino acid catabolism: unique segregation of pathway enzymes in organ systems and peripheral nerves. Am J Physiol Endocrinol Metab 286: E64-E76, 2004. First published September 9, 2003 10.1152/ ajpendo.00276.2003We have examined the localization of the first two enzymes in the branched-chain amino acid (BCAA) catabolic pathway: the branched-chain aminotransferase (BCAT) isozymes (mitochondrial BCATm and cytosolic BCATc) and the branched-chain ␣-keto acid dehydrogenase (BCKD) enzyme complex. Antibodies specific for BCATm or BCATc were used to immunolocalize the respective isozymes in cryosections of rat tissues. BCATm was expressed in secretory epithelia throughout the digestive tract, with the most intense expression in the stomach. BCATm was also strongly expressed in secretory cells of the exocrine pancreas, uterus, and testis, as well as in the transporting epithelium of convoluted tubules in kidney. In muscle, BCATm was located in myofibrils. Liver, as predicted, was not immunoreactive for BCATm. Unexpectedly, BCATc was localized in elements of the autonomic innervation of the digestive tract, as well as in axons in the sciatic nerve. The distributions of BCATc and BCATm did not overlap. BCATm-expressing cells also expressed the second enzyme of the BCAA catabolic pathway, BCKD. In selected monkey and human tissues examined by immunoblot and/or immunohistochemistry, BCATm and BCATc were distributed in patterns very similar to those found in the rat. The results show that BCATm is in a position to regulate BCAA availability as protein precursors and anabolic signals in secretory portions of the digestive and other organ systems. The unique expression of BCATc in neurons of the peripheral nervous system, without coexpression of BCKD, raises new questions about the physiological function of this BCAT isozyme. digestive system; human; leucine; monkey; rat IN THE BODY, the nutritionally indispensable branched-chain amino acids (BCAAs) serve a number of important metabolic functions. BCAAs are key nitrogen donors for the synthesis of the metabolically significant dispensable amino acids glutamine and alanine. Glutamine is an important energy substrate for the gastrointestinal tract (38). Glutamine and alanine are also the major carriers of nitrogen from amino acid oxidation in skeletal muscle to the liver (7,20,33,48,56). In the central nervous system, BCAAs are thought to participate in an intercellular shuttle between neurons and astroglia that provides nitrogen for synthesis of the excitatory amino acid glutamate (3,4,31,39,40,64). In addition to the role of BCAAs in nitrogen metabolism, the BCAA leucine serves as an anabolic nutritional signal. Leucine stimulates protein synthesis in selected tissues via activation of the ribosomal protein S6 kinase 1 (12,19,42,61). Furthermore, high physiological concentrations of leucine stimulate secretion of insulin, and it has been postulated that this effect occurs in part via activation of glutamate dehydrogenase (43, 52).The initial reaction in the de...

Summary. The vitamin K-dependent protein, matrix Gla protein (MGP) is a binding protein for bone morphogenetic protein-2 (BMP-2). Here we present additional evidence that the Ca 2þ -induced conformer of the vitamin K-dependent Gla region in MGP is involved in BMP-2 binding. Recombinant BMP-2 binds to the Gla-containing region of MGP in the presence of Ca 2þ . Immunohistochemistry showed that calcified lesions in the aortic wall of aging rats contained elevated concentrations of MGP that was poorly g-carboxylated and did not bind BMP-2. In contrast, we were able to identify glandular structures in the mucosa of the rat nasal septum that gave bright fluorescent signals with both antigens; confocal microscopy confirmed their colocalization. These results demonstrate that the BMP-2/MGP complex exists in vivo, consistent with a role for MGP as a BMP-2 inhibitor. Age-related arterial calcification may be a consequence of under-g-carboxylation of MGP, allowing unopposed BMP-2 activity.

Abstract:The causes of arterial calci®cation are beginning to be elucidated. Macrophages, mast cells, and smooth muscle cells are the primary cells implicated in this process. The roles of a variety of bone-related proteins including bone morphogenetic protein-2 (BMP-2), matrix Gla protein (MGP), osteoprotegerin (OPG), osteopontin, and osteonectin in regulating arterial calci®cation are reviewed. Animals lacking MGP, OPG, smad6, carbonic anhydrase isoenzyme II, ®brillin-1, and klotho gene product develop varying extents of arterial calci®cation. Hyperlipidemia, vitamin D, nicotine, and warfarin, alone or in various combinations, produce arterial calci®cation in animal models. MGP has recently been discovered to be an inhibitor of bone morphogenetic protein-2, the principal osteogenic growth factor. Many of the forces that induce arterial calci®cation may act by disrupting the essential post-translational modi®cation of MGP, allowing BMP-2 to induce mineralization. MGP requires gamma-carboxylation before it is functional, and this process uses vitamin K as an essential cofactor. Vitamin K de®ciency, drugs that act as vitamin K antagonists, and oxidant stress are forces that could prevent the formation of GLA residues on MGP. The potential role of arterial apoptosis in calci®cation is discussed. Potential therapeutic options to limit the rate of arterial calci®cation are summarized.

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.

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